Giardia duodenalis, a flagellated parasitic protozoan, the most common cause of parasite-induced diarrheal diseases worldwide. Codon usage bias (CUB) is an important evolutionary character in most species. However, G. duodenalis CUB remains unclear. Thus, this study analyzes codon usage patterns to assess the restriction factors and obtain useful information in shaping G. duodenalis CUB. The neutrality analysis result indicates that G. duodenalis has a wide GC3 distribution, which significantly correlates with GC12. ENC-plot result—suggesting that most genes were close to the expected curve with only a few strayed away points. This indicates that mutational pressure and natural selection played an important role in the development of CUB. The Parity Rule 2 plot (PR2) result demonstrates that the usage of GC and AT was out of proportion. Interestingly, we identified 26 optimal codons in the G. duodenalis genome, ending with G or C. In addition, GC content, gene expression, and protein size also influence G. duodenalis CUB formation. This study systematically analyzes G. duodenalis codon usage pattern and clarifies the mechanisms of G. duodenalis CUB. These results will be very useful to identify new genes, molecular genetic manipulation, and study of G. duodenalis evolution. Codon usage bias is a widespread feature among both prokaryotes and eukaryotes, which describes synonymous codons that are not used at the same frequency in the process of gene translation ,2,3. CUBTaenia saginata [Taenia multiceps [Plasmodium falciparum [Entamoeba histolytica [Microsporidia [Caenorhabditis, Drosophila, Arabidopsis [Streptomyces [Borrelia burgdorferi [Saccharomyces cerevisiae [Giardiasis is a worldwide epidemic water source diarrhea disease, which can infect various mammals, including human beings [G. duodenalis codon usage only analyzed with small number of genes [Analysis of synonymous codon usage bias has various important applications, such as degenerate primer design , heterolsaginata , Taenia ulticeps , Plasmodlciparum , Entamoetolytica , Microspsporidia , Caenorhbidopsis , Streptoptomyces , Borreligdorferi , and Sacrevisiae ,29. Giarn beings ,31,32. Pof genes ,34.G. duodenalis from transcriptome data, which provides useful information for elucidation of the mechanism of synonymous CUB, design of gene vaccine, and better control strategy of G. duodenalis.This study investigates the CUB of G. duodenalis (Giardia Assemblage A isolate WB) obtained from GiardiaDB were investigated based on the transcriptome profiling of Oscar Franzén et al. [Nine thousand, seven hundred and forty-seven coding sequences (CDS) of n et al. . To miniG. duodenalis was obtained from Codon Usage Database via Optimizer software [G. duodenalis genes was analyzed by Codon W 1.4.4 software and EMBOSS online tools . 17 CDS sequences codon usage of G. duodenalis highly expressed variant-specific surface proteins (VSPs) [https://www.bioinformatics.nl/emboss-explorer/, accessed on 26 July 2021). The analysis parameters included an effective number of codons (ENC), the codon adaptation index (CAI), relative synonymous codon usage (RSCU), GC content (guanine and cytosine content), A3s, T3s, G3s, C3s, and GC3s. Nucleotide composition and its frequency on the third synonymous codon can be used to reflect the codon usage preference of CDS sequences. The RSCU value is the observed codon frequency divided by the frequency expected under the same assumption for amino acid synonymous codons, and it is an index for studying differences in synonymous codon usage among genes. The RSCU values were measured according to the previous study [A codon usage table of software . CDS seqs (VSPs)  were anaus study . ENC valus study . CAI refus study .The effect of selection on CUB was usually measured by neutrality plot analysis. In this method, the average GC content of GC12 and GC3s was calculated. The scatter plot was drawn with GC3s as an independent variable and GC12 as a dependent variable. The points representing genes are distributed on or near the diagonal, indicating that the codon usage pattern is greatly affected by mutation; on the contrary, the smaller the slope of the scatter formation curve is, or even parallels to the horizontal axis, indicating that codon usage pattern is greatly influenced by environmental selection .The ENC plot of ENC values plotted against GC3s values is used to analyze the influence of base composition on the codon usage in a genome . A standParity rule 2 analysis, also known as parity rule analysis, is a method to study the base composition of codons. If the gene is not under the pressure of mutation or environmental selection, the internal composition of the base is A = T, C = G. However, due to the influence of gene mutation and environmental selection pressure, the usage of G and C in the genome coding sequence is often uneven, especially the third codon deviates from the rule of intrachain equivalence. In this method, amino acids encoded by four synonymous codons were analyzed, and the calculated results of G3/ (G3 + C3) and A3/ (A3 + T3) were plotted. The coordinates  represent the PR2 principle . The distance and position of the scattered points from the center indicate the degree and direction of the gene deviation from the rules .p < 0.01) in highly-expressed genes than those with low levels of expression were defined as optimal codons [Based on the CAI values, 5% of the total genes with extremely high and low CAI values were regarded as high and low datasets, respectively. Codon usage was compared using a Chi-squared contingency test of the two groups, and codons whose frequency of usage was significantly higher  .The indices of codon usage were analyzed by CondonW1.4.4 software. Microsoft Excel and SPSS 19.0 were used to analyze the correlation based on Spearman’s rank correlation.G. duodenalis CDSs (expressed as % GC) were shown in G. duodenalis genes exhibited a distinctly unimodal distribution. The GC contents of G. duodenalis genes varied from 39.1% to 81.1% (SD = 0.0523), and the GC contents of the 5968 CDSs were mainly ranged from 45% to 55%. To understand the distribution of nucleotides, we studied the content of GC and GC3s. GC1, GC2 and GC3 were 53.38%, 41.79% and 51.67%, respectively. This result suggested that the GC2 was different from GC1, and GC3—GC2 was the lowest among the three codon positions. The mean value of GC contents of all codons was 48.95%.The nucleotide contents of G. duodenalis gene was constructed. Our result demonstrated that the GC3s of G. duodenalis genes was widely distributed (26.17% to 99.32%), and there was a significant correlation between GC12 and GC3   of the  0.0001) , indicatG. duodenalis were shown in G. duodenalis genome was 49.1%, which indicated that the G. duodenalis genome was a little AT-rich. The total codon usage was biased towards G-and C-terminal codons . These results indicated that gene composition restriction played a significant role in the formation of codon usage variation in the G. duodenalis genome. In addition, we analyzed 17 CDS sequences codon usage of G. duodenalis highly expressed VSPs genes based on the actual protein levels [G. duodenalis was obtained from Codon Usage Database via Optimizer software, and 9/10 most frequently used codons were biased towards G-and C-terminal codons /ENCexp was calculated to estimate the observed and expected ENC values more accurately. We found that (ENCexp-ENCobs)/ENCexp was mainly located in 0–0.05, and the values of most genes were located in −0.05–0.15 . The resG. duodenalis genes were further studied by RSCU correspondence analysis. We determined four major contributors as axis 1–4. Axis 1 and axis 2 accounted for 14.81% and 5.30% of the total variance, respectively, while axis 3 and axis 4 accounted for 4.05% and 3.46% of the total variance, respectively, suggesting that axis 1 and axis 2 were the main contributors to G. duodenalis CUB. Axis 1 and axis 2 showed each gene in G. duodenalis genes could be separated by the primary axis. In addition, as shown in p < 0.0001). The location of axis 1 gene was positively correlated with GC3s  and negatively correlated with ENC . These results indicated that the genes with higher GC and GC3 contents and lower ENC content on the left side of the first axis showed stronger codon bias, which demonstrated that the main factor affecting the CUB of G. duodenalis was the nucleotide composition.The differences in synonymous codon usage among p < 0.01).To investigate different gene codon usages, we selected hydrophobic genes, aromatic genes, ribosomal genes, and other genes from 5968 genes. G. duodenalis preferred to use G and T rather than use C and A . As shown in p < 0.0001), while CAI value showed positive relationship significantly with GC3s and GC content . These results demonstrated that highly expressed genes had a strong CUB and preferred to choose G or C codons in synonymous positions.To investigate the relationship between CUB and gene expression level, the correlation coefficient between CAI and ENC was calculated and analyzed. The CAI values were used to evaluate p < 0.01) all significantly correlated, suggesting that genes with higher expression levels had a smaller size.As shown in G. duodenalis were represented by the average value of RSCU in high/low expressed genes (p < 0.01), which all end in G or C, indicating that G. duodenalis preferred to use G or C ending synonymous codons.Translational optimal codons of ed genes . Chi-squCUB widely exists among both prokaryotes and eukaryotes. This is an interesting and complex phenomenon in the process of biological evolution. Previous studies have proposed hypotheses trying to explain the origin of CUB, among which the selection-mutation-drift balance model and the neutral theory are the most influential ones ,45, whicG. duodenalis codon usage was restricted, which only considered eight genes, and yet it seems that the codon usage of G. duodenalis has been quite heterogeneous [G. duodenalis codon usage investigated 65 genes, and 21 codons were the optimal codons, which were all end in C or G and almost exclusively used in the highly expressed genes [G. duodenalis has not been fully studied yet. In the present study, we performed a more comprehensive analysis based on the whole transcriptome data and found that multiple factors influence shaping G. duodenalis CUB, such as mutation pressure, selection, gene expression, and compositional constraints, and protein size.A previous analysis of ogeneous . Anothered genes , which wOnchocerca volvulus, Mycoplasma capricolum, and P. falciparum [G. duodenalis genes was 49.1%. Although the genome of G. duodenalis seems to be slightly AT-rich, the usage of all codons is biased towards G or C-terminal codons (T. saginata [T. multiceps [Generally speaking, nucleotide composition is one of the most important effect factors in forming codon usage, while GC content reflects the overall trend of codon mutation . Some GClciparum ,64,65, hl codons , which isaginata  and T. multiceps .G. duodenalis genes ranged from 24–61, suggesting that the codon bias among these genes was significantly different.Previous studies have demonstrated that over-expressed genes are expressed more frequently, and produced more protein than other genes . MoreoveG. duodenalis indicated that highly expressed genes faced greater translation selection pressure in shaping CUB, which is similar to the codon usage in T. saginata [P. falciparum [CAI is a