Two abnormalities were identified. The optical coherence tomography analysis of the adult retina confirmed changes to the refractive properties caused by eye axial length reduction, leading to hyperopic shifts. The gjd2b/Cx35.1 depletion was also correlated with morphological changes to the head and body ratios in larvae. The differential expression of Wnt/ß-catenin signaling genes, connexins, and dopamine receptors suggested a contribution to the observed phenotypic differences. The alteration of visual-motor behavioral responses to abrupt light transitions was aggravated in larvae, providing evidence that cone photoreceptor cell activity was enhanced when gjd2b/Cx35.1 was depleted. The visual disturbances were reversed under low light conditions in gjd2b−/−/Cx35.1−/− larvae. Since qRT-PCR data demonstrated that two rhodopsin genes were downregulated, we speculated that rod photoreceptor cells in gjd2b/Cx35.1−/− larvae were less sensitive to bright light transitions, thus providing additional evidence that a cone-mediated process caused the VMR light-ON hyperactivity after losing Cx35.1 expression. Together, this study provides evidence for the role of gjd2b/Cx35.1 in the development of the visual system and visually guided behaviors.The zebrafish is a powerful model to investigate the developmental roles of electrical synapses because many signaling pathways that regulate the development of the nervous system are highly conserved from fish to humans. Here, we provide evidence linking the mammalian connexin-36 (Cx36) ortholog  The transient coupling of groups of neurons by electrical synapses (gap junctions) is a phenomenon documented in different regions of the mammalian central nervous system across all life stages . This coGjd2/Cx36)  . While td2/Cx36) , less isd2/Cx36) .gjd2b/Cx35.1 gene/protein  belongs with gjd1a/Cx34.1, gjd1b/Cx34.7, and gjd2a/Cx35.5, two closely related subgroups of connexins with a shared homology of ≈85% between each other and mammalian connexin-36 (Gjd2/Cx36)  larval zebrafish  . Previou tissues . A high-or cells . Additiol stages . Furtherebrafish .via loss-of-function studies. Research focusing on the Mauthner neural circuit, which mediates a fast escape behavior, demonstrated that the two Cx36 orthologs, gjd1a/Cx34.1 and gjd2a/Cx35.5, are required for synapse assembly and function at larval stages , and dopamine receptor expression suggested a contribution to the observed phenotypic differences. The alteration of visual-motor behavioral responses to abrupt light transitions was aggravated, providing evidence that cone-cone photoreceptor cell activity was enhanced when gjd2b/Cx35.1 was lost. Visual disturbances were reversed under low/mesopic light conditions in gjd2b−/−/Cx35.1−/− larvae. Since qRT-PCR data demonstrated that rhodopsin genes were downregulated, we speculated that rod photoreceptor cells (PRCs) in gjd2b−/−/Cx35.1−/− larvae were less sensitive to bright light transitions, thus providing additional evidence that a cone-mediated process caused the VMR light-ON hyperactivity after losing Cx35.1 expression. Together, the main results of this study provide evidence for distinct roles of gjd2b/Cx35.1 in the development of the visual system affecting visually guided behaviors.This study investigated the functional evidence linking eveloped . We idenDanio Rerio) of the strain Tubingen long fin (TL) were maintained in groups with mixed sex in a recirculation system  at 28°C on a 14h light/10h dark cycle. All animal work was performed at York University’s zebrafish vivarium and in a S2 biosafety laboratory in accordance with the Canadian Council for Animal Care guidelines after approval of the study protocol by the York University Animal Care Committee (GZ#2019-7-R2). The Cx35.1 mutant line was generated and characterized in-house.Zebrafish (gjd2b−/−/Cx35.1−/− line in-house using the CRISPR-Cas9 system (gjd2b/Cx35.1 gene were identified using the E-CRISP tool (www.e-crisp.org). A CRISPR sgRNA  targeting a XhoI restriction site in exon one of the gjd2b gene was selected (gjd2b−/−/Cx35.1−/− zebrafish line.The Tupfel longfin (TL) strain were used to generate the 9 system . Potentiselected . The seqgjd2b exon one region was amplified in a Mastercycler Nexus X1  using the primer information as follows: forward primer: 5′-GGTTCTCTGTGTTACATTCGCCTCC-3′ and reverse primer: 5′-CAATCATAGTAGAGTGCTGTTGGACAGC-3’. PCR primer pairs were ordered from IDT  and optimized at an annealing temperature of 58°C. The PCR reaction was carried out using the Q5 High Fidelity DNA Polymerase PCR Kit  in a final volume of 20 µL consisting of 1 µL DNA-template and 19 µL Taq master mix. PCR thermal cycling conditions were initiated at 94°C for 15 min, followed by 38 cycles of denaturing at 94°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 30 s. The final extension was carried out at 72°C for 10 min. To determine the presence of indel mutations, PCR amplicons were digested with XhoI using the Fast Digestion Top Fermentas Kit  as per the manufacturer’s protocol. Amplicons were subsequently visualized on a 2% agarose gel supplemented with 0.005% ethidium bromide using the Alpha Imager HP System . For Sanger sequencing, PCR amplicons were cloned into pJET1.2 using the CloneJET™ PCR Cloning Kit  as per the manufacturer’s protocols. Constructs were transformed into DH5α competent E. coli cells. Plasmids were purified using the QIACube in combination with the QIAPrep spin miniprep kit . Sequencing was performed by Eurofins Genomics .Zebrafish larvae (3dpf) or adult caudal fins were used for genotyping purposes. Adult zebrafish were anesthetized in pH-buffered 0.2 mg/mL tricaine methane sulfonate (MS-222)  prior to caudal amputations with surgical scissors. Total DNA was extracted by incubation in 100 mM NaOH for 15 min at 95°C using with vigorous shaking. DNA samples were diluted in a 1:2 ratio with TE buffer (pH 8.0) prior to polymerase chain reaction (PCR) analysis. The 18S, tuba1a, actb2,Total RNA (1 µg) was extracted and purified from pools of 30 zebrafish larvae at six to seven dpf using the RNeasy Plus Mini Kit  as per the manufacturer’s protocols. When appropriate, larvae were collected immediately following exposure to behavioral tasks, and the tissue was frozen at −80°C until further analysis. Fish were homogenized by bead beating in 1xTE buffer (pH 8.0). RNAs were reverse transcribed into cDNA using the iScript Reverse Transcription Supermix  as per the manufacturer’s instructions. Gene expression was analyzed by qRT-PCR using the SsoAdvanced universal SYBR Green Supermix  in the CFX96™ Real-Time PCR Detection System . Thermal cycling was carried out for 42 cycles of the following: 94°C for 30s, 52°C for 30s, and 72°C for 1min. Three housekeeping genes (gjd2b−/−/Cx35.1−/−) were humanely euthanized using MS222. The larvae were fixed in 4% paraformaldehyde (PFA) in 1xPBS overnight at 4°C, followed by cryoprotection in 30% sucrose in 1xPBS. After embedding in Tissue-Tek O.C.T, 10–15 µm sections were cut on a cryotome . Sections were washed three times for 5 min with 1xPBS containing 0.1% Tween-20 (PBST) at RT. Unspecific binding sites were blocked with freshly prepared 5% normal goat serum  in PBS-T for 1h at 4. Following blocking, samples were incubated with the primary mouse anti-Connexin 36 antibody  overnight at 4°C. Subsequent washes with PBS-T were for 1 hour at 4°C. Alexa 488 goat mouse secondary antibodies  were applied for 1 h at RT. After three washes with PBS-T followed by one wash with PBS, specimens were mounted on microscope slides using ProLong Antifade with DAPI . Confocal images were collected using LSM-ZEN2 software using a Zeiss LSM700 system  with a Plan-Apochromat 20x/NA0.8 or Plan-Apochromat 63x/NA1.3 oil DIC M27 objectives. The software optimized pixel resolution, line averaging, gain, and digital offset. The same imaging parameters were used to compare TL and gjd2b−/−/Cx35.1−/− tissues. The composite Six dpf larvae  system was built in Michelson configuration , employiBefore imaging age-matched adult zebrafish were humanely euthanized using MS222 and fixed in 4% paraformaldehyde (PFA) in 1xPBS overnight at 4°C. Fixed zebrafish (n = 26/genotype) were placed in a silicon mold to orient the eye toward OCT system’s objective lens. To minimize specular reflections from sample surface, a thin layer of PBS (∼80 µm) was placed over the eye before imaging. All captured OCT tomograms were interrogated in ImageJ software which enabled measurement of various geometrical parameters see  through Zebrafish embryos were raised in 10 cm ® system and the Zebralab analytical software  to obtain automated tracking data from video files. The recording chamber holding the plates was equipped with a water heating and flow system to maintain the temperature at 28°C. Illumination was from the bottom with LED and infrared lights. Unless otherwise stated, larvae underwent a dark-adaptation period for 2 hours prior to behavioral analyses. All experiments utilizing light were performed at 30% light intensity unless otherwise stated, where the Zebrabox® system was capable of 8,000 lux at 100%. Data was acquisitioned in 1s, 10s, 1 min, or 5min intervals for time periods ranging from 5 min to 4.5 h as outlined in figures. Data was acquisitioned in 1s, 10s, 1 min, or 5min intervals for time periods ranging from 5 min to 4.5 h as outlined in figures. The experimental designs for VMR , individual larvae were transferred into 48- or 96-well plates (Greiner)  filled with egg water. Transparent plates were utilized for all experiments. The cross-maze to assess color vision was generated in-house. Behavioral assays were performed using the Zebrabox for VMR , Color v for VMR , were ad for VMR .Spontaneous zebrafish larval locomotor activity was measured under constant illumination (30%) or darkness (0%). For constant illumination, larvae were acclimated in light (30%) for 2 h prior to behavioral analysis. Locomotor activity was tracked for 5 min or 1 h, depending on the type of data acquisition. Data were normalized accordingly.Responsiveness to light changes was measured and under mesopic conditions as described . Larvae The innate color preference test allowed for color bias and color-blindness to be assessed in zebrafish larvae. Here, a custom-made cross maze was milled from 5 mm transparent acrylic sheets. Each arm of the maze was of equal dimensions  and could accommodate removable sleeves of different colors . The test was performed in two variations. In the first set larvae were exposed to all four colors simultaneously. In the second test larvae were exposed to two colors and a neutral stimulus (white light). Up to 20 larvae of the same genotype were placed into the center of the cross maze. Larvae were dark-adapted to avoid pre-exposure to the colors, and color sleeves were rotated to avoid location bias. Locomotor activity was tracked for 40 min (35 min of illumination and 5 min prior to illumination). Data was measured manually from tracking videos by counting the number of fish in each arm every 2 min for the duration of the experiment.Responsiveness to light flashes was assessed with the FSTR assay. Various stimulus lengths were sequentially tested ; however, the final analysis utilized the 10 m and 1000 m experimental endpoints. The experimental design began with 2 h dark adaptation and was followed by alternating a light cue with a 