Dear Editor,Mycobacterium leprae, for peripheral nerves and skin. Of the 208,619 new cases detected worldwide in 2018, a total of 11,323 cases already had apparent deformities, of which 2,109 (18.6%) were from Brazil, a figure that was second only to India, with 3,666 new cases with deformities.Leprosy is a chronic infectious disease with a disabling potential due to the affinity of the agent, Neuropathy in leprosy can occur insidiously, more commonly as multiple mononeuropathy, with the simultaneous involvement of two or more peripheral nerves in different regions of the body, with the ulnar and posterior tibial nerves being the most frequently affected. Neural damage can also often occur during acute episodes of immunological reactions against bacillary antigens that may occur during the course of the disease or even after polychemotherapeutic treatment, in the form of neuritis with edema, vascular obstruction, ischemia, and strangulation in the passage through the osteofibrous channels of the limbs, leading to pain and deficit of function, the main cause of physical disabilities and stigma of the disease.3The basis for neuritis treatment is prednisone, used to control the inflammatory process and pain, preventing permanent neural damage. Neurolysis is surgery for nerve decompression through an incision in the epineurium and opening of the osteofibrous canal to release the nerve, at the level of the elbow and/or wrist.With the purpose of describing the clinical evolution of neural function and neuritis recurrences of leprosy patients with neuritis submitted to neurolysis, we present a series of 22 cases of ulnar nerve surgery performed in the years 2015 and 2016, at Fundação de Dermatologia Tropical e Venereologia Alfredo da Matta, located in the state of Amazonas, in the city of Manaus, Brazil.Immediately before the neurolysis and after it , the Simplified Neurological Assessment was carried out according to the protocol of the Ministry of Health of Brazil.The time between the first episode of neuritis and the performance of neurolysis was, on average, 25.3 months , with the procedure being performed after up to six months in 11 (50.0%) patients, which is related to the best results by other authors.Of 12 cases that already had altered sensibility before surgery, seven (58.3%) showed improvement and, of the six that had altered muscle strength, five (83.3%) maintained the same level, and only one (16.7% ) showed worsening. Fifteen (68.2%) patients did not have any neuritis in the operated nerve; however, three of these patients still had to use prednisone, because they had a leprosy reaction and/or neuritis in other nerves. Two patients had only a single episode of neuritis, which occurred three and four months after surgery. Another five patients (18.2%) had subentrant episodes of neuritis in the operated nerve, for an average of 52.6 months  after surgery, without necessarily developing loss of function .Table 1CThis series of cases emphasizes the clinical importance of the results in a routine situation, where the greatest contribution of neurolysis associated with the clinical treatment of leprosy neuritis was the non-recurrence or non-chronicity of the condition. Neurolysis also prevents prolonged corticosteroid therapy and its consequences, in addition to the possible evolution into physical disabilities. There was sensory function gain in most of the assessed cases. However, it is necessary to carry out studies with appropriate methodology to evaluate the effectiveness of neurolysis or its benefits in comparison to clinical treatment alone, even considering the studies that have been published for decades.10.13039/501100004916Fundação de Amparo à Pesquisa do Estado do Amazonas- undergraduate scholarship.Juliana Barroso-Freitas: Design and planning of the study; drafting and editing of the manuscript; collection, analysis, and interpretation of data; critical review of the literature.Pedro Arthur da Rocha Ribas: Critical review of the literature; collection, analysis, and interpretation of data.Paula Frassinetti Bessa Rebello: Design and planning of the study; critical review of the literature; analysis of data; critical review of the manuscript.Silmara Navarro-Pennini: Design and planning of the study; drafting and editing of the manuscript; critical review of the literature; analysis of data.None declared.


Cerebral ischemia and its sequelae, which include memory impairment, constitute a leading cause of disability worldwide. Micro-RNAs (miRNA) are evolutionarily conserved short-length/noncoding RNA molecules recently implicated in adaptive/maladaptive neuronal responses to ischemia. Previous research independently implicated the miRNA-132/212 cluster in cholinergic signaling and synaptic transmission, and in adaptive/protective mechanisms of neuronal responses to hypoxia. However, the putative role of miRNA-132/212 in the response of synaptic transmission to ischemia remained unexplored. Using hippocampal slices from female miRNA-132/212 double-knockout mice in an established electrophysiological model of ischemia, we here describe that miRNA-132/212 gene-deletion aggravated the deleterious effect of repeated oxygen-glucose deprivation insults on synaptic transmission in the dentate gyrus, a brain region crucial for learning and memory functions. We also examined the effect of miRNA-132/212 gene-deletion on the expression of key mediators in cholinergic signaling that are implicated in both adaptive responses to ischemia and hippocampal neural signaling. miRNA-132/212 gene-deletion significantly altered hippocampal AChE and mAChR-M1, but not α7-nAChR or MeCP2 expression. The effects of miRNA-132/212 gene-deletion on hippocampal synaptic transmission and levels of cholinergic-signaling elements suggest the existence of a miRNA-132/212-dependent adaptive mechanism safeguarding the functional integrity of synaptic functions in the acute phase of cerebral ischemia. Oxygen is a key determinant of mammalian life and severe oxygen deprivation is most often its antithesis. Oxygen deprivation accompanied by a sudden disruption of substrate delivery to brain tissue, as occurring in the form of ischemic stroke, triggers a devastating cascade of pathophysiological events, culminating in irreversible dysfunction and loss of CNS tissue . The resIn an effort to illuminate pathophysiological hallmarks of ischemic brain injury and ultimately establish novel therapeutic targets, micro-RNAs (miRNAs) have entered the spotlight of scientific inquiry , ameliorated ischemic tissue damage and fostered functional recovery, while the application of miRNA-132 inhibitors reversed mitigating effects of electroacupuncture (EA) on stroke . Collect−/−) adult female mice bred on a C57Bl/6 background (15–20 weeks old) were analyzed in this study. As female subjects are underrepresented in biomedical research in general and in neuroscience in particular [Wild-type (WT) C57Bl/6N and transgenic miRNAs 132/212 KO  were prepared as previously described [3, 2.5 CaCl2, 1 MgCl2, 25 D-glucose and 1 NaH2PO4 (pH 7.35–7.40). Coronal hippocampal slices  were cut while submerged in ice-cold, carbogenated aCSF (95% O2/5% CO2), using a vibrating microtome  at a frequency of 90 Hz, an amplitude of 0.75 mm and speed of 0.12 mm/s. Cutting was followed by separation of hemispheres and immediate transfer to a custom-build recovery chamber filled with carbogenated aCSF at 32 °C. Slices were allowed to recover for a minimum of 1.5 h before electrophysiological recording.Acutely dissected hippocampal slices obtained from miRNA 132/212escribed ,51,52,53−/− and WT mice. A total of n = 34 slices were included in the analysis. On average, n = 2–3 slices per animal and a total of n = 7 WT and 6 miRNA 132/212−/− animals were analyzed.Slices were transferred to a customized low volume (2 mL) submersion-type recording chamber perfused with carbogenated or nitrogenated aCSF solution at a constant flow rate of 4 mL/min. Slices were allowed to accommodate for at least 5 min before any further manipulation. Evoked field excitatory postsynaptic potentials (fEPSPs) were obtained using borosilicate glass micropipettes, using a pipette puller . Micropipette tips were backfilled with aCSF and yielded a tip resistance of (3 ± 1) MΩ. Teflon-coated tungsten wire bipolar stimulating electrodes (~50 μm diameter tip) connected to an ISO-STIM 01D isolator stimulator  were used to evoke fEPSPs. An AxoClamp-2B amplifier, Digidata-1440 interface  and pClamp-11 software  were used for data acquisition and analysis. As per previously established protocols ,54, stim3, 2.5 CaCl2, 1 MgCl2, 25 D-glucose and 1 NaH2PO4, was modified as follows to achieve an OGD-aCSF. Glucose was replaced with equimolar sucrose and the solution was continuously gassed with 100% N2 gas instead of a 95% O2/5% CO2 gas mixture, as previously established [3 and 25 mM HEPES. Firstly, evoked potentials were recorded for 15 min under euglycemic and normoxic conditions by superfusing the slices with standard aCSF, gassed with 95% O2/5% CO2, before any treatment. The slices were then superfused with OGD-aCSF (100% N2) for a total of three OGD 8 min intervals. Each OGD interval was followed by a recovery period of 10 min, in which the slices were superfused with standard, euglycemic, normoxic aCSF (95% O2/5% CO2).Standard aCSF, of the following composition (in mM): 125 NaCl, 2.5 KCl, 20 NaHCOablished . pH was 2/5% CO2), slices were superfused with OGD aCSF three times for 8 min with intervals of 10 min as recovery periods under normoxic and euglycemic aCSF superfusion (95% O2/5% CO2), following every OGD period (2SO3 in ddH2O) was prepared. Cobalt nitrate Co(NO3)2 standard solution (ρ(Co) = 1000 mg/L; concentration 0.5 mol/L in nitric acid) was used to catalyze oxygen desaturation. To estimate the maximal oxygen saturation of carbogenated aCSF , standard aCSF was bubbled with 95% O2/5% CO2 for at least 1 h before calibration. In accordance with previously published data [2 of aCSF in our experimental setup was estimated to be (720 ± 10) Torr. Oxygen saturation is reported as percentage of the maximal O2 saturation of continuously carbogenated aCSF. Oxygen saturation measured in the recording chamber ranged from 78.450 ± 0.024% during normoxia to 10.380 ± 0.046% under OGD treatment.  