method of recognizing differential gene expression by codon selection. Highly expressed genes exhibit a high tendency to use certain codons and tend to use them frequently ,69. In tsaginata  and P. flciparum .T. multiceps [T. saginata [S. cerevisiae [Silenelatifolia [Caenorhabditis elegans [Arabidopsis thaliana [G. duodenalis also showed a negative correlation between gene size and CUB, suggesting that selection restriction made G. duodenalis tend to produce smaller proteins with similar functions to larger proteins, thus reducing the energy consumption of producing specific functional proteins [It has been confirmed that various organisms, such as ulticeps , T. sagisaginata , S. cererevisiae , Silenelatifolia , Caenorh elegans , and Arathaliana , showed proteins .G. duodenalis [T. multiceps [T. saginata [G. duodenalis genome might provide valuable information for genetic engineering and evolutionary study.Identifying the optimal codons could provide an effective means for rational codon usage rearrangement and evolutionary molecular genetics research ,74,75. Todenalis  and otheulticeps  and T. ssaginata . IdentifG. duodenalis genome and its influencing factors. Our results indicated that the CUB of G. duodenalis seemed to be a complex equilibrium under different pressures: Natural selection, mutation, GC content, gene expression level, and protein size. Interestingly, all 26 optimal codons ended with G or C, which would be useful for cloning and expression of foreign genes in G. duodenalis. Together, our study elucidated the codon usage pattern of G. duodenalis and provided useful information for genetic engineering and evolutionary studies in this primitive eukaryote.Our study revealed the pattern of CUB in the G. duodenalis codon usage pattern and clarifies the mechanisms of G. duodenalis CUB, which will be very useful to identify new genes, molecular genetic manipulation, and study of G. duodenalis evolution.This study systematically analyzes


Balance impairment is a common consequence of chronic ankle instability (CAI). This study aimed to assess the discriminative validity of four clinical tests for quantifying balance impairment in individuals with CAI.N = 27 per group) were recruited and completed four clinical tests including the foot-lift test (FLT), the time-in-balance test (TIBT), the modified star excursion balance test in the posteromedial (mSEBT-PM) direction and the side-hop test (SHT). The receiver operating characteristics (ROC) curve coupled with Youden index were calculated to determine the optimal cut-off scores of each test.Participants were screened for their balance using the single-leg balance test (SLBT) and were assigned to either the positive or the negative SLBT groups. Fifty-four individuals with CAI . All four tests reached good to excellent sensitivity and specificity values and had significant cut-off scores to discriminate balance performance among CAI participants.All four clinical tests can be conducted with their respective cut-off scores to quantify balance impairment in individuals with CAI. A lateral ankle sprain (LAS) is a major injury that frequently occurs in athletes and physically active populations, and it has the highest recurrence rate of all musculoskeletal injuries . NotablyWithin 12 months of the initial injury, some individuals with CAI (i.e. copers) demonstrate successful sensorimotor adaptation and display no further symptoms . In contThe single-leg balance test (SLBT) is a clinical screening method for balance impairment . The SLBThe FLT is a commonly used static balance test that requires individuals to maintain 30 sec of single-leg balance and tracks the number of foot lifts and errors –11. LikeAccording to the current evidence, balance impairment is more commonly found in individuals with CAI than in control or coper individuals. However, the cut-off scores used to quantify the severity of balance impairment among the CAI population are still limited. Crucially, accurate cut-off scores provide clinical information that may help healthcare professionals and athletic trainers to identify CAI individuals with severe balance impairment and high risks of recurrent injury as well as potential copers. In this study, we examined the discriminative validity of the FLT, the TIBT, the mSEBT-PM and the SHT in CAI subjects. We screened their balance using the SLBT to divide them into positive and negative SLBT groups and then compared four balance outcomes. Finally, we established cut-off scores determining balance among the CAI group.A matched-pair case-control study was launched with physically active individuals and university athletes with CAI. The sample size was calculated using a formula that compares the means of two groups  and 27 p2,1 = 0.96)  , and it We excluded participants with any of the following conditions: i) a history of previous surgeries and/or any previous fractures in the musculoskeletal structures of the lower limb and/or spine; ii) an acute injury within the past 3 months in any musculoskeletal structure of the lower limb and/or spine; iii) any diagnosed visual disorders without correction; iv) any diagnosed vestibular disorders ; v) a hiAll participants performed the SLBT, FLT, TIBT, mSEBT-PM and SHT. We used the SLBT to screen for balance impairments . FollowiAll participants performed the SLBT first and then the other tests in a random order that we determined with simple random sampling. All tests were performed on a CAI limb. We assessed the most recent LAS limb in participants with bilateral CAI . We askeThe participants performed all tests except the mSEBT-PM without any practice sessions and they had a 1-min rest between the different tests . A raterThe participants began by balancing barefoot on a stable surface with the testing limb. They placed both of their hands on their hips and slightly bent their opposite hip and knee without allowing their foot to touch the ground or the testing limb. Next, the participants looked straight ahead, fixed their eyes on the wall, and then closed their eyes for 10 sec . The resThe participants assumed a single-leg stance by raising their opposite foot. The researcher then instructed them to touch the testing leg with it at the mid-calf level and monitored the number of foot lifts –11. The The participants maintained a single-leg stance using the same position as the SLBT  and the The participants placed their testing foot on the centre of a tape marker and their hands on their hips. The researcher then asked the participants to reach with their opposite leg as far as possible along the tape line, which extended 45° from the centre of the tape marker in the posteromedial direction , 13, 18.The participants were instructed to hop barefoot on the testing limb 10 times per trial in the mediolateral direction for 30 cm , 19. Thet-test (d: d = (μ1–μ2)/σ  for data analysis. To compare the balance performance results of the positive and negative SLBT groups, we conducted an independent sample t-test . To deteμ1–μ2)/σ . We inteectively .J values) throughout the ROC curve, which we calculated as follows: J = ([sensitivity+specificity]−1) × 100  curve, which represents the relationship between sensitivity and specificity, and the area under the ROC curve (AUC). These values indicated the discriminative performance of each test as well as their asymptotic significance (α = 0.05) . We inte1) × 100 –23.We determined the positive and negative likelihood ratios (LR) using the sensitivity and specificity at select cut-off points: LR+ = (sensitivity)/(1-specificity) and LR− = (1−sensitivity)/(specificity) . The posP = 0.015).Fifty-four individuals with CAI  voluntarily participated in the study. The average ages were 23.29 years old and 22.62 years old in the negative SLBT and positive SLBT groups, respectively. No group differences were demonstrated for demographic data, histories of LAS, giving way and/or feeling of ankle joint instability, except for participants’ CAIT scores . The negP < 0.001) coupled with large effect sizes (d > 0.80). Specifically, the negative SLBT group had superior balance results for all tests . This suggests that all tests had high diagnostic sensitivity and were able to quantify differences in balance performance among individuals with CAI. The ROC curves for all four tests are shown in n = 17 per group), with a large effect size (d = 0.94)   performe = 0.94) . However = 0.94) .d = 0.92). Their cut-off score was 25.89 sec  (g = 0.898) for the TIBT results of CAI and control groups.For the TIBT, we claimed that individuals with CAI who were unable to stand on the affected limbs with their eyes closed for 30 sec had severe balance impairment. Specifically, we found that the TIBT outperformed all other tests in quantifying balance. This finding is in line with that of a previous meta-analysis . It repo = 0.73) . Chrintz = 0.73)  studied  = 0.73) . Additio = 0.73)  found a To maintain static balance, individuals need proprioceptors that can transfer joint sense to the higher somatosensory area. The higher brain then fires a command to the targeted muscle of the lower extremities, particularly those muscles at the ankle joint, like the soleus and fibularis muscles, which help maintain a quiet stance , 25. Thed = 0.75) and even the CAI and coper groups (d = 0.95) (d = 0.66), a similar cut-off score  and the largest AUC value of all tested directions (g = 0.406)  . Our res = 0.95) . They corections . Rosen erections  also rep= 0.406) .d = 0.65) and a similar cut-off score  (P < 0.001), which may explain why their self-reported feeling of ankle joint instability was higher. Although both groups reported similar frequencies of feeling ankle joint instability in the past 6 months (P = 0.082), this data might explain the positive SLBT group’s worse results on the SHT.For the SHT, individuals in the negative SLBT group hopped faster than their counterparts. We asserted that individuals with CAI who were unable to complete 10 hops within 13 sec had severe balance impairment. Linens et al. ( = 0.70)  contrast = 0.70) . They fo = 0.70) . During  = 0.70) , 19. In Both dynamic balance measures, the mSEBT-PM and SHT, are related to sport movements, such as hopping, cutting and squatting. Hence, we recommend that clinicians selectively apply these tests with their respective cut-off points to suit each individual’s sport of choice. For instance, the SHT could be prioritised in basketball and soccer players.This study has some limitations. First, the baseline mean CAIT scores for both groups are different, and it is possible that the CAIT scores confounded the balance differences among groups. Unfortunately, there is no standard criterion to determine a CAIT score that accounts for the severity of balance impairment in the CAI population. Because all participants had CAIT scores ≤ 25 on the CAI limb, we attempted to control other factors to enhance the homogeneity of both groups. Namely, we matched participants based on their age, sex, weight, height and CAI limb . To imprThe present study presents diagnostic values that could be utilised in clinical diagnosis. Healthcare professionals and athletic trainers can gain benefits by recognising these cut-off scores to quantify and diagnose balance impairment among individuals with CAI. To improve the clinical benefits and meaningfulness of these tests, further research should apply these specific cut-off scores as indicators of injury risk or as monitoring tools after rehabilitation.


