Ferulago stellata) is Turkish medicinal plant species and used in folk medicine against some diseases. As far as we know, the data are not available on the biological activities and chemical composition of this medicinal plant. In this study, the phytochemical composition; some metabolic enzyme inhibition; and antidiabetic, anticholinergic, and antioxidant activities of this plant were assessed. In order to evaluate the antioxidant activity of evaporated ethanolic extract (EEFS) and lyophilized water extract (WEFS) of kınkor (Ferulago stellata), some putative antioxidant methods such as DPPH· scavenging activity, ABTS•+ scavenging activity, ferric ions (Fe3+) reduction method, cupric ions (Cu2+) reducing capacity, and ferrous ions (Fe2+)-binding activities were separately performed. Furthermore, ascorbic acid, BHT, and α-tocopherol were used as the standard compounds. Additionally, the main phenolic compounds that are responsible for antioxidant abilities of ethanol and water extracts of kınkor (Ferulago stellata) were determined by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Ethanol and water extracts of kınkor (Ferulago stellata) demonstrated effective antioxidant abilities when compared to standards. Moreover, ethanol extract of kınkor (Ferulago stellata) demonstrated IC50 values of 1.772 μg/mL against acetylcholinesterase (AChE), 33.56 ± 2.96 μg/mL against α-glycosidase, and 0.639 μg/mL against α-amylase enzyme respectively.Kınkor ( The plant kingdom is well known as a prolific and productive haven of phytochemicals with unmatched therapeutic potential. Moreover, 28,000 known plant taxa have been reported worldwide to have medicinal values. It has been reported that more than 3000 species have ethnomedical usage and applications against many diseases including cancer and diabetes mellitus . However1O2), hydrogen peroxide (H2O2), hydroxyl radicals (∙OH), and superoxide anion radicals (O2∙−) can easily occur as a result of normal aerobic metabolism, toxic agents, drugs, smoking, and burnt food 3−) to (Fe[(CN)6]4−) can easily occur. The addition of Fe3+ to the reduced product by addition of ethanol and water extracts of kınkor (Ferulago stellata) forms Fe4[Fe(CN)6], a complex in the Prussian blue color with sharp absorbance at 700 nm [Ferulago stellata), BHT, α-tocopherol, and ascorbic acid increased constantly when the concentration of sample was increased. Fe3+ reducing capacity of ethanol and water extracts of kınkor (Ferulago stellata) and standards exhibited the following order: ascorbic acid  > BHT  > water extracts of kınkor (Ferulago stellata)  > ethanol extracts of kınkor (Ferulago stellata)  > α-tocopherol  at 30 μg/mL. The results showed that ethanol and water extracts of kınkor (Ferulago stellata) had marked and powerful Fe3+ reducing effect  declined in the following order  > BHT  > ethanol extract of kınkor (Ferulago stellata)  > α-tocopherol  > water extract of kınkor (Ferulago stellata)  at 50 μg/mL. The FRAP method is carried out in an acidic environment to maintain iron solubility [Another putative and commonly used method is Feon assay . The FRAng order : ascorbilubility . 2+) reducing power of same concentration (30 μg/mL) of ethanol and water extracts of kınkor (Ferulago stellata) and standards is shown in 2+ reducing ability and different concentration of the ethanol and water extracts of kınkor (Ferulago stellata) (2+-reducing effect of ethanol and water extracts of kınkor (Ferulago stellata) increased with increasing concentrations (10–30 μg/mL). Cu2+-reducing ability of standards and both extracts at the same concentration (30 μg/mL) demonstrated the following order: BHT  > ascorbic acid  > α-tocopherol  > ethanol extract of kınkor (Ferulago stellata)  and water extract of kınkor (Ferulago stellata) .Copper is an important element in metallic form that can be found and used directly in nature. It is a very important cofactor for some endogenous and important metabolic enzymes including cytochrome c oxidase . In additellata) . It was 2 and transition metal ions, H2O2 can generate OH• via the Fenton reaction. In this way H2O2 is converted to a more reactive HO• by the Fenton reaction, which requires reduced iron ions (Fe2+), which had more reactivity than Fe3+ ions [Ferulago stellata) was evaluated using by two distinct metal chelator agents including ferrozine reagent. When the IC50 values of the binding effect of ethanol and water extracts of kınkor (Ferulago stellata) in the study were compared with the IC50 of the ethanol extract of kınkor (Ferulago stellata) and standard antioxidants, it was found to be as effective metal chelator with IC50: 31.5 ± 0.13 μg/mL (r2: 0.9030) (Ferulago