Background and aim: Vitamin D deficiency is frequently reported in patients with SARS-CoV-2 infection. The aim of this study was to correlate the 25OH-Vitamin D serum concentrations with clinical parameters of lung involvement, in elderly patients hospitalized for SARS-CoV-2 infection. Methods: Sixty-five consecutive COVID-19 patients (mean age 76 ± 13 years) and sixty-five sex- and age-matched control subjects (CNT) were analyzed. The following clinical parameters, including comorbidities, were collected at admission: type of pulmonary involvement, respiratory parameters , laboratory parameters . Results: Significantly lower vitamin D serum levels were found in COVID-19 patients than in CNT . Interestingly, a statistically significant positive correlation was observed between vitamin D serum levels and PaO2 (p = 0.03), SO2 (p = 0.05), PaO2/FiO2 (p = 0.02), while a statistically significant negative correlation was found between vitamin D serum levels and D-dimer (p = 0.04), C-reactive protein (p = 0.04) and percentage of O2 in a venturi mask (p = 0.04). A negative correlation was also observed between vitamin D serum levels and severity of radiologic pulmonary involvement, evaluated by computed tomography: in particular, vitamin D was found significantly lower in COVID-19 patients with either multiple lung consolidations (p = 0.0001) or diffuse/severe interstitial lung involvement than in those with mild involvement (p = 0.05). Finally, significantly lower vitamin D serum levels were found in the elderly COVID-19 patients who died during hospitalization, compared to those who survived . Conclusions: This study confirms that 25OH-vitamin D serum deficiency is associated with more severe lung involvement, longer disease duration and risk of death, in elderly COVID-19 patients. The detection of low vitamin D levels also in younger COVID-19 patients with less comorbidities further suggests vitamin D deficiency as crucial risk factor at any age. Vitamin D plays a central role in bone metabolism, as well as in immune function. Concerning the latter, it regulates the growth and differentiation of several cell types involved in the immune reactivity, displaying anti-inflammatory and immunoregulatory properties ,2,3. TheVitamin D serum concentrations are usually lower in elderly people and have been inversely associated with autoimmune inflammatory disease activity and severity, as well as with risk of pulmonary infections ,6. RecenBy considering the higher prevalence of COVID-19 in older people, the aim of this study was to correlate, in elderly patients hospitalized for SARS-CoV-2 infection, the 25OH-Vitamin D (25OHD) serum concentrations with clinical parameters of lung involvement.Sixty-five consecutive COVID-19 patients  and sixty-five sex- and age-matched non-COVID control subjects (CNT) were enrolled in the early months of the recent pandemic (March–April 2020) and retrospectively analyzed. All patients needed hospitalization in the COVID-19 dedicated medical unit and were on oxygen therapy for severe respiratory failure due to SARS-CoV-2 infection, but none needed intensive care treatment or intubation at entry. COVID-19 was confirmed in all patients by positive real-time reverse transcriptase polymerase chain reaction on nasal swabs during hospitalization. All patients and controls were Caucasian coming from the same region of the country. Control subjects were outpatients attending the Rheumatology Clinic in the same period. The study was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice and all patient data were collected during the routine medical visits. All the patients gave written informed consent to manage their clinical data and the study was approved by Regional Ethics Committee .The lung clinical parameters, along with comorbidities and laboratory parameters, were collected at admission. The type of pulmonary involvement  was assessed by computed tomography using a high-resolution CT imaging scanner .2, SO2, PaCO2) were measured by arterial blood gas analysis, and the ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2) was calculated to assess the alveolar injury and severity of hypoxemia. Blood samples collected from the radial artery were analyzed by ABL90 FLEX blood gas analyzer .Respiratory parameters  were assayed by routine standardized assessments in the hospital laboratories, and blood samples were taken at 8 a.m. on the first day of hospitalization. In particular, 25OHD serum levels were measured by standardized chemiluminescence , and the reported intra and inter assay coefficients of variation (%CV) were 5.4 and 10.6 respectively. Vitamin D serum levels were classified as sufficient, insufficient, deficient, or severely deficient, as suggested by the Endocrine Society guidelines .Duration of hospitalization and global duration of COVID-19 from first symptom until recovery were also recorded. p values equal or lower than 0.05 was considered statistically significant; correlation coefficients (r) are also reported throughout the manuscript. The results are reported as median along with interquartile range (IQR).Statistical analysis was carried out by StatView software. The Mann–Whitney U test was used to compare unpaired groups of variables, and the Kruskal–Wallis test to compare continuous variables with nominal variables with more than two levels. Possible associations between variables were assessed by Spearman rank correlation, simple, and multiple regression tests. Multiple linear regression models to estimate the associations between 25OHD and outcome measures were performed also after log-transformation of variables, while adjusting for possible confounders. The one-sample Kolmogorov–Smirnov test was used to check the normality assumption of the linear model residuals. Chi-square test was used to compare categorical variables. Any Clinical characteristics, comorbidities and baseline clinical parameters of enrolled patients are reported in p= 0.001)  see .Among COVID-19 patients, vitamin D sufficiency (>30 ng/mL), insufficiency (between 20 and 30 ng/mL), deficiency (between 10 and 20 ng/mL), and severe deficiency (<10 ng/mL) were observed respectively in 11, 11, 21, and 57% of patients. In CNT, the same vitamin D distribution occurred in 21, 21, 36, and 22% of subjects, respectively .p = 0.046)  . COVID-12 , SO2 , PaO2/FiO2 , while a statistically significant negative correlation was found between vitamin D serum levels and percentage of O2 in Venturi Mask   . No corr2/FiO2, SO2, and PaO2 while adjusting for age, sex, and number of comorbidities. Since the vitamin D levels were not normally distributed, we modelled the PaO2/FiO2 ratio with logarithmic transformation and used the log-transformed 25OHD levels as explanatory variable with age, sex and comorbidities as adjustments. The one-sample Kolmogorov–Smirnov test was used to check the normality assumption of the linear model residuals, and the multiple linear regression results are reported in Furthermore, we fitted three multiple linear regression models to estimate the associations between 25OHD and PaO2/FiO2 ratio and PaO2 was confirmed. The models show about 17% increase in PaO2/FiO2 every 1% increase of 25OHD, regardless of age, sex, and comorbidities , as well as for every 1% increase of 25OHD levels, PaO2 increases about 5 mmHg. No statistically significant association was detected between SO2 and 25OHD after adjustments.A statistically significant association between vitamin D serum levels and both PaOp = 0.0001) or diffuse/severe interstitial lung involvement (ground glass pattern) (p = 0.05) than in those with mild interstitial lung involvement. A negative association was found between vitamin D serum levels and severity of radiologic pulmonary involvement, as evaluated by CT: in particular, vitamin D was found significantly lower in COVID-19 patients with either multiple lung consolidations , C-reactive protein , and parathyroid hormone  . No correlation was observed between vitamin D serum levels and ferritin, LDH, blood cell count, hemoglobin, creatinine, and transaminases. In addition, vitamin D serum levels negatively correlated also with D-dimer ( = 0.05) , while s2/FiO2 ratio (dependent variable) showed vitamin D the only independent variable able to significantly interfere with this clinical parameter . Of note, the multiple regression analysis developed to identify possible effects of blood serum parameters  on PaOr = −0.49, p = 0.0009), but this correlation was not observed in COVID-19 patients (p = 0.77).A negative correlation was also observed between