as previously described, with minor modifications [g for 5 min in a bench top centrifuge , Austria) at 4 °C. After determination of sample protein concentrations using the BCA protein assay kit, as per manufacturer’s instructions , Austria), 25 μg of protein homgenate were loaded per lane in subsequent electrophoresis, performed on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel, followed by transfer to poly (vinylidene difluoride) (PVDF) membranes . PVDF membranes were blocked in 5% bovine serum albumin  and washed 3 times for 5 min with TBST. Next, membranes were incubated overnight, at 4 °C in solutions with diluted primary antibodies: Anti- α7-nAChR , Anti-Acetylcholinesterase (AChE) , Anti-Muscarinic M1 Acetylcholine Receptor (mAChR M1) , anti-MeCP2 . Goat anti-rabbit served as secondary antibody . Anti-GAPDH primary antibodies , with Goat anti-mouse secondary antibodies (1:5000 dilution in 5% Milk in TBST), were used as loading controls. Electrochemical luminescence (ECL) reagent  was used for blot development and the FluorChem HD2 system  was used for protein visualization. Protein quantification and densitometric analysis was performed using the downloaded ImageJ software .We extracted brains and isolated whole hippocampi from female miRNA 132/212ications ,51,52,53−/− and WT slices over time. For statistical analyses of data strictly from within subject designs, we performed one-way ANOVAs with repeated measures and subsequent Bonferroni’s multiple comparisons tests, where appropriate. p-values and the number of samples used (n) are reported in the main text and represented in the figures . All data is expressed as mean ± standard error. The threshold of statistical significance (α) was set at 0.05 in all analyses.Statistical analyses were performed using GraphPad Prism Software, Version 8.0 . Statistical analyses of more than two groups were conducted using analysis of variances (ANOVA), as appropriate. Specifically, we used two-way repeated measures ANOVA (or mixed model ANOVA) followed by Bonferroni’s multiple comparisons test to analyze normalized fEPSP slopes obtained from miRNA 132/212−/− and WT dentate gyri differed in their basal synaptic transmission properties. To this aim, we used standard Input/Output protocol . Recordings obtained from miRNA 132/212−/− and WT hippocampal slices showed virtually indistinguishable synaptic responses , but no significant main effect of genotype  or input voltage × genotype interaction . The dentate gyrus circuitry has the capacity to respond to fast repetitive stimulation via the perforant pathway with a decrease of neurotransmitter release, which can be assessed by delivering a succession of two pulses of increasing interstimulus intervals and quantifying the paired pulse inhibition (PPI) [Before implementing the OGD treatment protocols we assessed whether miRNA 132/212esponses A. Two-waon (PPI) ,58. Thison (PPI) . PPI canp < 0.0001, F3.990, 43.89 = 68.59), with no effect of genotype  and no significant inter-pulse interval x genotype interaction .Congruent with our previously published data  we found−/− mice and their respective WT littermates, who served as controls. We chose superfusion intervals with the OGD buffer for 8 min at a flow rate of 4 mL/min and a temperature of 32 °C, as in our pilot experiments this treatment robustly depressed synaptic transmission to ≤50% of its initial baseline, while allowing for recovery to 70–90% of the initial baseline within 10 min of normoxic/euglycemic reperfusion in WT slices. This protocol enabled us to study the effects of a series of multiple ischemia–reperfusion injuries on depression and recovery of synaptic transmission. We performed a two-way ANOVA analysis with repeated measures to compare synaptic transmission between the two genotypes over the course of the OGD protocol, which unveiled statistically significant main effects of time  and genotype  and a significant time x genotype interaction   and during the first minute of the first recovery in comparison to recordings in respective WT control slices min = 3.603; (t1518)max = 3.924, t-values referring to lowest to highest value in this time frame)   or the second recovery episode  , as well as the majority of the third recovery episode  with miRNA 132/212−/− slices recovering to significantly lower fEPSP slope values at the end of the last recovery period, compared to the WT controls  ,  see A. Subseqame) see B1. Howev707) see C1. Furth258) see B2,C1,C2.589) see A,B3,C3. GD), see A. Collec−/− and WT controls, respectively. Strikingly, fEPSP slopes obtained from miRNA 132/212−/− slices recovered to significantly reduced values at the end of each ischemia–reperfusion episode, in comparison to preceding recovery intervals, indicative of a cumulative effect of repeated OGD periods on the depression of synaptic transmission . Bonferroni’s multiple comparisons tests showed statistically significant differences between the fEPSP slope values at normoxic, euglycemic baseline in comparison to slope values at the end of each recovery interval . Furthermore, post hoc tests indicated statistically significant differences between the first vs. second recovery , first vs. third recovery  and second vs. third recovery .In order to further investigate the pronounced differences in the recovery of synaptic transmission at the last ischemia–reperfusion interval , we analsion see A. One-wap < 0.0001, F1.688, 10.13 = 81.83, with fEPSP slopes reaching lower values after all recoveries in comparison to the initial baseline . fEPSP slope values recovered to significantly lower values at the end of the third vs. the first recovery interval . However, comparing the fEPSP slope values reached at the end of the first vs. the second , as well as the second vs. the third  recovery intervals, no statistically significant difference was observed , no statistically significant main effect of treatment  .We subsequently examined whether OGD insults influence dentate gyrus short-term synaptic depression and whether this form of synaptic plasticity was differentially affected by OGD in miRNA 132/212275) see A, and a −/− slices comparing PPI ratios from before and after OGD treatment yielded a significant main effect of inter-pulse interval , as well as significant main effect of treatment (before vs. after) , with no statistically significant inter-pulse interval x treatment interaction . Bonferroni post hoc tests revealed a statistically significant increase in the PPI ratio after OGD in miRNA 132/212−/− slices in response to pulses at 40 ms interpulse intervals . PPI ratios collected before and after OGD treatment from miRNA 132/212−/− exhibited no statistically significant differences in response to paired pulse stimulation at any other inter-pulse interval   see B.p < 0.0001, F3.918, 43.09 = 16.15) and a statistically significant main effect of genotype , but no significant inter-pulse interval × genotype interaction , but no significant effect of treatment (before/after OGD treatment)  and also no significant input-voltage x treatment interaction , but not treatment (before vs. after OGD treatment) , nor a significant input voltage x treatment interaction   see B.−/− slices recorded after repeated OGD treatment, we observed a statistically significant main effect of input voltage , but no significant main effect of genotype , nor a significant input-voltage x genotype interaction  has been independently implicated in cognitive deficits in the aftermath of ischemic stroke . Intrigu−/− mice and their respective WT littermate controls and analysed using Western blots. Unpaired two-tailed Student’s t-test revealed significantly higher AChE protein expression in samples obtained from miRNA-132/212−/− mice, as compared to WT controls . On the other hand, lysates from miRNA-132/212−/− mice exhibited significantly lower mAChR-M1 expression levels, in comparison to WT controls , while no difference could be observed in the protein levels of nAChR-α7 , or MeCP2 .Cell lysates were derived from bilateral hippocampi collected from miRNA 132/212Studying the effects of OGD on synaptic transmission in brain slices ex vivo has proven to be a versatile approach in unveiling pathophysiological hallmarks of ischemia–reperfusion induced insults to neuronal circuits and identifying novel therapeutic targets, e.g., ,67,68,86We here observed that in the absence of miRNA 132/212 the depression of synaptic transmission in the dentate gyrus, elicited by repeated OGD, is aggravated and the recovery from these insults is impaired. Interestingly, we observed a steady decrease in the efficiency of the synaptic transmission recovery from OGD induced depression in the absence of miRNA 132/212, as compared to WT controls. We thus propose the existence of a miRNA 132/212 dependent rapid adaptive response to ischemia–reperfusion insults, which might be involved in safeguarding the functional integrity of synaptic transmission in the dentate gyrus from ischemia-induced disruption.2+ neuronal culture model [While many studies have examined the influence of OGD on synaptic transmission in the hippocampal CA1 region, e.g., ,88,89,90re model . Moreovere model . The conre model .Previous in vitro studies have implicated a neuroprotective and antiapoptotic role of miRNA 132 through the regulation of FOXO3 expression up to 48 h after an initial severe OGD insult . MoreoveHere, we focused on the previously unexplored role of miRNA 132/212 signaling in the immediate response of synaptic transmission to transient OGD in the intact dentate gyrus circuitry. Given the emerging role of the miRNA 132/212 cluster as a key regulator of cholinergic signaling in the nervous ,71,73,77Although miRNA 132 and miRNA 212 share identical seed sequences, exhibit similar mature sequences and hence considerable overlap in their mRNA targets, the degree of functional redundancy of the mature miRNAs 132 and 212 in OGD and other physiological and pathophysiological contexts has not yet been fully elucidated ,106. ThuOther authors  have alsAdditionally, compared to the wide range of information available in the scientific literature, here we explored only a very narrow section of putative targets directly and indirectly implicated in miRNA 132/212 dependent signaling. Further research will be therefore necessary to advance towards a mechanistic understanding of how synaptic transmission is influenced by the miRNA 132/212 cluster in response to OGD. Moreover, while the OGD protocol used for this study was optimized to allow for the induction of a sequence of repeated OGD intervals, with intermittent recoveries, this protocol does not model the irreversible loss of synaptic transmission  and accAn additional and very important limitation of the work described here pertains to the potential physiological relevance of the sex of the subjects. Here we have exclusively focused on the study of female subjects and, consequently, the scope of our findings must be toned down accordingly to avoid generalizations. We have previously reported that α7-nAChR expression is upregulated in the absence of miRNA 132/212 in the hippocampi of male mice . However−/− mice bred on C57BL/6N and 129/Sv backgrounds [−/− mice bread on different background strains might thus help to exclude the possibility of similar strain-specific roles of this cluster in the synaptic response to OGD.Moreover, previous research on the role of the miRNA 132/212 cluster in the modulation of central circadian rhythms unveiled highly strain- and light-regimen-specific effects of miRNA 132/212 deletion when comparing miRNA 132/212kgrounds . IndepenHippocampal synaptic plasticity events, including Long Term Potentiation (LTP) and Long Term Depression (LTD), have also been extensively investigated as potential mechanisms critical for the formation and regulation of learning and memory functions. We here observed that the miRNA 132/212 gene deletion promotes the vulnerability of short-term synaptic depression to repeated oxygen glucose deprivation. We, however, have not yet explored the susceptibility of LTP and LTD to OGD. As the miRNA 132/212 cluster has previously been shown by others (as well as by our group) to be involved in the regulation of hippocampal synaptic plasticity , the expHere we described for the first time an involvement of the miRNA 132/212 cluster in the acute response of synaptic transmission to repeated ischemia reperfusion insults in the mammalian hippocampus, using an ex vivo electrophysiological approach. While previous research has implicated anti-apoptotic and regenerative effects of the miRNA 132/212 cluster via the silencing of FOXO3  and SOX2This proposition might warrant further exploration of the manifold mechanisms regulated by miRNA 132/212 dependent signaling in the temporal course of ischemic challenges and their consequence on molecular signaling and cell viability.We further explored the miRNA 132/212 dependent regulation of key mediators in the cholinergic signaling underlying both the physiological basis of higher order cognitive functions as well as the adaptive response to ischemia. We propose that the herein observed alterations in AChE and mAChR-M1 expression and aggravation of ischemia induced synaptic dysfunction in the dentate gyrus elicited by miRNA 132/212 deletion might help in the establishment of a link between miRNA 132/212, cholinergic signaling and ischemia associated cognitive impairment. Specifically, we speculate that the previously established link of concomitant miRNA 132 downregulation and AChE upregulation to post-ischemic dementia might be partially mediated by the failure of a putative miRNA 132 mediated mechanism to safeguard the functional integrity of hippocampal cholinergic synaptic signaling from ischemic disruption. Future research, addressing how miRNA 132/212 dependent signaling is involved in short- and long-term synaptic plasticity under OGD in vivo in various brain circuits over time, might help in the exploration of this research question. Our findings furthermore invite the elucidation of whether the expression of key players of cholinergic signaling such as ACh recpetors, or enzymes mediating the functional availability of ACh might be regulated by the miRNA 132/212 cluster over different phases during and after ischemic insults, in the in vivo context.However, as the herein chosen paradigm can only illuminate a narrow aspect of miRNA 132/212 dependent signaling under OGD, any translational considerations must be phrased cautiously. For example, it has been recently shown that miRNA 132/212 overexpression is able to compromise blood–brain barrier integrity via regulation of tight junction properties in vitro , which m