Women diagnosed with cancer at reproductive age often face potential impairments in fertility due to cancer treatments and complex and uncertain fertility decisions. The complexity of the decision-making process is often associated with psychological distress and a potential for long-term decisional regret around a decision made at diagnosis, which may have a negative impact on patients’ Quality of Life (QoL). Some factors have been associated with the experience of regret, such as patients’ perceived quality and satisfaction with the fertility counseling received, the decision to undergo fertility preservation procedures, desire for children and decisional regret. Awareness of these factors is of utmost importance to support and guide this population through their fertility decision-making process.It is well established that fertility is an important issue for young women with cancer at reproductive age, as many have not initiated or completed their parenthood goals when diagnosed. Because cancer treatments may impair fertility, women face fertility decisions that are often complex and surrounded by uncertainty. This may put patients at risk for psychological distress and the experience of regret regarding decisions made at diagnosis, which may be associated with a negative impact on women’s QoL. This narrative review addresses current knowledge about decisional regret regarding fertility preservation decisions in adult female cancer patients at reproductive age. Electronic searches were conducted on Pubmed database for articles published in English from 1 January 2000 to 1 July 2021 that assessed decisional regret following fertility decisions in young women diagnosed at childbearing age. Of the 96 articles identified, nine provided information on decisional regret regarding fertility decisions. Studies reported that, overall, decisional regret regarding oncofertility decisions was low. Factors associated with the experience of decisional regret were patients’ perceived quality and satisfaction with fertility counseling received, the decision to undergo fertility preservation, desire for children and decisional conflict. Health providers should be aware of the factors that are potentially modifiable and prone to improvement in order to reduce decisional regret. All efforts should be made to improve availability of and access to tailored high quality fertility counseling and fertility preservation. Given the growing evidence that decision aids (DAs) are effective in increasing knowledge and reducing decisional conflict and regret, their use in a routine and timely manner to complement fertility counseling is recommended. The importance of fertility to young cancer patients and the wish to have biological children in the future is well established. Potential infertility due to the negative impact of a cancer diagnosis and gonadotoxic treatments on their fertility is a distressing aspect of their condition ,2. ChemoIn oncofertility, the complexity of the decision-making process, often associated with emotional consequences for the patient , leads, A better understanding of the experience of decisional regret in oncofertility will support clinical practice towards an optimal decision-making process and contribute to improved awareness regarding the needs of this patient population during their survivorship years. The aim of this narrative review is to address current knowledge about decisional regret regarding fertility preservation decisions in young adult female cancer patients at reproductive age. Factors associated with decisional regret in oncofertility decision-making are discussed. In addition, interventions to facilitate the decision-making process and reduce the probability of future decisional regret are presented. The main research question framing this review was: What is the experience of decisional regret in young women at reproductive age diagnosed with cancer?Despite a lack of consensus regarding its conceptualization , in geneIn cancer care, research has shown that decisional regret is a common consequence of preference-sensitive decisions , given tTo address the research question, a narrative review was conducted by searching peer-reviewed journals published in English in PubMed database from 1 January 2000 to 1 July 2021, limiting the search to human females. The search was conducted using combinations of these keywords: “cancer AND fertility OR oncofertility OR fertility preservation AND regret”. The following inclusion criteria were applied: (i) studies including young female adult patients of childbearing age with a diagnosis of any type of cancer; (ii) primary research reporting on regret following fertility decisions after a diagnosis of cancer as an outcome variable. Given the discrepancies in the literature about the definition of young women, for the purposes of this review, “young” refers to women 45 years of age or younger at the time of diagnosis. Review articles, conference abstracts, editorials, commentaries, correspondence or case reports were not evaluated for this review. In total, 96 articles were retrieved. Of those, nine articles met the inclusion criteria and were included in the review ,30,31,32The extent of regret following fertility related decisions has been evaluated by studies assessing perceptions and the quality of decisions and fertility counseling ,27,28,29Collectively, the reviewed studies indicated that, in general, decisional regret was low among young women at reproductive age after facing fertility decisions following a cancer diagnosis. However, it should be noted that there is no consensus on a specific cut-off point for clinically significant decisional regret based on the DRS scores . StudiesThree studies prospectively evaluating decisional regret in order to validate the effectiveness of a DA for fertility-related decisions (intervention group) in comparison with a consumer guide , brochurSome studies reported on the association between the provision of fertility counseling before cancer treatment and the decision to preserve fertility and the experience of regret . LetournThe desire for more children at the time of cancer diagnosis was associated with the experience of higher regret in a sample of women at reproductive age with gynecologic cancer .Factors associated with the decision-making process, such as decisional conflict, were associated with regret. A Dutch study using a sample of 64 women with different cancer diagnoses  found thAccording to the studies focusing on decisional regret in oncofertility decision-making, factors related to the decision-making process such as less decisional conflict, the existence of fertility counseling and the quality of the information provided in addition to patients’ satisfaction with the counseling received were significantly associated with women’s experience of reduced regret about their fertility decisions. This provides essential information to health care professionals involved in the care of women at reproductive age with cancer, as these factors are potentially modifiable and prone to improvement in order to help prevent, reduce or manage decisional regret . UnfortuFertility and fertility preservation are important issues to be considered during cancer management for patients at reproductive age. The possibility of undergoing fertility preservation procedures after potential infertility due to cancer treatments may offer women different rewards, such as, for example, the hope of bearing a child in the future that may represent, for some patients, a sense of normalcy and fulfilling life . As docuOverall, the decision-making literature in medical settings corroborates the view that the provision of comprehensive information by health care professionals that is relevant to patients’ individual needs is an essential component of a decision-making process . Given tThere is growing evidence for the use of educational resources to assist with fertility preservation decision-making, with DAs being regarded as a useful component of fertility counseling that increases fertility information satisfaction and knowledge and has the potential to lower decisional conflict and regret, thus helping patents to make better informed decisions . DAs areDespite the methodological heterogeneity across studies focusing on decisional regret following oncofertility decisions, findings revealed that a majority of women reported low levels of decisional regret regarding fertility decisions after a cancer diagnosis. However, a subset of women may be at risk of experiencing future regret that may compromise their long-term QoL. Current available literature suggests the need to carefully identify patients at risk for psychosocial distress, difficulties in coping with fertility decision-making and reduced QoL due to fertility loss or threat of infertility related to a cancer diagnosis and treatments. It is well-documented that fertility is an integral component of women’s QoL, and all efforts should be made to improve availability of and access to high-quality fertility counseling and fertility preservation. The quality of the fertility counseling provided correlates positively with better decision-making outcomes, such as decisional conflict and regret. Given the growing evidence that DAs are effective in increasing knowledge and reducing decisional conflict and regret, and the fact that patients value educational tools to inform their choices about fertility preservation, it is recommended that they are used routinely and in a timely manner to complement fertility counseling, to assist patients in their decision-making and to support clinicians and other health professionals in their daily practice. In addition, oncofertility knowledge and communication skills training must be compulsory for the clinical multidisciplinary teams that are involved in the care of young patients. This training should equip the teams with the confidence and knowledge of how to implement high standard oncofertility care and how to engage patients effectively in fertility counseling via shared decision-making.Given the currently limited body of research on this topic, more prospective longitudinal studies are needed to understand long-term regret following oncofertility decisions and its impact on psychological adaptation to cancer and QoL, as there is some evidence suggesting that regret may fluctuate over time. Furthermore, each cancer type brings unique clinical and psychological challenges to patients related to their decision-making experience, such as, for example, the experience of regret; therefore, it is important to understand specific needs related to a particular diagnosis in order to devise support strategies that are meaningful to these patients. In this context, qualitative studies could also provide meaningful insights regarding women’s experiences of regret and QoL following fertility decisions. Decisional regret is a complex emotion, and more knowledge needs to be gathered regarding patients’ perceptions of decision-making outcomes and consequences and their impact on patients’ adaptation to the different challenges of the cancer trajectory. Finally, this review supports the need for the development and validation of interventions designed to improve patient-provider communication and knowledge in oncofertility that may have a positive effect on the quality of decision-making and clinical practice.


Strongyloides stercoralis are co-endemic throughout much of their worldwide ranges resulting in a significant incidence of coinfection. Independently, HTLV-1 induces a Th1 response and S. stercoralis infection induces a Th2 response. However, coinfection with the two pathogens has been associated with the development of S. stercoralis hyperinfection and an alteration of the Th1/Th2 balance. In this study, a model of HTLV-1 and S. stercoralis coinfection in CD34+ umbilical cord blood hematopoietic stem cell engrafted humanized mice was established. An increased level of mortality was observed in the HTLV-1 and coinfected animals when compared to the S. stercoralis infected group. The mortality was not correlated with proviral loads or total viral RNA. Analysis of cytokine profiles showed a distinct shift towards Th1 responses in HTLV-1 infected animals, a shift towards Th2 cytokines in S. stercoralis infected animals and elevated TNF-α responses in coinfected animals. HTLV-1 infected and coinfection groups showed a significant, yet non-clonal expansion of the CD4+CD25+ T-cell population. Numbers of worms in the coinfection group did not differ from those of the S. stercoralis infected group and no autoinfective larvae were found. However, infective larvae recovered from the coinfection group showed an enhancement in growth, as was seen in mice with S. stercoralis hyperinfection caused by treatment with steroids. Humanized mice coinfected with S. stercoralis and HTLV-1 demonstrate features associated with human infection with these pathogens and provide a unique opportunity to study the interaction between these two infections in vivo in the context of human immune cells.Viral and parasitic coinfections are known to lead to both enhanced disease progression and altered disease states. HTLV-1 and  Strongyloides stercoralis are co-endemic throughout much of their worldwide ranges resulting in a significant incidence of coinfection. The coincident infections result in S. stercoralis hyperinfection and an alteration of the balance of the immune responses to the virus and to the worm. In this study, a novel model of HTLV-1 and S. stercoralis coinfection was established in humanized mice. Mortality was increased in the HTLV-1 and coinfected animals as compared to the S. stercoralis infected group. Analysis of cytokine profiles showed a distinct shift towards Th1 responses in HTLV-1 infected animals, a shift towards Th2 cytokines in S. stercoralis infected animals and elevated TNF-α responses in coinfected animals. Numbers of worms in the coinfection group did not differ from those of the S. stercoralis infected group, however, infective larvae recovered from the coinfection group showed a significant enhancement in growth. Humanized mice coinfected with S. stercoralis and HTLV-1 demonstrate features associated with human infection with these pathogens and provide a unique opportunity to study the interaction between these two infections.Human infections with the virus HTLV-1 and the nematode  Strongyloides stercoralis, an intestinal nematode parasite of humans, has an estimated global prevalence of 370 million people, mostly in tropical and subtropical regions of the world  . ErythroThe TCR gene segments were amplified from peripheral blood leukocytes following previously published protocols . DNA isoS. stercoralis infection) for flow cytometry analysis  suggests an interplay between the two pathogens affecting the viability of the infected host  , while i+ cell cytokine responses , and only 8% of that of the HTLV group . There was no significant difference in the IFN-γ levels between the dual infection group and the HTLV-1 group. On the other hand, for TNF-α the levels in the HTLV group was 8% of that of the dual infection group  and the levels of the S. stercoralis group was 8% of that of the dual infection group . Thus, differences in the cytokine levels between the groups of infected mice at 7-weeks post-infection showed humanized mice infected with HTLV-1 or coinfected with HTLV-1 and S. stercoralis had significantly higher IFN-γ levels than that seen in mice only infected with S. stercoralis. TNF-α levels were significantly enhanced in coinfected animals when compared to levels in mice infected with only S. stercoralis or only HTLV-1.Group-wise comparison of the 7-week infection-induced cytokine responses, regarding the day 0 baseline responses as an offset, were performed. Significant differences were only noted in IFN-γ and TNF-α responses between infection groups at week-7. The IFN-γ levels in the S. stercoralis. This analysis was performed at only one time point because of restrictions in blood volumes that could be obtained from individual mice. The cohorts of mice used in this analysis were comprised of humanized mice generated from multiple stem cell donors in an effort to diminish any donor bias and thus ensure that representative immune responses would be observed. At 2-weeks post infection with S. stercoralis, the CD45+ lymphocyte population in humanized mice was 14% ± 1.4 of the lymphocyte gate and granulocyte gate based on forward and side scatter, which was statistically lower than the 71% ± 35 in HTLV-1 infected and 87% ± 15 in HTLV-1/S. stercoralis coinfected animals. While no significant differences were observed in the total number of CD3+ cells at this time point, the number of CD4+ cells was significantly higher in HTLV-1/S. stercoralis coinfected mice as compared to mice infected with only HTLV-1. CD4+CD25+ T-cell populations were significantly elevated in HTLV-1/S. stercoralis coinfected mice [68% ± 6] as compared to HTLV-1 infected mice [37% ± 30] [Flow cytometry was performed on human PBMC isolated from blood recovered from infected humanized mice to determine the changes in T-cell subsets associated with infection with HTLV-1 and/or 7% ± 30] .+CD25+ cells represented a clonal proliferation possibly due to ATLL. The amplified variable regions of the T-cells isolated from the mouse peripheral blood were analyzed by gel electrophoresis for evidence of concise bands which may indicate the presence of clonal proliferation. In the absence of any concise bands, it was concluded that there was general T-cell proliferation and not clonal proliferation of these T-cells.Population clonality was examined by PCR to determine if the enhanced population of CD4S. stercoralis. Parasites were categorized as L3i, adult or L1 . L3i isolated from humanized-mice with hyperinfection caused by MPA were significantly longer [513 μm ± 42] than L3i recovered from untreated mice infected with S. stercoralis and mice coinfected with both S. stercoralis and HTLV-1 . The smaller L3i recovered from mice coinfected with S. stercoralis and HTLV-1 had a mean length of 419 μm ± 34 that was statistically equal to the L3i recovered from untreated humanized mice and different from L3i recovered from mice treated with MPA [p < 0.001]. It was therefore concluded that both MPA and coinfection with HTLV-1 enhance the growth of S. stercoralis L3i in humanized mice.One of the hallmarks of  animals . Only MPd length . Measured HTLV-1 . This obS. stercoralis coinfection has been associated with the development of S. stercoralis hyperinfection [S. stercoralis hyperinfection [S. stercoralis is described, utilizing NSG mice that have been humanized with human hematopoietic stem cells [S. stercoralis resulted in multiple facets of the clinical etiology that match with human disease progression, including variability within the proviral and total viral RNA loads of HTLV-1 depending upon host, strong Th1 cytokine responses to HTLV-1 infection, limited Th2 cytokine responses to S. stercoralis infection, mortality of humanized mice with S. stercoralis hyperinfection and enhanced development of the parasite in the coinfection model when compared to a solitary S. stercoralis infection [HTLV-1 and nfection –20,42,43nfection . Small anfection ,31. In tem cells ,45. Infenfection ,47. This+ humanized mice has functional elements as well as multiple deficiencies in immune components. The stem cells are capable of differentiation into an immature, partially functional human immune system consisting of human B-cells, T-cells, monocytes and dendritic cells. Although, the functionality of each of these cell types is often compromised, they do recapitulate many of the functions of the human immune system [S. stercoralis to determine if these mice were susceptible to infection and if the human immune system was capable of recognizing and controlling the infection. All stages of the S. stercoralis life cycle developed in humanized mice. There was, however, a significant decline in the number of L3i and adult worms that were recovered from the humanized mice as compared to NSG mice. These findings demonstrate that humanized mice are susceptible to infection with S. stercoralis and based on the decrease in numbers of L3i and adult worms recovered from humanized mice, it appears that the transplanted human immune system may play a role in controlling the repeated infection with L3i of S. stercoralis.The human immune system present in the CD34e system , 45. HumS. stercoralis with minimal mortality, closely approximating rates seen in humans during uncomplicated S. stercoralis infections. When humanized mice were infected with HTLV-1 alone, a 90% mortality rate was observed by week-7. Premature death was also found to be a limitation in the NSG-d1 model, in which premature death was related to the incomplete reconstitution of a normal immune system, and the rapid polyclonal proliferation of CD4+CD25+ T-cells that infiltrate organs causing weight loss and death [S. stercoralis coinfected mice exhibited longer survival as compared with HTLV-1-infected mice alone in two of three separate experiments using different donor CD34+ stem cells, with an overall tendency for slower progression and increased survival by 49 weeks [S. stercoralis in patients with HTLV-1 associated ATLL was associated with prolonged survival as compared with ATLL patients not infected with S. stercoralis [+CD25+ T-cells was not detected however, a general proliferation was observed. While the typical rate of mortality in humans due to early and uncomplicated HTLV-1 infection is low, a much higher rate was observed in the humanized mice. Mortality due to HTLV-1 infection is typically caused by the progression of infection particularly to ATLL, although increased mortality is observed associated with HAM-TSP, and even in asymptomatic infection [+ T-cell population with increased levels of cytokine production, eliminating the time needed for progressive clonal expansion associated with additional genomic mutations, as seen in ATLL [Humanized mice tolerated infection with nd death . Interes49 weeks . These drcoralis . Thus, tnfection ,50. The  in ATLL .S. stercoralis-infected humanized mice reached 30% at 4-weeks post-parasite infection, similar to the mortality rates seen in infected MPA treated NSG mice [The mortality rates for the MPA-treated + humanized mice [+ HSCs were used to engraft the groups of humanized mice used in the present study. This may explain the variability seen in levels of proviral DNA and total viral RNA seen between the three experiments. Regardless, proviral load does not appear to be associated with mortality in this model, as HTLV-1 proviral loads continued to increase in the surviving animals.The overall levels of proviral DNA and total viral RNA in circulating human PBMC in humanized mice were measured to investigate a possible cause for mortality in mice infected with HTLV-1. Similar to the CD133zed mice , the prozed mice , in anotzed mice ,27,52. Ized mice ,53–55. Ized mice . MultiplS. stercoralis results in increased HTLV-1 proviral load, clonal abundance and oligoclonal expansion of T-cells [S. stercoralis soluble antigen stimulates the oligoclonal proliferation of HTLV-1 infected CD4+ T-cells in HTLV-1 asymptomatic carriers, with proviral load being up to five times higher in HTLV-1 carriers coinfected with S. stercoralis than in those infected with HTLV-1 alone [S. stercoralis/HTLV-1 coinfected mice. It is possible that an interaction between HTLV-1 and S. stercoralis resulting in increased viral load would have been observed in humanized mice if the infections were allowed to persist in them for extended time periods, instead of being limited by the early lethality of the HTLV-1 infection.Coinfection of people with HTLV-1 and  T-cells ,27,52. S-1 alone . ContrarS. stercoralis skews the immune response towards Th2 responses [S. stercoralis, HTLV-1 or coinfected with HTLV-1 and S. stercoralis. The Th1 cytokines IFN-γ, TNF-α and IL-12p40 and the Th2 cytokines IL-10 and IL-13 were elevated in mice infected with HTLV-1, S. stercoralis and coinfected mice. The Th2 cytokine IL-5 was only modestly, yet significantly, elevated in mice infected with S. stercoralis alone and IL-4 was not measurable in any of the infected humanized mice. In the presence of HTLV-1 and S. stercoralis the increased IL-5 levels were abated which is consistent with that seen in human coinfections [HTLV-1 infection causes an enhancement in patient’s Th1 responses that includes increases in a number of cytokines ,39,56 anesponses . Human cfections .S. stercoralis had significantly higher IFN-γ levels than that seen in mice only infected with S. stercoralis. Interestingly, TNF-α levels were significantly enhanced in coinfected animals when compared to levels in mice infected with either S. stercoralis or HTLV-1. TNF-α may play a role in the control of S. strongyloides infection based on the observation that TNF-α blockers have been reported to induce hyperinfection in patients infected with S. stercoralis [Humanized mice have been shown to develop a limited human cytokine profile in response to specific stimuli ,59. To ercoralis . TNF-α brcoralis –65.S. stercoralis and HTLV-1 exhibit lower levels of TNF-α, IFN-γ, and an overall increase in the frequency of IFN-γ expressing CD8+ and CD4+ T-cells compared to patients infected solely with HTLV-1 [S. stercoralis coinfection. This increase may have been the result of the limited Th2 response induced by S. stercoralis. It is important to recognize that most of the reported human cases of coinfection with HTLV-1 and S. stercoralis appear to be the result of chronic infections whereas the infections analyzed in this study were acute infections. It is therefore unlikely that there will be harmony between the presentation of the chronic infections in humans and the acute infections in the humanized mice. Furthermore, study of the coinfections in humanized mice has allowed for the first time the observation of the initial interaction between these two pathogens in a human immune milieu.Patients coinfected with h HTLV-1 ,57. Thesh HTLV-1 . In the S. stercoralis coinfection have higher parasite burdens and increased proportions of regulatory T-cells [+ and CD4+ T-cells were observed, suggesting that they increased proportionately with other hematopoietic cells. CD4+CD25+ positive T-cells were significantly increased in frequency in both the HTLV-1 infection and HTLV-1/S. stercoralis coinfection when compared to S. stercoralis infection alone. The increase in CD4+CD25+ positive T-cells, which are presumably regulatory T-cells, aligns with observations in humans coinfected with HTLV-1 and S. stercoralis [Patients with HTLV-1 and  T-cells . Circularcoralis .