20-min rest period in darkness To avoid habituation to the stimuli, cue lengths were presented in forward and reverse order. Larval locomotor activity was tracked for 2.5hrs; the last 5 min of adaptation period prior to the presentation of the initial light cue was used in normalization calculations. Mean responsiveness, quantified as burst duration s), 1 s following each stimulus was measured. Data were normalized accordingly.post hoc test where appropriate. Normality was tested with the Shapiro-Wilk normality test. Statistical significance was defined by a p-value of ≤0.05. Statistical analyses were performed in GraphPad Prism 6 . Gardner-Altman estimation plots were created in a R-statistical program ; they show the mean difference between groups. Both groups are plotted on the left or top axes; the mean difference is plotted on floating axes on the right or bottom as a bootstrap sampling distribution. The mean difference is depicted as a dot; the 95% confidence interval is indicated by the ends of the vertical error bar. All remaining figures were created in GraphPad Prism 6.Unless otherwise stated, all data is presented as the mean ± SEM. The sample size for behavioral studies varied from n = 48 to n = 144 across a minimum of two experimental replicates; sample sizes are indicated in the figures. Statistical significance was determined by using a two-tailed unpaired Student’s t-test or its non-parametric equivalent Mann-Whitney, one-way ANOVA, or its non-parametric equivalent or Kruskal–Wallis, or two-way ANOVA with a Tukey gjd2b−/−/Cx35.1−/−) model was created utilizing a CRISPR-Cas9 genome engineering strategy and screening for indel mutations generated by the non-homologous end joining (NHEJ) repair system. Exon one of the gjd2b gene was targeted with a sgRNA located upstream of the start codon  larvae  larvae . The ant−/− genotype and partial loss of Cx35/36 immunoreactivity, we tested whether targeted ablation of the gjd2b gene causes a compensatory regulation of connexins in larvae. A previous RNA-seq analysis of 6dpf WT larvae had demonstrated the expression of two of the four zebrafish Cx36 orthologs; gjd1a/Cx34.1 and gjd2b/Cx35.1 were detected, but not gjd1b/Cx34.7 or gjd2a/Cx35.5 . Both gjd1b/Cx34.7 and gjd2b/Cx35.1 are co-expressed in cone photoreceptors  and gja3/Cx48.5  expression was detected. The expression of other connexins were indistinguishable from the WT. The regulation of gja2/Cx39.9 (Cx46 in human and murine models), and gja3/Cx48.5 (Cx50 in human and murine models) was of interest due to the role of both connexins in ocular lens homeostasis . The weight of TL controls and gjd2b−/−/Cx35.1−/−  were also measured; these measurements, however, were not used for normalization of measured metrics as they were deemed unreliable due to the inherent variations caused by the fixation process.In light of a recent report linking ataracts , the eyeataracts . Using Oe images , six prie images . To prevgjd2b−/−/Cx35.1−/− ; similar trend was observed in the measured retinal thicknesses of TL controls and gjd2b−/−/Cx35.1−/− . No evidence for the occurrence of cataracts were found in any of the TL or gjd2b−/−/Cx35.1−/− fish. The comparative analysis, however, demonstrated that mutants and TL differed significantly in terms of normalized eye axial length , lens radius , and retinal radius ; see Statistical analysis of quantified parameters in gjd2b−/−/Cx35.1−/− over the TL controls ; the positive values of RRE for the TL controls were due to the fixation process which makes the eye slightly hyperopic as reported by others  subtypes  as well as the dopamine transporter (dat) were quantified by qRT-PCR. The tyrosine hydroxylase (th) and the vesicular monoamine transporter (vmat2) genes were included in the analysis because of the roles in presynaptic dopamine production and in the regulation of packaging and subsequent release of dopamine vesicles into the synaptic cleft  and drd4a receptor genes , and an upregulation of vmat2   ; Table 1wnt8b , wnt11 , and fdz5  in gjd2b−/−/Cx35.1−/− larvae . The MD was indistinguishable between the two genotypes   length, head length, and midbrain diameter (MD) were detected . The bod = 0.95) .gjd2b/Cx35.1−/− larvae were objectively larger in their body and head length     F. In co= 0.008) . This sugjd2b−/−/Cx35.1−/− larvae suggested visual-motor deficiencies causing testable behavioral phenotypes. To address this question, spontaneous locomotor activity was quantified in 7dpf zebrafish in light-ON or light-OFF conditions. Both WT and gjd2b−/−/Cx35.1−/− larvae increased the swimming activity in light-ON conditions relative to light-OFF . However, the swimming distance of gjd2b−/−/Cx35.1−/− larvae and WT larvae was indistinguishable in either light-ON .The structural and genetic changes detected in light-ON  and lighlight-ON        C and li= 0.416) . Similar= 0.998) . However< 0.001) . The resgjd2b−/−/Cx35.1−/− altered retinal expression patterns, which was consistent with genetic profiles alluding to dysregulated eye development, we tested whether the retina was functionally intact using the standardized Visual Motor Response (VMR) assay    assay . Due to Whitney) . In the = 0.754) . The resgjd2b/Cx35.1-deficient larvae was caused by rapid temporal processing of visual stimuli. To determine the changes in visual processing speed, we developed the flash stimuli threshold response (FSTR) assay where the responsiveness of dark-adapted zebrafish was measured with transient light flashes at several different exposure times . The FSTR was resolved by calculating the burst duration 30s before and after light stimulation and determining whether there was a change in response , but was indistinguishable prior to the visual stimuli . In contrast to light-ON VMR, gjd2b−/−/Cx35.1−/− larval responses were attenuated following the 1000 m light flash; the burst activity increased by 178% for the WT, but only 138% for gjd2b−/−/Cx35.1−/− larvae (p < 0.001) . Upon stimulation with 10 m light-ON, WT and gjd2b−/−/Cx35.1−/− larvae increased their average burst duration by 112% and 109% respectively . The gjd2b−/−/Cx35.1−/− responses following the 10 m flash stimulus were again reduced in comparison to the WT (p = 0.002)  . Similar= 0.002) . As suchet al. have previously demonstrated that Cx35 electrical coupling facilitates both cone-cone and rod-cone photoreceptor crosstalk, the latter of which allows for saturated rod signaling to reroute to cones when exposed to the mesopic visual range    . Similar= 0.024) . Taken topn1lw2), green (opn1mw1 and opn1mw2), ultraviolet (opn1sw1), blue (opn1sw2), low-light rhodopsin (rho), and the second rhodopsin like (rhol) photopigments , whereas the remaining cone transcripts were indistinguishable from the WT  and rhol  was in line with the behavioral changes to low light.The behavioral studies were complemented with a quantification of opsin and rhodopsin gene expression. Our own RNA-seq analysis had demonstrated that 6dpf TL larvae express red (SE181853 ). The qRm the WT . The obsopn1mw2 expression in gjd2b−/−/Cx35.1−/− larvae was insufficient to impact color vision prominently. Using a cross-maze described previously . However, we found that the WT were indifferent towards to the red, green, and yellow colors . Similarly, gjd2b−/−/Cx35.1−/− larvae preferred blue  with no distinguishable preference for the other colors    . No stat> 0.999) .p = 0.011)  . In any gjd2b/Cx35.1 in the developing visual system of zebrafish larvae. We describe a new mutant line generated with Cas9/CRISPR technology, which differs from the gjd2bfh454 ) with an AB/Tu . This antibody was different to the anti-Cx35/36 antibody used previously to demonstrate Cx35.1 depletion . Here, tGJD2 (Cx36) is associated with refractive error (GJD2 (Cx36) have consistently been associated with refractive error (rs634990 and rs524952) and myopia. However, the coding region of GJD2 (Cx36) has not revealed coding variants explaining the genome wide association study results; it has been suggested that the identified variants might play a role through gene regulation. As pointed out by GJD2, facilitating the coupling of Cx36 (GJD2) gap junctions in the eye. Following this idea, the uncoupling of gap junctions could explain the opposite outcome.In humans, ve error . As discgjd2b/Cx35.1 in the TL strain. The robust expression of gjd2b/Cx35.1 is consistent with the reported single-cell RNA-seq data showing the expression of this gene in all major cell types of the developing retina. Interestingly, we found that gjd2b/Cx35.1 depletion caused a downregulation of gjd1b/Cx34.7. Previous work has demonstrated that both genes are expressed in cone photoreceptors . In mammals, five distinct types of dopamine receptors have been identified and are classified into two families; D1-like (D1R and D5R), which are stimulatory subsets to adenyl cyclase, and D2-like  which inhibit adenyl cyclase  or other retinal connexins could be phosphorylated. An alternative explanation could derive from the functional antagonism of dopamine and adenosine receptors, specifically the A2A and D2-like subtypes. In such circumstances, activation of A2A receptors reduces in dopamine affinity   . The Cx4/Cx48.5)  and the eye size . Interes mammals .gja2/Cx39.9 in the zebrafish does not affect the locomotory behavior but caused abnormal thigmotaxis and changes to muscle contraction ; the lack of access to a Cx35.1 specific antibody did not allow us to resolve this question. However, we also found Cx36 immunoreactivity in the optic nerve and the arborization fields (AF) where RGC axons contact dendrites of tectal neurons  that was exclusive to darkness, however, it is unclear whether this can be attributed to a motor-muscle deficiency, anxiety-like phenotype, or a combination of them both. Nevertheless, this served as our initial basis that processing of luminescence requires functional gjd2b/Cx35.1 expression.Considering the ly noted , and thedarkness , we antigjd2b/Cx35.1 was observed; gjd2b−/−/Cx35.1−/− larvae responded to sudden changes in luminescence, although this was met with an increased sensitivity to light-ON transitions. Under this condition, cone photoreceptor stimulation and subsequent downstream ON-bipolar cell activation is the primary signaling mechanism, providing evidence that gjd2b/Cx35.1 contributes to cone PRC activity. In support of this, exposing gjd2b−/−/Cx35.1−/− larvae to a mesopic visual range (5% light intensity) reversed the light-ON VMR hyperactivity, indicating that the recruitment of rod photoreceptor cells was able to reverse this response. Li et al. have previously demonstrated that Cx35 electrical coupling facilitates both cone-cone and rod-cone photoreceptor crosstalk, the latter of which allows for saturated rod signaling to reroute to cones when exposed to the mesopic visual range  assay  was usedal range . Taken tal range . We proprm cells .op1mw2. op1mw2 upregulation may have provided additional visual sensitivity, though we believe this is unlikely since we provide evidence that gjd2b/Cx35.1 uncoupling does not alter the spectral sensitivity. Previous reports have revealed the innate color preferences of adult zebrafish . In support of this result, gjd2b−/−/Cx35.1−/− measuring the B-wave responses in zebrafish were indistinguishable from the wildtype  than during FSTR. As such, this revealed an unexpected contribution of gjd2b/Cx35.1 expression to vision in mediating behavior based on the temporal features of the stimuli.The quality of visual processing can be quantified by the measurement of luminescence, chromaticity, and spatiotemporal properties of light stimuli. Our VMR studies established roles of wildtype . Though gjd2b/Cx35.1 depletion leads to changes in the refractive properties of the eye, leading to hyperopic shifts, and morphological changes to the head and body ratio of zebrafish larvae. The changes to Wnt/ß-catenin and dopaminergic signaling pathways, as well as retinal immunostainings, identify novel roles for gjd2b/Cx35.1. As such, the results provide evidence for the contribution of gjd2b/Cx35.1 in the development of the visual system and visually guided behaviors in zebrafish. To our knowledge, we are the first to describe these distinct behavioral responses in gjd2b/Cx35.1 deficient zebrafish. While the details surrounding junctional coupling in gjd2b−/−/Cx35.1−/− zebrafish remain unclear at present, we speculate that they are caused at the synaptic level by changes to the plasticity and functional asymmetry of electrical synapses. In conclusion, the evidence provided from phenotypic, molecular, and behavioral analysis support that significant functions of gjd2b/Cx35.1 in visual processing through the differential discrimination of luminescence and temporal properties, mediated by cone photoreceptors, start at the early stages of development.This study found that