vitamin D serum levels and age of patients in CNT as expected   .Multiple linear regressions using age, sex, and number of comorbidities (with vitamin D as the dependent variable) did not identify statistically significant effects of the independent variables on 25OHD level in COVID-19 patients. Present investigation underlines that serum vitamin D deficiency might influence the severity of lung involvement and the risk of deaths, in elderly COVID-19 patients hospitalized for SARS-CoV-2 infection.2-D] that as steroid hormone is involved in the regulation of growth and differentiation of various immune cell types [The results are likely linked to the role played by the biologically active metabolite of vitamin D [1,25(OH)ll types ,5,10.On this basis, the association between vitamin D deficiency and an increased incidence and higher activity of several autoimmune diseases  has been already demonstrated by several reports ,13,14,15On the other hand, low vitamin D levels have been associated with the increased risk of respiratory tract infections and pneumonia, whereas a two-fold increased risk of tuberculosis was observed in patients with vitamin D deficiency, with vitamin D levels predicting the tubercular disease risk in a serum concentration-dependent manner ,17,18,19In addition, vitamin D deficiency was found associated to longer acute respiratory infection course, while a recent meta-analysis showed that vitamin D supplementation could prevent respiratory infections ,21.Recently, vitamin D deficiency was associated with SARS-CoV-2 infection, in terms of higher risk of disease development, higher disease severity, higher frequency of intensive care unit hospitalization, higher risk of death, thus interfering with the prognosis of COVID-19 ,24,25,26SARS-CoV-2 may induce a severe acute respiratory distress syndrome (ARDS) requiring hospitalization, whose main risk factors, besides vitamin D deficiency, are old age, male sex, obesity, hypertension, diabetes, low ambient temperature, and high geographic latitude ,27. In particular, ageing correlates with progressively lower vitamin D serum levels, as known ,29. In oVitamin D deficiency (<20 ng/mL) was also found more frequent in COVID-19 (78% of patients) than in sex- and age-matched controls (57% of subjects). Even if the comparison of vitamin D serum levels detected into different studies is quite difficult, due to different cohorts of enrolled patients and various comorbidities that may influence the levels itself, as well as different methodologies, disease severity and age, the results of our study are quite similar to those of a recent study reporting vitamin D serum levels < 50 nmol/L (<20 ng/mL) in 61% of hospitalized patients (mean age 76 years) . In our The same authors observed a significantly greater prevalence of vitamin D deficiency (<50 nmol/L) in patients requiring intensive care treatment than in those without (81% of patients) . SimilarA recent systematic review analyzed seven studies on COVID-19 severity, intensive care treatment, and mortality (1368 patients were included) and detected a mean vitamin D level of 22.9 nmol/L (9.16 ng/mL), higher but similar to that of our cohort of patients (7.9 ng/mL) [8). Patients with good prognosis had significantly higher vitamin D levels compared with those with poor prognosis [2/FiO2 ratio that is commonly used for the diagnosis of alveolar injury (transfusion-related acute lung injury and acute respiratory distress syndrome) and for the assessment of pulmonary disease course and therapy. Practically, the ratio PaO2/FiO2 measures the severity of hypoxemia in patients with ARDS and has been included into the consensus definition of ARDS itself [Among clinical parameters, we included in our study the PaOS itself ,31.2/FiO2 ratio was detected as an independent risk factor for death in COVID-19 patients [2/FiO2 values . This observation is in line with the results of another study reporting a high prevalence of hypovitaminosis D in COVID-19 patients with low PaO2/FiO2 ratio [A low PaOpatients . Our stuO2 ratio .2-D plays an anti-viral role, regulating the inflammatory response by modulating toll-like receptor expression and NK cell function, and suppressing over-expression of pro-inflammatory cytokines [2-D enhances the defense also by inducing antimicrobial peptide release, like cathelicidin that lead to viral destruction and clearance and facilitates the recruitment of monocytes, macrophages, neutrophils and dendritic cells. Therefore, 1,25(OH)2-D may regulate the innate/adaptive responses and may interfere with the maturation of dendritic cells and their ability to present antigen to T-cells, shifting the T cell profile from the pro-inflammatory Th1 and Th17 subsets to Th2 and Treg subsets, thus inhibiting the pro-inflammatory processes [How vitamin D interferes with COVID-19 progression is not completely understood. The innate immune system represents the first line of defense against viruses depending on constitutive expression of pattern recognition receptors, like toll-like receptors. 1,25(OH)ytokines . 1,25 of ACE2 ,37,38,39 of ACE2 ,41,42,432-D induces type II pneumocyte proliferation and surfactant synthesis in the lungs [In the lung, several alveolar cell types express the ACE2 receptor. These cells play an important role in producing surfactant, able to regulate the alveolar surface tension. SARS-CoV-2 can infect the alveolar cells by ACE2 binding and suppress the production of surfactant. The loss of alveolar cells results in lung damage and respiratory insufficiency due to the loss of pulmonary surfactant . This dahe lungs ,46,47. Ohe lungs . Intereshe lungs . As knowhe lungs ,51. Thishe lungs . High D-he lungs . Our stuhe lungs . Finally, it must be said that hypovitaminosis D has been hypothesized to be the consequence, rather than the cause, of chronic inflammatory diseases, as well as current data are not yet sufficient to demonstrate a definite role for vitamin D in the modulation of adaptive immune system in humans ,56. HoweThis study has some limitations.The number of patients analyzed in this retrospective cross-sectional study is small, and included only elderly patients needing hospitalization and oxygen therapy due to severe respiratory failure in SARS-CoV-2 infection, but it did not include either patients with mild disease nor patients needing intensive care treatment or ventilation. Furthermore, due to small number of enrolled patients and large data variability, the sample size was not adequately powered to rule out the variation expected in the main outcome parameters: for this reason the correlation coefficients are relatively small. Therefore, robustly designed randomized clinical trials including a larger number of patients are needed, but are likely to further confirm these preliminary data. Enrolled patients did not perform ventilation/perfusion lung scan along with contrast enhanced computed tomography to investigate pulmonary microembolism/thrombosis, and this does not permit to surely associate the D-dimer abnormalities with the clinical condition. However, pulmonary embolism has been described in up to 37% of COVID-19 patients by radiological examinations and in 79% of patients after autopsy ,60. COVID-19 patients showed several different comorbidities that might interfere with some results of the study, however also sex- and age-matched healthy subjects had similar comorbidities, as reported in Several other studies have also linked low levels of serum vitamin D to SARS-CoV-2 infection . HoweverIt is already known that vitamin D serum level is usually low in patients with chronic inflammation or infections (including SARS-CoV-2), but this was previously demonstrated in younger people. This study reports and confirms similar findings also in older COVID-19 patients.Vitamin D deficiency is a risk factor for infections as well as for insufficient innate immune system reactivity, therefore the winter time testing for serum vitamin D status (lowest levels expected) should be mandatory as part of regular health check status. The test is even more mandatory in patients with chronic diseases, where we expect vitamin D deficiency during all the year and an increased burden of risk factors for further disease severity and even mortality. Deficiency must be treated with vitamin D administration until reaching the optimal range of 40–60 ng/mL during all the year.In conclusion, this study confirms that vitamin D deficiency is associated with more severe lung involvement, longer disease duration, and risk of death in elderly COVID-19 patients.