+CD25+ positive T-cells may be the result of clonal proliferation, or the development of ATLL or a regulation of the Th1 response [+CD25+ T-cells may simply be a response to alterations in the cytokine environment in mice caused by the coinfections. This finding does not preclude the future development of an ATLL-like progression, as was seen in the CD133+ humanized NRG mice at later stages of infection [The enhanced levels of CD4response . To explnfection . It is lnfection .S. stercoralis and treated with MPA develop a lethal hyperinfection, characterized by a significant increase in numbers of all stages of the parasite and the development of L3a [S. stercoralis did not result in an increase in the number of parasites or the development of L3a. Interestingly, L3i recovered from MPA treated animals were significantly longer than L3i recovered from untreated single infections in humanized mice. L3i recovered from coinfected animals were in two populations, one with the same length as L3i recovered from humanized NSG mice infected with only S. stercoralis and the other group of L3i the same length as seen in mice treated with MPA. Enhanced nematode growth is associated with the immune status of the host [S. stercoralis L3i through a shared immune-based mechanism. It has been hypothesized that MPA is effective at enhancing L3i growth through an alteration in the host immune response, which results in the develpment of hyperinfection. Infection with HTLV-1 induces equivalent enhanced growth in some L3i, yet not in others. MPA may induce a systemic response that enhances the growth of most of the L3i, whereas infection with HTLV-1 may induce a localized response that enhances the growth of L3i located in specific microenvironments of the host. The relative ability between MPA and HTLV-1 to enhance L3i growth may help explain why L3a are seen in mice treated with MPA but not in mice infected with HTLV-1. It is possible that hyperinfection would develop in mice coinfected with HTLV-1 and S. stercoralis if the infections were allowed to persist for extended time periods.NSG mice infected with t of L3a . Similarthe host –70 and MS. stercoralis. There was an increased level of mortality in the HTLV-1 and coinfected animals when compared to the S. stercoralis infected individuals. The enhanced mortality was not correlated with either the proviral loads or total viral RNA in the infected animals. Changes in the cytokine profiles in the HTLV-1 infected animals showed a distinct shift towards Th1 similar to the progression of human disease etiology, and Th2 cytokines were elevated in the S. stercoralis infected mice. Coinfection of humanized mice resulted in elevated TNF-α levels. The CD4+CD25+ T-cell population was significantly expanded in the HTLV-1 and coinfected groups, although this expansion was not clonal in nature. Worm counts in the coinfection group were not different from the S. stercoralis infected animals and they did not have any L3a in the L3 populations. There was, however, an enhancement in the lengths of the L3i recovered from the coinfection group, which suggest that there are interactions occurring between the two pathogens affecting worm development as seen in the MPA treated animals. Thus, humanized NSG mice provide a robust model to study the interactions that occur between HTLV-1, S. stercoralis and the immune system in vivo. Further development of this model with humanized mice that have been engineered to produce strong Th2 responses may further enhance the translational aspects of this model.In conclusion, humanized NSG mice are susceptible to coinfection with the human pathogens HTLV-1 and


The molecular mechanisms regulating differentiation of hepatic progenitor cells (HPCs), which play pivotal roles in liver regeneration and development, remain obscure. Autophagy and Wnt signaling pathways regulate the development and differentiation of stem cells in various organs. However, the roles of autophagy and Wnt signaling pathways in hepatic differentiation of HPCs are not well understood. Here, we describe the effects of autophagy and Wnt signaling pathways during hepatic differentiation of HPCs. We used a well-established rat hepatic progenitor cell line called WB-F344, which was treated with differentiation medium to promote differentiation of WB-F344 cells along the hepatic phenotype. Firstly, autophagy was highly activated in HPCs and gradually decreased during hepatic differentiation of HPCs. Induction of autophagy by rapamycin or starvation suppressed hepatic differentiation of HPCs. Secondly, Wnt3a signaling pathway was downregulated, and Wnt5a signaling pathway was upregulated in hepatic differentiation of HPCs. At last, Wnt3a signaling pathway was enhanced, and Wnt5a signaling pathway was inhibited by activation of autophagy during hepatic differentiation of HPCs. In summary, these results demonstrate that autophagy regulates hepatic differentiation of hepatic progenitor cells through Wnt signaling pathway. Chronic liver disease is a common clinical liver disease with high morbidity and mortality worldwide . The onlβ-catenin-dependent) and noncanonical Wnt (or β-catenin-independent) signaling pathways [Wnt signaling pathway plays a crucial role in the differentiation, proliferation, and self-renewal of stem cells in various organs , 10. Trapathways . The canpathways –15. A repathways .Autophagy is an evolutionarily conserved ubiquitous process in eukaryotic cells . AutophaIn this study, we investigated the roles of autophagy and Wnt signaling pathways during the hepatic differentiation process of HPCs. We found that autophagy was highly activated in HPCs and gradually decreased during hepatic differentiation of HPCs. Induction of autophagy by rapamycin or starvation suppressed hepatic differentiation of HPCs. Further study reported that Wnt3a signaling pathway was downregulated, and Wnt5a signaling pathway was upregulated in hepatic differentiation of HPCs. Wnt3a signaling pathway was enhanced, and Wnt5a signaling pathway was inhibited by activation of autophagy during hepatic differentiation of HPCs. These results demonstrate that autophagy regulates hepatic differentiation of hepatic progenitor cells through Wnt signaling pathway.β-catenin , Sox9 , Wnt5a , CaMKIIα , Phospho-CaMKIIα (Thr286) , and Actin  antibodies were used for Western blotting staining. HGF (315–23) and EGF (400–25) were obtained from Peprotech. OSM (495-MO) was purchased from R&D Systems. ITS-X (51500–056) was obtained from Lifetech. 2-Mercaptoethanol (21985023) was purchased from Thermo Fisher Scientific. VitC (A4403) and nicotinamide (N0636) were purchased from Sigma-Aldrich. Dexamethasone and gentamicin were purchased from Mengchao Hepatobiliary Hospital of Fujian Medical University.The P62 , LC3A/B , Alb , Aldob , Wnt3a , 2. WB-F344 cells were cultured with DMEM supplemented with 4.5 g/L glucose, 10% FBS, 0.1 mmol/L 2-mercaptoethanol, 1x ITS-X, 10 ng/mL HGF, 20 ng/mL EGF, 20 ng/mL OSM, 0.5 mmol/L VitC, 50 mg/mL gentamicin, 10 mmol/L nicotinamide, and 10−6 mol/L dexamethasone for 4 days for hepatocyte differentiation. We examined the effects of two autophagy stimuli, mTOR inhibitor rapamycin, and cell starvation, on hepatic differentiation of WB-F344 cells. WB-F344 cells were treated with hepatic differentiation medium for 2 days and cocultured rapamycin 200 nM for 24 h or serum-free medium for starve 24 h.WB-F344 cells were purchased from the Chinese Academy of Sciences, Shanghai. WB-F344 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 4.5 g/L glucose and 10% foetal bovine serum (FBS) at 37°C with 5% COβ-catenin (1 : 5000), Sox9 (1 : 1000), Wnt5a (1 : 1000), CaMKIIα (1 : 1000), Phospho-CaMKIIα (Thr286) (1 : 1000), and Actin (1 : 5000).Treated cells were washed with phosphate buffer saline (PBS) and lysed with RIPA buffer containing PMSF at a ratio of 100 : 1 to obtain total protein for Western blot analysis. Proteins were separated by electrophoresis on 10–12% SDS-polyacrylamide gels, transferred to polyvinylidene fluoride membrane, and incubated with corresponding primary antibodies overnight at 4°C. Following incubated with secondary antibody 1 h at room temperature. Immunoblots were developed using the BeyoECL (Beyotime) and Tanon 5200 system, and the blot was scanned and densitometric analysis with the Image J software. The primary antibodies are used in our experiment including P62 (1 : 1000), LC3A/B (1 : 1000), Alb (1 : 1000), Aldob (1 : 1000), Wnt3a (1 : 1000), 4 cells/mL. 20 μL CCK-8 solution was added into each well (containing 200 μL medium) and further cultured for 2 h at 37°C. The absorbance of each group at 450 nm was detected using an absorbance microplate reader.Cell proliferation was measured by Cell Counting Kit-8  assay according to the manufacturer's instructions. Experiments for CCK8 were performed in 96-well plates. Cells were seeded at a density of 1 × 10Total RNA from different groups of cells was extracted using TRIZOL reagent  according to the manufacturer's protocol. RT-PCR was performed using SYBR Green PCR Kit. The relative quantities of mRNAs were obtained by using the comparative Ct method and were normalized with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The primers were presented in Cells were fixed in 2.5% glutaraldehyde in PBS for 2 h at room temperature, then fixed in 1% osmium tetroxide in water for 1 h, dehydrated in an ascending series of ethanol, and at last embedded in Araldite . After solidified, 50 nm sections were cut on LKB-I ultramicrotome and picked up on copper grids, poststained with uranyl acetate and lead citrate, and observed in a Philips CM-120 TEM.t-test was used to compare between mean values of the two groups. P < 0.05 was considered statistically significant.All of the experiments were repeated at least 3 times. Data was expressed as mean ± standard deviation (SD). Statistical analysis of the data was done by using GraphPad Prism 6. The student's To investigate whether autophagy was involved in hepatic differentiation of HPCs, we used a well-established rat hepatic progenitor cell line called WB-F344, which was treated with differentiation medium to promote differentiation of WB-F344 cells along the hepatic phenotype. First, the morphology of WB-F344 cells was notably changed . Second,Next, we detected the level of autophagy (LC3-II/LC3-I and P62) during the progression of hepatic differentiation. We observed that the level of LC3-II/LC3-I was highly activated in WB-F344 cells; when treated with differentiation medium, LC3-II/LC3-I level was gradually decreased . On the To further investigate the effect of autophagy on hepatic differentiation of HPCs, we examined the effects of two autophagy stimuli, mTOR inhibitor rapamycin and nutrient deprivation medium, on hepatic differentiation of WB-F344 cells. WB-F344 cells were treated with differentiation medium for 2 days and cocultured DMSO, rapamycin, or starvation for 24 h. Western blot analysis indicated that LC3-II/LC3-I level was increased, and P62 level was decreased by rapamycin  or starvβ-catenin , Sox9, and Axin2  were downregulated during hepatic differentiation of WB-F344 cells  were increased  was suppressed, and noncanonical Wnt pathway (Wnt5a/Ca2+) was enhanced during hepatic differentiation of HPCs; activation of autophagy was enhanced Wnt3a/β-catenin signaling pathway and impeded hepatic differentiation. β-Catenin plays a key role in the transduction of Wnt pathway [β-catenin during hepatic differentiation of HPCs. Although the study of the mechanism underlying hepatic differentiation of HPCs is not sufficiently explained, the results will be helpful to further understand the mechanism of hepatic differentiation of HPCs.Autophagy is also reported to be related to Wnt signaling pathway in different cell models. Autophagy negatively regulates Wnt signaling by promoting Disheveled (Dvl) degradation in the late stages of colon cancer development . β-Catenpression . Autophapression . Autophantiation . However pathway . TherefoThe present results describe a direct link between autophagy and Wnt signaling pathways, both of which regulated hepatic differentiation. Firstly, autophagy was highly activated in HPCs and gradually decreased during hepatic differentiation of HPCs. Induction of autophagy by rapamycin or starvation suppressed hepatic differentiation of HPCs. Secondly, Wnt3a signaling pathway was downregulated, and Wnt5a signaling pathway was upregulated in hepatic differentiation of HPCs. At last, Wnt3a signaling pathway was enhanced, and Wnt5a signaling pathway was inhibited by activation of autophagy during hepatic differentiation of HPCs. These results demonstrate that autophagy regulates hepatic differentiation of hepatic progenitor cells through Wnt signaling pathway.