More than 70% of colorectal, prostate, ovarian, pancreatic and breast cancer specimens show expression of CD276 (B7–H3), a potential immune checkpoint family member. Several studies have shown that high CD276 expression in cancer cells correlates with a poor clinical prognosis. This has been associated with the presence of lower tumor infiltrating leukocytes. Among those, tumor-associated macrophages can comprise up to 50% of the tumor mass and are thought to support tumor growth through various mechanisms. However, a lack of information on CD276 function and interaction partner(s) impedes rigorous evaluation of CD276 as a therapeutic target in oncology. Therefore, we aimed to understand the relevance of CD276 in tumor-macrophage interaction by employing a 3D spheroid coculture system with human cells. Our data show a role for tumor-expressed CD276 on the macrophage recruitment into the tumor spheroid, and also in regulation of the extracellular matrix modulator PAI-1. Furthermore, our experiments focusing on macrophage-expressed CD276 suggest that the antibody-dependent CD276 engagement triggers predominantly inhibitory signaling networks in human macrophages. Despite being discovered almost 2 decades ago, the interaction partner(s) remains unknown. The short cytoplasmic tail lacks any known adaptor protein motif2, leaving the downstream signaling of CD276 unresolved. Initially, the studies mainly focused on modulation of T cell responses by CD276, yet the reported functions as co-stimulatory and co-inhibitory molecule remain controversial5. In addition to its debated immune modulatory functions, CD276 gained considerable attention in the tumor field. CD276 is expressed on fibroblasts6, epithelial cells7, endothelial cells8 as well as stromal cells9, and CD276 overexpression has been detected on a vast variety of solid tumors such as colorectal, breast and lung cancer10. The large body of data indicates a strong correlation between CD276 overexpression in tumor and poor patient prognosis10. For example, in hepatocellular carcinoma, CD276 expression associated with tumor aggressiveness and recurrence rate11. Nuclear localization of CD276 in colorectal cancer, high CD276 expression in pancreatic cancer and on tumor-associated endothelial cell in ovarian carcinoma were reported to be a predictive parameter for reduced survival rate14. Its pro-tumorigenic functions so far were associated to tumor growth, glucose and fatty acid metabolism, cell migration, metastasis, drug and chemotherapy resistance18.CD276/B7-H3 is a type I transmembrane protein and, based on the similarity in protein sequence, is considered as an immune checkpoint member of the B7 family. In mouse, the extracellular portion of CD276 is composed of two immunoglobulin (IgV–IgC) domains, whereas in human it has four Ig domains of two pairs of IgV–IgC due to exon duplication+ T cells, which implies an immune-modulatory role of CD27622. Analysis of tissue sections obtained from colorectal cancer patients indicated a positive correlation between CD276 expression on tumor cells and CD68+ tumor associated macrophages (TAM) infiltration20. Similarly, in hepatocellular carcinoma, CD276 expression was shown to positively correlate with infiltrated TAM and the same study indicated a potential role of tumor-expressed CD276 in polarization of the human monocytic cell line, THP-1 cells, to an anti-inflammatory M2-like phenotype23. Recently, a new study challenged this claim by demonstrating that higher CD276 expression on tumor cells positively correlated with increased number of CD8+ T cell and plasmacytoid dendritic cells (pDCs) in patients suffering from non-small cell lung cancer (NSCLC)24. Clearly, further studies are necessary to understand the connection between CD276 expression on tumor and tumor-infiltrating lymphocytes.Multiple studies suggest a possible connection between poor clinical prognosis in patients with high CD276 expression on tumor cells and lower tumor infiltrated lymphocytes such as CD8cis-partnership leading to phosphorylation of ITIM bearing LAIR-1, PECAM-1, Siglec-3, ILT2, ILT4 and ILT5 accompanied with kinase phosphorylation such as CREB, HSP27, ERK1/2, RSK1/2/3, c-Jun and STAT3. In conclusion, our data suggest that tumor-expressed CD276 is involved in regulating the recruitment of tumor-associated macrophages and provide insights, for the first time, in signaling mediated by CD276 in macrophages.However, to date there is no systematic study available investigating a possible role of CD276 on tumor in macrophage infiltration. Therefore, in this study, we aimed to understand whether CD276 modulates macrophage recruitment in a 3D tumor spheroid with human macrophage coculture system. Our data suggest that tumor-expressed CD276 contributes to macrophage recruitment in spheroid, possibly by influencing extracellular matrix (ECM) remodeling. In the absence of CD276 on tumor cells, plasminogen activator inhibitor-1 (PAI-1) and urokinase plasminogen activator (uPA)—two recognized players in ECM remodeling—are affected, indicating that CD276 could be an upstream regulator for PAI-1 in tumor. In addition to tumor cells, our data also provide insights in CD276 function on macrophages. CD276 is upregulated during monocyte differentiation to macrophages, and our data show that the intensity of CD276 expression depends on the cytokine milieu. Cross-linking of CD276 on macrophages with a specific antibody indicated a potential HCT116 colon cancer cells  were propagated and maintained in McCoy’s 5A (Modified) medium  with 10% FCS supplement.+CD16− Monocytes from frozen PBMC were magnetically isolated by using RoboSep according to the company’s protocol . Isolated cells were seeded in 6-well Nunc plates at 1 × 106 cells/ml with UpCell surface  in serum-free X-VIVO-10 media  and differentiated to macrophages by incubation for 7 days with 100 ng/ml M-CSF  or GM-CSF .Monocytes were isolated from frozen PBMCs obtained from either fresh blood donation or Leukopak concentrates. All studies on human donor blood were performed in accordance with the guidelines and regulations of German legislation and the experimental protocol was approved by the ethical committee of the Landesärztekammer Baden-Württemberg (Germany). Anonymized blood donors were healthy volunteers who provided written informed consent. PBMCs were isolated via density gradient centrifugation using Ficoll-Paque  and stored in − 160 °C until further use. CD14Neon Transfection System  was utilized to deliver the Cas9 —ribonuclear proteins (RNP) complex including guiding RNA against CD276  or scrambled gRNA  as control into the cell according to the company’s protocol with following modification.4 cells were electroporated with RNP complex (240 ng gRNA + 1.25 µg Cas9) in 10 µl reaction buffer with following setting on Neon: 1530 V, 20 ms, 1 pulse. After electroporation, cells were seeded in 24-well culture plate  in McCoy’s 5A (modified) medium with 10% FCS supplement and cultured for 4 days. CD276 expression on the cell surface was analyzed by flow cytometry.HCT116 cells were treated with accutase  to detach and washed with PBS. For each transfection, 5 × 105 cells were electroporated with the RNP complex (240 ng gRNA + 1.25 µg Cas9) in 10 µl reaction buffer with following setting on Neon: 1650 V, 10 ms, 3 pulse. After electroporation, cells were rested in X-VIVO-10 medium supplemented with 100 ng/ml M-CSF or GM-CSF for 7 days. CD276 expression was analyzed on day 7 by flow cytometry.Monocyte-derived macrophages (MDM) at day 7 were collected, washed with PBS and prepared for electroporation. For each transfection, 2 × 10WT and HCT116CD276KO cells were cultured and maintained in different culture flasks for further propagation. Five biological replicates were used per group for a total of 10 samples. 1 × 106 cells per sample were collected, centrifuged, washed two times with PBS. After removal of supernatant completely, cell pellets were immediately frozen at − 80 °C until further use. Total RNA extraction for Next-Generation Sequencing from cell pellets lysed in RLT plus buffer were performed by using the AllPrep DNA/RNA Mini Kit  according to the company’s protocol. An on-column DNA digestion was performed, and the final elution volume was 30 µL.HCT116Total RNA was quantitatively and qualitatively assessed using the fluorescence-based RNA Broad Range Qubit Kit (Thermo Fisher) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 48-channel Fragment Analyzer (Agilent), respectively. Concentrations averaged at 30 ng/µL while RIN were all > 8.5.10 HCT116-derived RNA samples were normalized and 200 ng of input was used for library construction using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760), together with the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490) upstream and the NEXNext Multiplex Oligos for Illumina (E7600) downstream . Sequencing libraries were quantified, normalized, pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered with the HiSeq 3000/4000 SR Cluster Kit on a cBot; and sequenced on a HiSeq 4000 Sequencing System (Illumina) with dual index, single read at 50 bp length , reaching an average depth of 40 million Pass-Filter reads per sample (11.1% CV).25: filtered reads were mapped against the Homo sapiens (human) genome hg38/GRCh38  using the STAR (v2.5.2b) aligner26 allowing for soft clipping of adapter sequences. For quantification, transcript annotation files from Ensembl (v86) corresponding to GENCODE 25 were used. Gene expression levels were quantified with the above annotations, using RSEM (v1.3.0)27 and featureCounts (v1.5.1)28. Quality controls were implemented using FastQC (v0.11.5)29, picardmetrics (v0.2.4)30 and dupRadar (v1.0.0)31 at the respective steps. Finally, differential expression analysis was performed on the human mapped counts derived from featureCount using limma/voom33. Processed data can be accessed in the NCBI GEO database under the accession code (GSE165610).Demultiplexing was performed using bcl2fastq v2.20.0.422 from Illumina. Sequencing reads from the RNA-seq experiment were processed with a pipeline building upon the implementation of the ENCODE “Long RNA-seq” pipelineWT or HCT116CD276KO were seeded in 96-well  plates  in McCoy’s 5A (Modified) medium and cultured for 5 days to form spheroid. On day 5, culture medium was replaced with serum-free X-VIVO-10 medium. In parallel, MDMs were collected, washed with PBS and resuspended in X-VIVO-10 medium. 