FLOWERING LOCUS T1 (FT1) and FLOWERING LOCUS T2 (FT2) were significantly enriched. Then, as a confirmatory experiment, the potted trees were uniformly sprayed with MV, N,N’-dimethylthiourea  plus MV, and water at medium-temperature. The results showed that the MV-generated ROS promoted flowering and changed related gene expression, but these effects were repressed by DMTU treatment. The results of our studies indicate that ROS could promote flowering and partly bypass chilling for litchi flowering.Litchi is an important subtropical fruit tree that requires an appropriately low temperature to trigger floral initiation. Our previous studies have shown that reactive oxygen species (ROS) are involved in litchi flowering. To identify oxidative stress-induced flowering related genes in leaves, ‘Nuomici’ potted trees were grown at medium low-temperature conditions . The trees were treated with the ROS generator methyl viologen dichloride hydrate (MV) as the MV-generated ROS treatment  and water as the control treatment . Sixteen RNA-sequencing libraries were constructed, and each library generated more than 5,000,000 clean reads. A total of 517 differentially expressed genes (DEGs) were obtained. Among those DEGs, plant hormone biosynthesis and signal transduction genes, ROS-specific transcription factors, such as AP2/ERF and WRKY genes, stress response genes, and flowering-related genes  Litchi is an evergreen fruit tree commercially cultivated in southern Asia, South Africa, and Australia. Floral initiation in litchi is triggered by low temperatures and enhanced by drought in autumn and winter ,2,3. LitWe have previously shown that reactive oxygen species (ROS) promoted flowering and decreased the chilling requirement for flowering  and obtaLitchi chinenesis cv. Nuomici) were used in this study. Twenty potted trees (1–1.5 m height) with leaves of the terminal shoots at the same maturity stage were selected. All trees were cultivated in the experimental orchard of South China Agricultural University, Guangzhou, China  and were grown in 30 L pots with loam, mushroom cinder and coconut chaff . Trees were transferred to a growth chamber at 18 °C/13 °C  with a relative humidity of 75%–85% and natural light. Ten trees were sprayed with 60 µM MV according to our previous studies [Five-year-old air-laying potted trees . Five replicated trees were sprayed with 40 µM MV as the medium-temperature plus MV treatment (MM). The other five trees were initially sprayed with 2 mM of N,N’-dimethylthiourea (DMTU) and then sprayed with 40 µM MV 12 h later as the medium-temperature plus DMTU and MV treatment (MMD). The remaining five trees were sprayed with water as the medium-temperature control (M). All the dosages for the chemicals were in accordance with our previous studies . Trees whttps://bigd.big.ac.cn/gsa.Total RNA was extracted using the RNAprep Pure Plant Kit (Polysaccharides and Polyphenolics-rich) . For RNA-Seq library construction, 5 μg of total RNA with an appropriate amount of Oligo-dT25 beads  was used. The mRNA was fragmented into short fragments and reverse transcribed into cDNA by random primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H and dNTPs. After that step, the cDNA fragments were purified by Agencourt AMPure XP . The purified cDNA fragments were end-repaired, modified with poly (A) tails, and ligated to Illumina sequencing adapters. The size-selected fragments were amplified and purified. Sixteen libraries were sequenced withIllumina HiSeq™ 2500 . The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive  in the Bhttps://pachterlab.github.io/eXpress/index.html). Differentially expressed genes (DEGs) were determined using OmicShare tools . The R package is edgeR (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html), and significant DEGs were restricted with false discovery rate (FDR) ≤ 0.05 and the absolute value of fold-change ≥ 2.Adapters were cut from raw reads, and the low-quality sequences were removed. rRNA sequences were filtered against ribosomal RNA using Bowtie2 (version 2.1.0). Then, cleaned reads were mapped to the litchi genome using HISAT2 (v2.1.0). The read counts and fragments per kilobase of transcript per million mapped reads (FPKM) of each gene were calculated using eXpress  (Tiangen Biotech). First-strand cDNA was synthesized from 1 μg of extracted total RNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen Biotech). Quantitative real-time RT-PCR (qRT-PCR) primers F/R  were desT method .According to the classification described by You et al. and Sudawan et al. ,16, DEGst-test and Duncan’s multiple range test. The expression of candidate genes was presented as a heat map diagram using the pheatmap package (http://www.r-project.org/) or a line chart using Origin .Data were analyzed by SPSS  using Student’s In our previous study, we found that after a period of low-temperature  treatment, ‘Nuomici’ litchi trees were induced to flower in 100% of cases, whereas under medium-temperature  conditions, litchi trees produced poor flowers in 20% of cases. However, when trees at medium-temperature were sprayed with an ROS donor MV, they were induced to flower in 80% of the trees . To furt6 raw reads and obtained 4.95–11.55 × 106 clean reads from the libraries with clean read ratios of more than 88%. We then mapped the clean reads to the litchi genome data, and the alignment rates were all above 85%.To identify genes potentially related to oxidative stress-induced flowering signals in leaves, we collected leaves from the ROS-treated and water-treated trees at 3 d, 33 d, 63 d, and 78 d of medium-temperature treatment. We constructed 16 RNA-Seq libraries for the four time points for the MM-treated and M-treated leaves. As shown in We remapped the unique match reads from the 16 libraries to the litchi genome data and normalized the unigene reads to FPKM values. Correlation analysis of the replicates of the two treatments (M and MM) and four time points  showed high repeatability of the sequencing samples . Then weFT1 (Litchi_GLEAN_10002963), FT2 (Litchi_GLEAN_10003608), SUPPRESSOR OF OVEREXPRESSION OF CO1 , and FLOWERING LOCUS D , were only found at the later time points. AP2C1 (Litchi_GLEAN_10024139), a protein phosphatase-encoding gene, and putative mitochondrial RNA helicase 2 , a helicase-encoding gene, were differentially expressed at the early and late time points.From the 517 DEGs, we identified those related to ROS signaling, stress response, and flowering-related genes. As a result, we found 48 DEGs at the 3 d time point, representing the early ROS responsive genes, and 60 DEGs at the 33, 63, and 78 d time points, representing the late ROS responsive genes. The DEGs were classified into 11 groups. These genes were ROS-scavenging-related, abscisic acid-related, brassinosteroid-related, calcium-associated, flowering-related, and other stress-responsive genes, as well as helicase, molecular chaperone, protein phosphatase, protein kinase, and transcriptional factor encoding genes . Interes2 = 0.8918), suggesting that