Ursolic acid (UA) is a pentacyclic triterpenoid found in many medicinal plants and aromas endowed with numerous in vitro pharmacological activities, including antibacterial effects. Unfortunately, UA is poorly administered in vivo, due to its water insolubility, low bioavailability, and residual systemic toxicity, thus making urgent the development of water-soluble UA formulations. Dendrimers are nonpareil macromolecules possessing highly controlled size, shape, and architecture. In dendrimers with cationic surface, the contemporary presence of inner cavities and of hydrophilic peripheral functions, allows to encapsulate hydrophobic non-water-soluble drugs as UA, to enhance their water-solubility and stability, and to promote their protracted release, thus decreasing their systemic toxicity. In this paper, aiming at developing a new UA-based antibacterial agent administrable in vivo, we reported the physical entrapment of UA in a biodegradable not cytotoxic cationic dendrimer (G4K). UA-loaded dendrimer nanoparticles (UA-G4K) were obtained, which showed a drug loading (DL%) much higher than those previously reported, a protracted release profile governed by diffusion mechanisms, and no cytotoxicity. Also, UA-G4K was characterized by principal components analysis (PCA)-processed FTIR spectroscopy, by NMR and elemental analyses, and by dynamic light scattering experiments (DLS). The water solubility of UA-G4K was found to be 1868-fold times higher than that of pristine UA, thus making its clinical application feasible. Medicinal plants have been used extensively for years in both folk and conventional medicine for years. Numerous studies have established that various beneficial bioactivities, including antibacterial effects, can result from the daily intake of natural products with a normal diet, or from the administration of isolated natural compounds found in plants . NowadayFructus mume, Gardeniae fructus, Fructus ligustri lucidi, Hedyotis diffusa willd  was added into each well and after 4 h, the medium and MTT were discarded and 100 µL dimethyl sulfoxide (DMSO) was added into each well. Finally, optical density at 490 nm was measured on a Termofischer Scientific microplate reader  to determine cells viability (%) paclitaxel was essayed in the same condition as a positive control. Determinations were made in triplicate and results were expressed as mean percentage of the control (untreated cells) ± standard deviation (SD).The cytotoxicity of G4K, UA and UA-G4K was evaluated in vitro on HeLa cell lines purchased by Termofischer Scientific . Briefly HeLa cells were increased in Dulbecco’s Modified Eagle Medium (DMEM) enriched with Fetal Bovine Serum , non-essential amino acids (1%) and antibiotics  and maintained in an atmosphere containing 5% CO® R-3000 , at 70 °C and reduced pressure. The solid residue was dissolved twice in the minimum volume of MeOH obtaining clear solutions which were precipitated in acetone, to leave in solution the eventual UA not encapsulated. After centrifugations at 3500 rpm the UA-loaded dendrimer NPs (UA-G4K) purified by free UA were obtained as a pale yellow, hygroscopic glassy solid which was stored under vacuum in a dryer (105.7 mg). Not encapsulated UA was recovered by evaporating the acetonic supernatant solutions and recrystallizing the solid residue .The UA-G4K NPs were prepared by the nanoprecipitation technique. Two miscible phases, both made of organic solvents were prepared. MeOH (7.7 mL) was used to solubilize the dendrimer G4K  and UA . Acetone (17 mL) was used as non-solvent. The clear solution of the two ingredients (G4K and UA) was added to the acetonic phase, drop-wise using a Pasteur, at room temperature and under moderate magnetic stirring (500 rpm), obtaining a milky suspension. The evaporation of the organic solvent was performed subsequently using a Rotavapor–1): 3500–3000 (NH3+ dendrimer), 3500–2500 (OH stretching UA), 1735 (C=O stretching esters of dendrimer), 1688 (C=O stretching carboxyl of UA), 1215, 1044 (C-O stretching esters of dendrimer).FTIR : δ = 0.75–0.98 , 1.00–2.40 , 2.95–3.16 , and deriving from a total of 22H, while not observable in the spectrum of empty dendrimer G4K (b), were detectable in the spectrum of UA-G4K (c), where they accounted for 726H, thus establishing for the presence of 33 UA moles per dendrimer mole (see below for more details). Furthermore, in panel (c), very intense signals due to the presence of UA were observable also at 1–2.5 ppm. Since they were provided by a higher number of proton atoms (694 vs. 423), these signals overlapped and hid the signals present in the same region belonging to G4K (b). Also, at 4.58–4.70 ppm and at 5.22 ppm, other signals peculiar to the UA structure only (a), not visible in the G4K spectrum (b) and counting for 33H each one, were detectable in the spectrum of UA-G4K (c), thus confirming the presence of 33 moles of UA. On the contrary, signals belonging to G4K only (b) and not to UA (a), known for accounting for 234H, were visible in the spectrum of UA-G4K at 4.10–4.30 (c). Finally, a signal at 2.95–3.16 ppm encompassing both signals of UA (a) and of G4K (b) was visible in the spectrum of UA-G4K, accounting for 129H. In particular, to determine the number of UA moles loaded per dendrimer mole (33) and then to compute the molecular weight (MW) of UA-G4K, the value of the integral of the signals belonging to the G4K structure only (4.10–4.30 ppm), which, as previously established, account for 234 proton atoms, was used as a reference value to determine the number of protons belonging to UA moles present in the complex, through appropriate ratios. MW obtained by the 1H NMR analysis was confirmed by elemental analysis.Unequivocally, the signals visible below 1.0 ppm in the spectrum of UA  and those of OH groups of UA (66H) were not detectable because the protons from these groups are exchanged with the proton of CD3OD.Note that the signals of NHThe SEM images of G4K and UA-G4K particles are shown in Moreover, a significant increase in the size of UA-G4K particles with respect to G4K particles is also observable, as confirmed by the DLS analyses results.mean ± SD) determined for each standard UA concentration injected, the standard concentrations of UA (CUA) used for the HPLC analyses, the UA concentrations predicted by the UA calibration model (CUAp), the residuals, and the absolute error percentages.mean) and CUA (µg/mL) data in UA) (µg/mL). Peak area (A2) was 0.9999, whose high value demonstrates the linearity of calibration. However, the linearity and sensitivity of the developed calibration model were evaluated by confirming the statistical significance of its slope, through the analysis of variance (ANOVA), performing the Fischer test. Statistical significance was established at the p-value < 0.05. By using equation Equation (3), the predicted UA concentrations (CUAp) were computed for each sample  computed by using equation Equation (3), the results concerning the concentration of UA in UA-G4K NPs and MW of UA-G4K, as well as the difference expressed as error % between the MW obtained by 1H NMR and that computed using HPLC results.By analysing six aliquots of the UA-G4K solution prepared as described in UA average ± SD) in the sample of UA-G4K analysed (333 µg/mL), resulted as 165.38 ± 1.95 µg/mL. Being the total amount of UA-G4K obtained by the encapsulation reaction 105.7 mg, the encapsulated UA resulted as 52.5 ± 6.2 mg. The DL% was calculated according to the formula reported in Area mean ± SD resulted as 29.165 ± 0.324 and the average UA concentration ± SD , and was further confirmed by elemental analysis. First, MW was estimated by considering the integral values of opportunely selected signals observable in the 1H NMR spectrum of UA-G4K. Briefly, non-overlapped signals belonging to G4K alone, whose structure and number of proton atom is known, were taken as reference. In particular, the peaks belonging to the CH2O groups of G4K and to the CHNH3+ groups of lysine (234 proton atoms) were considered. By making the proper ratios, the number of UA moles loaded by one mole of G4K was determined, which resulted in being 33. Then, the MW of UA-G4K was determined according to the following equation, Equation (4):The MW of UA-G4K was estimated by Secondly, MW of UA-G4K was estimated by using the results obtained by the HPLC analysis which provided the weight of UA present in the total weight of the complex, and therefore the number of UA moles loaded by a G4K mole, which resulted as 32.4 ± 3.8. In this case, the MW of UA-G4K was determined according to the following equation, Equation (5):1H NMR and elemental analysis.By comparing the results obtained  SLHN033NB filter , to remove eventual UA remaining in suspension and not solubilized. The filtrate was diluted (1:20) to have a final volume of 12 mL with MeOH and aliquots of 20 µL were analyzed by HPLC using the same apparatus and under the same conditions described in In vitro release of UA from UA-G4K was studied by the dialysis method, using PBS (pH = 7.4) both for dissolving UA-G4K and as receptor medium. The UA released at fixed points was expressed as UA cumulative released percentage (%) ratio between the UA released and the total entrapped UA, according to the computed DL%. The UA cumulative released (%) values were reported in a dispersion graph as a function of the incubation times. According to literature, the UA releases were monitored also for days ,49,50,51However, while the total amount of UA released by gelatin NPs at 24 h was limited (<35%), the UA released by UA-G4K was 55.7%. Note that while burst release can be useful to improve the penetration of the released bioactive compound in cells and tissues, sustained and protracted release provides a compound over an extended period with reduced toxicity. Different kinetics and related mechanisms can govern the drug release from a delivery system, including polymeric packing relaxation, polymer erosion, and molecular diffusion.2) of the linear mathematical models, which explains how the model is good for explaining the variability of data, was considered as a parameter to determine which model better fits the release data. As observable in 2 values were 0.8202 (zero order), 0.8924 (first order), 0.9211 (Korsmeyer–Peppas model), 0.8702 (Hixson–Crowell model) and 0.9507 (Higuchi model) thus establishing the UA cumulative release (%) dispersion graph best fitted with the Higuchi kinetic model.To determine the kinetics and the main mechanism which govern the release of UA from UA-G4K NPs, the UA release profile was analysed by fitting the cumulative drug release curve with zero order, first order, Higuchi, Hixson–Crowell and Korsmeyer–Peppas mathematical models ,55,56,57Q is the cumulative amount of drug release at time t, KH is the Higuchi constant, representing the slope of equation, t is the time in hours and express, and intercept should be zero. Its graphical representation and associated equation for a certain drug release profile can be obtained by reporting in a dispersion graph the cumulative (%) of the amount of drug released vs. the square root of time. Since the Higuchi model describes the drug release as a diffusion process based on the Fick’s law which is square root time (SQRT) dependent, we can assert that a Fickian diffusion was the main mechanism which governs the UA release from UA-G4K NPs thus establishing for an actual inner encapsulation of UA, rather than a simple surface absorption.The equation for Higuchi’s model is the following equation, Equation (6):For G4K the mean particle size was 333 nm and the mean PDI was 0.286, whereas for UA-G4K values were determined as 578 nm (size) and 0.235 (PDI). Concerning PDI, the value observed for UA-G4K was lower than that observed for the empty dendrimer G4K. Collectively, the PDI value determined for UA-G4K established for a low polydispersity, similar or lower than those previously reported for lipid-polymer- and dendrimer-based UA-formulations ,50,51. I1H NMR spectrum of UA-G4K provided a quantitative datum of the amount of UA trapped in G4K and allowed to compute the MW of UA-G4K. UA-G4K elemental analysis confirmed the 1H NMR data. Obviously, the drug loading (DL%) was also determined by HPLC analyses, which gave results in agreement with those obtained by NMR and elemental analysis and established a very high DL%, much higher than those previously reported. UA release experiments confirmed a sustained release profile, which fitted Higuchi’s kinetic mathematical model, and therefore governed by diffusion mechanisms. From dynamic light scattering experiments (DLS), it was established that the UA-G4K particles were nanosized and that the particle size was significantly increased compared to that of the empty dendrimer G4K, thus confirming that G4K was heavily stuffed with UA. The high content of UA (whose ζ-p is negative) was also confirmed by the ζ-p of UA-G4K which, unlike that of the G4K dendrimer reservoir (ζ-p = +66.1) was strongly negative (ζ-p =−42.6), thus also ensuring high stability in aqueous solution and low cytotoxicity, as confirmed by the viability cells experiments performed on HeLa cells. In particular, the results showed that UA-G4K NPs were not cytotoxic even at the high concentration tested and demonstrated the G4K’s ability to reduce the UA toxicity. However, among the many promising results obtained in this study, the most extraordinary remains the exceptional water solubility of the UA-G4K prepared here, which was found to be 1868 times higher than that of pristine UA, thus making possible its eventual application in clinics.Naturally occurring antimicrobials have attracted the attention of researchers in many industries, including pharmaceutical, food, and cosmetics due to their safety and non-toxicity, compared to that of several synthetic compounds. The natural constituents of several plants, including UA considered in this work, have been shown to be effective both against food spoilage and against infections by pathogenic microorganisms, but are often difficult to use as preservative ingredients and almost impossible to administer in vivo, due to their insolubility in water and low bioavailability, thus making the development of water-soluble formulations urgent. In this study, with the aim of developing a new UA-based in vivo administrable antibacterial agent, we physically entrapped UA in a biodegradable, not PAMAM structured and non-cytotoxic cationic dendrimer (G4K), thus obtaining dendrimer NPs loaded with UA (UA-G4K). A meticulous physicochemical characterization of UA-G4K NPs was performed and the interesting results were reported and discussed. In particular, FTIR spectral data, also processed by principal component analysis (PCA), and the NMR analysis confirmed the success of the encapsulation. The The overall merit of the present work consists in having prepared a highly water-soluble formulation of UA, stable in water solution, avoiding the use of co-solvents (PEG), surfactants, stabilizers, or emulsifiers, which can turn hazardous for humans. Only an easy and fast physical entrapment strategy, using G4K as reservoir, protector, and solubility enhancer has been implemented. Our UA-formulation could function as a prototype organic nanomaterial to inspire further studies in this direction.