Head and neck squamous cell carcinoma (HNSCC) is the sixth leading malignancy worldwide, with a poor prognosis and limited treatment options. Molecularly targeted therapies for HNSCC are still lacking. However, recent reports provide novel insights about many molecular alterations in HNSCC that may be useful in future therapies. Therefore, it is necessary to identify new biomarkers that may provide a better prediction of the disease and promising targets for personalized therapy. The poor response of HNSCC to therapy is attributed to a small population of tumor cells called cancer stem cells (CSCs). Growing evidence indicates that the Hedgehog (HH) signaling pathway plays a crucial role in the development and maintenance of head and neck tissues. The HH pathway is normally involved in embryogenesis, stem cell renewal, and tissue regeneration. However, abnormal activation of the HH pathway is also associated with carcinogenesis and CSC regulation. Overactivation of the HH pathway was observed in several tumors, including basal cell carcinoma, that are successfully treated with HH inhibitors. However, clinical studies about HH pathways in HNSCC are still rare. In this review, we summarize the current knowledge and recent advances regarding the HH pathway in HNSCC and discuss its possible implications for prognosis and future therapy. Head and neck squamous cell carcinoma (HNSCC) is the most common head and neck malignancy, with a global incidence of about 890,000 cases annually . HNSCC aAmong the many molecular alterations occurring in HNSCC, researchers have recently focused on signaling pathways that normally regulate several processes related to cell proliferation, differentiation, and stemness, while their aberrant activation has been detected in various cancers ,7. An esIn this paper, we aim to summarize the current knowledge and recent advances regarding the HH signaling pathway in HNSCC and discuss its possible relevance in tumorigenesis, progression, and future therapy, indicating new directions for further research.The poor prognosis of HNSCC is caused by the rapid dissemination of tumor cells to cervical lymph nodes, resistance to chemo/radiotherapy, and a high risk of local tumor recurrence . GeneralThe clonal evolution model of tumor development demonstrated by Peter Nowell suggests that cancer is an evolutionary process induced by the growth and expansion of subclones that carry selectively advantageous genetic alterations . The higIt was hypothesized that some types of cancer cells with unique features have been described as CSCs, which become highly malignant, leading to cancer recurrence, metastasis, and resistance to radio/chemotherapy ,7,12,20.The HH pathway is a strictly conserved evolutionary signaling cascade that controls embryonic development and regulates normal cell growth and differentiation under physiological conditions ,22. ThisActivation of HH signaling by the canonical mechanism requires the involvement of the primary cilium with its specialized transmembrane receptors . One of In the non-canonical mechanism of HH signaling, activation of GLI proteins can be performed without the participation of SMO, but this way is not yet fully understood . Up to nHH signaling is originally involved in the proper development of several organs, including head and neck tissues like teeth, lips, palate, and salivary glands . TherefoOrofacial cleft (OFC) is the most common birth defect, with incidence in about 1/500–700 living newborns . Up untiIn teeth, HH signaling plays a key role in physiological morphogenesis by controlling crown and root formation at multiple stages of odontogenesis . It was The importance of HH signaling in oncogenesis was first described in 1996 in Gorlin–Goltz syndrome, a rare autosomal dominant disease that is related to a high predisposition to OKC, basal cell carcinoma (BCC), medulloblastoma, and rhabdomyosarcoma . A mutatThe first analysis of the HH pathway in HNSCC was conducted by Schneider et al. They compared the expression of HH proteins in skin squamous cell carcinoma (SSCC) and HNSCC . SchneidThe correlation between the expression of HH proteins and prognosis in patients with HNSCC is still not well established. Most studies revealed that increased SHH, PTCH1, SMO, and GLI1 expression are significantly correlated with worse OS ,60,67,68The key reason for the aggressive clinical course of head and neck cancer (HNC) is tumor heterogeneity, which reflects differences within cell subpopulations existing in the tumor tissue ,7. It waCD44 is an adhesive, transmembrane glycoprotein located on the surface of normal and HNSCC cells as a receptor that is involved in extracellular matrix (ECM) communication ,7,78. InThe ALDH proteins are cytosolic enzymes that participate in numerous physiological and pathological processes in embryonic and adult tissue, including tumorigenesis ,97. The CD133 expression was detected in normal stem cells and CSCs ,7,78. ThHead and neck CSCs express the same profile of proteins as those that regulate embryonic stem cells (ESCs), in particular OCT4, SOX2, and NANOG, which were also reported to be expressed in various cancer tissues and were correlated with poor prognosis ,72,73,76OCT4 (octamer-binding transcription factor 4) has been involved in tumor development, invasion, and metastasis of many tumors, including OSCC ,73,78. ONANOG (Nanog Homeobox) has the ability to inhibit differentiation by maintaining ESC pluripotency, but its overexpression was also observed in HNSCC ,76,79. TSOX2 (Sex determining region Y-box 2) is a nuclear transcription factor that plays a crucial role in the development of pluripotent cells . SOX2 inCSCs also possess certain remarkable features that allow them to define their metastatic capabilities, drug resistance, invasiveness, and hierarchical differentiation ,79. SeveSeveral signaling pathways in normal stem cells contribute to their proliferation, differentiation, self-renewal, and survival. However, they are abnormally activated or repressed during tumorigenesis and facilitate CSC growth and self-renewal ,24,90. TGrowing evidence revealed that dysregulation of HH signaling is frequently observed in CSCs ,10,13,18Angiogenesis is a key process determining tumor growth and invasion that is balanced by numerous pro-angiogenic and anti-angiogenic factors . Newly fUpactivation of the HH signaling pathway in cancer cells induces GLI1 and SHH overexpression, which increases the expression of proangiogenic factors, including VEGF-A, MMP-2, MMP-9, heparanase, and cysteine-rich angiogenic inducer 61 (Cyr61), in different tumor cells ,124,126.The mechanisms of metastasis induced by HH signaling are understood. However, there are several reports documenting that the HH pathway induces invasion and metastasis through the activation and interaction between different signaling pathways, including HH ,118,123.There are data showing that high upregulation of the HH pathway mediates the function of GLI1, facilitates the CXCL12-induced migration of cancer cells, and increases the metastatic potential of tumor cells . The cruDownregulation of the GLI family mediates the activation of EMT, leading to the acquisition of a mesenchymal phenotype by epithelial cells ,123,131.The current treatment of patients with HNSCC includes a combination of surgery with radio/chemotherapy, which depends on the tumor location and its clinical stage . About oIt is well known that HH signaling controls complex mechanisms of DNA repair machinery in normal and tumor cells . This fuApoptosis is one of the main human defense mechanisms by which tumor cells are eliminated during anticancer treatment . HoweverThere is rising evidence that various viruses target HH signaling to facilitate its transcription, leading to effective and uncontrolled viral replication . The actIt is well documented that some of these viruses have oncogenic potential. They play a crucial role in the etiology of HNSCC. Infection with HPV is increasingly common in oropharyngeal squamous cell carcinoma (OPSCC), especially among young non-smokers from developed countries ,162. LikNowadays, it is not surprising that cell signaling pathways strictly influence themselves through activation or suppression . The croDespite the fact that the crosstalk between HH and other pathways is known, it is still not fully elucidated. HH signaling is closely related to the WNT pathway by producing WNT ligands like WNT2b, WNT4, and WNT7a, which are required for activation of the WNT pathway . It was The previous genetic analysis identified that NOTCH signaling modulates HH pathway activity by inducing key proteins belonging to the HH pathway, like GLI2, GLI3, and SMO . SeveralA better understanding of this complex crosstalk between signaling pathways involved in tumorigenesis is crucial for developing novel targeted therapies for HNSCC. Simultaneous targeting of several signaling pathways may provide more effective treatment for patients with HNSCC, but additional studies are necessary to confirm this hypothesis.The proven significance of HH signaling in a variety of human tumors has led researchers to target this pathway in anticancer therapy. The first discovered HH inhibitor was cyclopamine, which is a steroidal alkaloid that naturally originated from the corn lily . The posAs described in HH ligand inhibitors block the binding of HH ligands to the PTCH receptor, thereby inhibiting HH signaling at the first stage of this complex cascade. It is well-known from various tumors that SHH overexpression is associated with increased HH signaling . TherefoThe most common target for HH inhibitors is the SMO protein. The breakthrough moment in studies regarding HH signaling was the acceptance of vismodegib in 2012 by the Food and Drug Administration (FDA) for treating unresectable BCC . In the The positive effect of HH inhibition in HNSCC was first observed by Mozet et al., who studied the influence of cyclopamine, cisplatin, and docetaxel on HNSCC cell colony formation . It was Downregulation of the HH pathway in HNSCCs after the use of SMO inhibitors was represented by decreased expression of HH components in several preclinical studies ,184,185.Recently, interesting results were reported by Kleszcz et al., who presented a combined therapy with inhibitors of several signaling pathways in HNSCC . They ann = 9) and heterogeneous . Therefore, the authors concluded that further exploration of this combined therapy is recommended in a more numerous and homogenous cohort [Up to now, only two clinical trials have evaluated SMO inhibitors in HNSCC. The first survey performed by Bowles et al. (ID NCT01255800) studied the combination of IPI-926 (saridegib) with cetuximab in recurrent or metastatic HNSCC patients . This cls cohort .The revealed discrepancy between in vitro and in vivo studies might be a result of both genetic and epigenetic alterations leading to persistent GLI activation that allows tumors to evade inhibition of SMO. This contributes to the development of resistance to therapy and rapid tumor recurrence . A recenThe second clinical trial conducted by Mayo Clinic (ID NCT04007744) is still recruiting and has not been completed. This phase I clinical trial evaluates the combined use of sonidegib and pembrolizumab in various tumors, including metastatic HNSCC. However, the results are not yet available.The GLI transcription factor is a final protein of the HH pathway, which implies that it is directly involved in HH target gene expression. Therefore, inhibition of GLI seems to be a promising and more effective target for anticancer drugs, contrary to other HH proteins. To date, several GLI inhibitors have been developed, but only a few of them have been tested in vitro in HNSCCs. The most studied GLI antagonist is arsenic trioxide (ATO), which was approved in 2000 by the FDA for the treatment of acute promyelocytic leukemia . HoweverSimilar observations were reported in studies evaluating the influence of other GLI inhibitors—GANT61—on OSCC cell lines ,201. In To our knowledge, this is the first comprehensive review regarding the HH pathway in HNSCC. We made an effort to collect and summarize recent findings about HH signaling in HNSCC and pointed out the role of this pathway in the biology of HNSCC and its clinical outcome. Based on the latest studies, we may confirm that the HH pathway is aberrantly activated in advanced HNSCC; however, the clinical studies are very limited. As we summarized in this paper, HH signaling is involved in several crucial events during HNSCC oncogenesis, like tumor initiation, neoangiogenesis, cancer invasion, resistance to chemo/radiotherapy, recurrence, and metastasis. Furthermore, increased expression of proteins belonging to the HH pathway is correlated with worse survival and a poor prognosis in HNSCC patients, what may suggest the prognostic significance of HH signaling in HNSCC. Given the present lack of molecularly targeted therapies for patients with HNSCC and low survival rates, inhibition of HH signaling might be a promising intervention in future HNSCC therapies. Therefore, further clinical studies evaluating the efficacy and safety of HH inhibitors in HNSCC treatment are urgently needed. However, due to the strict and complex crosstalk between HH and other signaling pathways, it seems that combined therapy with simultaneous inhibition of several tumorigenic pathways may be required to provide breakthroughs in HNSCC treatment.


This paper focuses on the impact of the educational mismatch on workers’ fertility intentions and explores the underlying mechanisms. Empirical research based on the China Family Panel Survey (CFPS) data shows that undereducation has a negative effect on the fertility intentions of women aged 18–35, whereas overeducation reports no effect, a finding that remains stable in the robustness test and the IV model. An explanation for this phenomenon is that undereducated female workers have a wage premium and higher expectations of career development, which implies a greater opportunity cost of fertility. The heterogeneity analysis shows that women with low socio-economic status, especially those who are less educated, from low-income households, and who are employed in the private sector, are more affected. It is therefore necessary to reduce the substitution risk in the labor market for this group and to lighten the fertility burden and pressure on women. Since the beginning of the 21st century, numerous nations have undergone a demographic transition characterized by “aging” and “low fertility” , culturaIn the labor market, the demand for human capital and the supply of workers’ qualifications are not always perfectly matched. Scholars generally regard educational attainment as a proxy for human capital and define the above phenomenon as the educational mismatch of the labor force . If a woEducational mismatch inevitably leads to several consequences, the most obvious of which is the disparity in the returns on education ,10. For In this paper, we use data from the China Family Panel Survey (CFPS) to investigate the effect that educational mismatch has on workers’ fertility intentions. By summarizing the consequences of educational mismatch and the formation mechanism of fertility intention, we propose three potential channels. Subsequently, we conduct baseline analysis, robustness analysis, heterogeneity analysis, and mechanism analysis, and finally find a negative effect on the fertility intentions of undereducated women aged 18–35, which could be explained by childbearing opportunity cost and career development desire.Our study broadens the established literature in three ways. First, we supplement the literature on fertility intentions. Compared with prior literature that focuses on education factors, we take individual work performance into consideration and examine the effects of overeducation and undereducation, which push the boundaries of the research. Second, we shed new light on the consequences of educational mismatch. Most of the studies find the impact of educational mismatch on manufacturers and workers. We extended the perspective from the workplace to the family and observed variances in fertility intentions caused by an educational mismatch. Third, this study is related to the literature on the opportunity cost of childbearing. By discussing the potential mechanisms, we capture not only the tangible pecuniary opportunity cost but also the intangible opportunity cost on career development and find that young undereducated women with low socio-economic status are more vulnerable.Fertility behavior has both consumption and investment attributes. These two aspects correspond to two values: “altruism” and “intergenerational exchangeism” . AccordiAt the same time, fertility intentions are subject to economic budget constraints. The cost–benefit theory divides the cost of childbearing into direct and indirect cost . Direct The degree of matching between workers’ education and their job demand reflects, to a certain extent, the quality of employment. It influences individuals’ wage level, psychological health, and career prospects, thus linking to the formation mechanism of fertility intention. In the following section, we construct a research framework that includes three potential channels through which educational mismatch may affect labor fertility intention .There is a consensus that educational mismatch causes deviations in the returns on education. According to the hedonic/assignment models proposed by Sattinger , productThese income disparities may affect fertility intentions through budget constraints and value proposition: income levels determine a family’s financial well-being, affecting whether parents can afford the direct cost of childbirth and child-rearing. If children are considered normal goods, an increase in income typically results in a positive impact on fertility intentions . However, income levels also impact the opportunity cost of childbearing, leading to a negative substitution effect, especially for women, who are directly involved in childbearing ,29. MoreHypothesis 1.Education mismatch leads to income disparities, thereby influencing workers’ fertility intentions. However, the direction of this influence remains ambiguous due to the interaction of wealth effect, substitution effect, and precautionary saving.Educational mismatch also has a significant impact on the cognitive and psychological well-being of workers. Overeducated individuals usually experience feelings of exploitation, frustration, and financial insecurity. This results in lower levels of subjective well-being and an increased risk of depression compared to properly matched workers with the same number of years of schooling ,32. PartAs implicit emotional capital, subjective well-being and mental health status are closely related to fertility intentions. On the one hand, according to Becker’s theory, children are perceived as a source of psychological comfort and joy in the household . ParentsHypothesis 2.From the psychological channel, the fertility intentions of undereducated (overeducated) workers are lower (higher) than those of the properly matched workers with the same number of years of schooling.In the long term, educational mismatch compels workers to make adjustments to their career plans. In general, undereducated individuals are considered to have long-term career development goals  and mostOverall, both undereducated and overeducated individuals experience career development expectations or pressures, leading to increased career mobility and uncertainty. Researchers find that job uncertainty can reduce individuals’ fertility intentions or motivate them to delay their childbearing plans . AdditioHypothesis 3.From the career development channels, the fertility intentions of undereducated and overeducated individuals are lower than those of the properly matched workers with the same number of years of schooling.Previous literature highlights gender inequalities in labor supply and childbearing. As women usually take on the irreplaceable childcare responsibilities such as pregnancy and breastfeeding, their labor behaviors tend to be more fragile . While aHypothesis 4.There is a significant gender difference in the impact of educational mismatch on fertility intentions, with female workers being more susceptible than their male counterparts.The data used in this paper are from the China Household Panel Studies (CFPS). This survey has been running since 2010, tracking and collecting data at the individual, household, and community levels. The database covers 25 provinces with a target sample size of 16,000 households. We use data from the 2014 and 2018 surveys in which individuals were asked in detail about their fertility intentions.In this study, we retain samples aged 18–50 who had full-time jobs and exclude the samples with missing values in core variables such as occupation, education, and fertility intention. Finally, 17,532 valid samples are obtained. Of these, 55% are male and the remainder are female. The mean and standard deviation of the sample age are 34.73 and 8.72, respectively. More specifically, the 18–30, 31–40, and 41–50 age groups account for 37.70%, 31.80% and 30.5%, respectively, which means that the age distribution of the sample is relatively balanced.edu) and the level of education required by the job (redu). When an individual’s education falls below the job requirements, they are considered undereducated (underedu); conversely, if an individual’s education exceeds the job requirements, they are deemed overeducated (overedu).Whether there is an educational mismatch is determined by two indicators: the worker’s educational attainment (redu) over the education attained by the individual (edu), while the degree of overeducation is the excess of the education attained by the individual (edu) over the education required by the job (redu), both of which are nonnegative.The degree of mismatch can be measured in years. Specifically, the degree of undereducation is the excess of the education required by the job , which relies on the respondents’ subjective descriptions of whether their educational attainment matches their current occupation. Given the estimation bias caused by respondents’ vanity or subjective concealment, this is not used in this study. The third is the realized matches (RM), which calculate the average educational level of all practitioners within the occupation to estimate the redu, consisting of the mean method and the mode method. The former uses the mean as the criterion. If a person’s edu is one standard deviation higher (lower) than their redu, he is considered overeducated (undereducated). The latter uses the mode as the criterion and classifies one as overeducated (undereducated) if his edu is above (below) his redu. Since the realized matches effectively circumvent the drawbacks of the above two approaches, we adopt the RM estimation. In the latter part of this paper, we use the mean method to perform the baseline analysis, and the mode method is used to perform the robustness test.There are three main methods to measure educational mismatch . The firIn the calculation process, four points are noted:redu that contain less than 30 samples under the same major category. If there is no similar category, the small sample will be excluded.Occupational classification. The CFPS uses the National Standard Occupational Classification and Code of the People’s Republic of China (GB/T6565-2009) , which dredu of the two generations.Generation differences. In the late 1990s, China began to implement the college enrollment expansion plan. Since then, higher education has become popularized, and the scale of college students has expanded rapidly. Correspondingly, the labor market also imposes strict requirements on the education of employees. It is unreasonable to directly take the average educational level of all-age employees as the criterion required for occupation. In this regard, we divided the sample into two generations born around the 1980s and measured the Regional differences. There are significant differences in the educational attainment of workers from different regions. According to data from the Seventh National Population Census in 2020, the average number of years of schooling for people aged 15 and over is 9.91 years nationwide. However, there are significant disparities, with an average of 12.64 years in Beijing and 8.75 years in Guizhou. There is also a significant regional economic divide between China’s eastern, central, and western regions, which leads to different demands on human capital. For example, when looking at the education level of purchasing and sales personnel, the averages in the eastern, central, and western regions are 10.42, 10.10 and 9.57 years, respectively. We therefore divide the sample into three segments based on their geographical location.IQV) [K is the number of education categories, N is the number of individuals within the occupation, and f is the number of individuals within each education category within the occupation.Heterogeneity of education within occupations. Considering the technological refinement and organizational structure settings, the same occupation requires workers with different qualifications to work together, which is ignored by the RM approach . AccordiIQV)  into theredu estimates within each group are shown in Based on the above principles, we divide all the workers into several groups, calculate the average education level within each group, and determine whether each sample falls into the categories of undereducated, overeducated, or educationally matched  at Peking University. In 2010, the CFPS officially launched baseline surveys in 25 provinces, municipalities, and autonomous regions across the country, eventually interviewing 14,960 households and 42,590 individuals. All family members identified in the baseline survey, along with their future biological or adopted children, are considered CFPS genetic members and serve as permanent subjects for CFPS surveys conducted every two years.CFPS uses computer-assisted personal interviewing (CAPI), this the first time this approach has been used in a large-scale longitudinal project in China. During each round of the survey, the CFPS project team recruits a significant number of local interviewers in the sample areas, brings these interviewers to Peking University for standardized training, and conducts a nationwide household interview survey using the CAPI mode. Throughout the survey, interviewers are required to send data back to ISSS on a daily basis after completing the household interviews in the field.This study uses Stata version 16.0 for data analysis. We sequentially perform data description, baseline regression analysis, robustness test, mechanism analysis, and heterogeneity analysis to explore how educational mismatch affects workers’ fertility intentions.j in year t. The independent variable IQV index. The regression also includes fixed effects for industry, county, and year. The empirical model is as follows: β, which we are interested in, captures the differences in fertility intentions among undereducated (overeducated) workers compared to those who are properly matched with the same number of years of schooling. Given that educational matches seldom change in the short term, the variation over time may not be significant enough to be captured by the fixed-effect model [When examining the effects of undereducation (overeducation), we exclude overeducated (undereducated) workers and include only those who are properly matched as the control group. As a result, the estimation of ct model . We use The benchmark results are shown in As we can see from the estimations of the whole sample, educational mismatch does not affect workers’ fertility intentions. However, the results show significant heterogeneity after sample grouping. One is gender heterogeneity, and the other is age heterogeneity. Specifically, educational attainment has a negative effect on the fertility intentions of female workers aged 18–35. Moreover, if they are undereducated, fertility intentions will be further reduced. At the 5% significance level, the fertility intentions of women aged 18–35 decrease by 0.53% for each year of undereducation , while overeducation reports no significant effect on fertility intentions.The benchmark result may have serious endogeneity. First, measurement error, using different measures obtains different estimates of educational mismatch. Second, omitted variable bias, fertility intentions imply individual feelings and values that are difficult to quantify or introduce into the model. Third, due to self-selection bias, some individuals take their fertility intentions into consideration, thus having a preference for specific occupations and choosing jobs that do not fit their educational background. We adopt a series of robustness tests to ensure that the findings are robust and reliable.The baseline regression restricts the sample to only two groups above and below the age of 35, which cannot identify the effect of educational mismatch across age groups in detail. Taking 5 years as a stage, we divide the sample age into six generations and set dummy variables for each generation group –(2) of Mode method. Furthermore, we replace the educational mismatch measured by the mean method with the measurement of the mode method for verification. The results in columns (3)–(4) of Traditional gender norms. Gender norms can significantly influence both women’s labor participation and childbearing behaviors. On one hand, traditional gender norms often dictate that women should assume greater responsibilities for household work rather than engaging in productive labor. On the other hand, these norms emphasize the importance of family continuity, placing a significant burden on women for reproduction. The decline in fertility intentions among undereducated women can be attributed to the increasingly egalitarian gender norms. These changing norms encourage women to pursue higher positions within the workplace, potentially leading them to prioritize their careers over childbearing. The CFPS survey asked, “How important are family and reproduction, scored from 1–5?” We introduce this answer as the proxy of the views on traditional gender norms.Fertility status. Current fertility status may influence both employment choices and future fertility intentions. Previous studies have shown that when women have children to care for, they tend to be overeducated in order to balance their family and career . In addiMigration status. Migration status can also be an omitted variable in the benchmark regression. Workers often migrate in search of better job prospects, especially in the face of the rapid expansion of the market economy and accelerated urbanization. There are more opportunities for migrant workers to advance their careers in the place of relocation. Nevertheless, ensuring equitable access to public resources and services for the migrant population remains a challenge, which may contribute to lower fertility intentions among this group. To eliminate the interference brought by migration, we further control the relevant variables in the benchmark model.In The instrumental variable (IV) is an alternative approach for endogeneity. In this paper, we select the proportion of province-intergenerational technical personnel and the operational stock of the intra-industry robots ) as instrumental variables. The reasons are as follows.First, the proportion of province-intergenerational professionals reflects the demand for highly skilled workers among different regions, and this demand is closely linked to the educational distribution of workers. In general, regions with a higher proportion of technical professionals tend to attract a more technology-intensive workforce, which is more likely to be undereducated rather than overeducated.Second, the wave of artificial intelligence and robotics has fundamentally reshaped the labor market. According to the task-based model, scholars contend that the implementation of automation technology affects labor demand by either replacing human resources or generating new job opportunities . From anThird, educational mismatch depends on demand–supply interactions. From the perspective of the supply side, individual employment decisions are strongly influenced by self-induced factors, which makes it difficult to remove self-selection bias. It is necessary to select instrumental variables from the more exogenous demand side. The two instrumental variables selected in this paper reflect the demand for human capital from two macro levels, namely, the employment structure across regions and the technological trend within the industry. They determine the probability of being undereducated or overeducated in the labor market, but are generally unaffected by individuals’ fertility intentions.p-values of Hansen J statistics in the overidentification test are all above 0.05, indicating that the instrumental variables are exogenous. The first-stage regression results show that a higher proportion of province-intergenerational technical personnel is related to more undereducation and less overeducation. Robotics mainly substitutes for female workers over 35 years old. More precisely, it reduces the probability of undereducation and enhances the possibility of overeducation. The second-stage estimations show that the fertility intentions of 18–35 years old undereducated women are significantly lower than those of the properly matched workers with the same number of years of schooling, which confirms the benchmark.In summary, the above instrumental variables satisfy the conditions of relevance and exogeneity. As mentioned above, sample self-selection challenges the research findings. Individuals with stronger fertility intentions and a higher commitment to family life tend to gravitate towards less demanding jobs. Conversely, those with lower fertility aspirations and higher career aspirations are more likely to seek more lucrative employment opportunities. In this case, it is difficult to discern the effect of educational mismatches on fertility intentions.In this paper, we use propensity score matching (PSM) to construct a counterfactual test. The principle of PSM is to match the treatment group with the control group that has similar characteristics to guarantee comparability. The difference between the two groups is the average treatment effect. In the analysis, the educationally mismatched workers are taken as the treatment group, while the properly matched workers are taken as the control group. We adopt k-nearest neighbor matching, radius matching, local linear regression matching, and kernel matching to match the treatment group with the control group and calculate the average treatment effects. On the basis of previous findings, we mainly studied women aged 18–35. As can be seen in Why does undereducation have a negative impact on young women’s fertility intentions? This section verifies the three potential channels listed in the theoretical analysis.The income channel hypothesis suggests that there is a “wage premium” for undereducation, which increases the opportunity cost of childbearing or increases precautionary saving for old age. Column (1) of The psychological channel hypothesis suggests that undereducated workers generally have a high sense of achievement and job satisfaction, which strengthens their perception of their role at work and weakens their desire to have children. As shown in column (3) of The career development hypothesis posits that undereducation influences women’s fertility intentions by reshaping their career plans. To examine this, we introduced a dummy variable indicating workers’ promotion intentions. This variable takes the value of one if the worker aims to attain a technical title or seek promotion to an administrative role; otherwise, it is set to zero. Columns (5)–(7) of This paper finds that women aged 18–35 are affected by educational mismatch. Within this group, what kind of women are more likely to be affected? In this section, we discuss the heterogeneity in the results depending on the characteristics of the female worker and her family.First, we divided the samples into two groups, low-educated and high-educated, based on whether the workers had completed high school education or more. As confirmed in columns (1) and (2) of Then, based on household income quantiles, we divided the sample into three groups: low-income, middle-income, and high-income. As depicted in Columns (3)–(5) of Furthermore, as can be seen in columns (6) and (7) of Using CFPS data, we first examine the impact of educational mismatch on workers’ fertility intentions. Our research confirms that undereducation has a negative impact on the fertility intentions of female workers aged 18–35. Specifically, fertility intentions decrease by an average of 0.53% per year of undereducation, while overeducation reports no significant effect. These findings remain robust when accounting for factors such as measurement error, omitted variable bias, self-selection bias, and the use of IV and PSM methods. Previous studies have generally found that higher education levels are associated with lower fertility intentions among women ,59. Our Why are women aged 18–35 years old influenced by the educational mismatch? According to the 2015 National Population Sample Survey, the fertility rate among women of childbearing age in China is 30.93%, with an average fertility rate of 53.92% for women aged 18–35. This demographic group is at a pivotal point in their lives, marked by both childbearing and career development. In contrast to men, women often experience double pressure, both from the labor market and family. They bear the responsibility for material production, such as paid employment, while also shouldering the role of social reproduction, which includes unpaid domestic work. Educational mismatch in the labor market may further intensify the conflict between family and work.We then explore the potential channels for the negative effect of undereducation on the fertility intentions of women aged 18–35.On the one hand, it is the tangible opportunity cost  that contributes to the decline in fertility intentions among undereducated women. We find that compared with individuals whose employment is properly matched with the same number of years of schooling, undereducated individuals have a wage premium. Numerous studies find that women may experience career interruptions and wage decline after childbirth, which is also known as a maternal penalty . MoreoveOn the other hand, the intangible opportunity cost (in the form of limited career advancement) plays a key role. Compared with individuals whose employment is properly matched with the same number of years of schooling, undereducated individuals have higher expectations of career development. Previous research indicates that childbirth often disrupts the accumulation of work experience and professional skills, diminishing women’s competitiveness for career progression ,61. In eThe above underlying mechanism is confirmed through heterogeneity analysis. We find that women with low socio-economic status are more likely to reduce their fertility intentions due to undereducation.In terms of educational attainment, low-educated women are more affected. Since low-educated female workers face a higher risk of substitution and face more severe challenges when returning to work after childbirth, they are more reluctant to give up the good employment opportunities that undereducation brings, thereby reducing their fertility intentions.In terms of family income, women from low-income families are more affected. This confirms the hypothesis that undereducated women tend to have weaker fertility intentions due to the opportunity cost of childbearing. Given that low-income families often have limited financial resources, they may struggle more to accommodate the loss of wage premium resulting from an undereducated family member’s temporary departure from the workforce.In terms of employment type, women who are employed in the private sector are more affected. This finding may be attributed to the fact that, compared to the self-employed, employees face the risk of being dismissed due to childbearing. Furthermore, unlike the family-friendly public sector, the private sector tends to be less accommodating to female employees who are pregnant or have children ,63.This study has several limitations. First, our model fails to include work experience due to data constraints. Previous research has indicated that undereducation and work experience are often mutually substitutable, which may explain wage premiums. In the future, when the data is available, the inclusion of work experience as a control variable could potentially increase the strength of the conclusions. Second, our analysis primarily focuses on discussing the impact of educational mismatch on workers’ fertility intentions. It might be more meaningful to examine the effect of educational mismatch on actual fertility behavior, including the number of children and the timing of childbirth. However, we could only obtain information about workers’ current jobs and lack historical work trajectories before childbirth. Consequently, conducting such research is challenging, as many people, especially women, change jobs after giving birth.Our study finds a negative effect on the fertility intentions of undereducated women aged 18–35, which could be explained by childbearing opportunity cost and career development desire. Specifically, women with low socio-economic status are more affected. For these groups, the undereducation premium holds even greater significance. At the same time, they face a higher likelihood of labor market replacement after childbirth.Our findings have the following implications. On the one hand, online education, distance education, and other flexible ways can be adopted to provide learning opportunities for the undereducated group to enhance the degree of matching between their qualifications and job requirements, thus alleviating their substitution risks in the labor market. On the other hand, the protection of women’s rights and interests in the labor market, such as the establishment of diversified maternity allowance systems and the provision of post-natal employment counselling, should be accelerated to remove the reproductive burden and opportunity cost faced by women.


Inappropriate urine cultures (UCs) lead to misdiagnosis and unnecessary antibiotic use and can impact surveillance. The absence of pyuria on a urinalysis (UA) has a high negative predictive value for urinary tract infections (UTIs) in certain settings and can reduce unnecessary UCs. UCs performed without UA or with a negative UA indicate opportunities for diagnostic stewardship. Understanding facility variability and patient characteristics associated with inappropriate UCs can inform improvements in acute care hospitals (ACHs).Using clinical microbiology data from 279 ACHs reporting UC and UA data to the PINC AI Healthcare Database, we assessed appropriateness of the first UC collected during adult hospitalizations, excluding pregnant and urologic procedure patients identified using ICD-10-CM diagnosis codes. Each UC was categorized as follows: (1) UC with at least 1 positive UA  24 hours before/after UC (UA+ UC), (2) UC with negative UA in 24 hours (UA- UC), and (3) UC without UA in 24 hours (no-UA UC). Overall percentages of UC categories were described by year and patient characteristics. Monthly facility-specific percentages of UCs were calculated, and facility-level variability was described by medians, quartiles 1 and 3 (Q1:Q3).1.2 million incident UCs were included. Of those, 65% were UA+, 21% UA-, and 14% no-UA UCs. From 2017 to 2020, UA+ UCs increased from 60.5% to 68.2%, no-UA UCs decreased from 19.3% to 10.5%, and UA- UCs stayed at 21%. UA- and no-UA UCs were more common among male, younger age, and Medicaid patients . No-UA UCs were more commonly obtained on day 4 or later. The median monthly facility-level percentages of UA+, UA-, no-UA UCs were 75 , 15 , and 5.3 , respectively .Overall Percentages of Urine CulturesVariability in Hospital Monthly Percentages of Urine Cultures (UC) in Each Category, 2017-2020, N hospital-months = 9,863Hospital-level VariabilityDuring the study period, urine culturing practices improved; however, 35% of incident UCs had no UA or negative UA. The high proportion of these UCs and facility-level variability of the monthly proportions call for diagnostic stewardship to identify and address causes of the potentially suboptimal UC practices. This will support the efforts to decrease UTI overdiagnosis and antibiotic overuse.All Authors: No reported disclosures


Hepatozoon spp. in ticks infesting asymptomatic domestic animals and to provide insight into their potential spillover from wild to domestic animals. In total, 537 tick specimens were collected in Khyber Pakhtunkhwa, Pakistan, and morphologically identified. The most prevalent tick species was Haemaphysalis cornupunctata , followed by Haemaphysalis kashmirensis , Rhipicephalus microplus , Haemaphysalis montgomeryi , Rhipicephalus sanguineus , each Haemaphysalis bispinosa and Haemaphysalis sulcata , each Hyalomma anatolicum and Rhipicephalus turanicus , Rhipicephalus haemaphysaloides  Hyalomma scupense , and Hyalomma isaaci . The extracted DNA from a subset of each tick species was subjected to PCR to amplify 18S rRNA fragments of Hepatozoon spp. By BLAST analysis, the Hepatozoon sp. detected in Hy. anatolicum infesting cows and in Ha. sulcata infesting sheep showed 99.7% maximum identity with Hepatozoon colubri. Similarly, the Hepatozoon sp. detected in R. haemaphysaloides infesting goats shared 99.49% maximum identity with Hepatozoon ayorgbor, and the Hepatozoon sp. detected in R. sanguineus infesting dogs exhibited 99.7% identity with Hepatozoon canis. Having an overall infection rate , the highest infection rate was recorded for each H. canis, and H. colubri , followed by H. ayorgbor . In the phylogenetic tree, H. colubri clustered with corresponding species from Iran, H. ayorgbor clustered with the same species from Croatia, Ghana, and Portugal, and H. canis clustered with the conspecifics from Iran, Israel, Romania, and Zambia. Regarding the potential spillover of Hepatozoon spp. from wildlife through ticks, free ranging animals was at higher risk compared to confined animals (RR = 3.05), animals consuming food from wildlife habitats were at higher risk compared to those consuming domestic food (RR = 3.06), and animals residing in farm buildings located in wildlife habitats were at higher risk compared to those residing in farm buildings located in villages (RR = 3.28). In addition to the first report on H. canis in R. sanguineus in Pakistan, this is the earliest data showing H. ayorgbor in R. haemaphysaloides and H. colubri in Ha. sulcata and Hy. anatolicum. These preliminary findings suggest a potential spillover of Hepatozoon spp. from wild to domestic animals via ticks under certain risk factors.This study aimed to detect  Hepatozoon is a diverse genus of apicomplexan, primarily comprised of haemoparasites transmitted by arthropods, especially by ticks to all classes of terrestrial vertebrates, including wild and domestic animals  granted ethical permission (Dir/A&R/AWKUM/2018/1410) for the conduction of this study. Additionally, the owners of the animals also gave their oral permission to collect ticks from their animals.This study was conducted in four districts of Khyber Pakhtunkhwa, including Bajaur , Malakand , Mohmand  in the north-western and Lakki Marwat  in the south-eastern. Global Positioning System was used for finding the actual coordinates of collection points in the studied area and subsequently used in designing the map using ArcGIS v 10.3.1 , tick specimens were morphologically identified based on various morphological features, and validated up to the species level using the standard taxonomic identification keys  were selected and subjected to DNA extraction individually. Prior to DNA extraction using the phenol-chloroform method with minor modifications , the homHepatozoon spp., the extracted genomic DNA samples were subjected to conventional PCR  using specific primers: HEP2 144-169-F (5′-GGTAATTCTAGAGCTAATACATGAGC-3′) and HEP2 743-718-R (5′-ACAATAA AGTA AAAAACAYTTCAAAG-3′), and PCR experimental conditions were set as previously described (DreamTaq green MasterMix (2X). All PCR procedures employed PCR water as a negative control and H. canis DNA as a positive control. The obtained amplicons were run on a 2% agarose gel followed by observing via Gel Documentation  and purified using the GeneClean II Kit .For the amplification of 18S rRNA fragments of escribed . Each PCThe purified amplicons were submitted for DNA sequencing  by Sanger sequencing method using an ABI 373XL system. Low-quality nucleotide sequences and contaminated reads were eliminated during trimming and assembling of raw sequences through SeqMan version 5.0 . With the aim to determine the closest identities with other sequences previously deposited in the GenBank, the cleaned sequences were subjected to BLAST  at NCBI . For the subsequent phylogenetic tree using Molecular Evolutionary Genetics Analysis (MEGA-X) , the hom2), selecting significance at P < 0.05, and relative risk (RR), with the 95% confidence interval.The questionnaire data along with PCR data were collected and assembled in spreadsheets using Excel . Using GraphPad Prism version 5.0 , the assembled data was subjected to chi-square test , followed by Haemaphysalis kashmirensis , Rhipicephalus microplus , Haemaphysalis montgomeryi , R. sanguineus , each Haemaphysalis bispinosa and Haemaphysalis sulcata , each Hyalomma anatolicum and Rhipicephalus turanicus , Rhipicephalus haemaphysaloides  Hyalomma scupense , and Hyalomma isaaci . The highest prevalence of tick infestation was recorded on dogs (68.4%: 13/19), followed by cows (64.7%: 22/34), goats (58%: 29/50), and sheep (56.3%: 27/48), resulting in overall prevalence . The details about each tick species, including life stage, host association, and collection site, are provided in Altogether, 537 ticks were morphologically identified, which were belonging to three distinct genera of hard ticks, including Hepatozoon sp. detected in Hy. anatolicum from cows and Ha. sulcata from sheep shared a maximum identity of 99.66% with Hepatozoon colubri, whereas Hepatozoon sp. detected in R. haemaphysaloides from goats revealed a maximum identity of 99.49% with Hepatozoon ayorgbor, and Hepatozoon sp. detected in R. sanguineus from dogs showed a maximum identity of 99.66% with Hepatozoon canis. With a total infection rate , the highest rate was recorded for each H. canis, and H. colubri , followed by H. ayorgbor . Hepatozoon spp., including the associated vertebrate hosts and the corresponding locality.According to BLAST analysis, OR241141 (H. ayorgbor), OR241161 (H. colubri), and OR241147 (H. canis).The obtained sequences for 18S rRNA were submitted to GenBank under the following accession numbers; Hepatozoon sp. amplified from the genomic DNA of Hy. anatolicum and Ha. sulcata were identical to each other, resulting in a consensus sequence of 590 bp. Similarly, all sequences of Hepatozoon sp. amplified from the genomic DNA of R. haemaphysaloides were identical, while all sequences amplified from the genomic DNA of R. sanguineus were identical, yielding a consensus sequence of 588 and 596 bp, respectively. The Ha. sulcata and Hy. anatolicum based sequences showed the highest identity of 99.66% with H. colubri (MN723844), while the R. haemaphysaloides based sequence displayed the highest identity of 99.49% with H. ayorgbor (EF157822.1), as well as with two undetermined Hepatozoon spp. (MT919387 and MT919388), and R. sanguineus based sequence revealed maximum identity of 99.66% with H. canis (KX712126 and KX880505). The details regarding the infection rate and the association of Hepatozoon spp. with tick species in each district are shown in A sum of 64 sequences  were obtained from all amplified fragments. All sequences of Hepatozoon spp. detected in the present survey. Hepatozoon ayorgbor clustered with the same species previously detected in Apodemus flavicollis, Python regius, and an unknown tick species from Croatia, Ghana, and Portugal, respectively. Hepatozoon colubri clustered with the corresponding species found in Zamenis lineatus from Iran, and H. canis clustered with conspecifics detected in Canis lupus, Canis aureus, and Canis familiaris from Iran, Romania, and Israel and Zambia, respectively , free ranging animals were at greater risk compared to those confined in shelters (RR = 3.05), animals getting food from wildlife habitat were at greater risk compared to those getting domestic food (RR = 3.06), and animals living in farm buildings located in wildlife habitats were at greater risk compared to those living in farm buildings located in villages (RR = 3.28).Different factors were noted which could lead to the spillover of gathered . Herein,Hepatozoon spp. from wildlife to domestic animals, ticks have been widely overlooked as vectors of Hepatozoon spp., and there is a scarcity of knowledge regarding Hepatozoon spp. of wildlife origin in ticks infesting domestic animals. However, it is crucial to address this knowledge as these parasites pose a potential zoonotic threat, which, in turn could negatively impact food security within ecosystem , H. ayorgbor in snakes (Python regius) and the closely related Hepatozoon spp. in rats and gerbils , and H. canis in jackals (Canis aureus) and dogs (Canis lupus familiaris)  in Brazil, and Amblyomma fimbriatum infesting monitor lizard (Varanus panoptes) and snake (Liasis fuscus) in Australia (Ixodes tasmani infesting Tasmanian devils (Sarcophilus harrisii) in Australia and Ha. sulcata (host-seeking) in Turkey  , 52–57, ustralia , 59, Ixon Turkey , 61. Somin Japan . Unlike he world , 20, 63,e region , 64–66, Hepatozoon spp. spillover could be determined by their abilities such as infecting a wide host range  can be found in the article/supplementary material.The animal studies were approved by the owners of the animals also gave their oral permission to collect ticks from their animals. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.AAli: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Writing—original draft, Writing—review and editing. HT: Formal analysis, Investigation, Methodology, Visualization, Writing—original draft, Writing—review and editing. MK: Data curation, Investigation, Methodology, Software, Validation, Visualization, Writing—original draft, Writing—review and editing. MA: Data curation, Funding acquisition, Investigation, Methodology, Project administration, Writing—original draft, Writing—review and editing. AAlo: Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Writing—original draft, Writing—review and editing. HA: Data curation, Software, Writing—original draft, Writing—review and editing. TT: Data curation, Formal analysis, Methodology, Project administration, Software, Writing—review and editing. K-HT: Data curation, Formal analysis, Funding acquisition, Methodology, Resources, Software, Visualization, Writing—original draft, Writing—review and editing.


This study was conducted to evaluate the antitumor activity of WRH-2412 in HCC induced in rats through affecting the TGF-β/β-catenin/α-SMA pathway. Antitumor activity of WRH-2412 was evaluated by calculating the rat’s survival rate and by assessment of serum α-fetoprotein. Protein expression of TGF-β, β-catenin, E-cadherin, fascin and gene expression of SMAD4 and α-SMA were determined in hepatic tissue of rats. WRH-2412 produced antitumor activity by significantly increasing the rats’ survival rate and decreasing serum α-fetoprotein. WRH-2412 significantly reduced an HCC-induced increase in hepatic TGF-β, β-catenin, SMAD4, fascin and α-SMA expression. In addition, WRH-2412 significantly increased hepatic E-cadherin expression.Hepatocellular carcinoma is considered one of the most lethal cancers, which is characterised by increasing prevalence associated with high level of invasion and metastasis. The novel synthetic pyrazolo[3,4-  Hepatocellular carcinoma (HCC) is ranked as the sixth most frequent cancer in the world. It occupies the fourth leading cause of cancer-related death. Hepatocarcinogenesis process is considered as a complex multistep process that is affected by a group of risk factors leading to tumour progression and metastasis.b] pyridine compounds, which are small molecules proved as antitumor,,b]pyridine compounds is 4–(4-hydroxyphenyl)-1,3,6-triphenyl-1H-pyrazolopyridine-5-carbonitrile, WRH-2412  value equals 2.16 μM.WRH-2412 against HCC hasn’t been investigated in vitro or in vivo.One of the candidate therapeutic agents against HCC is pyrazolopyridine compounds, such as WRH-2412 with many therapeutic activities as antiviral and anticancer. Treatment of HCC rats with WRH-2412 proved promising antitumor activity. This can be proved by WRH-2412 ability to reduce HCC-induced elevation in serum α-fetoprotein and number of nodules in the liver. Furthermore, the percent of HCC rats survival was doubled by using WRH-2412. In addition, WRH-2412 produced many improvements in the structure of hepatic histopathological features as indicated by attenuation of destroyed cells, reduction of hyperplastic nodules and heteromorphism. It is the first time to prove the ability of WRH-2412 to produce anticancer activity against experimentally induced HCC.The first line therapy is represented by sorafenib and lenvatinib, while regorafenib is considered the second line of treatment.WRH-2412 reversed all of these effects in HCC rats without affecting the control rats. It it the first time to report that WRH-2412 protects against HCC through blocking TGF-β/β-catenin/SMAD4 axis.TGF-β is a pleiotropic, potent, regulatory cytokine and a major component that leads to EMT. Its signaling pathway has a great role in many cellular processes as cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance and survival.α-SMA activation is linked to activation of myofibroblasts. It is associated with progression of several types of malignancy as osteosarcomas, lung adenocarcinoma, head and neck squamous cell carcinoma.α-SMA associated with reduction in E-cadherin expression. Treatment of HCC rats with WRH-2412 reversed these effects in HCC rats without affecting the control rats.Vascular invasion is present in about 25–50% of HCC patients and represents a great risk factor that enhances tumour recurrence and leads to poor overall survival among patients with HCC.WRH-2412 has an antitumor effects and may suppress HCC development via inhibition of the TGF-β/β-catenin/α-SMA pathway axis, which may suppress vascular invasion markers. Therefore, WRH-2412 stands out as a promising starting point and as a novel potential therapeutic drug for improving the outcome of patients with HCC.The present study indicated that


Cranial neural crest cells (NCCs) are the origin of the anterior part of the face and the head. Cranial NCCs are multipotent cells giving rise to bones, cartilage, adipose-tissues in the face, and neural cells, melanocytes, and others. The behavior of cranial NCCs  are well regulated by several signaling pathways; abnormalities in their behavior are often reported as causative reasons for craniofacial anomalies (CFAs), which occur in 1 in 100 newborns in the United States. Understanding the pathological mechanisms of CFAs would facilitate strategies for identifying, preventing, and treating CFAs. Bone morphogenetic protein (BMP) signaling plays a pleiotropic role in many cellular processes during embryonic development. We and others have reported that abnormalities in BMP signaling in cranial NCCs develop CFAs in mice. Abnormal levels of BMP signaling cause miscorrelation with other signaling pathways such as Wnt signaling and FGF signaling, which mutations in the signaling pathways are known to develop CFAs in mice and humans. Recent Genome-Wide Association Studies and exome sequencing demonstrated that some patients with CFAs presented single nucleotide polymorphisms (SNPs), missense mutations, and duplication of genes related to BMP signaling activities, suggesting that defects in abnormal BMP signaling in human embryos develop CFAs. There are still a few cases of BMP-related patients with CFAs. One speculation is that human embryos with mutations in coding regions of BMP-related genes undergo embryonic lethality before developing the craniofacial region as well as mice development; however, no reports are available that show embryonic lethality caused by BMP mutations in humans. In this review, we will summarize the recent advances in the understanding of BMP signaling during craniofacial development in mice and describe how we can translate the knowledge from the transgenic mice to CFAs in humans. Wnt1-Cre mice and P0-Cre mice, are largely used for tracing NCCs and NC-specific activation or deletion of targeted genes by the Cre-LoxP system  expression is more abundant in premigratory neural crest cells that post-migratory cells  are birth defects affecting the shape of the head and the face. The incidence is approximately 1 in 100 newborns . OrofaciP system . Both trCre mice . Detailen region . Wnt1  are members of the Transforming Growth Factor Beta superfamily. Upon BMP ligands  binding, BMP receptors form hetero-multimers consisting of BMP type II receptors (BMPR2 and ACVR2A and ACVR2B) and BMP type I receptors  then transduce the signaling through phosphorylation of SMAD1/5/9 (SMAD-dependent pathway) or TAK1/p38 MAP kinases (SMAD-independent pathway) . The sigBone morphogenetic proteins (BMPs) were originally discovered as bone inducers . InteresOrofacial cleft  is the most common CFAs that show splitting lip and/or splitting shelves of the mouth  . BecauseBmp7-deficient mice (Bmp7Δ/Δ mice) develop cleft palates with the shorter Meckel’s cartilage  develop cleft palate with lower proliferation and suppressed osteogenic condensation at the palate  develops cleft palate as a consequence of the failure of tongue descent  in mice  also develop cleft lip and cleft palate. Wnt1-Cre;pMes-caBmpr1a mice showed reduced proliferation and ectopic cartilage formation at the palatal mesenchyme  develops cleft palate association with a higher level of fibroblast growth factor (FGF) signaling that is a known causative reason for Apert syndrome, a subset of patients with the syndrome develops cleft palate  develops cleft palate while P0-Cre;caBmpr1afl/+ mice and P0-Cre;Tak1fl/fl mice did not develop a cleft palate.  in NCCs in mice  develops midfacial defects including orofacial cleft  develop large calvarial defects in the frontal bones as expected; however, NC-specific deletions of all four alleles in Msx1/Msx2 in mice  develop ectopic bones at the calvaria  demonstrated that Msx1 and Msx2 at E10.5 to E11.5 are required to suppress the ectopic bone formation while those at E11.5 to E12.5 are required to prompt calvarial formation  develops premature fusion of the anterior frontal suture and the naso-premaxillary suture, which leads to craniosynostosis  develop craniosynostosis  develops brachycephaly with unilateral coronal craniosynostosis in humans with a slight elevation of phospho-SMAD levels  and ACVR2A  are found in patients with lambdoid craniosynostosis  were found in a Smad6 exon and a putative enhancer region of BMP2, and frameshift mutations in Smad6 were found . SMAD6 iThere are still uninvaded niches, especially, why missense or nonsense mutations in genes related to BMP signaling are rarely found in patients with CFAs. In the future, it is an essential effort to analyze BMP mutations in preterm and stillbirth babies. It is also essential to identify gene mutations in patients with non-syndromic CFAs. Genome-wide association studies (GWAS) and exome sequencing are powerful tools to determine whether they have mutations in BMP ligands, BMP receptors, Smads, noncanonical signaling of BMP signaling, and downstream target genes of BMP signaling, especially identifying SNPs in regulatory regions. We also need to examine whether these mutations change signaling levels of BMP signaling by testing the phosphorylation of SMAD1/5/9 and non-SMAD signaling levels, which is less understood at this moment. These efforts could provide novel therapeutic options for non-syndromic patients with CFAs.


The mechanistic/mammalian target of rapamycin (mTOR), a member of the phosphoinositide 3-kinase (PI3K) related kinase family, integrates intracellular and environmental cues that coordinate a diverse set of cellular/tissue functions, such as cell growth, proliferation, metabolism, autophagy, apoptosis, longevity, protein/lipid/nucleotide synthesis, and tissue regeneration and repair . AlthougmTOR is the core component of two structurally and functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 integrates various stimuli and signaling networks to promote anabolic cellular metabolism, but it blocks catabolic processes, such as autophagy, by regulating each step of the autophagy process, including induction, nucleation, elongation, and the formation of a double-membrane autophagosome, the formation of autolysosomes, and the recycling of autophagosome-sequestered substrates . The dysThe PI3K/mTOR signaling pathway plays a pivotal role in regulating cellular homeostasis; therefore, the dysregulation of signaling is often involved in aging and in age-related pathologies, including cancer, cardiovascular diseases and diabetes, among others. Thus, understanding this highly non-linear system pathway—involving intricate regulatory mechanisms and crosstalk with neighboring—is mandatory to advancing biology and the development of new therapeutic approaches. Making use of the computational and experimental studies available in the literature, Ghomlaghi et al. provided a nice overview of the complex dynamic mechanistic network of PI3K/mTOR signaling, highlighting its interaction/interdependence with other major signaling pathways, competitive inhibition and epigenetic alterations properties, and associated interconnecting positive/negative regulatory loops (feed-back/feed-forward mechanisms) . In thisAs second messengers, the intracellular levels of cyclic nucleotides, cAMP (cyclic adenosine monophosphate), and cGMP (cyclic guanosine monophosphate) control all major cellular signaling pathways in cell proliferation, cell cycle regulation, and metabolic functioning. cAMP and cGMP are produced by activated adenylyl-cyclase and guanylyl-cyclase, respectively, and degraded by phosphodiesterases (PDEs). cAMP/cGMP and their associated kinases, including protein kinase A (PKA) and protein kinase G (PKG), directly/indirectly regulate mTOR signaling by phosphorylating different components of mTOR complexes under various physiological and pathological conditions. In the review article by Shi and Collins, the recent findings about the regulation of the mTOR signaling network by cAMP and cGMP, as well as by their corresponding protein kinases, PKA and PKG, were briefed . They alc-myc and Notch1 genes, phosphatase, and tensin homolog (Pten), as well as the RNA binding protein LIN28B, among other potential mechanisms and targets [Despite the growing number of people suffering from a severe hearing impairment, hearing aids or cochlea implants are the most effective and common treatment options. Currently, no curative intervention has been clinically used to treat sensorineural hearing loss (SNHL) because of the lack of understanding about the potential mechanisms underlying SNHL. New pharmacological strategies have been proposed that can promote cell survival pathways in sensory hair cells. A growing number of studies have illustrated the potential regenerative benefits of using progenitor cells of the chochlea, which can stimulate the differentiation of these progenitor cells into the hair cells and neurons to alleviate hearing impairment . A coupl targets . mTOR si targets . The autThis Special Issue also features a few original research articles on the therapeutic potential of mTOR inhibitors in different pathological conditions. Several lines of evidence implicate that mTOR signaling is critical for physiologic wound healing and pathologic fibrogenesis . AberranIn the heart, glycogen synthase kinase-3 beta GSK3β has been shown to play a major role in cardiac ischemic injury and hypertrophy. The activity of GSK-3β is inversely regulated by phosphorylation at serine-9 (S9) residue via AKT, which has been shown to regulate hypertrophic cardiomyocyte growth and viability and protects hearts against myocardial ischemia/reperfusion injuries ,17. HowePatients with diabetes have a poor prognosis after myocardial infarction (MI), which leads to the development of heart failure with a higher risk of mortality  due to aEndothelin-1 (ET-1), a potent vasoconstrictive peptide produced by the endothelial cells, stimulates cell proliferative, fibrosis, and inflammation through binding to the endothelin receptors, ETA-R and ETB-R, which are abundantly expressed in cardiac tissues. Several lines of evidence suggest that the ET-1 and ET-R expression levels are elevated in the plasma and hearts of diabetic patients, and endothelin receptor antagonists protect against diabetes-induced cardiac injuries ,25,26. ACaenorhabditis elegans.Preclinical studies and randomized clinical trials established that pharmacological inhibitors of mTOR counteract age-associated pathogenesis to extend the patient’s longevity. Employing artificial intelligence (AI)-assisted approaches, Vidovic et al. identified a potent mTOR inhibitor, TKA001, with low toxicity, favorable physicochemical properties, and a preferable ADMET  profile from 1000 small molecules predicted to target mTOR . They mo