1 × 104 MDMs were distributed per spheroid and cocultured for additional 4 days. At the end of coculture, supernatants were collected and stored at − 20 °C until further use. To quantify the viable cells per spheroids, 5–10 spheroids were pooled into one sample for each group and before dissociating the spheroids, a defined number of negative compensation flow cytometry beads  were added. Afterwards, spheroids were dissociated, and single cell suspensions were processed for surface and intracellular staining. Upon analysis by flow cytometry, ratio of recorded bead count to the initial bead amount was used to back calculate viable cells. For the experiments with uPA, recombinant uPA protein —2 ng/ml or 20 ng/ml—was added to the spheroid-MDM coculture on day 5. For the experiments with αCD276 antibody treatment, only HCT116WT spheroids were used and cocultured with MDMs. The antibody treatment experiment was performed in the presence of 20 µg/ml Fc Block , and cells received either 20 µg/ml αCD276 antibody  or 20 µg/ml monoclonal rabbit IgG isotype  for 4 days.4000 cells from HCT116Single cell suspension was washed with 0.2% BSA-PBS and directly incubated with Pacific Orange  for 10 min on ice to exclude dead cells for the follow-up data analysis. After live/dead staining, surface staining was carried out with the listed antibodies in the presence of αhFcγ‐receptor  in 0.2% BSA‐PBS for 10 min on ice. After washing, to proceed with intracellular CD68 staining, cells were fixed and permeabilized with FACS‐Lysing  and FACS‐Perm2 Solution , respectively, according to the company’s protocol. Cells were analyzed using the flow cytometer FortessaX20 (BD Bioscience).Following antibodies with indicated dilutions were used: BD Bioscience; CD14 , CD16 , CD163 , CD206 , CD36 , CD86 , CD68 , CD209 , CD11b , CX3CR1 (565801: 1:50), CD83 , Siglec1 ; HLA-DR . BioLegend; CD200R , CCR2 , CD11c . Miltenyi Biotec; CD300E  and CD276 . eBioscience; Marco .For the samples in which unconjugated rabbit αhCD276  was added to the culture, the flow cytometric analysis of CD276 surface expression was performed with mouse αhCD276 .6 cells/ml. MDMs were reseeded into 48-well or 24-well Nunc cell culture (UpCell) plates. After 2 h resting, following stimuli were added to the culture: 20 ng/ml LPS  + 50 ng/ml IFNγ ; 10 ng/ml IFNα ; 20 ng/ml IL-4  + 20 ng/ml IL-13 ; 20 ng/ml IL-10 . 24 h after stimulation, supernatants were collected and stored at − 20 °C until further use.MDMs generated for 7 days were collected, washed with 0.2% BSA‐PBS, and resuspended in serum-free X-VIVO-10 medium with a cell density of 1 × 10To quantify the protein amount in supernatants, following ELISA kits and arrays were used according to the company’s protocol: PAI-1 ELISA ; uPA ELISA ; Proteome Profiler Human XL Cytokine Array .WT and HCT116CD276KO cells were formed as described above and on day 5, culture medium was exchanged to serum-free X-VIVO-10 medium. From day 6 on, metabolic activity was assessed using the WST-1 assay kit according to the company’s protocol . Media was aspirated and 150 μl of 10% WST-1 solution was added to each well and cells were incubated for 3 h. To measure the absorbance at wavelength 450 nm, 100 µl from the supernatant was transferred into a 96-well F-bottom plate and immediately was recorded using microplate reader .Spheroids from HCT1166 in 500–750 µl X-VIVO-10 medium. After 3 h resting, cells were treated with 20 µg/ml Fc Block  for 10 min at 37 °C. Afterwards, cells were stimulated with either 20 µg/ml αCD276 antibody  or 20 µg/ml monoclonal rabbit IgG isotype . For Phospho-Immunoreceptor array , antibody treatment was for 10 min at 37 °C and 30 min for Phospho-Kinase Array . After incubation, cells were immediately transferred onto ice, collected and washed 2 times with PBS. Cell lysate preparation was carried out according to the company’s protocol. To inhibit the proteinase activities, lysis buffers for all samples were supplemented with 1% Halt Protease Inhibitor Cocktail (EDTA free) . Cell lysates were stored at − 80 °C until further use. Protein quantification was performed with Pierce BCA Protein Assay  and measurement was carried out with microplate reader . Equal amount of cell lysates (between 200 and 250 µg) was used per assay, and membranes were developed with ImageQuant LAS 4000 .MDMs were generated as described above. On day 7, cells were collected, washed with 0.2%BSA-PBS and reseeded in 24-well UpCell plates with a density of 1–1.5 × 10t test was used to calculate p values. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.GraphPad Prism was used to perform statistical analyses. Paired student CD276KO cells by employing Crispr-Cas9 gene editing technology. The loss of CD276 on the cell surface was monitored and HCT116CD276KO cells were sorted by flow cytometry to generate single cell clones  feature without additional support such as matrigel. HCT116 spheroids are rigid enough for transfer without disruption and permissive enough for monocytes and macrophages to infiltrate. HCT116 cells naturally express CD276, therefore we generated HCT11635. We could confirm that freshly isolated human monocytes from frozen PBMCs lack CD276 but upregulate it while differentiating to macrophages in vitro when cultured in the presence of GM-CSF or M-CSF , PAI-1, urokinase-type plasminogen activator receptor (uPAR). Signal for PAI-1 was higher in supernatant from SpheroidCD276KOcompared to Spheroid WT while the signals for DKK-1 and uPAR were lower  in the presence of αCD276 antibodies or isotype controls for 4 days. Based on quantification analysis done by flow cytometry, we detected increased macrophage infiltration upon αCD276 treatment   and as shown in Supplementary Fig. 16. If CD276 induces downstream signaling, this could indicate a cis-acting transmembrane protein partner. Immunoreceptors are among the main modulators for macrophages, therefore αCD276 treated MDMs were analyzed with Phospho-Immunoreceptor array  produced detectable PAI-1 only under LPS + IFNγ condition with no clear impact from αCD276 antibody treatment  is becoming increasingly clear. In addition to promoting angiogenesis, tumor cell invasion and motility, TAM can suppress the anti-tumor T cell responses45. In this matter, CD276 as a member of the B7 family of immune checkpoint proteins is a good candidate, especially because the tumor express much higher CD276 compared to healthy tissue10. In addition to tumor cells, CD276 expression on macrophages was shown in non-small-cell lung cancer (NSCLC) patient samples24 as well as on TAM in in triple-negative breast cancer patient samples46. However, insufficient data about the CD276 function is an impediment to evaluate CD276 as a therapeutic target. According to correlation studies, patients with high CD276 expression are reported to have lower tumor infiltrating lymphocytes, without giving any insight about the underlying mechanism10. In this study, we employed 3D human tumor spheroids to recapitulate solid tumor structure and cocultured with human macrophages to monitor tumor-macrophage interactions, including macrophage infiltration into the tumor spheroid. Tumor spheroids can contain regions of proliferating, oxygenated, hypoxic and necrotic cells and thus are a good translational model for the situation in vivo47. Additionally, and as would be expected, 3D structure influences gene expression, cell motility and adhesion48. Therefore, using tumor spheroids allowed us to interrogate the correlation analysis results in an in vitro experimental setting with human cells. Our data suggest that CD276 on tumor cells is a significant contributor for macrophage recruitment. In the absence of CD276 on tumor cells, significantly lower macrophage infiltration was observed  compose up to 50% of tumor mass and are often supporters for tumor growth51. Therefore, we focused on uPA and PAI-1. Proteolytic activity of macrophage-produced uPA was demonstrated to be essential for matrix degradation and adhesion leading to macrophage infiltration in a 3D matrigel experimental setting52. However, in our 3D spheroid setting, addition of exogenous uPA neither restored the macrophage recruitment nor affected the PAI-1 concentration. Moreover, besides uPA, PAI-1 interacts with other proteins including the low-density lipoprotein receptor-related protein 1 (LRP1), and vitronectin. Indeed, the LRP1-PAI-1 interaction was proposed to be responsible for macrophage recruitment in an in vivo tumor model54, yet a recent publication challenged the previous observation by demonstrating that increased PAI-1 in skeletal muscle in aged mice resulted in delayed macrophage recruitment55. Despite using different models, these contradictory data indicate the necessity of further investigation to elaborate the involvement of PAI-1 in macrophage mobility. On the other hand, studies performed in cancer patients have been providing supportive data. A pan-cancer analysis of coagulome and TME environment across multiple tumor samples highlighted the positive correlation of CD276 to PAI-1 and uPA56. As in line with our data, a role of CD276high TAM in triple-negative breast cancer patients was attributed to ECM modeling for immune cell infiltration46. However, whether PAI-1 acts as a checkpoint for macrophages to infiltrate in our setting requires further investigation.When we screened for soluble proteins in the supernatant of these cultures, three major soluble factors were affected: Dkk-1, PAI-1 and uPA. The involvement of Dkk-1, negative regulator for Wnt signaling, in cell adhesion and migration has been implied but currently in debate regarding its role as PAI-1 modulatorWe decided to further investigate CD276 biology by targeting it with specific antibodies. While using one of the antibodies currently tested in clinical trials would be more clinically relevant, accessing these antibodies are not easy. Hence, we used commercially available antibodies in our assays and this antibody might have different features than the ones in clinical trials. Targeting CD276 with specific antibodies in our spheroid-macrophage coculture resulted in increased macrophage numbers in spheroids, suggesting that CD276 favors in our setting for tumor-supportive macrophage. In this experiment, one striking result was the selective effect of αCD276 antibody treatment on CD276 expression on macrophages. In the presence of αCD276 antibody, its overall expression on macrophages was reduced. We ruled out the epitope competition between staining antibody and treatment antibody, since CD276 staining was not complete demolished, rather a reduction in expression was observed. Additionally, this shift in expression was only observed on macrophages but not on tumor cells in the same sample. As a result, we wanted to explore further the impact of targeting CD276 on macrophages. Since GM-CSF generated MDMs significantly expressed higher CD276, we included these cells as well in the further experiments.16, therefore we speculated that any induction of downstream signaling might occur via a cis-partnership and one or more of the immunoreceptors expressed on macrophages are potential candidates. According to our phospho-immunoreceptor array experiment performed on macrophages, αCD276 treatment on M-CSF and GM-CSF MDMs induced different patterns of immunoreceptor phosphorylation. ILT2, ILT4 and ILT5 phosphorylation tend to be stronger in M-CSF MDMs, while LAIR-1, PECAM-1 and Siglec-3 phosphorylation was only detected in GM-CSF MDMs. All these receptors are inhibitory receptors, supporting the idea of CD276 being an inhibitory molecule. In both MDM subset varieties, the phosphorylation of FcγRIIα raised a question about Fc binding of the antibody. All of the experiments were conducted in Fcγ Receptor blocking conditions, yet still to clarify the unspecific involvement of Fcγ receptors, THP1WT and THP1CD276KO cells were treated with αCD276 antibody and FcγRIIα phosphorylation was only detected in THP1WT cells indicating that crosslinking for this antibody is essential for its function (Supplementary Fig. The cytoplasmic tail of CD276 is short and has no known signaling motif, yet CD276 overexpression on tumor cells was reported to increase Jak2 and STAT3 phosphorylationOverall, as summarized in Fig. Supplementary Tables 1.Supplementary Figures 2.