This review summarizes recent studies revealing the complexity of D1 signaling and its clinical implications, and suggests functional selectivity as a promising strategy for drug discovery to magnify the merit of D1 signaling. Functional selectivity/biased receptor signaling has become a major research front because of its potential to improve therapeutics through precise targeting. Retrospective pharmacological review indicated that many D1 ligands have some degree of mild functional selectivity, and novel compounds with extreme bias at D1 signaling were reported recently. Behavioral and neurophysiological studies inspired new methods to investigate functional selectivity and gave insight into the biased signaling of several drugs. Results from recent clinical trials also supported D1 functional selectivity signaling as a promising strategy for discovery and development of better therapeutics.Research progress on dopamine D Duce cAMP . Nobel L1Rs stimulate adenylate cyclase primarily through Gαs. The Gαs family is comprised of Gαs and Golf, the latter named for its predominant expression in the olfactory system. Studies on Gαolf knock-out mice suggested that Gαolf may play an essential role in D1R-mediated cAMP accumulation [olf knock-out mice showed no hyperlocomotor response to the D1 agonist SKF81297, and their striatal D1Rs had decreased affinity for dopamine. The adenylyl cyclase response to dopamine in the caudate/putamen and nucleus accumbens were also decreased dramatically, but prefrontal cortex signaling remained unaffected. Studies on the striatum of Parkinson’s disease patients also suggested that Gαolf plays the essential role in D1R-mediated cAMP [olf in this region is less abundant in Parkinson’s patients, whereas Gαs is highly expressed. The combination of dopamine deficiency and Gαolf being sparsely expressed suggested that the coupling of D1Rs to adenylate cyclase is mediated primarily by Gαolf or the switch/shift between Gαs and Gαolf.G proteins are a family of proteins made up of subunits Gα, Gβ, and Gγ. Dmulation ,11. Gαol7 subunit mRNA was detected within rat brain areas that aligned with striatum-enriched adenylyl cyclase, dopamine receptors, and Gαolf. This suggested that γ7 may be part of the Gαolf-containing complex that couples dopamine receptors selectively to adenylyl cyclase [7 subunit in HEK 293 cells stably expressing the human D1Rs, and revealed a significant attenuation of D1 agonist SKF81297-stimulated adenylyl cyclase activity [7 subunit on D1R-mediated cAMP signaling.The mRNA of other G proteins are also expressed in the striatum. For example, G protein γ cyclase . Anotheractivity . This ev1Rs and each G protein subunit, there is ongoing progress regarding functional selectivity of G protein subtype-specific signaling. Although there are currently no D1 ligands being developed to target specific G protein subunits, such compounds have been reported for the C-C chemokine receptor 5 (CCR5) [q/11 activation, whereas other analogs displayed a signaling bias for Gi/o. Their results demonstrated that ligands can elicit G protein subtype-specific signaling bias and cause receptors to couple preferentially to one subtype of G protein signaling over others. This is inspiring for the pharmacology field. A timely study is needed to investigate this interesting phenomenon since it is reasonable to assume that some dopamine ligands may also engage each G protein subtype differently, providing a more targeted action.Along with improved understanding of the interaction between D5 (CCR5) . Lorenze2-like receptors (D2Rs) [1Rs, there are a few reports that suggested the existence of an interaction between D1R and β-arrestin and indicated the involvement of MAP kinase phosphorylation , or links GPCR to elements of internalization machinery. Starting in the 1970s, Nobel Laureate Robert Lefkowitz and his colleagues made several discoveries related to β-adrenergic receptors, and β-arrestin related signaling is one of their concomitant discoveries. To date, it has been reported widely in several receptor systems, including β-adrenergic , opioid s (D2Rs) ,23. Regarylation , but man1/2) formed stable heterotrimeric complexes with the D1R and β-arrestin2. In cells transfected with the dominant negative mutant of β-arrestin2, however, the formation of such complexes was inhibited substantially [1R knockout mouse model and demonstrated that formation of the β-arrestin2 and ERK1/2 complexes was also blunted [1Rs in vivo or in vitro with the D1 selective agonist SKF38393 and showed ERK1/2 was phosphorylated [1R-mediated β-arrestin signaling and its function through ERK1/2. In addition to ERK1/2, several studies indicated the involvement of other MAP kinases. Zhen et al. showed SKF38393 increased activation of p38 MAP kinase and c-Jun amino-terminal kinase in SK-N-MC neuroblastoma cells that endogenously express D1Rs, whereas ERK activity was not affected [1R-related MAP kinase phosphorylation could be cell-type specific.Chen et al. used co-immunoprecipitation to show that extracellular signal-regulated kinases 1 and 2 , was critically involved in D1R-mediated ERK1/2 phosphorylation. A mutation on the phosphorylation site of DARPP-32 reduced activation of ERK, whereas sensitization of DARPP-32 led to increased activation of ERK1/2 [1/2, p38 MAP kinase, and c-Jun amino-terminal kinase [1R-mediated cAMP and β-arrestin-related signaling.Another interesting finding is that Df ERK1/2 ,30. The l kinase ,29. In cl kinase ,31. Thes1R involvement in the regulation of ion channels. Voltage-dependent Ca2+ channels  play critical roles in balancing intracellular Ca2+ concentrations that are key for neurotransmitter release and synaptic plasticity [1 agonists reduced N- and P-type Ca2+ currents, but enhanced L-type currents. The differential regulation of Ca2+ currents by D1 agonists helps to explain the diversity of effects that D1Rs have on synaptic integration and plasticity [1R effects on L-type Ca2+ current. They examined the impact of D1 agonists at depolarized and hyperpolarized membrane potentials and showed that D1R activation either can inhibit or enhance evoked activity, depending on the level of membrane depolarization. Interestingly, the effects on evoked activity at membrane potentials were blocked by the L-type Ca2+ channel antagonists nicardipine or calciseptine, and were occluded by the agonist BayK8644. These data indicated that the D1R-mediated effects occurred through the L-type Ca2+ channel [2+ channels, coimmunoprecipitation showed the existence of a D1R-N-type Ca2+ channel signaling complex in the prefrontal cortex. This complex had a direct receptor-channel interaction. D1 agonists not only regulated N-type Ca2+ channel distribution but also inhibited influx Ca2+ current. Consequently, neuronal transduction was attenuated [2+ channels, as cyclic AMP analogs mimicked the effects of D1 agonists [Many studies have indicated Dasticity . Using wasticity . Hernand channel . For N-ttenuated ,36. The agonists .1 agonist SKF81297 or SKF82957 in combination with the D2 agonist quinpirole increased spike firing of nucleus accumbens neurons via inhibition of a slow A-type K+ current. This enhancement was prevented by inhibitors of PKA or Gβγ and enabled by intracellular perfusion with Gβγ. These data suggested that the underlying mechanism of D1R and D2R cooperativity in mediating the slow A-type K+ current was by activation of specific subtypes of adenylyl cyclases released by Gβγ from the Gi/o-linked D2R in combination with Gαs-linked D1R [1 agonists SKF81297 or dihydrexidine induced prolonged membrane depolarization and excitability of fast-spiking interneurons in the prefrontal cortex. Voltage-clamp analyses revealed that this mimicked dopamine-suppressed inward rectifying K+ current and can be reduced by the D1 antagonist SCH23390 [1R-mediated K+ current changes has not been fully understood, studies have suggested possible options: the direct interaction of cAMP with K+ channels and the involvement of D1R-mediated cAMP/PKA signaling. The first reason is that the effect of D1R stimulation on K+ current can be mimicked by the adenylyl cyclase activator forskolin and the active cAMP analog Sp-cAMP. The second reason is that the inhibition of PKA with either PKI, Rp-cAMP, or the protein phosphatase inhibitor okadaic acid abolished D1R modulation [The Dnked D1R . The D1 SCH23390 ,39,40,411Rs also appear to impact Na+ channels. For example, the D1 agonist SKF38393 reduced the peak Na+ current amplitude in rat striatal neurons and subsequently depressed striatal neuron excitability. These effects were reversed by the D1 antagonist SCH23390 [1R-mediated Na+ current inhibition, and diffusion of the PKA inhibitor PKI into the cytosol of neurons blocked it, suggesting the involvement of PKA [+ current amplitude [1R regulation of ion channels may be a subsequent event of D1-mediated G protein-dependent cAMP signaling. On the other hand, Cantrell et al. reported that phosphorylation of Ser573 on the Na+ channel α subunit was critical for D1R-mediated effects on the Na+ current since this site was phosphorylated by D1R activation [1R and ion channel. A timely study is needed to investigate this interesting hypothesis. More importantly, if some dopamine ligands can engage each ion channel differently, they may provide a more targeted action and potentially lead to better therapeutic implications.DSCH23390 ,43. Intrt of PKA . Schiffmmplitude . This litivation . Since t1R-mediated PLC signaling was once proposed as a novel target, but controversies occurred, and it now is considered to be purported. The possibility that D1Rs may function through PLC first was reported in a series of studies on adenylate cyclase type 5 (AC5), a dopamine sensitive-adenylate cyclase. Genetic disruption of the AC5 isoform led to loss of adenylyl cyclase activity after administering the D1 agonist SKF38393, and this was accompanied by a decrease in the expression of Gαs. AC5 null mice also showed parkinsonian-like motor dysfunction. Interestingly, administration of the partial D1 agonist SKF38393 improved some of the symptoms, suggesting compensation of D1 signaling outside the Gαs mechanism and beyond adenylate cyclase [q-mediated PLC activation and subsequent Ca2+ elevation as a non-cyclase signaling for D1Rs was then proposed to explain D1R-mediated motor behaviors of the null mice. SKF38393 increased Gαq protein binding to the D1R in the striatum, suggesting the possible role of the Gαq protein in D1R-mediated PLC activation [1 antagonist SCH23390. In addition, dopamine-induced inward Ca2+ current was mimicked by the administration of SKF38393 and blocked by SCH23390 [D cyclase ,48. Gαq-tivation . SeveralSCH23390 ,51,52.1R-mediated PLC signaling came from studies using the D1 ligand SKF83959 that has small effects on adenylate cyclase but strong efficacy for PLC activation. Interestingly, it induced contralateral rotations in the unilateral 6-OHDA-lesioned parkinsonian rat model, and the rotations were completely blocked by the D1 antagonist SCH23390 [1R in PLC signaling, however, is still controversial [1Rs and D2Rs form a D1/D2 heterodimer. Heterodimers have been shown to play a role in functional selectivity in several other GPCR systems [1/D2 heterodimer led to PLC signaling. D1Rs and D2Rs, however, are seldom co-expressed in striatal neurons [1Rs actually may be independent of PLC activation.The most supportive evidence for DSCH23390 ,54. The oversial . Second,oversial . Third,  systems ,58, incl systems . It is v neurons ,63,64,651Rs (including D1 and D5) is higher than that of D2Rs , especially in the cortex where there is a significantly higher overall density and different laminar patterning of D1R compared to D2R. The D1R is preferentially distributed in deeper cortex layers and is proportionally more widespread and expressed within local GABAergic interneuron populations. In the basal ganglia where the density of dopamine receptors is the highest, the segregation of D1R and D2R is more distinct, with <10% overlap. The GABAergic medium spiny projection neurons of the striatum express D1R in the direct pathway and D2R in the indirect pathway. These two parallel and segregated pathways form the outflow of the basal ganglia to regulate thalamocortical circuitry [1Rs for therapeutic intervention is attractive. In this section, we try to differentiate D1 signaling by highlighting some reports that in retrospect have contributed to the understanding of functional selectivity.Dopamine receptors are highly expressed in the brain. The densest area is forebrain where the major dopaminergic terminal fields occur including caudate-putamen and nucleus accumbens. The midbrain  also has a high density of dopamine receptors. Olfactory, limbic, and brainstem areas have moderate densities of dopamine receptors. The cerebral cortex has a relatively light density of dopamine receptors but those that are there have important functional implications. In general, the density of Dircuitry . Dysfunc1R-mediated cAMP and Parkinson’s disease. AC5 is highly concentrated in the striatum. Genetic ablation of the AC5 gene eliminated adenylate cyclase activity stimulated by D1 agonists in the striatum, and induced parkinsonian-like motor dysfunction. These findings supported the involvement of D1R-mediated AC5 activation in the motor symptoms of Parkinson’s disease [1-mediated behavioral effects is still unclear, these downstream molecules are involved in the regulation of gene expression [There has been a long history between D disease ,48,67. A1R-mediated β-arrestin have yielded several impressive clinical implications. Urs et al. reported that the D1R-dependent, β-arrestin-related ERK signal cascade affected morphine-induced psychomotor activation but not reward [1R-mediated β-arrestin signaling through ERK may effect sex-specific depression [1 agonists with distinct signaling profiles at β-arrestin recruitment to evaluate rodent behavior in a working memory-related T-maze task [1 agonists on cognitive improvement. Interestingly, there were also clinical implications reported from ligands that bias against β-arrestin. Jiang et al. and other groups reported that β-arrestin activation was related to β-amyloid-induced cognitive impairment [1 agonists that have less β-arrestin activity may be novel therapeutics. Functionally selective D1 agonists that have less β-arrestin activity may have less side effects in Parkinson’s disease because ERK1/2 activation correlated with levodopa-induced dyskinesia, whereas blocking ERK1/2 activation significantly decreased it [1 agonists with almost no β-arrestin activity have shown highly positive clinical indications [Recent studies on Dt reward ,25, suggpression . Severalpression , more dypression , enhancepression , and imppression . Recentlaze task . We showpairment ,73,74. Seased it . Indeed,ications ,78,79,801R signaling has several clinical implications, even though some results seemed to contradict each other. In fact, the concept of functional selectivity was born in part to deal with the fact that different types of bias at a signaling pathway lead to benefits or disadvantages depending on different conditions. Essentially, functional selectivity was founded on the idea of precise targeting. In other words, the bias at one receptor’s whole signaling complex could be adjusted based on different applications to magnify merit.It is encouraging to see D1Rs. The crystal structure of D1R complexed with a G protein and a non-catechol agonist was reported only recently [2Rs, the binding site, and binding affinities for agonists and antagonists have been around since the 2000s [2R, D3R, D4R, and D2R-Gi complexes were reported [2LR functional selectivity [Progress on the structural biology of dopamine receptors has a large impact on the theory of functional selectivity and the discovery of subtype selective ligands, although it is rather slow for Drecently , althoughe 2000s . Over threported ,84,85. Bectivity . Using dectivity .2R/neurotensin NTS1 receptor heterodimer. The results indicated a strong, predominantly NTS1 receptor-mediated β-arrestin-2 recruitment in D2R/NTS1 receptor-co-expressing cells [1Rs and D2Rs in the same neuron [1/D2 heterodimer plays a critical role in cell signaling or overt behaviors.The heterodimer theory was also enhanced by structural biology progress. Crystal structures of receptor–ligand complexes allow for rational design of novel molecular probes and drugs. Following this strategy, bivalent ligands were synthesized and investigated for the Dng cells . There hng cells ,58 but be neuron ,63,64,652Rs [1-related studies are sparse, it will not be a surprise that future studies show D1 functional selectivity in different organelles. Location bias is an emerging paradigm in GPCR biology and drug discovery. This is particularly true for endosomes regarding D1Rs. D1Rs start to internalize through endosomes where β-arrestin is recruited. It has been shown that endocytosis promotes rapid D1 signaling [1 has signaling bias toward β-arrestin at endosomes, but more studies are needed for this topic.It is commonly known that dopamine receptors function differently in distinctive brain areas. Although there is not yet many focused studies to specifically investigate this, the possibility is high that functional selectivity is involved as a part of the underlying mechanism. More intriguingly, recent studies have shown that besides the primary effects in the central nervous system, dopamine also acts in the pancreas as a peripheral regulator of metabolism, and this action is functionally biased at D2Rs . This su2Rs . Althougignaling . This coignaling . Therefo1 ligands were reported [1 agonists [1 signaling pathway—the intrinsic activity at stimulating adenylate cyclase and producing cAMP. It is noteworthy that a retrospective review of some “traditional/classical” D1 ligands may have revealed that they are functionally selective even if this was not appreciated at the time of publication, as highlighted by our group [Functional selectivity is currently a hot topic in the drug discovery field. Recently, several novel, functionally selective Dreported ,93. Theyagonists . Full anur group .1 agonists and their ability to cause functional desensitization [1 agonists  caused homologous desensitization of the D1R in vitro to the same extent as dopamine, whereas two other full agonists  and all the partial agonists  caused only partial desensitization that was homologous, but not associated with PKA-induced phosphorylation. Ryman-Rasmussen et al. later tested 13 D1 agonists and showed that internalization efficacy was independent of either agonist affinity or chemotype [1Rs that are distal from the ligand-binding domain [Lewis et al. compared the intrinsic activities at adenylate cyclase for a series of structurally dissimilar full and partial Dtization . Surprisg domain . Since r2 ligands that were full agonists at adenylate cyclase but antagonists at other functions [2 agonists [2Rs and possibly 5-HT1A receptors [1R-mediated cAMP and β-arrestin signaling. Using a pair of D1 agonists with distinct signaling profiles, we evaluated rodent behavior in a T-maze task and examined how this was associated with neural activities in the prefrontal cortex [1R functional selectivity. It is encouraging that the field has discovered more ways to investigate functional selectivity—not only by pharmacological means but also behavioral and neurophysiological methods. These interdisciplinary approaches improve the innovation and development of more functionally selective D1 ligands as better therapeutics.Functional selectivity was first reported as a pharmacological phenomenon. Later studies using behavioral and neurophysiological methods, and recent clinical trials, have led to improved understanding of functionally selective drugs and provided significant insight on how to further investigate this “so called” pharmacological phenomenon. The earliest example of functional selectivity was for a series of Dunctions ,98,99. Tagonists ,101. Coneceptors ,103, coneceptors , angioteeceptors , and opieceptors  systems,eceptors ,108,109.l cortex . We show1Rs, studies related to grasping the breadth of D1R functional selectivity are expanding. Even though some reports at the time of publishing did not focus on functional selectivity, a retrospective review of their findings indicate they contributed to this field. More importantly, many studies showed positive implications for each unique D1 signaling pathway, suggesting that functional selectivity could be a promising strategy for drug discovery. Furthermore, retrospective pharmacological review revealed that many D1 ligands have some degree of mild functional selectivity. Moreover, novel compounds with extreme bias at D1 signaling were reported recently. Collectively, these data show that the development of precision medicine with the use of functionally selective D1 ligands is a promising direction to pursue.The understanding of functional selectivity has evolved over the years in conjunction with advances in improved knowledge of fundamental receptor signaling and complexes. With the discovery of many novel signaling or sub-pathways related to D