the transcriptome analysis by RNA-Seq is reliable.To confirm the accuracy of the transcriptome analysis results, we randomly selected 10 genes for real-time quantitative reverse transcription-PCR (qRT-PCR) confirmation. As shown in LcFT1, LcFT2, Ferritin 1 , 18.2 kDa Heat Shock Protein , Metallothionein 3 , Cyclin-dependent kinase D1 , Cold, circadian rhythm, and rna binding 2 , Chaperone-domain superfamily protein dnaJ 11 , Glutathione S-transferase 6 , CBL-interacting protein kinase 12   . We compCIPK12, AP2C1, calcium-binding protein CML45 (Litchi_GLEAN_10022624), ERF domain protein 9 , microsomal glutathione S-transferase 3 , PMH2, DnaJ11 (Litchi_GLEAN_10044599), nine-cis-epoxycarotenoid dioxygenase 3 , disease resistance family protein (Litchi_GLEAN_10036444), and brassinosteroid-insensitive 4  encoding genes showed high connectivity in each of the 11 categories. The protein kinase genes LcCIPK12 and LcCDKD;1 showed a low degree (4 and 3) but high connectivity (15.5 and 12). Among the three flowering related genes, LcFT2 had the highest connectivity, which was connected with four C2H2 zinc finger-type transcription factor (TF)-encoding genes. LcFLD was associated with LcDnaJ, LcMT3, and LcPP2C39. LcFT1 was related to LcHSP18.2 . Ninety-three DEGs were selected among the 517 DEGs. As shown in cHSP18.2 .Stress is defined as a situation in which the vegetative growth of plants is suppressed . FloweriIn this study, we focused on the leaves from which flowering signals can be produced. ROS treatment and control at four time points  under medium-temperature conditions were carried out. We constructed 16 RNA-Seq libraries. After mapping to the litchi genome database, we found that the alignment rate of the 16 libraries was above 85%, indicating the excellent quality of the libraries. A total of 517 DEGs were obtained, among which 175 DEGs were from the 3 d time point, and 358 DEGs were from later time points. By selecting those related to ROS signaling, stress responsive and flowering-related genes, 48 out of 175 DEGs (27.27%) at 3 d of treatment, and 60 out of 358 DEGs (16.76%) at 33, 63, and 78 d of treatment were identified. Interestingly, more transcription factor-encoding genes were found at the 3 d time point, the early stage, while flowering-related genes were found at later time points. The results suggested that TFs might play roles in the early response of ROS. It might be that the early responsive TFs trigger floral induction activity and then initiate the expression of flowering related genes.LcFT1, LcFT2, LcFER1, LcMT3, LcCDKD;1, LcCCR2, LcDnaJ11 and LcGST6. The genes might be related to ROS-promoted flowering, for example, LcFT1 and LcFT2.To further confirm the effects of ROS on the expression of the candidate genes, we used an ROS scavenger DMTU to trap ROS ,19. In aLcFT1 and LcFT2 are proven to play critical roles in flowering, and overexpression of the two genes could promote flowering [LcFT1 is also involved in drought-enhanced flowering [LcFT1 and LcFT2 was induced by ROS treatment, but the induction was trapped by the ROS scavenger DMTU, suggesting that they might play crucial roles in ROS-promoted flowering. In litchi, lowering . LcFT1 ilowering . In the FER1 plays an essential role in floral development and fertility. The ferritin-lacking triple mutant fer1-3-4 shows that the loss of ferritins reduces growth and impairs flower development [LcFER1 was induced by ROS treatment, but the induction was repressed by the ROS scavenger DMTU, suggesting that it might be involved in ROS-promoted flowering in litchi. To date, we have not obtained any overexpression information regarding LcFER1, and further functional studies on LcFER1 should be carried out. In Arabidopsis, elopment . Our resLcCCR2 is the homolog of AtGRP7. In Arabidopsis, AtGRP7 promotes flowering, its mutants and AtGRP7i plants are late flowering, and the transcript levels of FLC are affectced, which reveals the involvement of vernalization and autonomous pathways [AtGRP7 in rice (Oryza sativa) under drought stress conditions showed higher recovery rates, and grain yields compared to the control wild-type plants [AtMT2a and AtMT3 overexpressed in Vicia faba guard cells could increase resistance to cadmium [GhMT3a could function as an effective ROS scavenger in cotton, transgenic tobacco, and yeast, and its expression was induced by various abiotic stresses through ROS signaling. In rice, OsMT-3a plays a crucial role in salinity and heavy metal tolerance via ROS scavenging [LcCCR2 and LcMT2 was induced by ROS treatment at the early stage of floral induction, but the induction was trapped by the ROS scavenger DMTU, suggesting that they might be involved in ROS-promoted flowering at an early stage of floral induction. At present, we do not know whether overexpression of LcCCR2 or LcMT2 can promote flowering. Further functional studies on LcMT2 should be carried out. Similarly, other candidate genes, such as LcCDKD;1, LcCCR2, LcDnaJ11 and LcGST6, should be subjected to functional studies.pathways . Interese plants . Metalloe plants . AtMT2a  cadmium . Accordi cadmium , GhMT3a avenging . In our LcCIPK12 and LcCDKD;1 showed low degree but high connectivity, mostly because of the higher weight value compared to protein phosphatases, calcium-associated genes, and the other genes. LcFT2 among the flowering genes had the highest connectivity, which was connected with four C2H2 zinc finger-type TF-encoding genes. These results suggested that the protein kinase genes LcCIPK12 and LcCDKD;1, and the flowering-related gene LcFT2 may play central roles in ROS-promoted flowering in litchi.For further studies of the interaction between the ROS responsive genes, we established a co-expression network and divided those 11 groups into four categories according to the classification method from You et al. and Sudawan et al. ,16. InteFT1 and FT2, were significantly enriched. A confirmatory experiment in which potted trees were uniformly sprayed with MV, N,N’-dimethylthiourea  plus MV, and water at medium-temperature was carried out. The results showed that the MV-generated ROS promoted flowering and changed the expression of related genes, but these effects were repressed by DMTU. We constructed 16 RNA-Seq libraries of ROS treatment and control samples at four time points  under medium-temperature conditions, and 517 DEGs were obtained. Among those DEGs, plant hormone biosynthesis and signal induction genes, ROS-specific transcription factors, such as AP2/ERF and WRKY genes, stress response genes, and flowering-related genes Our previous studies have shown that the ROS promoted flowering and decreased the chilling requirement for flowering at the ecological and anatomical levels and obtained an inner network of chilling- and ROS-promoted flowering in the apical meristem of litchi. The studies revealed that ROS could promote flowering and partly bypass chilling requirements for litchi flowering, suggesting a possible litchi flowering promotion method for addressing climate change and global warming.