stellata). Additionally, relatively higher IC50 values were found for α-tocopherol , ascorbic acid , and BHT .In the presence of Oe3+ ions ,49. In t 0.9030)  using fe∙+ and DPPH∙ scavenging methods are fast, simple, selective and repeatable procedures. So, they are widely used to define the radical elimination abilities. It is easy to use the violet DPPH∙ and green-blue ABTS∙+ chromogens that have high sensitivity [50 values for ethanol and water extracts of kınkor (Ferulago stellata) had less effective DPPH∙ scavenging effect and were found to be 34.7 ± 0.22 μg/mL (r2: 0.9965) and 57.8 ± 0.07 μg/mL (r2: 0.9993), respectively when compared to α-tocopherol , ascorbic acid , and BHT , which are food additives used as preservative ingredients in some foods  had effective ABTS radical removing effects. The IC50 value of ABTS∙+ scavenging activity for ethanol and water extracts of kınkor (Ferulago stellata) was calculated as 7.8 ± 0.01 μg/mL (r2: 0.9844) and 19.3 ± 0.04 μg/mL (r2: 0.9419), respectively. Furthermore, this value was calculated as 26.7 ± 0.08 μg/mL (r2: 0.9717) for BHT, 15.4 ± 0.03 μg/mL (r2: 0.9825) for α-tocopherol, and 23.1 ± 0.01 μg/mL (r2: 0.9998) for ascorbic acid. The results clearly demonstrated that the ethanol and water extracts of kınkor (Ferulago stellata) have effective ABTS∙+ scavenging ability when compared to all standard antioxidants  have a rich content of small molecules such as chlorogenic acid, rutin, and orientin. Additionally, ethanol extract of kınkor (Ferulago stellata) effectively inhibited AChE with IC50 values of 1.772 μg/mL (r2: 0.9831) for AChE. On the other hand, tacrine was used as positive control for AChE inhibition and had Ki value of 0.124 µM (r2: 0.9804) against AChE. AChE is the primary cholinesterase at mainly neuromuscular junctions and in chemical synapses in the body. However, it was observed that water extract of kınkor (Ferulago stellata) does not have any modulatory effects against the used metabolic enzymes.An important metabolic enzyme is acetylcholinesterase (AChE), which had been associated in some neurodegenerative diseases including AD . The AChFerulago stellata) to modulate the activity of enzymes related to AD (AChE) and diabetes (α-glucosidase and α-amylase) was also investigated. Recently, diabetes is one of the fastest growing, serious, and costly health problems worldwide. A complete form of treatment and effective drugs for diabetes are still not found [Ferulago stellata) had IC50 values of 0.826 μg/mL (r2: 0.9491) toward α-glycosidase and 0.639 μg/mL against α-amylase enzyme (r2: 0.9580). The results show that ethanol extract of kınkor (Ferulago stellata) as a crude extract exhibited efficient α-glycosidase and α-amylase inhibition effect when compared to acarbose as a starch blocker, which had IC50 of 10.00 μM for α-amylase and 22.80 μM for α-glycosidase [Scientists have been extensively investigating the potential of medicinal plants to inhibit certain metabolic enzymes associated with some global diseases due to the various undesirable side effects of synthetic drugs. In this study, the ability of ethanol and water extracts of kınkor (ot found . Plant eot found . In thisot found . EthanolFerulago stellata) were determined using the Folin-Ciocalteu reagent. Gallic acid, which is easily obtained in large amounts by acid or alkaline hydrolysis of tannin, was used for a standard graph (r2: 0.9840). Plants, vegetables, and fruits, which include polyphenols, are important sources of phenolic compounds in human diet. Accordingly, the consumption of foods containing phenolics, especially polyphenols, is of great importance in terms of natural antioxidants [Ferulago stellata) was determined using the equation taken from standard gallic acid graph and found as 31.36 and 56.36 gallic acid equivalents (GAE/mg extract), respectively. On the other hand, for determination of total flavonoids content of ethanol and water extracts of kınkor (Ferulago stellata), a standard gallic acid graphs was used. The flavonoids quantity in both extracts was determined as 35.98 and 28.50 μg quercetin equivalent (QE), respectively. The most favorable structural properties characterizing the antioxidative potential of phenolic compounds are the presence of hydrogen-donating substituents and the ability for delocalization of the resulting free electron for stability. The most active form of antioxidant molecules is the one that has more than one active group  in the ortho-position, which plays an important role in the structure-activity relationship of antioxidants [The amount of total phenolic and flavonoids in medicinal plant extracts has been associated with their antioxidant capacity. