This protective effect was explained by the increased nuclear translocation of nuclear factor erythroid 2-like 2 (Nrf2), consequently upregulating the production of heme oxygenase-1 (HO-1), a critical cellular instinct antioxidant enzyme that neutralizes excessive oxidative stress. Furthermore, bee venom treatment activated the tropomyosin-related kinase receptor B (TrkB)/cAMP response element-binding (CREB)/brain-derived neurotrophic factor (BDNF), which is closely related to the promotion of cellular antioxidant defense and neuronal functions. A mouse model with cognitive deficits induced by Aβ1–42 intracerebroventricular (ICV) injections was also used. Bee venom enhanced animal cognitive ability and enhanced neural cell genesis in the hippocampal dentate gyrus region in a dose-dependent manner. Further analysis of animal brain tissue and serum confirmed that bee venom reduced oxidative stress, cholinergic system activity, and intercellular neurotrophic factor regulation, which were all adversely affected by Aβ1–42. Our study demonstrates that bee venom exerts antioxidant and neuroprotective actions against neural oxidative stress caused by Aβ1–42, thereby promoting its use as a therapeutic agent for neurodegenerative disorders.Honeybee venom has recently been considered an anti-neurodegenerative agent, primarily due to its anti-inflammatory effects. The natural accumulation of amyloid-beta (Aβ) in the brain is reported to be the natural cause of aging neural ability downfall, and oxidative stress is the main route by which Aβ ignites its neural toxicity. Anti-neural oxidative stress is considered an effective approach for neurodegenerative therapy. To date, it is unclear how bee venom ameliorates neuronal cells in oxidative stress induced by Aβ. Here, we evaluated the neuroprotective effect of bee venom on Aβ-induced neural oxidative stress in both HT22 cells and an animal model. Our results indicate that bee venom protected HT22 cells against apoptosis induced by Aβ Maintaining the yin-yang balance has been a holistic approach in Korean medicine and traditional East Asian medicine to foster stable health, and a tilt in this stability would emerge as a disease condition in the human body . OxidatiBee venom medicine has long been used in Korea to relieve pain symptoms and treat inflammatory diseases. Recent clinical and experimental studies have demonstrated that bee venom and its derived active components are applicable to a wide range of neurodegenerative disorders. The effects of bee venom on neural disease are known primarily through the modulation of inflammatory responses .2+ influx and destabilizing intercellular electrochemical balance [It has been well established that neuro-oxidative stress ignited by naturally produced Aβ deposited in the aging brain is a main factor in the development of neural disorders ,12. Aβ i balance ,20,21. A balance ,23. Meta balance . In neur balance , neuron  balance ,27,28. Recent studies have indicated that antioxidant properties may provide a more holistic strategy to the anti-inflammatory approach. Antioxidant and detoxification genes are believed to preserve cellular homeostasis and eliminate toxins before they can cause damage and activate inflammatory responses ,30,31. NAlthough previous studies have depicted bee venom as a potent anti-neurodegenerative agent owing to its anti-inflammatory ability , the priTherefore, we propose that bee venom’s neuronal protective effects might be accountable due to its potential to buttress cellular redox balance by upregulating cellular instrinsic antioxidative stress barriers. Clarifying this uncharted aspect of bee venom mechanisms plays a key role in promoting this drug candidate as a holistic and effective neurodegenerative agent.Apis mellifera, Aβ1–42, and Fluoromount™ Aqueous Mounting Medium were purchased from Sigma–Aldrich . The Quanti MAXTM WST-8 Cell Viability Assay Kit was purchased from BIOMAX . The specific mouse primary antibodies  and secondary anti-mouse IgG were purchased from Cell Signaling Technology, Inc. . BDNF, mAChR1, and lamin B antibodies were purchased from Merck KGaA . The Pro-PREP protein extraction solution was purchased from iNtRON Biotechnology . The first doublecortin antibody from Santa Cruz Biotechnology  and Alexa Fluor 488 secondary antibody  were used. Cellular ROS, superoxide dismutase (SOD), lipid peroxidation (MDA), lactate dehydrogenase (LDH), and protein carbonyl activity required acetylcholine esterase (AChE) assay kits, AIF first antibody, CytoC first antibody, and CleaCas3 first antibody, annexin V, and propidium iodide (PI) from Abcam . A nuclear extraction kit was purchased from Cayman Chemical Company, Inc. . An acetylcholine (Ach) level assay kit was purchased from Cell Biolabs .Dulbecco’s Modified Eagle Medium (DMEM), PBS, and TBS were purchased from Welgene Inc. . Trypsin-EDTA (0.25%), FBS, 2,7-dichlorofluorescein diacetate (DCFDA), and penicillin–streptomycin were obtained from Invitrogen . Bee venom from 2 and 37 °C in 96-well plates. The cells were cultured with the following conditions: 1 × 104 cells per well in 0.1 mL media, and 6-well plates with 3 × 105 cells per well in 3 mL media. Twenty-four hours after cell seeding, further drug administration was carried out as described in the following sections.The mouse hippocampal HT22 cell line was a gift from Professor Na Chang Su, College of Korean Medicine, Dongshin University. Cells were cultured in DMEM supplemented with FBS (10%) and penicillin–streptomycin (1%). The incubation conditions were 5% CO1–42 was added. At different time points—0 h, 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h after Aβ1–42 was added—cell availability was measured by adding 100 μL WST-8 and incubating for 1 h. Absorbance at 450 nm was measured using a Versa Max Microplate Reader . The experiments were repeated three times, and 2 mM Triton X was used to achieve absolute cell death as a reference for the availability percentage calculation.To assess cell viability after seeding for 24 h in 96-well plates, bee venom was added as follows: 0.15–50 μg/mL for a safety concentration test; for the cellular protection activity, 0.15–5 μg/mL, and 1 h later, 5 μM of Aβ1–42. All cells (floating and attached to the well bottom) were harvested and washed twice with PBS. Cells (1 × 105) were collected and resuspended in 500 µL of 1× binding buffer. The staining process was carried out by adding 5 µL of annexin V (detected by the FITC channel) and 5 µL of propidium iodide  channel). For ROS detection, 500 µL of 25 µM DCFDA (detected by the FITC channel) was used. The cells were incubated at 37 °C for 15 min in the dark. Approximately 10,000 cells from each sample were used to produce the data. Analysis was carried out using a Cytoflex flow cytometer  according to a previous study protocol [In 6-well plates, the experiment was performed 8 h after treatment with Aβprotocol .1–42 in black 96-well plates with clear bottoms. For the apoptosis assay, the cells were washed once with 200 µL assay buffer, after which 1 µL of CytoCalcein 450 solution was added  + 1 µL of 7-AAD solution , + 2 µL of Apopxin Green Indicator solution , and incubated at room temperature in the dark for 45 min. Subsequently, the cells were washed once, and the wells were filled with 100 µL assay buffer and observed under a 50× objective. For the cellular ROS assay, the cells were washed twice with assay buffer, and then each well was filled with 200 µL of 25 µM DCFDA solution (Ex/Em = 490/525 nm) and incubated at 37 °C in the dark for 15 min. The DCFDA solution was then removed and replaced with 100 µL of assay buffer and observed with a 30× objective. An Invitrogen EVOS FL Auto Imaging System fluorescence microscope  was used.For apoptosis, and ROS immunofluorescence imaging, the experiment was performed 8 h after treatment with Aβ1–42; cell lysate and assay performance were carried out according to the manufacturer’s instructions. The data were collected using a VersaMax microplate reader .To measure SOD, MDA, LDH, and protein carbonyl levels in 6-well plates, the experiment was performed 8 h after treatment with Aβ1–42 treatment, cells were harvested. Part of the cell pellet was lysed immediately to collect whole-cell proteins. The remaining part was added to a hypotonic buffer containing phosphatase and protease inhibitors and incubated for 10 min at 4 °C. Subsequently, the 10% Nonidet P-40 assay reagent was introduced to facilitate phase separation. Centrifugation was performed , and supernatant with cytosolic content was carefully collected and stored at −80 °C. The remaining mixture was introduced into the nuclear extraction buffer and incubated for 30 min at 4 °C. This was then centrifuged , and the supernatant was collected as the nuclear extract. The blot membranes of cytosolic and nuclear proteins were processed similarly, and the imaging exposure time was used to express the true scale of protein expression between nuclear and cytosolic proteins. To confirm the accepted separated nuclear and cytosolic contents, the total cytosolic lamin B signal must be less than 5% of that in the nuclear extract. Similarly, the total cytosolic β-actin signal must be less than 5% of that in the nuclear extract.Whole-cell, nuclear, and cytosolic proteins were collected to study the nuclear translocation of Nrf2 . In a 6-Male ICR mice (7 weeks old) were purchased from Samtaco  and housed with a maximum of five mice per cage under pathogen-free conditions , with a 12-h light/dark cycle and free access to standard food  and water. Acclimation was carried out for five days before the experiments were started. Behavioral experiments were conducted in accordance with the Guide for Care and Use of Laboratory Animals of the National Research Council  and were approved by the Committee of Animal Care and Experiment of Dongshin University, Korea (DSU2019-04-02).1–42 was dissolved in 100 mL sterile physiological PBS in a tube, which was sealed and incubated for four days at 37 °C [1–42 ICV injections were performed as described, and the prepared Aβ1–42 solution , was injected into the right cerebral ventricle using a 28-gauge needle with stereotaxic coordinates (in mm), A: −0.22, L: 1.0, V: 2.5, from the bregma, flow rate of 3 μL/5 min [1–42. A bee venom maximum safe dosage of approximately 13.19 mg/kg was previously determined [n = 6):To stimulate aggregation, 1 mg Aβat 37 °C . After aμL/5 min ,42,43. Otermined . Anothertermined . Therefotermined —to avoidGroup 1—PBS ICV + PBS SC;1–42 ICV + PBS SC;Group 2—3 μg Aβ1–42 ICV + bee venom 0.1 mg/kg SC;Group 3—3 μg Aβ1–42 ICV + bee venom 1 mg/kg SC.Group 4—3 μg AβThe spatial learning and memory capacity of the mice were assessed by MWM as previously described, with minor modifications . BrieflyThe ANY-maze  animal behavior monitoring software was used to record the indexes and form a distribution heat map for the last probe test.After the MWM experiment, on day 11, all mice were anesthetized, their chests were opened, and blood was collected from the hearts. After collection, whole blood was allowed to clot by leaving it undisturbed at room temperature for 40 min. Subsequently, serum was separated by centrifugation at 3000 rpm for 10 min at 4 °C. Hippocampuses were also collected from the brains immediately. Within the groups, the hippocampuses of a subset of animals were used for immunofluorescence analysis ; the hippocampuses of the other animals were immediately subjected to biochemical or Western blot analysis the same day.2 ice powder and sliced into 30-μm sagittal sections before being transferred to gelatin-coated slides [Post-fixed brains were incubated with 30% sucrose in PBS for 1 d at 4 °C, which was repeated once with 30% sucrose in PBS solution. The brains were frozen using dry COd slides . At room1–42 treatment, cell pellets were collected and lysed. For the in vivo samples, the mouse hippocampuses were homogenized with lysate buffer immediately after collection and proceeded to the next steps on the same day. Western blotting was performed as described previously [For the in vitro experiments, cells were grown in 6-well plates, and after 8 h of Aβeviously . The AmeHippocampal lysate or serum samples were prepared, and assays were carried out according to the manufacturer’s instructions. Data were collected using a VersaMax microplate reader .p < 0.05.Data are presented as the mean ± standard deviation (SD) for each group. Differences in quantitative measurements were assessed by one-way analysis of variance followed by the Kruskal–Wallis test using Prism software version 8.1. The number of repetitions was given in this method. Differences were considered significant with 1–42-induced cytotoxic in HT22 cells, 0.15–5 μg/mL bee venom was screened. As a result, bee venom at 0.5–5 μg/mL exhibited a dose-dependent effect on HT22 cell availability at 12 to 24 h after Aβ1–42 challenge; therefore, this dosage was selected for later in vitro experiments  significantly reduced cellular ROS levels  pre-treatment dose-dependently enhanced Nrf2 nuclear translocation. At 5 μg/mL, bee venom treatment markedly increased Nrf2 nuclear translocation and HO-1 production by nearly three-fold compared to Aβ1–42 treatment alone  dose-dependently recovered this index  , as wellpression C, a 1.6-pression B.1–42-only-treated group but were significantly upregulated in the bee venom-treated groups.Immunohistochemistry imaging revealed the capacity of bee venom to enhance hippocampal neurogenesis. We further confirmed this via the upregulation of key neurotrophic proteins, such as BNDF and p-CREB. As shown in 1–42 [1–42 (Inflammation is a crucial link that promotes neurodegeneration and is induced by Aβ1–42 ,16,17,1842 [1–42 C.Natural aging neurodegenerative diseases, including Alzheimer’s disease, result in decreased human cognitive function, reduced human life span, and an overall decline in well-being . The braDuring aging, Aβ is a byproduct of the degradation of the amyloid-beta precursor protein—an important functional protein that is concentrated in neuronal synapses. Accumulated Aβ naturally forms aggregates over time and alters the intercellular redox balance of neurons . Neural Previous cardiology and renal studies have suggested that melittin, the major component, makes up more than half of bee venom’s dry weight, stimulates the nuclear translocation of Nrf2 ,35, and We previously published an article that suggested melittin to upregulate both Nrf2/HO-1 and BDNF/TrkB/CREB, and slow down amyloid-beta neuro damage. Nrf2/HO-1 pathway normalization, is a strong pillar in anti-neurodegeneration strategies ,5,7. Kno1–42. To further explain these phenomena, we examined the effects of bee venom on the activation of the Nrf2/HO-1 signaling pathway. HO-1 is an intrinsic antioxidant enzyme that is considered the first line of cellular defense against oxidative damage [1–42-induced HT22 cell injury was attributed to its antioxidant properties. As mentioned, the ability of bee venom to normalize depleted HO-1 levels and combat ROS production was mentioned in other steatohepatitis , wound he damage . Nrf2 is1–42 significantly depleted the TrkB/CREB/BDNF pathway. Remarkably, bee venom pre-treatment stimulated the production of p-TrkB and p-CREB, and in turn, increased the expression of BDNF [The TrkB/CREB/BDNF pathway is commonly studied due to its antioxidant properties, owing to its capacity to significantly reinforce Nrf2/HO-1 upregulation ,59,61,62 of BDNF . Moreove of BDNF ,65. As m of BDNF , wound h of BDNF , and hep of BDNF , bee ven1–42 into mouse brains was established as a means of inducing oxidative stress and neuroinflammation, suppressing neurogenesis, and promoting cognitive impairments. This model depicts significant grades of Alzheimer’s progression symptoms and neurodegeneration results in general [For in vivo research, the ICV injection of Aβ general ,16,17,18In this study, bee venom significantly restored the memory and learning capabilities of animals with cognitive impairment induced by Aβ. The neuroprocessing proficiency of mice is directly associated with hippocampal functioning, and Alzheimer’s disease patients also show signs of significant hippocampal dysfunction . The hip1–42 ICV, and bee venom pre-treatment clearly decreased these oxidative stress markers. Bee venom also reduced the amount of NO buildup and iNOS protein expression in the hippocampus, which implies a reduction in neuronal-derived nitric oxide, a vital factor promoting neural diseases [The overexpression of oxidative stress level is closely related to pathological progression. To confirm the antioxidant effects of bee venom on the in vivo model, we measured ROS and MDA levels in the hippocampus and serum; these parameters were sharply increased due to Aβdiseases ,68.1–42. To confirm the effects of bee venom on neuron biochemistry, we assessed the intercellular neurotrophic factor pathway CREB/BDNF [The balance of the cholinergic system is the foothold in cognitive function . PreviouREB/BDNF , which wREB/BDNF . ConsistIn this study, we exhibited bee venom to upregulate the Nrf2 into the nucleus and to upregulate HO-1 production, which, as mentioned, is a phenomenon observed by many researchers ,37,38. TIn neuro-aging, oxidative stress provoked by naturally deposited Aβ is thought to initiate neurodegenerative diseases and cognitive downfalls. The cellular antioxidant system is the first line of defense in many disorders, including neurodegeneration. Here, we revealed for the first time that the wide neuroprotective effects of bee venom are primarily possible due to its ability to promote Nrf2 nuclear translocation and increase HO-1 antioxidant enzyme production, a process that is also closely reinforced by the TrkB/CREB/BDNF pathway. This ability of bee venom to help regain cellular redox balance promotes its use as a holistic anti-neurodegeneration agent.


FOXP1 syndrome is an autosomal dominant neurodevelopmental disorder characterized by intellectual disability, developmental delay, speech and language delays, and externalizing behaviors. We previously evaluated nine children and adolescents with FOXP1 syndrome to better characterize its phenotype. We identified specific areas of interest to be further explored, namely autism spectrum disorder (ASD) and internalizing and externalizing behaviors.Here, we assess a prospective cohort of additional 17 individuals to expand our initial analyses and focus on these areas of interest. An interdisciplinary group of clinicians evaluated neurodevelopmental, behavioral, and medical features in participants. We report results from this cohort both alone, and in combination with the previous cohort, where possible.Previous observations of intellectual disability, motor delays, and language deficits were confirmed. In addition, 24% of the cohort met criteria for ASD. Seventy-five percent of individuals met DSM-5 criteria for attention-deficit/hyperactivity disorder and 38% for an anxiety disorder. Repetitive behaviors were almost universally present (95%) even without a diagnosis of ASD. Sensory symptoms, in particular sensory seeking, were common.As FOXP1 syndrome is a rare disorder, sample size is limited.These findings have important implications for the treatment and care of individuals with FOXP1 syndrome. Notably, standardized testing for ASD showed high sensitivity, but low specificity, when compared to expert consensus diagnosis. Furthermore, many individuals in our cohort who received diagnoses of attention-deficit/hyperactivity disorder or anxiety disorder were not being treated for these symptoms; therefore, our findings suggest that there may be immediate areas for improvements in treatment for some individuals.The online version contains supplementary material available at 10.1186/s13229-021-00469-z. Deep phenotypic characterization of individuals with functional disruptions in high-confidence autism spectrum disorder (ASD) and/or intellectual disability (ID) risk genes is a key first step in enhancing clinical care and advancing therapeutic development , 2. SuchFOXP1 (OMIM 605515) is a highly conserved transcription factor of the forkhead box P (FOXP) subfamily of FOX transcription factors: mutations in this gene are associated with an autosomal dominant neurodevelopmental syndrome, first described over a decade ago . Where possible, data for the 17 participants (“cohort 2”) were combined with results from the 5 individuals previously seen at the Seaver Center (“cohort 1”)  , the MulThe Peabody Picture Vocabulary Test, 4th Edition (PPVT-4)  and the Motor skills were assessed through the relevant subdomains from the Vineland-3, the Beery–Buktenica Developmental Test of Visual-Motor Integration, Sixth Edition (VMI-6) , and theThe psychiatric evaluation was conducted by a child and adolescent psychiatrist with expertise in neurodevelopmental disorders and included a caregiver interview and direct assessment of the participant. The evaluation placed emphasis on the developmental history, and the assessment of externalizing behaviors, anxiety, compulsive behaviors, and ASD symptomatology Table . EvaluatBehavioral observations were documented during the administration of the assessment battery. The ADOS-2 systematically collects information on “Other Abnormal Behaviors” (E codes): overactivity (E1); tantrums, aggression and negative or disruptive behaviors (E2), and anxiety (E3). Only scores of 2 or 3 were considered problematic. In addition, observation of behavioral symptoms during the administration of other clinician-administered assessments  were documented for all participants. Examples of behaviors considered problematic include: “significant anxiety in response to requests,” “constantly moves,” “unable to settle for evaluation,” “hitting,” or “biting.” Behaviors were interpreted in the clinical context of the evaluation.Empirical instruments  were used to assess behavioral symptoms. ASD symptomatology was assessed using autism-specific measures, including the Autism Diagnostic Observation Schedule, Second Edition (ADOS-2) , and theBehavioral symptoms were assessed through (1) comprehensive psychiatric evaluation, (2) behavioral observations, and (3) administration of empirical instruments. Following the evaluation, all clinicians involved in the assessment (at least three) met to assign a Diagnostic and Statistical Manual for Mental Disorders Fifth Edition (DSM-5)  consensuComprehensive medical histories were collected by clinician interview of caregivers and medical records review. In addition, a clinical geneticist performed dysmorphology and physical exams. Semi-structured neurological evaluations were completed by a pediatric neurologist. Results from clinical electroencephalograms (EEGs) and brain magnetic resonance imaging (MRI) were reviewed when available, as was prior medical testing on organs of interest in the syndrome .After completion of the evaluation, all participants received a clinical research summary detailing evaluation results.p < 0.05.All analyses were performed using SPSS Statistics Version 24 . DescripFOXP1, and the remaining 20 individuals carry sequence variants. One individual has a de novo deletion that includes approximately 30 genes, including the entirety of FOXP1. The deletion includes two other genes associated with autosomal dominant disorders: PROK2 associated with hypogonadotropic hypogonadism 4 with or without anosmia (MIM 610628) and ROBO2, associated with vesicoureteral reflux 2 (MIM 610878). Individual 1477 carries a deletion that includes four exons of FOXP1. De novo status is unknown for this patient. Sequence variants included 7 missense, 6 frameshift, 5 nonsense, and 2 splice site variants. Variants were confirmed de novo in 18 individuals; in cases where the second parent was not available for testing, maternal inheritance was ruled out (n = 3). The cohort includes individuals with known recurrent variants, including p.Leu414Aspfs*26, p.Arg525Gln, p.Arg514His, and p.Arg503*  = 5.68, p < 0.01). The Vineland-3 Adaptive Behavior Composite scores  = 0.086, p = 0.6). Socialization scores for participants with a DSM-5 diagnosis of ASD were lower than for participants without a diagnosis of ASD (t(14) = 1.77, p < 0.05). Scores for the Communication subdomain were significantly lower than Socialization (t(15) = 6.39, p < 0.01) and Daily Living scores (t(15) = 3.85, p < 0.01) across the sample. Figure Intellectual quotient (IQ) scores Fig.  were calres Fig.  ranged ft(18) = 0.66, p = 0.51).Individuals’ scores on the PPVT-4 and EVT-2 and Vineland-3 subscales were used to compare receptive and expressive language skills , scores ranged from 1 to 13 (6.67 ± 3.29). Vineland-3 subdomains indicated that 9/16 (56%) had significantly higher receptive than expressive language abilities (t(8) = 8.66, p < 0.01).The Vineland-3 Communication domains yield subdomain scores and v-Scale scores (mean 15 and SD 3) for Receptive, Expressive, and Written language. v-Scale scores in the Receptive and Expressive subdomains ranged from 1 to 11 (6.25 ± 3.42) and 1 to 12 (3.50 ± 3.48), respectively. In the Written subdomain  (310.12 ± 123.10) and the number of Words Produced ranging from 0 to 396 (of 396) (254.76 ± 159.02). Total Gestures ranged from 5 to 63 (43.59 ± 16.97), combining Early Gestures, which ranged from 5 to 18 (of 18) (13.71 ± 4.24), and Later Gestures, which ranged from 0 to 45 (of 45) (29.88 ± 13.31).Language milestones were significantly delayed across the sample. The average age of first word and first phrase was 29 months and 44 months, respectively. The single participant in our cohort that did not use words at the time of evaluation was 25 months old.Motor milestones were assessed by the ADI-R. The age of first sitting ranged from 5 to 18 months (9.50 ± 3.11), age of first crawling ranged from 9 to 25 months (14.47 ± 54.56) where one individual (age 25 months at time of exam) had not yet reached this milestone, and age of first walking independently ranged from 13 to 38 months (21.75 ± 5.49). Again, the same individual had not achieved this milestone at the time of the evaluation.t(10) = 1.13, p = 0.28). Gross Motor scores ranged from 1 to 14 (6.45 ± 4.37), and Fine Motor scores ranged from 1 to 11 (5.55 ± 3.98).Eleven of the 16 individuals who completed the Vineland-3 were administered the Motor Domain with standard scores ranging from 20 to 85 (53.09 ± 23.82). V-Scale scores in the Gross and Fine Motor subdomain were comparable (The VMI-6 standard scores ranged from 43 to 85 (61.05 ± 15.23) where 7 individuals (33%) met the cutoff for the impaired range. Lastly, caregivers completed the DCDQ (score range 15–75), where all individuals received scores indicating the presence of a developmental coordination disorder (DCD). Total scores ranged from 15 to 57 (30.60 ± 10.69); subdomain scores in the Control During Movement domain ranged from 6 to 29 (14.00 ± 5.91), in the Fine Motor/Handwriting domain ranged from 4 to 10 (6.55 ± 2.14), and in General Coordination domain ranged from 5 to 19 (10.00 ± 3.95).X2  = 0.24; p = 0.62].Forty-eight percent of participants (10/21) met criteria for autism, and 10% (2/21) for autism spectrum on the ADOS-2. In regard to the ADI-R, 47% (10/21) of participants were above the cutoff scores in all domains, 61% (13/21) met the cutoff for the Social domain, 76% (16/21) met the cutoff for the Communication domain, and (71%) 15/21 met the cutoff for the Restricted and Repetitive Behavior domain. Nonetheless, only 24% (5/21) of participants received a diagnosis of ASD based on expert clinical consensus . Average subdomain scores fell in the moderate impairment range in four of the five subscales, specifically, Social Awareness 70.14 ± 13.03), Social Cognition (69.95 ± 11.61), Social Communication (65.95 ± 12.94), and Restricted and Repetitive Behavior (69.18 ± 14.86). Interestingly, average scores for the Social Motivation subscale were in the typical range (56.50 ± 13.37). When individual total scores were analyzed, 31% (7/22) of participants scored in the severe range, 27% (6/22) of participants scored in the moderate range, 14% (3/22) of participants in the mild range, and 27% (6/22) in the typical range. As expected, mean SRS-2 scores across domains for participants with a consensus diagnosis of ASD had a tendency toward greater severity than those of individuals without ASD  = 0.91, p = 0.37) or the restrictive and repetitive behavior subscale of the SRS-2 (t(19) = 1.44, p = 0.16), did not differ between those with and without a clinical diagnosis of ASD.Caregivers completed the RBS-R. Individual total scores ranged from 0 to 62 (19.13 ± 16.22). To facilitate graphical comparison to the ASD population, RBS-R scores were Z-normalized against ASD normative data . The pren = 54)  controls n = 5 . Additional file p > 0.5).The most common behavioral comorbidity observed in our cohort was ADHD; 15/21(71%) participants received a consensus diagnosis of ADHD, with 13 meeting DSM-5 diagnostic criteria for ADHD-combined presentation and two for predominantly inattentive presentation. In addition, anxiety  and aggressive behavior  were reported and/or observed in a noteworthy proportion of individuals  medical comorbidities are shown in Table The most common medical finding was hypotonia, present in 21 out of 22 individuals. Ocular abnormalities followed (17/22); of the ocular abnormalities the most common findings were strabismus (10/22), esotropia 6/22), nystagmus (3/22), and amblyopia (3/22). Recurrent infections were present in 17/22 individuals. Otitis media was the most common recurrent infection (11/22), followed by respiratory tract infections (9/22), urinary tract infections (2/22), and skin infections (1/22). Gait abnormalities were observed in 12 out of 22 participants. Lastly, feeding issues were present in approximately half of the sample (12/22). Common medical comorbidities include gastrointestinal problems (9/22), sleep disturbance (7/22), neonatal problems (7/22), ptosis (6/22), and structural brain abnormalities (5/19). Of the gastrointestinal problems, the most common finding was reflux (6/20). Of the neonatal problems, 4/22 individuals required a neonatal intensive care unit (NICU) stay, 3/22 reported meconium aspiration, and 2/22 reported hyperbilirubinemia. Structural brain anomalies were present in 5 individuals out of the 19 who had reported completing an MRI. These included two individuals with periventricular leukomalacia, and one individual each with prominent lateral ventricles, mild thickening of the corpus callosum, hypoplastic vermis, and Chiari malformation type 1 , seizures (3/22), hearing problems (2/22), and obstructive sleep apnea (1/22). Congenital heart defects were present in three participants, characterized by (1) patent ductus arteriosus and an atrial septal defect, (2) atrial septal defect and a patent foramen ovale, and (3) atrial septal defect, pulmonary stenosis, enlarged right ventricle, deformed pulmonary valve, and dilated main pulmonary artery. This latter individual carried a pathogenic variant in KCNQ1. Clinical neurological evaluation further described hypotonia, gait abnormalities, and dysarthria in all participants  of individuals in our cohort had ID, with the majority falling in the mild to moderate ID range. One individual’s IQ scores, who was previously described by Siper et al. [11], fell in the average range. However, in contrast to our previous observation, we didn’t observe a correlation between age and performance on cognitive testing or adaptive functioning. This reinforces the notion that individuals with FOXP1 syndrome present with a range of cognitive abilities. Furthermore, we observed some discrepancies in terms of adaptive functioning and IQ scores. For example, the participant with a FSIQ of 93  scored 69 on the Vineland Adaptive Behavior Composite. Similarly, the participant with the highest adaptive behavior scores (Vineland-3 Adaptive Composite standard score of 80) had a FSIQ of 50 . This observation aligns with the notion that adaptive functioning is influenced by a number of factors, including the presence of associated behavioral symptoms and/or response and availability of therapeutic interventions.Assessment of adaptive functioning showed a tendency for comparatively higher socialization skills than daily living or communication abilities Figs. , 3. MotoLanguage and communication varied widely across participants, with some individuals scoring three standard deviations below normative scores and one individual presenting with nearly typical receptive and expressive language ability Fig. . ImportaBased on previous reports, we paid careful attention to ASD symptomology. The majority of individuals ~ 60%) fell above cutoff scores on standardized instruments. However, only 24% of participants met criteria for a consensus diagnosis of ASD. Although a large number of participants (95%) presented with difficulties in developing and maintaining friendships, a smaller subgroup presented with core deficits in social communication and interactions   = 2.73, p = 0.041)]. Although these observations could suggest that ASD is associated with a more severe phenotype, there are multiple confounding factors that prevent us from drawing conclusions. Among them, the small sample size and proportion of individuals with ASD and the wide age range of participants. Importantly, these difference in norm-referenced measures did not extend to statistically significant differences in adaptive behavior scores between groups. This latter observation emphasizes the need for larger longitudinal studies adequately powered to evaluate the impact of ASD and comorbid behavioral symptoms on daily functioning of individuals with FOXP1 syndrome.Further exploratory analysis found that mean IQ, PPVT-4, and EVT-2 scores were significantly lower for the ASD group [Overall, standardized measures of ASD symptoms in this sample showed high sensitivity, but low specificity. Indeed, all participants in the ASD consensus group met cutoff scores on ASD clinician-administered and caregiver-reported instruments (Fig. Psychiatric comorbidities were commonly noted by clinicians and a prevalent area of concern for caregivers. Consensus diagnosis of ADHD, based on caregiver report, direct observation by expert clinicians and results from empirical instruments, was assigned to 71% of participants. Furthermore, anxiety symptoms were observed in 47% of participants, and concerns for aggressive behavior were reported in 42% of participants. However, CBCL subscales only partially captured these observations, which may suggest the need to apply different tools to quantify psychiatric symptoms in populations such as this.Clarifying the nature and prevalence of psychiatric symptoms stands to have important implications for treatment and overall quality of life. For example, aggressive behavior can reflect a mediative response in children with anxiety disorders and/or ADHD symptoms, in particular in the presence of ID and language impairments . FurtherGiven our small sample size and limitations in the interpretation of missense variants without functional studies of the protein, we are unable to report genotype–phenotype associations. This limitation in the sample size is compounded by the wide age range of participants, hampering our ability to draw conclusions on symptom variability across the lifespan. Furthermore, study participants came from across the USA and Canada, where service availability can vary significantly by geographical location, which makes it difficult to systematically report on current educational settings, service utilization, and therapeutic interventions. Finally, our study did not include academic achievement testing, which could strengthen sample characterization and support the development of educational and additional therapeutic recommendations.Individuals with FOXP1 syndrome present with a complex and variable neurobehavioral profile characterized by mild to moderate intellectual disability, speech delays, motor impairments and high rates of neurobehavioral symptoms, which include ASD features, ADHD, and anxiety disorders. Our findings also highlight the inherent limitations of standardized instruments when it comes to assessing individuals with complex neurobehavioral profiles and underscore the importance of clinical judgment when interpreting results. Consistent with previous studies, aggressive behavior was observed in a proportion of individuals; however, we conclude that aggression appeared to be secondary to poorly treated ADHD and anxiety symptoms compounded by limited language abilities. Finally, our observations offer an opportunity for immediate intervention as both ADHD and anxiety symptoms can respond to available evidence-based pharmacological and behavioral treatments that could improve long-term outcomes.Additional file 1. Supplemntal Table 1: Genetic variants.Additional file 2. Supplemental Table 2: Neuropsychological assessments.Additional file 3. Supplemental Table 3: Medical comorbidities.Additional file 4. Supplemental Table 4: Dysmorphic features in the cohort.Additional file 5. Supplemental Table 5: Neurological exam.Additional file 6. Supplemental Table 6: Autism spectrum disorder symptoms.


Oyster shells are rich in calcium, and thus, the potential use of waste shells is in the production of calcium phosphate (CaP) minerals for osteopathic biomedical applications, such as scaffolds for bone regeneration. Implanted scaffolds should stimulate the differentiation of induced pluripotent stem cells (iPSCs) into osteoblasts. In this study, oyster shells were used to produce nano-grade hydroxyapatite (HA) powder by the liquid-phase precipitation. Then, biphasic CaP (BCP) bioceramics with two different phase ratios were obtained by the foaming of HA nanopowders and sintering by two different two-stage heat treatment processes. The different sintering conditions yielded differences in structure and morphology of the BCPs, as determined by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Brunauer–Emmett–Teller (BET) surface area analysis. We then set out to determine which of these materials were most biocompatible, by co-culturing with iPSCs and examining the gene expression in molecular pathways involved in self-renewal and differentiation of iPSCs. We found that sintering for a shorter time at higher temperatures gave higher expression levels of markers for proliferation and (early) differentiation of the osteoblast. The differences in biocompatibility may be related to a more hierarchical pore structure (micropores within macropores) obtained with briefer, high-temperature sintering. Taiwan has a history of oyster breeding on its western coast, producing large quantities of oyster shells that have been abandoned/discarded as a waste product . Oyster Biodegradability is a critical property of artificial bone fillers, which must not hinder the growth of new bone; therefore, the degradation rate has to adapt to (or at least be compatible with) the formation of new bone . To imprMany apatite filling materials with porous granules have been developed and applied clinically . These mMost biological apatites are nonstoichiometric and contain many essential trace elements such as Mg, Mn, Zn, Na, and Sr. Many studies have synthesized calcium phosphate doped with trace elements; for example, the synthesis of Zn-doped BCP fine powders  or incor2/g for BCP1 and BCP2, respectively, by the Brunauer–Emmett–Teller (BET) adsorption equation [The nitrogen gas adsorption–desorption curves and pore size distribution curve are shown in equation . Accordiequation , both BCequation . Figure The differentiation of human iPSC-derived mesenchymal stem cells (iMSCs) for bone regeneration has recently been reported . iMSCs cWu et al. reviewed transforming growth factor beta (TGF-β) and bone morphogenetic protein (BMP) signaling in osteoblast skeletal development, and in bone formation, homeostasis, and disease . Figure In 4)2HPO4  aqueous solution was added dropwise; aqueous ammonia (NH4OH)  was then used to adjust the pH value to 10, and stirring was continued resulting in the precipitation of calcium phosphate in an hour. This precipitate was centrifuged and washed in deionized water and dried in a 70 °C oven for 48 h. Coconut oil  was added (1.5 mL/g) to the above-prepared powders while stirring for 15 min to induce foaming. The foam containing powder was squeezed onto a crucible and dried in a 70 °C oven for 2 h.Oyster shells, collected from Dongshi beach, Taiwan, were carefully washed, crushed, and calcined at 1100 °C for 4 h; then, they were ground to fine powders using an agate mortar and sieved using a 325 mesh screen. The calcined oyster shell powders were added to an aqueous acetic acid solution and stirred with a magnetic stirrer at 320 rpm for 6 h at 55 °C. Next, 0.06 M (NHThe foamed powders were then subjected to a two-stage heat treatment process to obtain porous granules with two different phase ratios (HA/β-TCP), BCP1 and BCP2. The granules were first calcined at 700 °C for 2 h at a heating rate of 2 °C/min and cooled to room temperature in the furnace . To form BCP1, the granules were heated to 1300 °C at a heating rate of 2 °C/min, kept at 1300 °C for 20 min, and then slowly cooled in the furnace. To form BCP2, granules were heated to 1000 °C at a heating rate of 2 °C/min, kept at 1000 °C for 25 h, and then slowly cooled in the furnace.Nitrogen adsorption measurements were performed with a NOVA 1000e, and Brunauer–Emmett–Teller (BET) analysis was performed with the Autosorb program . BET analysis was carried out using nitrogen gas at a relative vapor pressure of 0.05–0.3 at 77 K. The sintered porous granules were observed with a field emission scanning electron microscope . An X-ray powder diffractometer  was used for crystalline phase analysis, operating with a Cu Kα radiation generated at 40 kV and 40 mA. The diffraction angles (2θ) of each sample were measured from 20° to 50° at a scan speed of 5°/min and a step size of 0.02°. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/episomal_hipsc_man.pdf accessed on 31 August 2021). iPSCs were cultured in 98% Essential 8™ basal medium with 2% Essential 8™ supplement (50×), 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) , sodium bicarbonate  1.743 g/L at 37 °C and 5% CO2. For the cytotoxicity experiments about 50,000 iPSCs per well were seeded in 24 well tissue culture polystyrene (TCPS) culture plates and incubated with various concentrations of BCPs. The CCK-8 cell counting kit  was used for assessing the viability of cells after exposure. Briefly, CCK-8, (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium, monosodium salt) solution (50 µL) was added to each well and incubated for 3 h, and then the optical density was measured at 620 nm with a plate reader . Cell viability (%) was calculated as a percentage with respect to untreated cells on TCPS. After the optical density measurements, iPSCs were washed with 400 µL Dulbecco’s phosphate-buffered saline (DPBS) in each well. The cells were fixed in 350 µL of 3.7% formaldehyde in PBS (pH 7.4) for 15 min at room temperature. After washes with 350 µL DPBS in each well, cells were stained with the nuclear dye  for 5 min. Finally, the cells were washed with DPBS and examined with an inverted fluorescence microscope .Induced pluripotent stem cell line (iPSC A18945), 98% Essential 8™ basal medium (A1517001), Essential 8 supplement (A1517001), DMEM/F12+ and GlutaMAX (10565018), Geltrex (A1413301) were purchased from Gibco ; the culture protocol can be found in (The sequences (5′-3′) of primers for GAPDH, OCT4, SOX2, c-Myc, Klf4, Akt1, Akt2, β-catenin, STAT3, NANOG, HRAS, KRAS, NRAS, ERK1, and ERK2 and MKK6, MAPK11, MAPK12, MAPK13, MAPK14, RAC1, CDC42, c-Abl, TAK1, MKK3, SMAD2, SMAD3, SMAD4, SMAD1, SMAD5, SMAD8, LIMK1, PKC are listed in t-test. Statistical significance was set at a p-value of less 0.05 and highly significant as p < 0.01.All experiments were carried out in triplicate, and data are expressed as means ± standard deviation. The gene expression data were analyzed with Student’s This study has provided strong evidence that oyster-shell-derived microporous apatites are suitable to be used successfully for osteopathic, biomedical applications involving hard tissue regeneration. Two materials were synthesized, differing in sintering duration and temperature. BCP1, sintered for a shorter time at a higher temperature, was the more promising of the two: iPSCs cultured with BCP1 particles showed higher expression levels of markers for proliferation and (early) differentiation of the osteoblast  than did


The mechanical homeostasis of tissues can be altered in response to trauma or disease, such as cancer, resulting in altered mechanotransduction pathways that have been shown to impact tumor development, progression, and the efficacy of therapeutic approaches. Specifically, ovarian cancer progression is parallel to an increase in tissue stiffness and fibrosis. With in vivo models proving difficult to study, tying tissue mechanics to altered cellular and molecular properties necessitate advanced, tunable, in vitro 3D models able to mimic normal and tumor mechanic features. First, we characterized normal human ovary and high-grade serous (HGSC) ovarian cancer tissue stiffness to precisely mimic their mechanical features on collagen I-based sponge scaffolds, soft (NS) and stiff (MS), respectively. We utilized three ovarian cancer cell lines  to evaluate changes in viability, morphology, proliferation, and sensitivity to doxorubicin and liposomal doxorubicin treatment in response to a mechanically different microenvironment. High substrate stiffness promoted the proliferation of Caov-3 and SKOV3 cells without changing their morphology, and upregulated mechanosensors YAP/TAZ only in SKOV3 cells. After 7 days in culture, both OVCAR3 and SKOV3 decreased the MS scaffold storage modulus (stiffness), suggesting a link between cell proliferation and the softening of the matrix. Finally, high matrix stiffness resulted in higher OVCAR-3 and SKOV3 cell cytotoxicity in response to doxorubicin. This study demonstrates the promise of biomimetic porous scaffolds for effective inclusion of mechanical parameters in 3D cancer modeling. Furthermore, this work establishes the use of porous scaffolds for studying ovarian cancer cells response to mechanical changes in the microenvironment and as a meaningful platform from which to investigate chemoresistance and drug response. The microenvironment surrounding cancer cells has been shown to contribute to tumor progression at both biological and physical levels ,2. IndeeThe field of cancer biomechanics aims to better understand not only how cancer cell behavior is affected by mechanical changes in the microenvironment, but also how tissue mechanical features can be exploited to detect specific disease stages, while enabling the discovery of potential new diagnostic tools and/or therapies . The rolSimilar to breast cancer, epithelial ovarian cancer (EOC) evaluation with MPM and SHG imaging has revealed altered collagen fibril density and topology linked to increased stiffness and fibrosis, and associated with both primary and disseminated EOC ,23,24. SCarboplatin with paclitaxel represents the standard first-line chemotherapy regimen for ovarian cancer patients; however, only 40–60% of patients will achieve complete remission, with a high risk of neurotoxicity, which can persist for more than a year after the treatment ,37. ConsIncreased matrix stiffness is closely linked to tumor progression ,43; howeTissue engineering (TE) describes the process of fabricating functional 3D tissues using a combination of scaffolds and/or devices with cells to facilitate essential cellular functions, such as growth, differentiation, migration, and organization . In the For its high biocompatibility and bioactivity, the natural polymer collagen was selected for scaffold fabrication in this research. Collagen is the most abundant structural protein in the connective tissues, and its homology across species provides low antigenicity and high biocompatibility ,61; in hUsing a sponge scaffold 3D collagen-based culture system, we investigate the role of substrate stiffness in affecting EOC cell behavior and chemoresistance in vitro. Following the assessment of fresh OC tissue to define the stiffness parameters of both peritoneum/cancer and normal ovarian mechanical features, we mimic the stiff  and soft  tissue properties using 3D porous matrices, testing their utility and suitability for reproducing in vivo tissue mechanics while serving as platform for drug testing. Detailed mechanical tests are performed using multiple technologies, from atomic force microscopy (AFM), easily run on the cell/tissue surface , to rheow/v  and 1% w/v (metastatic scaffold) BDDGE at 4 °C for 24 h. Finally, the slurry was washed with Milli-Q water  and casted onto a 48-well plate and freeze-dried via an optimized freezing and heating ramp  to obtain the desired pore size and porosity.Chemicals were purchased from Sigma-Aldrich. The scaffolds were synthesized from type I bovine collagen  and fabricated with the freeze-dry technique. Briefly, we prepared an acetic collagen slurry . The wet slurry was crosslinked in an aqueous solution of 0.1% http://nanophys.kth.se/nanophys/facilities/nfl/afm/icon/bruker-help/Content/SoftwareGuide/NanoScope815CoverPage.htm, accessed on 3 February 2022). For the AFM experiment, both biopsies and scaffolds were embedded in OCT and cryosectioned at 20 µm section thickness. The samples were pre-coated on a glass slide and kept at −80 °C. Then, samples were carefully moved into a 60 mm dish on the AFM scanning stage. A volume of 3 mL phosphate-buffered saline (PBS) or media was pre-injected into the dish after sample incubation for 5 min at room temperature. For force measurement, we kept the ramping size at 10 µm. All experiments were conducted at room temperature (22 °C). Young’s modulus was calculated from the force curves with NanoScope Analysis 1.40 , with 3 to 5 spots randomly tested per sample and recorded, and at least 50 force curves acquired from each spot. For Young’s modulus calculations, extended ramp force curves, and a linearized model  were used.The atomic force microscope used in this experiment was the Bio-Catalyst AFM . A spherical cantilever  was used for the force measurement. For biopsy samples, the QNM in fluid mode was used with a borosilicate tip (5 µm in diameter). For scaffolds, a silica bead (5 µm in diameter) was mounted onto the end of the cantilever. The spring constant of the cantilever was 0.06 N. Prior to the AFM experiment, both the spring constant and sensitivity of the cantilever were calibrated under thermal tune conditions with the controlling software  was used to verify the range of linear viscoelasticity. Frequency response was measured by frequency sweep tests in the range 1000 to 0.1 rad per s . Storage modulus and loss moduli measures were reported as 3-sample averages collected at 1 rad per s angular frequency.NS and MS scaffolds of 0.5 cm thickness were soaked in PBS and loaded onto a UniVert Mechanical Test System. A load cell of 1 N was calibrated and used to perform a compression test with a stretch magnitude of 35%, stretch duration of 60 s, and relaxation time of 60 s. For each condition, 3 replicates were analyzed.https://imagej.nih.gov/ij/, accessed on 3 February 2022).The morphology of the scaffold was characterized by SEM and the pore size determined by ImageJ . Scaffolds were coated with 7 nm of Pt/Pl  for SEM examination. The pore diameter of scaffolds was measured from SEM images, and 3 images from each of 3 areas were used for each scaffold at the same magnitude. For each image, porosity analysis was performed using ‘analyze particles’ measurement in ImageJ software , version 1.41, −1 resolution 64 times over the range of 500–4000 cm−1 using a Nicolet 6700 spectrometer . The ATR/Fourier transform infrared spectroscopy (FTIR) spectra were reported after background subtraction, baseline correction, and normalization on Amide I. Graphs reported a range of 500–1800 cm−1 wavelength.The samples were analyzed in attenuated total reflection (ATR) mode at 2 cmOVCAR-3, Caov-3, and SKOV3 cells were purchased from ATCC. Cultures were established in standard growth medium, as suggested on the ATCC website, composed of RPMI-1640 Medium with 0.01 mg per mL bovine insulin and fetal bovine serum to a final concentration of 20% for OVCAR-3, Dulbecco’s Modified Eagle’s Medium with fetal bovine serum to a final concentration of 10% for Caov-3, and McCoy’s 5a Medium modified with fetal bovine serum to a final concentration of 10% for SKOV3. All growth media were supplemented with 1% penicillin (100 UI per mL)–streptomycin (100 mg per mL).2. Medium was changed twice per week. When seeded onto scaffolds, ovarian cancer cells were harvested and re-suspended in cell culture medium. A 20 µL drop of medium containing 1 × 105 cells was seeded on the center of each scaffold and kept in an incubator for 30 min. Culture medium was then added to each well.Adherent cells were detached from plates using trypsin before reaching confluence (80%) and subsequently re-plated for culture maintenance. For maintenance of cultures, cells were incubated at 37 °C in a humidified atmosphere (90%) with 5% COEthical approval for this study was obtained from NHS HRA Wales6 REC (15/WA/0065) to collect tissue samples from ovarian cancer patients and non-cancer controls. Formal written consent was obtained from all patients at the time of recruitment into the study.A total of 6 ovarian biopsies were collected for this study and processed for AFM analyses. The histological evaluation of the ovarian biopsies and the cancer diagnosis was confirmed by the pathology department as part of the patient’s routine clinical care. Three were used as normal ovary controls as they were obtained from normal contralateral ovaries of patients diagnosed with benign pathologies. The remaining 3 ovarian biopsy tissue samples represented HGSC stage IIIc  in Leica Autostainer ST5010 XL. Paraffin was melted prior to staining by heating the slides at 60 °C for 30 min, then slides were deparaffinized by performing 3 × 2 min washes in xylene, 3 × 1 min washes in 100% ethanol, and 1 × 1 min wash in 95% ethanol, before rinsing with tap water. Slides were incubated for 30 s in Hemalast, for 5 min in hematoxylin, and were rinsed for 1 min with tap water. Next, slides were incubated for 30 s in a differentiator and 1 min in bluing agent, with each step followed by a tap water rinse for 1 min then 95% ethanol for 1 min. Slides were stained with eosin for 30 s, dehydrated in 95% ethanol for 1 min, 4 min in 100% ethanol, and 2 × 1 min in 100% ethanol, and cleared for 3 × 2 min in xylene. Every step after the initial heating of the slides was conducted at room temperature.After 7 days of culture, scaffolds with cells were incubated with 2 µL of 50 µM calcein AM working solution and 4 µL of ethidium homodimer-1 stock and incubated for 20 min at 37 °C protected from the light. After several washes with warm media, cells were analyzed by Keyence BZX800 using a 4× objective and a final stich process to show the entire scaffold surface.After 7 days of culture, scaffolds were collected and washed with 1% PBS. After fixation with 4% paraformaldehyde for 10 min at room temperature, cellularized scaffolds were washed twice with PBS +0.1% Tween and permeabilized using Triton X-100 0.1% in PBS for 10 min at room temperature. Incubation with phalloidin-555  and Draq-5 5 Mm  was performed for 2 h at room temperature protected from light. After washing with PBS +0.1% Tween twice, imaging was conducted with the Nikon A1 confocal imaging system, using a 20× objective. Volume recording was performed on z-stacks of 200 μm and step size of 10 μm.After 7 days of TGFβ1 treatment, scaffolds were incubated for 10 min in trypsin under shaking conditions; subsequently, scaffolds were removed, and cell pellets were collected after centrifugation and washed with PBS. Cells were incubated with 2 µL of 50 µM calcein AM working solution and 4 µL of ethidium homodimer-1 stock, and incubated for 20 min at 37 °C protected from the light. After several washes with warm media, cells were analyzed by flow cytometry using BD FACS Fortessa.g for 15 min at 4 °C. The aqueous phase was transferred to a 1.5 mL tube and 500 µL of isopropyl alcohol added, before incubating for 10 min at 4 °C and centrifuging 12,000× g for 10 min at 4 °C. After washing the pellet twice with 1 mL 70% ethanol, it was aspirated and allowed to dry before resuspending in 20 µL of water. Total RNA (500 ng) was reverse transcribed into cDNA using the Bio-Rad iScript™ cDNA Synthesis Kit. Quantitative PCR was performed using the TaqMan™ Fast Advanced Master Mix on a StepOnePlus Real-time PCR System . Expression of YAP1 (Hs00902712) and WWTR1  was detected using TaqMan® Gene Expression Assays. 18S ribosomal RNA was used as an internal reference for normalization. Analysis was performed using the relative ΔΔCT method.Reverse transcription PCR was performed on cells grown in 2D culture and on 3D scaffolds after 7 days. Scaffolds with cells were washed in PBS and incubated with 1 mL TRIzol RT for 10 min under shaking conditions. After removing the scaffolds, 200 µL of chloroform was added and samples were inverted for 15 min, incubated on ice for 2 min, and centrifuged at 12,000× To perform this test, SKOV3 and Caov-3 cells were seeded at a concentration of 8000 cells per well in 96-well plates, while 20,000 OVCAR-3 cells were seeded per well in a final volume of 100 µL per well. MTT was added to achieve a final concentration of 0.5 mg per mL MTT in normal media. After incubating 2 h at 37 °C, MTT was removed and 100 µL DMSO added before mixing contents for 30 min on an orbital shaker protected from light. Absorbance was measured at 590 nm.® Reagent  equal to the volume of cell culture medium present in each well/scaffold was added. Contents were mixed for 10 min on an orbital shaker to induce cell lysis, protected from light. The plate was incubated at room temperature for 25 min to stabilize luminescent signal, before transferring 100 µL  in white opaque-walled 96-well plates to measure luminescence.For the proliferation assay, ovarian cancer cells were grown on scaffolds for 10 days and analysis performed at 4 time points . For cytotoxic evaluation of doxorubicin, ovarian cancer cells were grown on scaffolds for 7 days, and then treated with free doxorubicin (DOXO), doxorubicin-loaded liposomes (DOXO-LIPO), or empty liposomes for 72 h. To perform the analysis, a volume of CellTiter-Glov/v) to a final volume of 1 mL. The solvent was evaporated via a rotary evaporator  for 20 min at 45 °C to form a thin lipid film. The film was hydrated with 1 mL sterile water to assemble empty liposomes or 250 mM ammonium sulfate for liposomes to be loaded with doxorubicin. The 1 mL solution was incubated for 3 min at 45 °C followed by 3 min vortexing. Lipid suspension was forced through a polycarbonate filter  10 times under nitrogen gas pressure at 45 °C (filter was replaced after 5 extrusions). Size, zeta potential, and polydispersity index (PDI) were measured using dynamic light scattering. After the nanoparticles (NPs) were fabricated, they were loaded into dialysis floaters in order to exchange the outside buffer with 0.9% NaCl overnight. Lipid formulation was then incubated at 1:1 v/v for 2 h at 45 °C with 1 mL of 2 mg per mL doxorubicin hydrochloride  dissolved in DDW. Only DOXO-LIPO NPs were loaded again into dialysis floaters in order to exchange the outside buffer with 0.9 NaCl overnight. NanoSight NS300  for both empty and loaded NPs was performed as the final step to measure lipid NPs concentration.To assemble liposomes, 20 mg of total lipids including DPPC, DSPC, DOPX, and cholesterol (molar ratio 5:1:3:1) were dissolved in methanol–chloroform solution (1:3 v/v) with 0.2% (v/v) triton x-100  or water (released doxorubicin) in a black 96-well plate for 5 min at room temperature under shaking conditions. Doxorubicin fluorescence was read at excitation 480 nm/emission 590 nm and cyclophosphamide (Cy) fluorescence at 5.5 excitation 650 nm/emission 700 nm.Doxorubicin encapsulation and drug release analysis was performed using a Tecan Microplate Reader. For the doxorubicin release experiment, DOXO-LIPO NPs were incubated with PBS +10% FBS (50:50) at 37 °C under shaking conditions and samples were collected and analyzed after 0.5, 1, 1.5, 2, 4, 5, 8, 24, 48 h. For DOXO encapsulation, DOXO-LIPO NPs were diluted in water to 1:200 and mixed .All data were obtained from at least 3 independent experiments and expressed as mean ± standard deviation, with p < 0.05; w/v BDDGE crosslinkers achieved a higher stiffness for the metastatic scaffolds  compared with the normal scaffolds , respectively  stage III and normal ovary tissues, were analyzed at the nanoscale using AFM. A 5.5-fold increase in stiffness was observed between HGSC IIIa (0.11 ± 0.034 MPa) and normal ovary (0.02 ± 0.016 MPa) tissues  and Amide II (1600–1500 cm−1), related to the stretching vibration of C=O bonds, and to C–N stretching and N–H bending vibrations, respectively, for both scaffold types. The samples contained C=O, C–N, and N–H bonds. The Amide III region (approximately 1200–1300 cm−1) is related to C–N and C–C stretching, N–H bonds, and CH2 wagging from the glycine backbone and proline side chain.FTIR was used to characterize scaffold composition after crosslinking. FTIR spectra, reported in We employed three cell lines derived from the ovary  or peritoneal ascites (SKOV3) of EOC  to test ®. The OVCAR-3 proliferation rates showed no differences between MS and NS scaffolds until day 10 of culturing, at which point, OVCAR-3 proliferation was observed to be higher on NS scaffolds when compared to their MS counterparts  (Cancer cell proliferation rate was assessed by CellTiter-Gloctively) A. The Cactively) B. The SKctively) C.p < 0.01 and p < 0.05); for OVCAR-3-cultured scaffolds, these were 0.093 ± 0.041 MPa for MS and 0.012 ± 0.002 MPa for NS; for Caov-3, these were 0.032 ± 0.005 MPa for MS and 0.012 ± 0.004 MPa for NS; and for SKOV3, these were 0.039 ± 0.011 MPa for MS and 0.012 ± 0.0004 MPa for NS. A decrease in MS storage moduli was observed at day 7 for the OVCAR-3 culture  and SKOV3 culture  (p < 0.01). Regarding SKOV3 no significant differences were recorded for loss moduli at day 7. At day 1, loss moduli for OVCAR-3 were MS 0.015 ± 0.010 MPa, NS 0.002 ± 0.0001 MPa; for Caov-3 were MS 0.004 ± 0.001 MPa, NS 0.002 ± 0.001 MPa; and for SKOV3 were MS 0.005 ± 0.002 MPa, NS 0.002 ± 0.0001 MPa . These values at day 7 were: OVCAR-3 MS 0.004 ± 0.001 MPa, NS 0.001 ± 0.001 MPa; Caov-3 MS 0.004 ± 0.001 MPa, NS 0.002 ± 0.0001 MPa (p < 0.05); SKOV3 MS 0.003 ± 0.0001 MPa, NS 0.001 ± 0.0001 MPa  A,C,E; in0001 MPa B,D,F.Yap1 and Taz, which are essential for triggering cancer initiation and growth of most solid tumors [Mechanical changes in the microenvironment can strongly affect cells at the transcriptomic level through a process called mechanosensing . We evald tumors . Among td tumors , and itsd tumors , for rapd tumors .To evaluate the link between stiffness of the substrate, mechanosensing, and resistance to chemotherapy, we employed our 3D model platforms to test ovarian cancer cell sensitivity to mainline chemotherapeutic treatments, doxorubicin (DOXO) and doxorubicin-loaded liposome (DOXO-LIPO). Similar formulations of doxorubicin have been reported to be effective, with tolerable side-effects in either combination therapy with carboplatin or in monotherapy for recurrent or platinum-resistant ovarian cancer .The physicochemical features of conventional liposomes ,93 were ®. After normalization against untreated cells, we observed a similar effect of free DOXO and DOXO-LIPO in reducing all ovarian cancer cell viability in 2D, while empty liposomes did not affect cell viability after either 48 or 72 h of treatment. Interestingly, OVCAR-3 cells in 2D were the most resistant to treatment after 48 and 72 h in the lower dose range, showing reductions in cell viability at 72 h of 64, 43, and 35.8% for DOXO, and 75, 46, and 42.6% for DOXO-LIPO, at concentrations of 0.0625, 0.125, and 0.25 µM, respectively  , and we chose to focus our analysis on a low and high dose close to the IC per mL) . Overall per mL) A,B. Inte per mL) C,D. Fina per mL) E,F. Empt per mL) .From a biophysical standpoint, solid tumor cancers have been shown to share physical characteristics; however, these are affected by specific mechanical cues linked to the tissue of origin ,94,95,96The NS and MS scaffold 3D in vitro model mimics the difference in stiffness between normal ovarian tissue and pathological HGSC stage III-derived tissue, respectively. After successfully mimicking in vivo stiffness on scaffolds MS (0.144 ± 0.010 MPa) and NS (0.015 ± 0.0003 MPa), we zoomed out from the AFM analysis, to evaluate the scaffolds’ bulk mechanic characteristics using rheology. A systematic mechanical characterization of the bulk tissue mechanics of patients’ biopsy samples, or having the diagnostic tools able to detect changes in patients’ tissue mechanics, could provide guidelines for clinical mechanopathology evaluation , ultimatOur results are in line with previous research that analyzed tumor stiffness in vivo. For example, researchers employed supersonic shear wave elastography in a patient-derived xenograft (PDX) mouse model engrafted with HGSOC tumors isolated from patients, recording a significant increase in tumor stiffness (120 to 140 kPa) over time in mesenchymal HGSOC, while stiffness remained low (60 kPa maximum) in non-mesenchymal tumors. In high-grade serous ovarian cancers (HGSOC), representing the vast majority (75%) of total ovarian cancers, the “fibrosis” or “mesenchymal” HGSOC molecular subtype has been identified in all studies, and is systematically associated with poor patient survival. It is characterized by high stromal content composed of myofibroblasts and ECM proteins, such as collagen and fibronectin, which are major causes of tumor stiffness . In the To test the mechanoresponsive potential of ovarian cancer malignancy, we selected three adenocarcinoma ovarian cancer cell lines: OVCAR-3 and Caov-3, derived from the ovary, and SKOV3, derived from ascitic fluid from post-chemotherapy patients. OVCAR-3 and Caov-3 possess TP53 mutations and substantial copy-number changes, which are key characteristics of HGSC, whereas SKOV3, based on its genetic profile, is categorized as non-serous ,107. AltYorkie-homologues YAP (Yes-associated protein) and TAZ  are transcriptional coactivators pervasively activated in human malignancies. Their activation in cancer cells impacts the behavior of cancer cells themselves by regulating their capacity to proliferate and adjust their metabolism to the altered cellular context, promoting the acquisition of stem-like properties, drug resistance, and migratory capacity that allow tissue invasion and metastatic dissemination. Interestingly, in the present study, YAP/TAZ are sensors of the structural and mechanical features of the cell microenvironment, including changes in mechanotransduction, inflammation, oncogenic signaling, and inhibition of the Hippo pathway ,115. ManAside from intrinsic molecular mechanisms , tumor cFirst-line management for ovarian cancer consists of surgery plus platinum-based combination chemotherapy, typically cisplatin or carboplatin, with the addition of a taxane, either paclitaxel or docetaxel . NeverthApproved by the US Food and Drug Administration (FDA) in 1995, Doxil  was the first nanodrug marketed in the United States for the treatment of ovarian cancer in women for which the disease has progressed or recurred after platinum-based chemotherapy . Many clIn line with these results, we tested the cytotoxic effects of free DOXO and DOXO-LIPO on cis-platinum-resistant SKOV3 , OVCAR-3To summarize, the inclusion of physical parameters in 3D in vitro model design will help configure a microenvironment closer to native tissues, which could sustain meaningful cell culture conditions for cancer research and drug testing. Envisioning a clinical application, the use of patient-derived primary cells in combination with a 3D biomechanical scaffold, could be used to assess the likelihood of a favorable outcome to tumor treatment, and eventually suggest possible alternative patient-tailored options.


Here, we performed proteomics and metabolomics analyses of plasma and bone marrow supernatant, together with transcriptional analysis of CD8+ T cells from SAA patients and healthy donors, to find key pathways that are involved in pathogenic CD8+ T-cell activation. We identified 21 differential proteins and 50 differential metabolites in SAA patients that were mainly involved in energy metabolism, complement and coagulation cascades, and HIF-1α signaling pathways. Interestingly, we found that these pathways are also enriched in T cells from SAA patients by analyzing available single-cell RNA sequencing data. Moreover, CD8+ T cells from SAA patients contain a highly activated CD38+ subset, which was increased in the bone marrow of SAA patients and a murine model of SAA. This subset presented enriched genes associated with the glycolysis or gluconeogenesis pathway, HIF-1α signaling pathway, and complement associated pathways, all of which were of importance in T-cell activation. In conclusion, our study reveals new pathways that may regulate CD8+ T-cell activation in SAA patients and provides potential therapeutic targets for SAA treatment.Severe aplastic anemia (SAA) is an autoimmune disease characterized by immune-mediated destruction of hematopoietic stem and progenitor cells. Autoreactive CD8 Severe aplastic anemia (SAA) is a bone marrow (BM) failure syndrome that is characterized by the destruction of hematopoietic stem cells (HSCs) and empty BM. Most patients respond to immunosuppressive therapies (ISTs), suggesting an immune mechanism of AA pathogenesis . However+ T cells are key mediators of progressive BM failure, which inhibits hematopoiesis in AA  and then dissolved in 100 μl of MeOH:H2O . After centrifugation at 14,000 g for 10 min at 4°C, the supernatant was collected for further detection.Proteins and metabolites were extracted as previously described (−1. Mobile phase A was 0.1% aqueous formic acid (v/v), and mobile phase B was methanol. Gradient elution was optimized as follows: 0–2 min, 2% B; 2–20 min, 2–100% B; 20–24 min, 100% B; 24–24.1 min, 100–2% B; and 24.1–30 min, 2% B. The column temperature was maintained at 35°C, and the injection volume was 5 µl.The metabolites in plasma and BM samples were separated on a high-performance liquid chromatography (HPLC) system  with a C18 column  at a flow rate of 0.25 ml minm/z 70–1,050 with an MS resolution of 70,000. The other MS parameters included the following: auxiliary gas flow, 10 arbitrary units; sheath gas flow, 35 arbitrary units; automatic gain control (AGC) target, 3e6 ions; capillary temperature, 320°C; S-lens radio frequency (RF), 50%; maximum injection time, 100 ms; and spray voltage, 3.5 kV.The high-resolution MS for metabolite analysis was performed on a Thermo Scientific Q Exactive Plus mass spectrometry  with an electrospray ionization (ESI) source. Both negative and positive ion scan modes were carried out. The scan range was set at m/z) in the experimental samples with those of the chemical standards in the reference databases of Compound Discoverer 2.1.0.401 software (Thermo Fisher Scientific). The LC-MS/MS raw data were processed using Compound Discoverer 2.1 software to obtain the QC-normalized peak area. Then, the data were further processed in the MetaboAnalyst platform  including normalized by median and transformed by log(base 10) without scaling. Then, the normalized data were analyzed by partial least squares discriminant analysis (PLS-DA) and a volcano plot.Metabolites were identified by automatically comparing the precursor and product ions features  and microarray data (GSE3807) were obtained from the Gene Expression Omnibus (GEO). As for single-cell sequencing data quality control, cells that have more than 4,000 or less than 200 unique genes or have more than 25% mitochondrial genes were removed. Only genes expressed in 20 or more cells were used for further analyses. Then ribosome and mitochondria genes were filtered out. The pre-processed data matrix was transformed to a Seurat object using CreateSeuratObject function and normalized and log-transformed, and the log-transformed matrix was used for all downstream analyses in Seurat package (version 4.0.1). Principal component analysis (PCA) was used for dimensionality reduction using highly variable genes. With the use of the first fifteen principal components as input, the data in two dimensions were visualized with Uniform Manifold Approximation and Projection (UMAP). The gene set variation analysis (GSVA) and gene set enrichment analysis (GSEA) were performed using the GSVA package (version 1.36.3) and ClusterProfiler package (version 3.16.1), respectively. Gene sets were downloaded from the MSigDB database or collected from document literatures. The differences in pathway enrichment scores between different clusters were calculated using LIMMA package (version 3.44.3). As for microarray data analysis, the expression matrix was downloaded by GEOquery (version 2.10), and gene annotation was performed to transform the probe name to symbol; then expression matrix was normalized with log2. The normalized matrix was analyzed for differential expression, and then heatmap was obtained by pheatmap package (version 1.0.12).7 mixed lymph node cells and splenocytes from age- and sex-matched C57BL/6 mice or phosphate-buffered saline (PBS) intravenously. Mice were sacrificed at 3–4 weeks after cell transfer. All mice were housed and bred in Laboratory Animal Research Center, South China University of Technology (SCUT), under a specific pathogen-free condition.C57BL/6 and BALB/c mice were purchased from Hunan SJA Laboratory Animal Co., Ltd (China). Male C57BL/6 mice were crossed with female BALB/c mice to generate CByB6F1 mice. To induce immune-mediated BM failure, CByB6F1 mice aged 8–12 weeks received 5 × 10BM mononuclear cells were isolated, and red blood cells were depleted using red blood lysis buffer . For surface markers staining, cells were incubated with purified anti-CD16/CD32 antibody  for 15 min at 4°C and then stained with fluorochrome-conjugated antibodies for 20 min at 4°C. The antibodies against Ter119 (TER-119), CD45 (104), CD11b (M1/70), CD19 (6D5), NK1.1 (PK136), CD3 (17A2), CD8α (53–6.7), FasL (MFL3), and CD44 (IM7) were purchased from BioLegend; the antibodies against CD4 (GK1.5) and CD62L (MEL-14) were purchased from BD Biosciences . For intracellular cytokine staining, cells were resuspended in RPMI-1640 with 10% fetal bovine serum and stimulated with Cell Stimulation Cocktail (plus protein transport inhibitors)  at 37°C for 3 h. After surface marker staining, cells were fixed and permeabilized with Cytofix/Cytoperm™ Fixation/Permeabilization Kit (BD Biosciences) and then stained for anti-IFN-γ and anti-TNF-α (BioLegend). Flow cytometric analysis was performed on LSR Fortessa (BD Biosciences), and data were analyzed with the Flowjo software (BD Biosciences).p > 0.05), statistics were analyzed using a two-tailed unpaired t-test with Welch’s correction; otherwise, a nonparametric test was used. p-Values less than 0.05 were considered significantly different: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.Data were analyzed using R  and GraphPad Prism 8 . Results in all figures were expressed as mean ± standard error of the mean. For the comparison of two groups, if they pass homogeneity of variance test   .Upregulated proteins in SAA patients are enriched in immunoglobulin-mediated immune response  and complement activation pathways by pathway enrichment analysis . MoreoveSeveral significantly changed proteins between SAA patients and healthy donors, such as SHBG, APOA1, and PGK1, were closely associated with the metabolic state of patients . Therefon = 5) to profile the metabolome. PLS-DA plot perfectly clustered BM supernatant and plasma  or impact (top four) were indicated the following pathways: glycolysis or gluconeogenesis, fructose, and mannose metabolism, purine metabolism, caffeine metabolism, nicotinate and nicotinamide metabolism, staphylococcus aureus infection, complement and coagulation cascades, HIF-1 signaling, cholesterol metabolism, and phenylalanine metabolism pathways  of CD8+ T cells from AA patients and healthy controls  genes in BM T cells from AA patients by analyzing microarray data (GSE3807) . Previou+ T cells in SAA. We defined CD38+ cells by the expression value of CD38 > 0 in the single-cell transcriptome data (GSE145669) and observed that CD38+CD8+ T cells are mainly grouped within clusters 1, 5, and 7  in AA patients , leading to an increased susceptibility of SLE patients to infections  and STAT3 signals  development (Androgen has been used for the treatment of AA (+ T-cell activation in SAA. These findings will promote our understanding of AA pathogenesis, therefore providing potential therapeutic targets for SAA treatment.However, there are limitations to this study. First, more samples and another patient cohort are needed to validate findings. Second, mechanisms of how these molecules and pathways are involved in AA pathogenesis, which need further investigation. Nevertheless, our study provides a proteome and metabolome map of AA plasma and BM and reveals several pathways that are associated with CD8