The Ca2+-conducting transporters located on the endoplasmic reticulum (ER) membrane shoulder the responsibility of constructing the global Ca2+ signaling landscape. These transporters gate the ER Ca2+ release and uptake, sculpt signaling duration and intensity, and compose the Ca2+ signaling rhythm to accommodate a plethora of biological activities. In this review, we explore the mechanisms of activation and functional regulation of ER Ca2+ transporters in the establishment of Ca2+ homeostasis. We also contextualize the aberrant alterations of these transporters in carcinogenesis, presenting Ca2+-based therapeutic interventions as a means to tackle malignancies.Ca Ho. Ho2+ rear space . Importa2+ release into the cytosol begins with signals generated at the plasma membrane. Stimulation of G-protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) activates phospholipase C (PLC) beta and gamma, respectively. PLC then hydrolyzes the plasma membrane-enriched phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3)  on the ER membrane, causing them to open, ultimately leading to the release of ER Ca2+ into the cytosol (2+ transporters.ER Cate (IP3) A. The ne cytosol B 20]. T. T2+ rel3R is a macroscopic (~1.3 MDa), six-pass transmembrane ER Ca2+-release transporter, collectively functioning as a homo- or hetero-tetrameric assembly . I. I3R is isoforms . Using gmembrane ,36. Uponct sites ,38,39. P sensing ,41. Furtgramming ,43. Morefficking ,45. In skinetics . Howeverlic Ca2+ ,48. Intrresponse ,50. On tansients ,52. This2+ release via CICR [2+] ranging from 100–200 nM. Once the rising cytosolic [Ca2+] reaches a certain threshold, it begins to act on the RyRs, triggering the opening of the homo-tetrameric channels. Extensively characterized in excitable tissues, RyRs exhibit optimal opening probability at sub-micromolar cytosolic Ca2+ concentration  gradient between the ER and cytosolic compartments . The dynamic orchestration of Ca2+ uptake and release contributes to the maintenance of ER Ca2+ homeostasis, protecting the ionic integrity of the ER for cellular survival and physiological functions.To prevent ER Cad spleen ,92. Struembranes ,94,95. T of BI-1 . In addimembrane . Mechani2+ transporters are manifested clinically in a diverse array of human cancers. As tumor pathogenesis varies with each malignancy, it is important to be aware of the highly context-dependent nature of the methods through which cancer cells hijack the ER Ca2+ signaling. The rest of this review will cover some of the mechanisms employed by cancer cells to sabotage ER Ca2+ signaling and the current therapeutic strategies being investigated as potential treatments for cancer patients.The pitfalls of abnormal levels of activity of the ER Ca3Rs to emerge as crucial determinants of cell fate  via SERCA over-expression, rapidly proliferating cancer cells strategically endure and alleviate cytotoxic stress associated with their hyperactive protein synthesis and folding machineries [2+ re-uptake to sustain their specific oncogenic needs. Hence, it is within reason that different SERCA isoforms demonstrate varying expression kinetics throughout distinct stages of tumorigenicity [SERCA activity represents a nodal point of cellular survival and has been extensively exploited in carcinogenesis . The expn cancer ,163,164.pillomas . Furtherpillomas ,167. An survival ,170,171.+ influx ,173. Sim release . Nonethe release ,176. Of nduction . Importa+ uptake . Intrigu80 cells . Moreoveferation . In compferation . Furtherferation . Mechanihineries . Since chineries . The purgenicity .2+ signaling is indispensable for cell development, movement, metabolism, survival, and signal transduction, this, therefore, poses a challenge for a specific and efficacious Ca2+-based drug design. Similar to the “undruggable” Ras and MAPK, targeting ER Ca2+ alone seems impractical due to its ubiquitous presence and integral contribution to cellular physiology [2+ at the levels of channels/transporters/pumps and downstream effector molecules that decipher Ca2+-encoded messages seems to be more feasible [3R channel activity alone as an isolated molecular entity, research is now directed towards gaining collective understandings of the fate-determining pathways following IP3R -mediated Ca2+ release, such as the IP3R-VDAC1-MCU-signaling axis bridging ER Ca2+ release and mitochondrial Ca2+ uptake [2+ signals, inhibiting tumor cell survival [2+ signaling, however, we will briefly describe the use of ER Ca2+ transporter-based chemical drug conjugate, computational pharmacology and extrapolate the immunotherapeutic potential of CRAC channels in the design of novel anti-neoplastic therapy.As ER Caysiology . Howeverfeasible . Rather + uptake . Indeed,survival . It is n2+ through prolonged inhibition of SERCA activity. This depletion of the ER Ca2+ store, in and of itself, induces ER stress, and causes elevated cytoplasmic Ca2+ that can activate intrinsic apoptotic pathways through calmodulin/calcineurin-mediated signal transduction [2+ signaling and generating ER stress to induce apoptosis.Conjugating tumor-specific marker with seemingly unlikely drug target offers new hope in drug delivery. For instance, thapsigargin (TG) is widely used in research laboratories to deplete ER Casduction . Despitesduction . Recognisduction . This pr2+ binding kinetics during SOCE. Found up-regulated in glioblastoma multiforme (GBM), STIM1 and Orai1 are positively associated with GBM invasiveness [2+ binding to the active sites of Orai1, inhibiting ORAI-mediated Ca2+ influx with relatively ideal pharmacokinetics [As an alternative investigative tool, computational pharmacology has been utilized to explore Casiveness . Throughkinetics . However2+ transients required for the proliferation, migration, recruitment of T lymphocytes as well as the subsequent degranulation of lytic vesicles into the tumor cell, CRAC channel activity is key to the initiation and maintenance of a potent anti-tumor immune response [Another interesting area for clinical implementation of CRAC-channel based drug design is immunotherapy. Within a tumor mass, the tumor microenvironment (TME) is produced by the functional crosstalk among miscellaneous cell types, such as the cytotoxic T-lymphocytes (CTLs), B-lymphocytes, Natural Killer (NK) cells, tumor-associated macrophages (TAMs), tumor-associated neutrophils, regulatory T cells, pericytes, vascular endothelial cells, and cancer-associated fibroblasts ,196,197.response ,199,200.response . However2+ signaling for cancer therapy are the different facets of drug design. What are the precise pharmacophore and mechanisms of action? Are there any non-specific interactions with other drug molecules, targets, or enzymes? What are the pharmacokinetic properties, namely absorption, distribution, metabolism, and excretion associated with the drug? Are there adverse side effects? How to reconcile and fit thermodynamic stability, drug bioavailability, and solubility all into the diverse pharmacogenomics of the patients? Nonetheless, we are entering an exciting era of biomedical research where basic mechanistic understandings of ER Ca2+ and its homeostatic regulation are vigorously pursued for the development of new anti-cancer therapies.Adding to the complexity of targeting ER Ca2+ transporters and their aberrant functional alterations in cancer. We described the homeostatic regulation of ER Ca2+ store and its connection with the global Ca2+ signaling transduction network. We also appreciated the many ways ER Ca2+ signaling manifests itself through its receptor distribution, isoform expression, downstream effector landscape and how these processes could be hijacked in malignancies. We examined the dynamic modulation of these transporters in the context of organellar crosstalk as well as endogenous regulation by oncoproteins and tumor suppressors. We then culminated the review with pharmacological interventions of ER Ca2+ transporters. As future design of anti-cancer therapy continues, it is awe-inspiring to reflect on the width and depth of Ca2+ signaling as its regulatory networks have evolved since early prokaryotic life. Without a doubt, learning to comprehend and communicate in a beautiful yet universal language spoken by both prokaryotic and eukaryotic lives through the intricate flow of Ca2+ across cellular compartments is a useful and powerful way in combating cancer and more.In this review, we have summarized the major ER Ca


Prunus pedunculata Pall, the deciduous shrub of Amygdalus subgenus in Rosaceae, is a new kind of desert oil-bearing tree. It has a long story of being planted in the West and North of China for sand fixation and desert control. In addition, the seeds of P. pedunculata are rich of oil, especially the monounsaturated fatty acid and polyunsaturated fatty acid. However, little is known about the molecular mechanisms of oil accumulation during the seed development of P. pedunculata.P. pedunculata from three independent plants at 10, 18, 24, 31, 39, 45, 59 and 73 days after flowering (DAF) were obtained and the oil compositions were evaluated. It showed that oleic acid was the dominant type of oil content in the mature seeds (from 32.724% at 10DAF to 72.06% at 73DAF). Next, transcriptome sequencing for the developing seeds produced 988.795 million high quality reads and TRINITY assembled 326,271 genes for the first transcriptome for P. pedunculata. After the assembled transcriptome was evaluated by BUSCO with 85.9% completeness, we identified 195,342, 109,850 and 121,897 P. pedunculata genes aligned to NR, GO and KEGG pathway databases, respectively. Then, we predicted 23,229 likely proteins from the assembled transcriptome and identified 1917 signal peptides and 5512 transmembrane related proteins. In the developing seeds we detected 91,362 genes (average FPKM > 5) and correlation analysis indicated three possible development stages – early (10 ~ 24DAF), middle (31 ~ 45DAF) and late (59 ~ 73DAF). We next analyzed the differentially expressed genes (DEGs) in the developing seeds. Interestingly, compared to 10DAF the number of DEGs was increased from 4406 in 18DAF to 27,623 in 73DAF. Based on the gene annotation, we identified 753, 33, 8 and 645 DEGs related to the fatty acid biosynthesis, lipid biosynthesis, oil body and transcription factors. Notably, GPAT, DGD1, LACS2, UBC and RINO were highly expressed at the early development stage, ω6-FAD, SAD, ACP, ACCA and AHG1 were highly expressed at the middle development stage, and LACS6, DGD1, ACAT1, AGPAT, WSD1, EGY2 and oleosin genes were