The hedgehog (Hh) pathway is a sophisticated conserved cell signaling pathway that plays an essential role in controlling cell specification and proliferation, survival factors, and tissue patterning formation during embryonic development. Hh signal activity does not entirely disappear after development and may be reactivated in adulthood within tissue-injury-associated diseases, including idiopathic pulmonary fibrosis (IPF). The dysregulation of Hh-associated activating transcription factors, genomic abnormalities, and microenvironments is a co-factor that induces the initiation and progression of IPF. Cell signaling is a multifactorial system that represents the knot-like schematics of the signaling cascades that are used to transfer messages from the first messenger to the receptor and decoded through the signaling intermediates of the second messengers ,2. SignaThe dysregulation of the Hh signaling network in controlled cell growth and division induces autocrine and paracrine function distortions, leading to the development of tumorigenesis and cancer progression ,7. MyofiVertebrate genome duplication categorized Hh genes into three different types of hedgehog proteins: the Desert Hedgehog (Dhh), Indian Hedgehog (Ihh), and Sonic Hedgehog (Shh) proteins .Hh proteins undergo multiple processing steps that are required for the generation and release of the active ligand from the producing cell. The core components that mediate the Hh signal response in vertebrae are two patched receptors , a key signal transducer smoothened receptor (Smo), three glioma-associated oncogene (Gli) transcription factors , the suppressed fusion homolog (Sufu), and kinesine protein 7 (Kif7) .Hh genes are automatically cleaved into a 20 kDa N-terminal protein (Hh-N) and a 25 kDa C-terminal protein (Hh-C) . After tHh ligands start to trigger signaling in the target cells by binding a 12-pass integral membrane, the Ptch protein (complex Hh-Ptch). Vertebrates have two Ptch genes, Ptch1 and Ptch2, but Ptch1 is the primary signaling regulator . Ptch1 iThe G protein-coupled receptor (GPCR), the Smo protein, which is predominantly located in the membrane of intracellular endosomes, functions as a co-receptor and a positive regulator of the Hh signaling pathway. Smo consists of an amino-terminal cysteine-rich domain (CRD), three extracellular and three intracellular loops (ECL and ICL), seven transmembrane domains (TM), and an intracellular carboxyl-terminal tail that is able to undergo a range of post-translational modifications .The Gli family of latent zinc-finger proteins function as transcriptional mediators and are implicated in the activation and repression of the Hh target genes . In detaSufu is an essential intracellular negative regulator of Hh signaling and acts by binding and modulating the Gli transcription factors . In the Kif7 is a cilia-associated protein that regulates signaling from the Smo to the Gli . HoweverThe Hh activates canonical  and non-canonical (ligand-independent interaction) signaling pathways .The canonical signaling pathways focus on the mechanism by which Hh regulates the Gli . This pa+ and membrane cholesterol to regulate Smo [In the absence of Hh ligands, Ptch1 functions to suppress any inactive Smo that is inside the cell and inhibits the migration of Smo to the membrane . The meclate Smo . Furtherlate Smo .The inhibition of Smo activity is an essential step for activating this pathway in mammals . The fulThe Ptch1 protein stops inhibiting Smo after binding the Hh ligand and limits the half-life of the ligand . Smo act2+ and actin skeleton [Contrary to canonical complex signaling network resulting in activation of the Gli family of transcription factors, some Hh signaling proceeds through Gli independent activation. In detail, non-canonical Hh delivers signals via (1) Ptch1 in the presence of Hh ligand, (2) Ptch1 in the absence of Hh ligand, and (3) Smo-dependent and Gi protein modulating Caskeleton ,46,47,48After embryogenesis, the Hh pathway continues to signal to discrete populations of stem and progenitor cells within various organs in order to maintain tissue homeostasis and repair . In the It is already known that the Hh signaling pathway plays a critical role as the principal regulator in the normal development of many tissues such as those in the lung. Lung morphogenesis relies on intricate interactions and the coordinated development of the endoderm layer-derived epithelial cells into the surrounding mesoderm-derived mesenchyme .Embryonic lung development follows the principle of branching morphogenesis into five phases; the first four phases  result in a typical branching structure that ends with alveolar sacs with a surrounding stromal scaffold and vascular structures where during the final  alveolar phase, the terminal sacs give rise to mature alveolar ducts and alveoli . The firDuring embryogenesis, the Shh that is secreted by the epithelial cells during the early steps of embryogenesis, act as a spatial regulator of bronchial bud formation and are essential for the mesenchymal–epithelial cross-talk that guides branching and epithelial tube elongation, as well as smooth muscle cell/myofibroblast differentiation . Other eIn contrast with its crucial roles during embryonic development, Hh signaling has more restricted roles after birth. Postnatally, mature lung development begins with the formation of the alveolar septum  followed by secondary septa and microvascular maturation . The Hh Recent studies indicate that the growth signaling pathways may be reactivated in tissue remodeling and cancer development. IPF and lung cancer share similar cellular and molecular pathological processes, including aberrant embryological pathways . Hh signSeveral studies showed the involvement of the Hh pathway during fibroblast activation and during myofibroblast transformation in biliary and liver fibrosis ,63,64,65IPF is a chronic, progressive, irreversible disease that is characterized by the pathogenic cellular fibroblast to myofibroblast transition, the plasticity of alveolar epithelial cells, the recruitment of fibroblasts, cell–matrix interactions, and immune system activation within the alveolar wall . The patThe reactivation and deregulation of the Hh signaling pathway and its cross-talk with tumor growth factor-β (TGF-β) contribute to IPF pathogenesis by inducing myofibroblast differentiation and epithelial–mesenchymal transition (EMT) and by producing excessive amounts of extracellular matrix (ECM) .The histopathological hallmark of IPF is usually interstitial pneumonia (UIP), which is characterized by variations in temporospatial heterogeneity fibrosis, the accumulation of fibroblasts (fibroblast foci), and subpleural and paraseptal honeycombing . AlthougThe alveolar epithelium consists of type I (AECI) and type II cells (AECIIs). AECIIs are capable of self-renewing and self-differentiation into AECIs . HoneycoThe differentiation of airway fibroblasts to myofibroblasts (myofibroblast transdifferentiation) leads to the secretion of excessive amounts of ECM . HoweverBidirectional interactions between AECIIs and fibroblasts drive the progression of IPF. AECIIs regulate the immune response to ameliorate lung injury by stabilizing host immune competence and by repairing most of the damaged epithelium . RepetitMonocytes and tissue-resident macrophages are innate immune cells that also play a critical role in driving tissue repair, regeneration, and fibrosis . While tRecent studies have reported a dysregulation of Hh signaling in IPF. The inhibition of the Hh signaling pathway by up-regulating Sufu prevents lung fibrosis in mouse models . AnotherTaken together, the successful use of Taladegib, a small-molecule inhibitor of the Hh signaling pathway that has been approved for the treatment of cancers, might inspire the use of Hh as a targeted therapy for IPF. The reactivation of Hh signaling drives pulmonary fibrosis via the dysregulation of epithelial injury, EMT, fibroblast activation and myofibroblast differentiation, and macrophage polarization .The pathogenesis of IPF is started by a usually unknown epithelial injury. Recurrent injuries lead to epithelial apoptosis that is mediated by misfolded proteins and an unfolded protein response . EpithelIn normal conditions, Hh is one of the signaling cascades that regulate and maintain the balance of wound healing, chronic fibrosis, and cancer . FollowiThe dysfunction of Hh-associated lung epithelial repair and regeneration induce fibrogenesis. Normally, epithelial cells preserve the mesenchymal quiescence through paracrine Hh signaling, simultaneously as negative feedback to maintain epithelial quiescence . Hh mainInjury to the AECIIs causes the release of many soluble factors that participate in epithelial repair, including growth factors (TGF-β), cytokines, chemokines, stromal cell-derived factor-(SDF-)1, vasoconstrictor endothelin-1 (ET-1), interleukins, prostaglandins, and matrix components ,101,102.In response to injury, the airway epithelial cells induced TGF-β activation through αvβ6 and αvβ8 . Zhang eIn general, the various products that are created following the lung epithelial injury-related activation of the Hh signaling pathway is involved in the following fibrogenesis process, including myofibroblasts differentiation, macrophage polarization, and EMT.Fibroblast activation and myofibroblast differentiation are the central pathogenesis of pulmonary fibrosis ,113,114.It is known that control of Hh-associated lung fibrogenesis is varied in each Hh gene. Horn et al. demonstrated that the activation of the Hh pathway in patients with systemic sclerosis (SSc) was increased, which was characterized by the accumulation of Gli1-2, Smo, and Ptch1-2 . ConsequThe Hh signaling pathway regulates myofibroblast function in parallel with other pro-fibrogenic proteins and cytokines. The cross-talk linking connective tissue growth factor (CTGF) and TGF-β induced myofibroblast differentiation and ECM production . OsteopoIn addition, targeting the RAS axis decreased collagen deposition, myofibroblast differentiation, and α-SMA expression via the inhibition of the Hh genes that are involved in silicosis . Next, CA hypoxic microenvironment drives fibrosis progression. Wang et al. showed that hypoxia significantly enhanced the expression of Hh signaling in pulmonary vascular smooth cell proliferation . Next, hEven though the specific roles of EMT in IPF have been widely hypothesized; its precise mechanisms are not entirely understood. Kalluri and Weinberg classified three classes of EMT. Type 1 EMT regulates embryogenesis and organ development. Next, type 2 EMT determines routine wound healing, tissue regeneration, and inflammation-associated organ fibrosis. On the other hand, type 3 EMT has been linked with the epithelial cells, which underwent genetic and epigenetic changes and then transformed into cancer cells .The Hh pathway regulates the EMT mechanism of lung fibrosis through cross-talk with various EMT-activating transcriptional factors, such as zinc finger E-box–binding homeobox (ZEB) and Snail 1/2, and by responding to signals from the microenvironment . FurtherEMT might also be induced by various growth factors. Blocking the Hh pathway in the primary cilium abrogated the TGF-β1-induced mesenchymal differentiation of the AECIIs . FurtherThe macrophage balance in the absence of injury, in the early phases of injury, and in the fibrotic phase is different. Misharin et al. revealed that the monocyte-derived alveolar macrophages (Mo-AMs) that persevere in the lung after injury resolution express higher proinflammatory and profibrotic genes than tissue-resident alveolar macrophages (TR-AMs) . HoweverHh-associated macrophage function promotes fibrogenesis. Several pieces of evidence have demonstrated Hh genes were expressed in human monocytes and macrophages; therefore, Hhs act as potent chemoattractants ,144. AddSince Hh resembles the Achilles’ heel in IPF, targeting the Hh signaling pathway and Hh-related tumor microenvironments can help achieve better therapy outcomes. Ligustrazine reduced the expression of the profibrotic factor, suppressed total collagen production, and ameliorated oxidative stress, resulting in attenuating paraquat (PQ)-induced lung fibrosis . RecentlA variety of Smo antagonists that target Hh signaling have been developed for cancer and fibrosis diseases. Vismodegib is the first oral Hh inhibitor that has been approved to treat patients with locally advanced or metastatic basal cell carcinoma . HoweverFurthermore, targeting the Gli transcription factor in the nucleus could be a therapeutic option. The administration of the Gli inhibitor GANT61 reduced lung fibrosis and lung collagen accumulation in a mouse model . A studyThe aberrant activation of Hh signaling is one of the core pathways that is involved in the development of pulmonary fibrosis. Recent evidence has shown high Hh signaling expression in the epithelial cells, fibroblasts, myofibroblasts, and macrophages, as well as in other IPF cells. When uncontrolled, the activation of Hh signaling is responsible for the development growth factors and other various products following a tissue-associated epithelial injury, the progressive accumulation of ECM by the myofibroblasts, crosstalk between the fibroblasts and EMT, and M2 macrophage polarization.Even though IPF progression could potentially be reversed through the targeting of Hh genes, the detailed mechanism of the Hh pathway in lung fibrosis and other organ fibrosis remains to be investigated in more detail. Hh signaling regulates mesenchymal and epithelial quiescence/proliferation during normal repairs. In IPF patients, the presence of a repetitive injury induces chronic epithelial proliferation more than mesenchymal quiescence. Instead, it seems different in the ischemic heart; Hh signaling reduces fibrosis. These discrepancies might be because Hh is acutely and not chronically overexpressed in the ischemic heart.Furthermore, a concept “apoptosis paradox” in IPF, where epithelial apoptosis can stop collagen deposition, but apoptosis resistance in myofibroblasts leads to increased fibrosis, may correlate with Hh non-canonical signaling. Therefore, further research is still needed to discover the detailed Hh signaling pathway-related inflammation and abnormal repairment mechanism in IPF.


The effects of high hydrostatic pressure (HHP) compared to thermal pasteurization (TP) were studied in healthy citrus-maqui beverages. The impact of the processing technologies on the microbiological and phytochemical profile was assessed by applying two HHP treatments at 450 and 600 MPa for 180 s and TP at 85 °C for 15 s. The shelf life under refrigeration (4 °C) and room temperature (20 °C) was monitored for 90 days. All treatments ensured microbiological stability at both storage temperatures. Aside from that, the physicochemical parameters were not significantly different after processing or throughout the storage period. Regarding color parameters, an increase in the reddish coloration was observed during storage for those beverages treated by HHP. In general, phenolic compounds were little affected by the processing technique, even when treatment under HHP was more stable than by TP during storage. On the other hand, vitamin C showed great degradation after processing under any condition. It can be concluded that HHP is an effective alternative to thermal treatments, achieving effective microbial inactivation and extending the shelf life of the juices by contributing to a better preservation of color and bioactive compounds. Nowadays, there is a growing trend focused on the implementation of healthy and balanced diets by consumers, minimizing the consumption of processed foods. In this respect, recent studies revealed that over 70% of consumers associate emerging non-thermal technologies with better nutrient or sensory quality , since tAristotelia chilensis (Mol.) Stunz), a native plant from Chile and Argentina particularly studied as a potential healthy food ingredient [This study follows previous studies focused on the design of healthy plant-based soft drinks, which showed that thermally treated beverages based on maqui and citrus fruits are an excellent source of bioavailable bioactive compounds ,8,9, maigredient ,13. The gredient , in addigredient  as well gredient  or cancegredient ,18. Thatgredient  and thosgredient .On the other hand, citrus fruits are a source of nutraceutical and bioactive phytochemicals  such as Concerning the different beverage processing methods, conventional thermal methods have been capable of ensuring food safety and achieving a high degree of enzymatic inactivation in the fruit- and vegetable-based beverage industry. However, thermal processing (TP) tends to degrade the overall quality in juices, such as the sensory and phytochemical profiles , affectiGiven these antecedents, the purpose of the present study was the optimization of the processing design for healthy maqui-citrus soft drinks using HHP in order to minimize the loss of bioactive compounds and, consequently, increase the healthy properties previously reported. Thereby, a comparison between HHP (at 450 and 600 MPa) and TP  for their quality parameters  and bioactive compound contents (flavonoids and vitamin C) over 90 days of storage at 4 °C and 20 °C was set. Moreover, to our knowledge, this is the first study comparing the effect of high hydrostatic pressure relative to TP on the levels of functional compounds and shelf life in juices containing a mixture of bioactive compounds from berry and citrus fruits.O-glucoside was purchased from TransMIT . Acetonitrile, formic acid, methanol, and ethylenediaminetetraacetic acid disodium salt 2-hydrate (EDTA), were obtained from Panreac . Buffered peptone water, Plate Count Agar (PCA), Rose Bengal Agar (RBA), Brilliant Green Bile 2% Broth (BGBB) and Man Rogosa Sharpe Agar (MRSA) were purchased from Scharlab . L-ascorbic acid (AA) and dehydroascorbic acid (DHAA) were acquired from Acros Oganics  and Sigma-Aldrich , respectively. All solutions were prepared with ultrapure water from a Milli-Q Advantage A10 ultrapure water purification system .Hesperidin was obtained from Merck , and cyanidin 3-Fresh dry maqui powder organic was provided by Maqui New Life S.A. , citrus juices by Cítricos de Murcia S.L.  and AMC Grupo Alimentación S.A.  and sucrose by AB Azucarera Iberia S.L. .Juice preparation was conducted according to previous studies of the group . BrieflyThe juices were poured into PET clear bottles  with plastic screw caps  prior to HHP processing and subsequently in the case of TP. The beverages were pressurized using a commercial Hiperbaric 135 for HHP . This equipment has a capacity of 135 L, which applies a maximum treatment of 6000 bares (600 MPa). The samples underwent two HHP treatments at 450 and 600 MPa for 3 min at 20 °C. The times to reach 450 MPa or 600 MPa were 120 and 165 s, respectively. The initial water temperature in the vessel ranged from 8–10 °C, and the rate of temperature rise during compression was 3 °C/100 MPa, while the decompression of the beverages was almost instantaneous (less than 5 s). Selection of processing parameters was mainly based on HHP common industrial processing conditions for ensuring microbial safety in juices with low pH levels.The thermal treatment was carried out with a Mastia thermoresistometer . The inin = 3), and all analytical determinations were performed in triplicate (n = 3). The samples were analyzed at 0, 7, 15, 30, 45, 60, and 90 days. Moreover, untreated samples were stored and analyzed as controls against the treated ones for day 0.All treated beverages were stored immediately after processing at 4 and 20 °C in darkness for 90 days. The samples were labeled according to the codification specified in ® System; Biotech SL, Barcelona, Spain) at 20 °C, with values being expressed as °Brix [The pH values were measured using a GLP 21 pH meter . The TA was determined using a 794 Basic Titrino (Metrohm) by titrating 2 g of juice (up to 30 g of Milli-Q water) with 0.1 mol/L NaOH to an end point of pH 8.1. The results were expressed as grams of citric acid per 100 mL of the sample (g CA/100 mL). The TSS contents of the samples were recorded in a refractometer . They were analyzed at day 0 and after 7, 15, 30, 45, 60, and 90 days of storage at 4 °C and 20 °C. Microbial analyses were carried out in order to measure the short- and long-term effectiveness of HHP processing and TP at day 0 just after processing and throughout 90 days of shelf life. The quantifications of the molds and yeasts were determined by plating the samples in Rose Bengal Agar (RBA), followed by incubation for 5 days at 25 °C. The counts for aerobic mesophilic and psychrophilic bacteria were performed using the Plate Count Agar (PCA) medium, incubated for 48 h at 30 °C and for 10 days at 5 °C, respectively. Enterobacteriaceae were determined using the more probable number method with Brilliant Green Bile 2% Broth (BGBB) after incubation for 24–48 h at 37 °C. Lactic Acid Bacteria (LAB) were determined using Man Rogosa Sharpe Agar (MRSA) after incubation in anaerobic conditions for 5 days at 37 °C. Microbial counts were expressed as colony-forming units per milliliter (CFU/mL).O-glucoside at 520 nm. The concentration of phenolic compounds was expressed as mg per 100 mL of juice.Juice samples were processed following the method previously described . The ideThe content of vitamin C was found by applying the UHPLC-ESI-QqQ-MS/MS-based method recently developed  and calcL*a*b* system with reference to a visual angle of 10° and a light source set on D65. Three measurements of each sample were performed, and the values were averaged. The color parameters determined were the luminosity (CIEL*), redness (a*), and yellowness (b*) using the CM-5 spectrophotometer in reflection mode. The Hue angle (H), Chroma (C) and total color differences (ΔE) were calculated from tan−1 (b*/a*), (a*2 + b*2)1/2, and (da*2 + db*2 + dL*2)1/2, respectively [The color was determined using a Konica Minolta CM-5 Chroma Meter . The results were expressed in accordance with the CIEectively .n = 3). A paired t-test was developed to compare two parameters, and analysis of variance (ANOVA) and Tukey’s multiple range tests were carried out to compare three or more conditions. All statistical analyses were performed using SPSS 19.0 software . The level of statistical significance was established at p < 0.05.The results are presented as the means ± SD (p > 0.05) in all treated drinks  in the aftermath of processing  in comparison with the untreated beverage (ocessing . These cp < 0.05) were noticed after all treatments (p < 0.05) in the pasteurized juices just after processing related to HHP and the unprocessed beverages. In this sense, TP managed to enhance the content and, consequently, bioaccessibility of these bioactive molecules up to 55% more, while both HHP treatments were capable of maintaining the initial flavanone content at a rather stable value compared with the untreated juice. This increase in flavonoids after thermal treatment may have been associated with the increased extractability of them as they were released when applying moderate temperatures during thermal treatment due to vegetable cell wall disruption, as previously reported [Regarding flavonoids (anthocyanins and flavanones), slight significant changes (eatments . These rreported . It shoureported .p < 0.05) compared with the unprocessed citrus-maqui drink by 35% on average after all the processes   values were similar in all treated beverages ( > 0.05) , with onThe microbiological profiles of the citrus-maqui juice samples treated by HHP (450 and 600 MPa/3 min) and TP (85 °C/15 s) were analyzed during storage for 90 days at both 4 and 20 °C. In the current study for all the processed juices, no signs of microbial growth were observed throughout the 90 days of storage, with microbial counts remaining below the detection limit for all microorganisms analyzed, indicating that the citrus-maqui blends were microbiologically safe and stable at both refrigerated and room temperatures. Previous studies of similar technologies have also reported efficacy in preserving microbiological safety over the storage period in vegetable- and fruit-based juices. In this respect, Chen et al. reported that both HHP and thermal treatment ensured microbiological stability during 90 days of storage at 4 °C in pomegranate juice . Moreovep < 0.05) of this compound was found  between the HHP-treated and thermally treated juices, since beverage processing by TP managed to extend a portion of the content of vitamin C up to 30 days more than juices subjected to HHP for both storage conditions during their shelf lives. Among other factors, these results may be due to the degradation of the active forms of vitamin C (AA and DHA) by enzymes with oxidase activity [Regarding individual treatments, there were significant differences , narirutin (naringenin 7-O-rutinoside), and hesperidin (hesperetin 7-O-rutinoside), characterized in preliminary studies by Salar et al. [Regarding the flavanones of the citrus-maqui beverage, they were provided by citrus juices, being the most abundant eriocitrin (eriodyctiol 7-p < 0.05) were found between both treatments submitted to HHP compared with TP  at the end of storage. This indicates that even if a higher concentration of these compounds is reached after treatment, phenolic compounds are more stable after both HHP treatments. Again, there were no significant differences between both HPP conditions.O-tryglycosil-naringenin nor eriocitrin and narirutin displayed significant losses over 90 days of storage (data not shown), whereas hesperidin was quite affected under all processing treatments, as previously reported [Moreover, considering the contribution of individual flavanones to the total concentration of the phenolic compounds, neither reported ,85. OverA. Chilensis [The range of anthocyanins present in the citrus-maqui beverages was due to hilensis , widely hilensis . In thishilensis ,87.After the first fortnight of the storage period, the rate of anthocyanin degradation was gradual for all treatments, decreasing with time and without significant differences between both HHP processing treatments, reaching 30% loss on average at the end of the beverage’s shelf life regardless of the treatment under refrigerated storage at 4 °C A. These Moreover, anthocyanin stability in plant-based products depends on the interaction of various factors such as temperature, light, pH, presence of oxygen, metal ions, and solvents, among others ,94. In tL*a*b* color parameters. The reddish coloration of the new citrus-maqui blend was due to the total content of anthocyanins, which contribute to providing a more attractive appearance in the beverages for consumers.The color changes during 90 days of shelf life at 4 and 20 °C were determined by measuring the CIEL*) value, statistically significant differences were found between individual treatments (p < 0.05) throughout the storage period  was observed for most of the samples as the shelf life increased  in citrus-based juices [Furthermore, an overall trend to decrease redness , as this parameter increased along with the shelf life time for most of the drinks, mainly in HHP-treated beverages at 450 MPa relative to the HHP-treated ones at 600 MPa or under thermal treatment  increased along the 90 days of storage , indicatOn the whole, the shelf life had a significant impact on the color parameters monitored for beverages processed under either HHP or TP, although those samples processed by TP retained their original colors better than HHP, in accordance with previous reports ,116. FinIn this study, the application of HHP showed interesting results related to the microbial safety and stability of all parameters analyzed in the citrus-maqui beverages. Neither the processing nor storage conditions had an impact on the physicochemical parameters. Moreover, both HHP and TP guaranteed the microbiological safety throughout the shelf life. Regarding bioactive compounds, although TP promoted an increase in the content of flavonoids immediately after processing, HHP displayed a higher protective effect on bioactive flavonoids (flavanones and anthocyanins), giving rise to a lower rate of degradation during storage and preserving the healthy properties for the beverages. On the other hand, vitamin C underwent a rapid degradation for all processing conditions. Aside from that, the color parameters remained rather stable for all treated samples, keeping an attractive reddish coloration, although the use of HHP and refrigeration better preserved the intensity of the color during storage. It is noteworthy that color was the only studied parameter slightly affected by the HPP conditions (450 or 600 MPa). Finally, the storage temperature was shown to be the most critical parameter for the degradation of all the studied bioactive compounds, with 4 °C being the most suitable temperature for storage. Due to these results, HHP could be considered an effective alternative to conventional TP in the food industry for the production of high functional quality fruit-based beverages.


Malus domestica cultivars and a M. asiatica cultivar were collected in 2016 and 2017. The phenotype segregated considerably with high broad‐sense heritability of 83.85% and 83.64% for flesh firmness and crispness retainability, respectively. Fifty‐six candidate genes were predicted from the 62 QTLs identified using bulked segregant analysis and RNA‐seq. The genotype effects of the markers designed on each candidate gene were estimated. The genomics‐predicted values were obtained using pyramiding marker genotype effects and overall mean phenotype values. Fivefold cross‐validation revealed that the prediction accuracy was 0.5541 and 0.6018 for retainability of flesh firmness and crispness, respectively. An 8‐bp deletion in the MdERF3 promoter disrupted MdDOF5.3 binding, reduced MdERF3 expression, relieved the inhibition on MdPGLR3, MdPME2, and MdACO4 expression, and ultimately decreased flesh firmness and crispness retainability. A 3‐bp deletion in the MdERF118 promoter decreased its expression by disrupting the binding of MdRAVL1, which increased MdPGLR3 and MdACO4 expression and reduced flesh firmness and crispness retainability. These results provide insights regarding the genetic variation network regulating flesh firmness and crispness retainability, and the GAP models can assist in apple breeding.Retention of flesh texture attributes during cold storage is critical for the long‐term maintenance of fruit quality. The genetic variations determining flesh firmness and crispness retainability are not well understood. The objectives of this study are to identify gene markers based on quantitative trait loci (QTLs) and to develop genomics‐assisted prediction (GAP) models for apple flesh firmness and crispness retainability. Phenotype data of 2664 hybrids derived from three  However, optimal flesh firmness and crispness are important fruit quality traits determining consumer choice. The retention of these texture attributes during cold storage is critical for long‐term maintenance of fruit quality , a number of QTLs for fruit firmness and flesh texture were repeatedly mapped on chromosomes 1, 5, 6, 8, 10, 12, 14, 15, 16 and 17, which explained 10–49% phenotypic variance  and ACC oxidase (ACO) are the two well‐studied ethylene production‐related genes. QTL‐based markers on MdACO1 and MdACS1 have been shown to be responsible for apple fruit firmness and shelf life  was developed from a QTL for fruit post‐harvest firmness  was associated with apple fruit crispness and mealiness in a genome‐wide association study (GWAS) , bind to the MdACS1 promoter and oppositely regulate its transcription during post‐harvest ripening in apple  for apple crispness has been practically used in breeding programmes, enabling breeders to design the perfect hybrid cross and to select the best seedlings from large populations  was higher than that of genomic best linear unbiased prediction (G‐BLUP) model (0.72–0.77)  was 75.24–83.06% and 86.91–94.43% for the initial flesh firmness and crispness, respectively  and crispness (≥0.7 kg/cm2) retainability was higher in the population of ‘Zisai Pearl’ × ‘Red Fuji’ than the other two , followed by that between initial flesh firmness and crispness   Figure . The cor) Figure . The hyb) Figure . Among t) Figure . However) Figure . These d2) or crispness (0.7 kg/cm2) lost already at harvest. Type II: acceptable flesh firmness or crispness maintained until the sixth month after a rapid softening during the first month of cold storage. Type III: acceptable flesh firmness or crispness maintained after the sixth months of cold storage with constantly slow softening rate , MdBGAL (MD15G1220900 and MD15G1221000) and MdBGLU (MD03G1215300) , MdGLCAT14B (MD02G1033100), MdGWD1 (MD16G1097800) and a cellulolyase gene (MD17G1249600)  were detected between the three types of flesh firmness and crispness retainability Table . As expe) Figure . On the ) Figure .MdZAT12 (MD01G1123300 and MD07G1192900), MdERF110 (MD15G1334900) and MdACO1 (MD15G1205100) was 0.01‐ to 0.48‐fold lower in type II and type III hybrids , MdACO3 (MD02G1050800), MdEIN3 (MD15G1441000) and MdERF98 (MD06G1208700) was significantly low (0.01‐ to 0.47‐fold difference) in flesh crispness retainable type III and type II hybrids  in firmness type III and type II hybrids than that in firmness type I hybrids, such as MdAGL24 (MD15G1384500), MdACO4 (MD16G1019900), MdACO4‐like (MD16G1019800), and MdALD1 (MD15G1412200 and MD15G1412400).Several ethylene‐related DEGs were detected between flesh firmness type III or type II and type I hybrids. The expression of s Figure . Similars Figure . A groupMdSAUR71 (MD10G1202100), MdSAUR40 (MD01G1106100) and MdARGOS (MD02G1000700), was observed in flesh firmness type I hybrids  of some auxin‐related genes, s Figure . In addis Figure . There ws Figure . In summMdERF3 (MD03G1194300) and MdERF118 (MD16G1043500), were predicted as candidate genes for both flesh firmness and crispness retainability  from −323 to −330 (Del323) prior to the ATG codon of g Figure . The DelMdERF3 in ‘Red Fuji’ and three flesh firmness and crispness retainable hybrids  with Del323:del323 genotype was significantly higher (2.1‐ to 2.9‐fold difference) than that in ‘Zisai Pearl’ and three flesh firmness and crispness non‐retainable hybrids  with Del323:Del323 genotype during 6 to 18 weeks of cold storage  leaves and the relative expression of GUS in MdERFpro‐Fdel323:GUS and pro‐Fdel323‐2:GUSMdERF3 indicated that both the presence of Del323 from ‘Zisai Pearl’ and truncating off the corresponding fragment from ‘Red Fuji’ destroyed the promoter activity of MdERF3  than in pro ‐F:GUSMdERF3 (del323)  staining of transient transformed tobacco  Figure .MdERF3 may bind to the GCC‐box or DRE motif on the promoter of genes such as MdPGLR3, MdPME2 and MdACO4  of ‘Red Fuji’ containing the CTTAACTG element, but did not bind to MdERF3 (Del323) of ‘Red Fuji’  co‐transformed tobacco leaves, the MdERF3 promoter activity was higher than that co‐transformed with a pro‐F:MdERF3GUS (Del323)  Figure . These rMdERF3 was then transiently overexpressed via Agrobacterium‐mediated infiltration or silenced via pTRV virus‐induced gene silencing (VIGS). Compared to that with the PRI101 empty vector transformants control, a significant increase in MdERF3 expression and distinct changes in fruit skin colour were observed in ‘Golden Delicious’ apples five days after transient transformation with PRI‐MdERF3  at −229 to −231 (Del229) upstream of the ATG codon of ’ Figure , which ds Figure . The GUS8 Figure . Y1H and4 Figure .MdRAVL1 was predicted from QTL F‐Z16.2 (Table MdERF118 (del229) but not MdERF118‐D (Del229)  Figure . ‘Goldeny Figure .N. benthamiana leaves further verified that MdERF3 interacted with MdERF118 in the nucleus  assay revealed the protein–protein interaction between MdERF3 and MdERF118 Figure . Then, as Figure .MdERF3 and MdERF118  of flesh firmness retainability of 473 hybrids and GPV of flesh crispness retainability of 394 hybrids were shown in Tables et al., et al., et al., et al., Flesh firmness retainability and crispness retainability are the critical properties affecting fruit cold storability. The heritability of flesh firmness and crispness retainability was as high as 83.85% and 83.64%, respectively, which was slightly higher than the estimate (70.0%) for firmness and crispness at harvest or after two month cold storage  and MdACO1 (MD15G1205100) did not segregate in the ‘Zisai Pearl’ × ‘Red Fuji’ population .The plant materials were 2,664 bi‐parental hybrids derived from ‘Zisai Pearl’ × ‘Red Fuji’ (1185), ‘Zisai Pearl’ × ‘Golden Delicious’ (967), and ‘Jonathan’ × ‘Golden Delicious’ (512). ‘Zisai Pearl’ is a Chinese apple cultivar that belongs to MdERF3 and MdERF118 relative expression. The fruit flesh and leaf samples were collected, immediately frozen in liquid nitrogen and stored at −80 °C until use.In 2016–2017, fruit samples were harvested at commercial maturity based on fruit background colour and starch degradation   and crispness (≥0.7 kg/cm2) was recorded as the retainability of flesh firmness and crispness, respectively  till which the apples maintained acceptable flesh firmness . One millilitre of gas was sampled from the headspace in the container using a BD syringe . The ethylene concentration of gas samples was measured with a gas chromatography (HP 5890 series II)  equipped with a flame ionization detector. The fruit ethylene production was calculated as described previously   from each phenotype type, III, II, and I of flesh firmness or crispness retainability were respectively sampled at 0, 6, 12 and 18 weeks of cold storage. Totally, 72 samples were collected. The Illumina HiSeq 2000 platform was used to generate 100‐bp paired‐end reads. Bioinformatic analysis of RNA‐seq data was performed as previously described  may affect its promoter activity, 2000‐bp promoter fragments were amplified using PCR from the genomic DNA of ‘Zisai Pearl’ and ‘Red Fuji’. After Sanger sequencing, the reporter constructs containing the promoter sequences of MdERF3 and MdERF118  were prepared as previously described  to generate the effector constructs. The reporter and effector vectors were constructed and injected into tobacco leaves. After three days infection, the transgenic leaves were dyed using the GUS reporter gene staining kit  in three biological replicates and four technical replicates, as described by Zhang et al.  was infiltrated into ‘Golden Delicious’ apple fruit (140 days after anthesis) . These included pro‐F:GUSMdERF3 (Del323), pro‐F:GUSMdERF3 (del323), pro‐Z:GUSMdERF118 (Del229), and pro‐Z:GUSMdERF118 (del229). All constructs were transiently transformed into apple calli (from hypanthium of ‘Orin’ cultivar) using the method described by Jia et al.  was ligated into the pGBKT7  binding domain (BD) vector using the EcoRI and SalI sites. The primers used are shown in Table The CDS region of MdERF3 or MdERF118 without stop codon was amplified and ligated into pSPYNE‐35S and pSPYCE‐35S vectors containing green fluorescent protein (GFP), using the one‐step seamless cloning kit (Aidlab Biotechnologies); the constructs were checked by sequencing and then transformed into A. tumefaciens strain GV3101. Two plasmids were co‐transformed into the abaxial side of 4‐ to 6‐week‐old N. benthamiana leaves to detect specific interactions as described previously . GFP signals were imaged under 488 nm excitation wavelength.The full‐length CDS of et al., et al., Three hundred hybrids were randomly selected from each F1 population of ‘Zisai Pearl’ × ‘Red Fuji’, ‘Zisai Pearl’ × ‘Golden Delicious’, and ‘Jonathan’ × ‘Golden Delicious’. All these hybrids were genotyped for the 56 markers (29 for flesh firmness retainability and 27 for flesh crispness retainability) using KASP assay. The 200‐bp sequences flanking the interested SNPs were used for KASP primer design . Asterisks denoted significance: *P < 0.05, **P < 0.01. Error bars indicate standard deviation of three biological replicates, each containing at least three technical replicates.Significant differences were analysed using the independent samples Student’s https://www.uniprot.org/ and https://www.rosaceae.org/species/malus/malus × domestica, GDR). All BSA‐seq and RNA‐seq raw data have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number PRJNA650592. The accession numbers for the genes in this study are as follow: MdPME2 (MD02G1207900), MdPGLR3 (MD03G1260700), MdACO4 (MD16G1019900), MdDOF5.3 (MD11G1123400), MdRAVL1 (MD16G1047700), MdERF3 (MD03G1194300) and MdERF118 (MD16G1043500).Sequence data were based on online databases  × ‘Red Fuji’ (F), ‘Zisai Pearl’ × ‘Golden Delicious’ (G) and ‘Jonathan’ (J) × ‘Golden Delicious’ in 2016‐1017.Figure S2 Frequency distribution of apple flesh firmness and crispness retainability of hybrids from ‘Zisai Pearl’ (Z) × ‘Red Fuji’ (F), ‘Zisai Pearl’ × ‘Golden Delicious’ (G) and ‘Jonathan’ (J) × ‘Golden Delicious’ in 2016‐1017.Figure S3 Flesh firmness and crispness of the segregant bulks at the sixths month of cold storage from hybrids of ‘Zisai Pearl’ × ‘Red Fuji’. (a) The bulks for type III, type II and type I of flesh firmness retainability. (b) The bulks for type III, type II and type I of flesh crispness retainability.Figure S4 Quantitative trait loci (QTLs) for apple flesh firmness and crispness retainability identified via BSA‐seq using hybrids from Malus asiatica Nakai. ‘Zisai Pearl’ × M. domestica Borkh. ‘Red Fuji’.  QTLs for flesh firmness retainability by pairwise comparisons between phenotype type III (FFIII) and type I (FFI) (a), type III and type II (FFII) (b), or type II and type I (c).  QTLs for flesh crispness retainability comparison between phenotype type III (FC‐III) and type I (FC‐I) (d), type II (FC‐II) and type I (e), or between type III and type II (f). The solid horizontal lines represent significant thresholds for QTLs on the maternal (red), paternal (blue) and both (black) parents.Figure S5 Overlapping of QTLs on chromosome 3 (a) and 16 (b) for flesh firmness and crispness retainability during cold storage in hybrid populations from Malus asiatica Nakai. ‘Zisai Pearl’ × M. domestica Borkh. ‘Red Fuji’. FFIII, FFII and FI represent the type III, type II and type I of flesh firmness retainability; FCIII, FCII and FCI indicate type III, type II and type I of flesh crispness retainability, respectively. The solid horizontal lines represent significant thresholds for QTLs on the maternal (red), paternal (blue) and both (black) parents. The position of candidate genes MdERF3 (MD03G1194300) and MdERF118 (MD16G1043500) were marked with red solid dots.Figure S6 Heatmaps showing differentially expressed genes of flesh firmness (a) and crispness (b) retainability detected by RNA‐seq of hybrids from Malus asiatica Nakai. ‘Zisai Pearl’ × M. domestica Borkh. ‘Red Fuji’. FFIII, FFII and FI represent type III, type II, and type I of flesh firmness changes; FCIII, FCII and FCI indicate type III, type II, and type I of flesh crispness changes, respectively. The numbers following the sample names indicate time points of sampling.Figure S7 Gene Ontology (GO) term enrichment  and KEGG pathway analysis  of the flesh firmness changes type III, type II and type IFigure S8 Gene Ontology (GO) term enrichment  and KEGG pathway analysis  of the flesh crispness changes type III, type II and type IClick here for additional data file.Table S1 Phenotype segregation of initial flesh firmness/crispness at harvest and their retainability in hybrids ‘Zisai Pearl’ × ‘Red Fuji’, ‘Zisai Pearl’ × ‘Golden Delicious’ and ‘Jonathan’ × ‘Golden Delicious’ in year 2016–2017.Table S2 QTLs of apple fruit firmness/crispness retainability in hybrids of ‘Zisai Pearl’ × ‘Red Fuji’ identified by using BSA‐seq.Table S3 The fragments per kilobase of exon per million fragments mapped (FPKM) value of differentially expressed genes by RNA‐seq.Table S4 Differentially expressed genes of flesh firmness retainability in ethylene, cell wall degradation, ABA, GA, JA, and auxin synthesis and response pathways.Table S5 Differentially expressed genes of flesh crispness retainability in ethylene, cell wall degradation, ABA, GA, JA, and auxin synthesis and response pathways.Table S6 Candidate genes predicted from QTLs for apple flesh firmness and crispness retainability.Table S7 Kompetitive allele‐specific PCR (KASP) markers for apple flesh firmness and crispness retainability designed on candidate genes within QTL intervals.Table S8 The marker genotypes, observed phenotype values (OPV) and genomics‐predicted values (GPV) of apple flesh firmness retainability in hybrids randomly chosen from populations of 'Zisai Pearl' × 'Red Fuji', 'Zisai Pearl' × 'Golden Delicious', and 'Jonathan' × 'Golden Delicious'.Table S9 The marker genotype effect estimations of apple flesh firmness retainability in 473 hybrids randomly chosen from populations of 'Zisai Pearl' × 'Red Fuji', 'Zisai Pearl' × 'Golden Delicious', and 'Jonathan' × 'Golden Delicious'.Table S10 The marker genotypes, observed phenotype values (OPV) and genomics‐predicted values (GPV) of apple flesh crispness retainability in hybrids randomly chosen from populations of 'Zisai Pearl' × 'Red Fuji', 'Zisai Pearl' × 'Golden Delicious', and 'Jonathan' × 'Golden Delicious'.Table S11 The marker genotype effect estimations of apple flesh crispness retainability in 394 hybrids randomly chosen from populations of 'Zisai Pearl' × 'Red Fuji', 'Zisai Pearl' × 'Golden Delicious', and 'Jonathan' × 'Golden Delicious'.Table S12 Pearson's correlation coefficient (r) between observed phenotype values and genomics‐predicted values of apple flesh firmness retainability in fivefold cross‐validation.Table S13 Pearson's correlation coefficient (r) between observed phenotype values and genomics‐predicted values of apple flesh crispness retainability in fivefold cross‐validation.Table S14 Simulative selection by genomics‐assisted prediction model for apple flesh firmness retainability in 473 hybrids randomly chosen from ‘Zisai Pearl’ × ‘Red Fuji’, ‘Zisai Pearl’ × ‘Golden Delicious’ and ‘Jonathan’ × ‘Golden Delicious’.Table S15 Simulative selection by genomics‐assisted prediction model for apple flesh crispness retainability in 394 hybrids randomly chosen from ‘Zisai Pearl’ × ‘Red Fuji’, ‘Zisai Pearl’ × ‘Golden Delicious’ and ‘Jonathan’ × ‘Golden Delicious’.Table S16 The primer sequences used for qRT‐PCR, molecular interaction and gene cloning.Click here for additional data file.