highly expressed at the late development stage.The seeds of P. pedunculata and our findings will provide a valuable resource for future studies. More importantly, it will improve our understanding of molecular mechanisms of oil accumulation in P. pedunculata.This is the first time to study the developing seed transcriptome of The online version contains supplementary material available at 10.1186/s12870-021-02921-x. Prunus pedunculata Pall (P. pedunculata), belongs to the deciduous shrub of Amygdalus subgenus in Rosaceae, is a new kind of desert oil-bearing tree, which is also known as wild cherry, almond with stem and hairy cherry  9-desaturase 6), ACP (acyl carrier protein 1), ACCA , LACS (long chain acyl-CoA synthetase), ACAT1 , DGD1 , GPAT (glycerol-3-phosphate acyltransferase), AGPAT (1-acyl-sn-glycerol-3-phosphate acyltransferase 1), AHG1 (Probable protein phosphatase 2C 75) and UBC (ubiquitin-conjugating enzyme) during the seed development. According to their expression patterns during the seed development, these genes can be divided into three groups. The first group of DEGs consisted of FAD, SAD6, ACCA and ACP, which peaked at the middle stage of the seed development of P. pedunculata  peaked at 31DAF  were overexpressed at late stage of the seed development. Like LACS genes, UBC1/UBC6 and UBC28 were overexpressed at early and late stages, respectively  were highly expressed at the early stage and gene encoding TF-B3 domain-containing protein peaked at 45DAF  in the seeds of ta Table  and we ita Table . Interes4 in the P. pedunculata seeds and 645 were differentially expressed during the seed development . As a result, we identified 450, 133, 6, 743 and 103 genes from blue, brown, grey, turquoise and yellow modules, respectively , ACOX4, LACS6, oleosin, O-acyltransferase WSD1-like and various TFs . Interestingly, the other fatty acid types, including palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid and linolenic acid, were found to be associated with the turquoise module genes , 3.1.2.14 , 3.1.2.21 , 1.14.19.2 , 6.2.1.3 , FabD, FabF, FabH, FabI, FabG and FabZ. Their products are either substrates, intermediates or important enzymes. Next, we proposed the working model of fatty acid synthesis in plastid and endoplasmic reticulum , involved in the overall conversion of pyruvate to acetyl-CoA, was found to be highly expressed in the seeds of 24DAF and 31DAF , FATB, DGAT, PAH (phosphatidic acid phosphohydrolase), CPT (diacylglycerol cholinephosphotransferase), PLA (phospholipase), AGK (acylglycerol kinase) and GLPK (glycerol kinase). However, some genes like KASI (ketoacyl-ACP synthase I), KASII (ketoacyl-ACP synthase II), KASIII (ketoacyl-ACP synthase III), WRI, LPCAT (lysophosphatidylcholine acyltransferase), SDP (sugar-dependent protein) and LPAT (acyl-CoA:acylglycerol-3-phosphate acyltransferase) were not detected in the developing seeds of P. pedunculata. The reason for their absence requires further experiments to be explored.To propose a proper pathway involved in the fatty acid biosynthesis in the developing seeds of ion Fig. a. It shoP. pedunculata. A total of six genes  were randomly selected and the 18S rRNA was used as the internal control. The primers can be accessed in the Additional file LACS6) were confirmed by qRT-PCR at the late seed development stage and the high expression of TR61099|c1_g5  was validated in the early seed development stage  was found to be highly expressed at 31DAF by the sequencing and at 39DAF by qRT-PCR  were highly expressed at the early stage (10 ~ 24DAF) of the seed development  genes highly expressed  catalyze the desaturation of fatty acids attached to phospatidylcholine (PC) from PC-18:1 to PC-18:2 (FAD2) and from PC-18:2 to PC-18:3 (FAD3) [FAD6 has the potential of introducing the second double bond in the biosynthesis of 16:3 and 18:3 fatty acids, which are important constituents of plant membrane, and functions on fatty acids esterified to galactolipids, sulfolipids and phosphatidylglycerol through ferredoxin [P. pedunculata seeds) by SAD, which is one of the regulators for the proportion of fatty acids in seeds [At the middle stage of seed development 31 ~ 45DAF), we identified 1 ~ 45DAFungeanum , Camelina sativa , Linum utissimum , Eucommiulmoides , Glycinecine max  and Paeo section . FAD2 an3 (FAD3) –30. FAD6in seeds . The higLACS6, DGD1, ACAT1, AGPAT, WSD1, EGY2 and oleosin  of the seed development, such as DAF of thsin Fig. . Using pbidopsis . AGPAT ibidopsis . The o-abidopsis , 40. In he seeds . Oleosinhe seeds . The expa sativa  soybean a sativa , Paeonia section  and Periutescens . OverallP. pedunculata , ω6-FAD, SAD, ACP, ACCA and AHG1 were highly expressed at the middle seed development (31 ~ 45DAF) while LACS6, DGD1, ACAT1, AGPAT, WSD1, EGY2 and oleosin genes were abundant at the late seed development. The genes expression changes were confirmed by qRT-PCR. This is the first time to study the transcriptome of P. pedunculata developing seeds and our findings will provide a valuable resource for future studies. More importantly, it will improve our understanding of the oil accumulation for P. pedunculata.In conclusion, we assembled the first transcriptome for the developing seeds (eight time points) of Prunus pedunculata Pall which were planted in the experimental fields of Inner Mongolia Breeding Center, China. The original seeds were obtained from the wild place of Baotou, China  and maintained in the Inner Mongolia Seed Museum as Mengbian-1. The days after flowering (DAF) of fruits were marked with tags to track the seed development. From 10th April of 2019, we collected the fruits of Prunus pedunculata at 10, 18, 24, 31, 39, 45, 59 and 73 DAF with three biological replicates (n = 3). Every development stage we collected a total of ~ 100 seeds at each timepoint and peeled off the peel and stone. Then, the nucleolus was flash frozen in liquid nitrogen and stored at − 80 °C until further use. As the ovules at 0 DAF were hard to collect, we used seeds at 10 DAF as the control.We selected three six-year-old plants of P. pedunculata seeds was determined using the Gas Chromatograph  according to the manufacturer’s protocol, as described [The oil content of escribed . The CP-Total RNA was extracted from the nucleolus using the TRIzol reagent (Invitrogen), as previously described . After thttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to evaluate the clean reads for quality control. Then, clean reads of each time point were subjected to the TRINITY software for de novo assembly, as described previously [We used Trim_galore to remove low quality reads, reads with adapters and contamination reads from the raw reads. FASTQC , UniProt and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to obtain the Gene Ontology (GO) and pathway annotation, as previously described . After tp-value < 0.5 and false discovery rate (FDR) < 0.1 [We used Bowtie2 , 50 and p-value using the Fisher’s Exact Test to present the enrichment significance and calculated the corrected p-value  using the qvalue package in R platform. Significantly enriched GO terms and KEGG pathways were selected as follows: q-value < 0.05 and non-plant associated terms/pathways were filtered.We performed functional analysis for the differentially expressed genes using the GO and KEGG pathway annotation results. To identify significantly enriched GO terms and pathways, we first calculated the Prunus pedunculata. We randomly selected six genes  and used the 18S rRNA as the internal control. Forward and reverse primers were predicted for the candidate genes and control using the Beacon Designer 7.9. After the total RNA was extracted from the seeds at all eight time points (as described above), an aliquot (2 mg) of the total RNA was used for the first-strand cDNA synthesis using the TRUEScript First-Strand cDNA Synthesis kit . Then, the cDNA (1 μL) was used to build the qRT-PCR reaction mixture (10 μL) together with 2 × SYBR Green Supermix , forward primer (0.5 μL), reverse primer (0.5 μL) and ddH2O (3 μL). The qPCR mixture was run on the qTOWER 2.2 qRT-PCR Thermal Cyclers  following the protocols. Then, Ct value of each gene in each sample was obtained and ΔCt was calculated. The 10DAF samples were used as control and the expression levels of all genes in the other samples were calculated present using the relative normalized expression: RNE = 2−ΔΔCt. Three reactions were performed for each gene in every single replicate, thus we obtained nine replicates (n = 9) for each gene at each timepoint.We performed quantitative real-time PCR (qRT-PCR) to validate the gene expression changes during the seed development of Additional file 1. Differentially expressed genes in the developing seeds of P. pedunculata.Additional file 2. Differentially expressed genes related to fatty acid in the developing seeds of P. pedunculata.Additional file 3. Numbers of differentially expressed genes of fatty acid related pathway/biological process.Additional file 4. Differentially expressed genes related to lipid biosynthesis in the developing seeds of P. pedunculata.Additional file 5. Differentially expressed genes related to oilbody in the developing seeds of P. pedunculata.Additional file 6. WGCNA identifies co-expressed genes and their relationship with the oil contents.Additional file 7. Forward and reverse primers for qRT-PCR.


Human naïve pluripotent stem cells (PSCs) represent an optimal homogenous starting point for molecular interventions and differentiation strategies. This is in contrast to the standard primed PSCs which fluctuate in identity and are transcriptionally heterogeneous. However, despite many efforts, the maintenance and expansion of human naïve PSCs remains a challenge. Here, we discuss our recent strategy for the stabilization of human PSC in the naïve state based on the use of a single chemical inhibitor of the related kinases CDK8 and CDK19. These kinases phosphorylate and negatively regulate the multiprotein Mediator complex, which is critical for enhancer-driven recruitment of RNA Pol II. The net effect of CDK8/19 inhibition is a global stimulation of enhancers, which in turn reinforces transcriptional programs including those related to cellular identity. In the case of pluripotent cells, the presence of CDK8/19i efficiently stabilizes the naïve state. Importantly, in contrast to previous chemical methods to induced the naïve state based on the inhibition of the FGF-MEK-ERK pathway, CDK8/19i-naïve human PSCs are chromosomally stable and retain developmental potential after long-term expansion. We suggest this could be related to the fact that CDK8/19 inhibition does not induce DNA demethylation. These principles may apply to other fate decisions.  The extent to which human PSCs can be captured in a stable naïve state, its similarity to rodent naïve pluripotency, and even its clinical usefulness, remain in question . InhibitThe Mediator complex operates at enhancers and is a pivotal orchestrator of the transcriptional program of cell identity ,9 Fig. . Moreove2Each cell-type contains a unique repertoire of active enhancer complexes at specific DNA regions, which arise by high concentration of lineage-specific transcription factors . CollectSuper-enhancers (SEs) constitute a relatively novel concept that refers to a small fraction of unusually large and powerful enhancers ,13. SE's3There have been great advances in recent years regarding the structure and mechanistic functioning of Mediator . The 30 in vitro biochemical assays have collectively revealed how Mediator undergoes extensive structural re-arrangements to achieve this [The Mediator complex must associate with the many different transcription factors that define an enhancer locus. This constitutes one arm of the Mediator bridge at the enhancer. Recent evidence hints at how this may be achieved. Mediator subunits in its tail sub-module have been shown to engage in a high number of low specificity and weak interactions via phase separation, thereby achieving a “fuzzy” interaction between Mediator and the activation domains of many different transcription factors . The seceve this ,9,18,19.eve this . Preciseeve this  and phoseve this  of multi4The CDK8/19-kinase module (CDK8/19-CCNC-MED12/L-MED13/L) associates with core-Mediator and plays a central role in the regulation of how Mediator recruits RNA Pol II during each transcription cycle . The detAs mentioned above, currently it is not possible to fully understand the mechanism of CDK8/19 during the transcription cycle. Nevertheless, it is useful to consider the temporal order of events when interpreting the literature, and we summarize the role of CDK8/19 below in this order. CDK8/19 can sterically hinder the initial approach of Pol II to the Mediator complex based on several data and crystal structures ,19,36, ain vitro [in vivo [Later in the transcription cycle, following recruitment of RNA Pol II to Mediator, the pre-initiation complex is completed, and CDK8/19-Mediator undergoes further structural re-arrangements to participate in the release of RNA Pol II into productive elongation ,19. CDK8in vitro ,37, and [in vivo . Also, C[in vivo . Here, i[in vivo , CDK8/19[in vivo .In summary, CDK8/19 has an overall effect of repressing RNA Pol II recruitment and, thereby, CDK8/19 kinase inhibition increases recruitment of RNA Pol II by Mediator and stimulates global hyperactivation of transcription of enhancer target genes ,35.5RNA Pol II is known to participate in discrete membrane-less protein aggregates previously described as transcription factories . More re6in vitro which faithfully reflect developmental stages in the early embryo [in vivo around the E4.5 mouse pre-implantation embryo epiblast (Pluripotent stem cells (PSCs) transition between cell states y embryo ,2,6,7 , while the aggregate shift in cell identity  appears relatively slower and incremental. We use as a model the developmental window of naïve-to-primed pluripotency, outlined above. Extensive analyses over the past ~15 years have shown how this path in embryos is followed in close parallel by PSCs in vitro ,6,7. Pluin vitro ,57. Subsin vitro ,6,7. Thie states  of plurie states ,6,59. HoUltimately, around E5.5–6.5, mouse embryo epiblast cells exit from pluripotency, and choose between three competing transcriptional programs for lineage-specification, into one of the embryonic germ layers: ectoderm, mesoderm, and endoderm ,6,7,60 . This deIn vivo, this is transient, and resolves in a few hours, with each cell choosing a single germ-layer program. In vitro, it is possible to capture this decision-making process, as a population of primed PSCs, with heterogenous and fluctuating gene expression programs, literally “priming” themselves to fully-activate ectoderm, mesoderm, or endoderm [Crucially, lineage-specifying transcriptional programs attempt to reinforce themselves, and repress other competing programs . The antendoderm ,2,6,7. Iendoderm ,72. Thus8We recently hypothesized that manipulation of the transcriptional machinery during states of plasticity, might toggle cellular identity toward one path or another. We chose PSCs in the naïve-primed pluripotency window as our model of plasticity. As an actionable strategy, we began by targeting the transcriptional CDKs, CDK7, CDK8/19, and CDK9, since these kinases represent direct regulators of the transcriptional machinery, they are amenable to pharmacological inhibition, and indeed, several highly selective small molecule inhibitors exist . We founWe explored a possible mechanism, following the role of CDK8/19 as a transcriptional CDK, located within the Mediator complex . Also, wAs mentioned before, the induction of naïve identity using 2i or CDK8/19i treatments can stimulate Mediator function, which we detect by a global increase in RNA Pol II recruitment, global hyper-activation of existing mouse PSC enhancer loci, and upregulation of enhancer-driven transcription. We propose that this reinforces the pluripotency network underlying naïve PSC identity. In agreement with a recent report , we obse9As outlined above, CDK8/19i recapitulates a significant proportion of 2i-associated effects on mouse naïve cell identity, however, global DNA hypomethylation was an exception  Fig. 4)Fig. 4). In contrast to 2i, CDK8/19i recapitulates the same transcriptional changes as 2i, but does not trigger global DNA hypomethylation  Fig. 4)Fig. 4). 10Species-specific differences exist between human PSCs and our prior understanding of pluripotency based on rodent models ,5. In pa11in vitro has proven to be challenging and remains to be optimized [Stabilization of the human naïve pluripotent state ptimized ,74,94. Iptimized ,94,95. Tptimized .Similar to our observations in mouse PSCs, CDK8/19i does not induce MEK-inhibition or global DNA hypo-methylation in human PSCs . Accordi12in vitro, we have explored the possible role of CDK8/19 in the embryo, where the naïve state arises naturally in the absence of chemical inhibitors [(i)CDK8 is required during 1C to morula development, where its expression is high. In support of this, CDK8-knockout is embryonic lethal before the 4C stage , and CDK(ii)During morula to blastocyst pre-implantation development, CDK8 and cyclin C expression declines. This coincides with the emergence of the E4.5 pre-implantation naïve epiblast and, accordingly, small molecule CDK8/19i does not interfere with naïve epiblast specification . Also, i(iii)in vitro [in vivo, a feature which can be exploited to stabilize naïve PSC culture by CDK8/19i in vitro.During the subsequent developmental transition of pre-implantation naïve epiblast to the post-implantation primed state, CDK8 expression becomes increased and its activity is required for the morphogenic events during this transition . Moreovein vitro . In sum,Since CDK8/19 kinase inhibition favours up-regulation of naïve pluripotency hibitors  CDK8 In further support of these data, mouse PSCs are known to include an additional form of transcriptional heterogeneity involving infrequent, but dramatic and transient, re-activation of the gene expression program of the 2C embryo stage . It has 13ex vivo. The high degree of overlap in phospho-changes and RNA Pol II regulation induced by 2i and CDK8/19i kinase inhibitors immediately suggests that these chemical inducers of the naïve state might operate within the same pathway [Better understanding of the downstream signaling from MEK-ERK, and how it links to the transcriptional machinery, may improve our control over cellular plasticity and aide stabilization of cell identity  pathway . Importa pathway .Significant evidence supports the direct input of MEK-ERK signaling into Mediator and the control of the core transcriptional machinery in PSCs. Consistent with this model, it has been recently reported that MEK signalling during exit from the naïve state, directly or indirectly, results in phosphorylation of Mediator subunits and alter eRNA transcription within PSC super-enhancers . All cur14trans-activating potential of existing enhancers and Mediator complexes become globally hyper-activated [A better understanding of the intracellular competition between transcriptional programs may apply widely to human disease. In this regard, global enhancer hyperactivation was recently identified as a common feature across all human cancers tested , while activated . This ractivated . For exactivated . Within ctivated ,114,115,ctivated . Thus, uctivated .15We note that a similar mechanism of Mediator hyperactivation via CDK8/19 inhibition has been reported in cancer cells . HoweverBinary fate decisions are common throughout development and regeneration. We speculate that other cell fate decisions may operate via processes in the transcriptional machinery similar to the naïve-primed pluripotency equilibrium. Thus, we suggest that other examples of CDK8/19i influencing cell identity may exist. Recently, CDK8/19-kinase inhibition was found to produce an anti-inflammatory effect in mouse tissues. This was attributed to the ability of genetic and small molecule CDK8/19-inhibition to promote the production of immuno-suppressive regulatory T cells (T-regs) from their progenitor population of naïve T cells . We hypo16In vitro, we identify that CDK8/19i shifts the equilibrium between naïve and primed pluripotent states, favouring naïve features in mouse and human pluripotent cells Fig. 4). in vivo.Molecular analyses reveal how the RNA Pol II transcriptional machinery is reorganized by CDK8/19i to coordinate cell identity conversion . In the in vivo [The extent to which CDK8/19i mimics 2i suggests a central role of Mediator during the induction of naïve pluripotency, and it provides a mechanism by which naïve pluripotency may arise in vivo . Lastly,Cian J. Lynch: Methodology, Conceptualization, Writing - original draft, participated in all discussions. Raquel Bernad: Methodology, Conceptualization, helped to improve the text and figures. Isabel Calvo: Methodology, Conceptualization, helped to improve the text and figures. Manuel Serrano: Methodology, Conceptualization, helped to improve the text and figures, Funding acquisition.


Sc) and are accompanied by spongiform vacuolation of brain lesions. Approximately three centuries have passed since prion diseases were first discovered around the world; however, the exact role of certain factors affecting the causative agent of prion diseases is still debatable. In recent studies, somatic mutations were assumed to be cause of several diseases. Thus, we postulated that genetically unstable cancer tissue may cause somatic mutations in the prion protein gene (PRNP), which could trigger the onset of prion diseases. To identify somatic mutations in the PRNP gene in cancer tissues, we analyzed somatic mutations in the PRNP gene in cancer patients using the Cancer Genome Atlas (TCGA) database. In addition, to evaluate whether the somatic mutations in the PRNP gene in cancer patients had a damaging effect, we performed in silico analysis using PolyPhen-2, PANTHER, PROVEAN, and AMYCO. We identified a total of 48 somatic mutations in the PRNP gene, including 8 somatic mutations that are known pathogenic mutations of prion diseases. We identified significantly different distributions among the types of cancer, the mutation counts, and the ages of diagnosis between the total cancer patient population and cancer patients carrying somatic mutations in the PRNP gene. Strikingly, although invasive breast carcinoma and glioblastoma accounted for a high percentage of the total cancer patient population , somatic mutations in the PRNP gene have not been identified in these two cancer types. We suggested the possibility that somatic mutations of the PRNP gene in glioblastoma can be masked by a diagnosis of prion disease. In addition, we found four aggregation-prone somatic mutations, these being L125F, E146Q, R151C, and K204N. To the best of our knowledge, this is the first specific analysis of the somatic mutations in the PRNP gene in cancer patients.Prion diseases are caused by misfolded prion protein (PrP Sc) derived from normal prion protein (PrPC) [Prion diseases are fatal and irreversible neurodegenerative diseases caused by a deleterious form of the prion protein (PrPn (PrPC) ,2. Thesen (PrPC) ,10,11,12PRNP), which encodes PrP, induce familial forms of human prion diseases, including familial CJD, GSS, and FFI [de novo mutations in the PRNP gene were detected in several types of prion diseases, including sporadic CJD and GSS [PRNP gene can be assumed to be a cause of prion diseases. In addition, previous studies have been reported that cancer tissues showed more genetic instability and elevated expression of the PRNP gene. Thus, we postulated that cancer tissue may induce somatic mutations in the PRNP gene, which can trigger the onset of prion diseases [It has been approximately three centuries since scrapie in sheep was first discovered in 1732 . However and FFI ,16,17. I and GSS ,19,20,21diseases ,23,24,25PRNP gene in cancer tissues, we searched the information on somatic mutations in the PRNP gene in cancer patients using the Cancer Genome Atlas (TCGA) database [PRNP gene in cancer patients with that of previously reported pathogenic mutations associated with prion diseases and with the total cancer patient population. Furthermore, to assess whether the somatic mutations in the PRNP gene in cancer patients are deleterious, we performed in silico annotation using PolyPhen-2, PANTHER, PROVEAN, and AMYCO [To find somatic mutations in the database . In addind AMYCO ,30,31,32PRNP gene. In addition, FFI has been reported to carry D178N-129M mutations in the PRNP gene. Finally, GSS has been reported to carry P102L, P105L, P105T, P105S, A117V, G131V, Y145*, Q160*, V176G, H187R, F198S, D202N, Q212P, Q217R, Y226*, Q227*, and M232T mutations in the PRNP gene. Unique phenotypes of dementia have been reported to carry S17G, P39L, Y163*, D167N, D187fs, and R208C mutations in the PRNP gene.Genetic forms of human prion diseases include CJD, FFI, and GSS. Previous pathogenic mutations involved in genetic forms of prion diseases are described in PRNP gene in 10,953 cancer patients. The detailed information is described in Using cBioPortal, we identified a total of 48 somatic mutations in the PRNP gene with that of the total cancer patient population. The detailed information is described in PRNP somatic mutations carriers (subgroup 1) and 8 PRNP pathogenic somatic mutation carriers of prion disease (subgroup 2). Subgroup 1 accounts for 0.43% of the total cancer patient population assessed, while subgroup 2 accounts for 0.07% of the total cancer patient population. Subgroup 2 accounts for 17.02% of subgroup 1. The age of diagnosis in the total cancer patient population was 59.1 ± 14.5 years, in subgroup 1 was 66 ± 12.8 years, and in subgroup 2 was 66.3 ± 7.8 years. Notably, compared to the age of diagnosis in the total cancer patient population, those of subgroups 1 showed significant differences with p-values of 0.000742. In addition, there were also significant differences in the mutation count distributions between the total cancer patient population and subgroups 1 and 2, with p-values < 0.00001 and 2.3 × 10−11, respectively. Specifically, a large number of the total cancer patient population displayed mutation counts <100 (67.3%). By contrast, subgroups 1 and 2 primarily displayed mutation counts >280 . The distribution of cancer type between the total cancer patient population and subgroup 1 showed statistical significance. (p = 6 × 10−12). Specifically, the percentage of non-small cell lung cancer patients in subgroup 1 (16.7%) was increased by 1.7-fold compared to the total cancer patient population (9.6%). The percentage of endometrial carcinoma patients in subgroup 1 (33.3%) was increased by 6.3-fold compared to the total cancer patient population (5.3%). The percentage of non-small cell lung cancer patients in subgroup 2 (12.5%) was increased by 1.3-fold compared to the total cancer patient population (9.6%). The percentage of colorectal adenocarcinoma patients in subgroup 2 (12.5%) was increased by 2.3-fold compared to the total cancer patient population (5.4%). The percentage of endometrial carcinoma patients in subgroup 2 (25.0%) was increased by 4.7-fold compared to the total cancer patient population (5.3%). Notably, although invasive breast carcinoma and glioblastoma account for a high percentage of the total cancer patient population , somatic mutations in the PRNP gene have not been identified in these two cancer types. Strikingly, distributions of six major cancer types  of the patients with somatic mutations of the PRNP gene were shown to be significantly different from distributions of the six major cancer types of total patients  [PRNP gene in cancer tissues are very important. Of the 48 somatic mutations, 8 were previously confirmed pathogenic mutations of prion diseases. Although we cited pathogenic mutations of the PRNP gene from previous studies, the penetrance on the pathogenic mutations of the PRNP gene was still debatable. In a previous study, several mutations including V180I, V210I, and M232R were found in healthy controls and these mutations showed low penetrance under 10%. Thus, these results suggest that these mutations were benign variants or low-risk variants [in silico programs. Notably, V180I, V210I, and M232R mutations were predicted to be low amyloid propensity and benign by some programs  was much older than that of the total cancer patient population (59.1 ± 14.5 years)  and glioblastoma (5.4%) account for high percentage of the total cancer patient population, somatic mutation of the PRNP gene has not been identified in these two cancer types. Specifically, because somatic mutations of the PRNP gene in glioblastoma, which occurs in the brain, can be masked by a diagnosis of dementia, the absence of somatic mutations in the PRNP gene in glioblastoma can be explained. Previous study has been reported that transgenic mice carrying a pathogenic mutation of the PRNP gene can lead to spontaneous prion disease [Sc were also detected in healthy people, who have not been diagnosed with prion diseases [PRNP gene may produce a basal level of PrPSc in peripheral tissues and may not be diagnosed with prion disease. However, to date, there is no clinical evidence for this hypothesis. Since PrP protects cancer cells against apoptotic stress and contributes to tumorigenesis, the difference on distribution of somatic mutation of the PRNP gene may be induced depending on the degree of contribution of PrP to different cancer types [In the present study, we identified a total of 48 somatic mutations in the s (GWAS) ,34. Thusvariants . In the programs , Table 4programs . Since Ptastasis ,37. Furt5 years) . Since se future ,37,39,40 disease ,42. In adiseases . On the PRNP gene in cancer patients by PolyPhen-2, PANTHER, and PROVEAN. Notably, six somatic mutations—P26L, G34R, G119R, G131V, C214G, and Y218C—and five pathogenic mutations—P102L, P105L, G114V, H187R, and E200G—were considered to be damaging to PrP function by all three programs. Since PrPC has also been reported to be related to the survival signaling pathway and copper ion-associated enzyme activity, these somatic mutations may impact the normal function of PrP [PRNP gene in cancer patients. Notably, four somatic mutations—L125F, E146Q, R151C, and K204N—were newly identified amyloid-prone somatic mutations in the PRNP gene in cancer patients. In addition, we found several nonsense somatic mutations. These nonsense mutations induced PrP cleavage, which can affect the survival signaling pathway or the aggregation of PrP [We also analyzed somatic mutations of the n of PrP . In addin of PrP ,45. ThusSc has not been investigated thus far, the similarity of cross-seeding activities in other misfolded proteins suggests that aggregated p53 may act as a seed to induce the aggregation of PrP [Sc in cancer patients using Western blot or protein misfolded cyclic amplification (PMCA) is highly desirable in the future.In recent cancer studies, p53 has been demonstrated to play a pivotal role in several types of cancer and showed prion-like aggregation. Aggregation of p53 causes additional deleterious effects in cancer patients ,47,48. In of PrP . Thus, tPRNP gene in cancer patients were extracted from TCGA database using cBioPortal  [PRNP gene, germline mutations were excluded using the filtering option. Somatic mutations were analyzed using reference sequence from GenBank at the NCBI (NM_000311.5). Clinical data including somatic mutations, age of diagnosis, sex, mutation count, cancer type, and allele frequencies were retrieved from cBioPortal and the data were read by R (https://www.r-project.org/).The protein sequence of human PrP in al.org/) . Among t2 test. The age of diagnosis was reported as the mean values ± standard deviation (SD). Statistical significance based on p-values was calculated with a one-tailed Student’s t-test for single comparison.Statistical analyses were performed using SAS version 9.4 . The differences in the distributions of sex, mutation count, and cancer type between the total cancer patient population and the subgroups were compared using the χPRNP gene in cancer patients were evaluated by PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), PANTHER (http://www.pantherdb.org/), PROVEAN (http://provean.jcvi.org/index.php), and AMYCO (http://bioinf.uab.es/amycov04/) [Somatic mutations in the ycov04/) ,28,29,30PRNP gene in 10,967 cancer patients. Among them, eight somatic mutations are pathogenic for prion diseases. We identified significantly different distributions of cancer type, mutation count, and the age of diagnosis between the total cancer patient population and cancer patients carrying PRNP somatic mutations. We found six somatic mutations, these being P26L, G34R, G119R, G131V, C214G, and Y218C, and five pathogenic mutations, these being P102L, P105L, G114V, H187R, and E200G, which can affect PrPC function, along with four aggregation-prone somatic mutations—L125F, E146Q, R151C, and K204N. To the best of our knowledge, this is the first specific analysis on the somatic mutations of the PRNP gene in cancer patients.In conclusion, we identified 48 somatic mutations in the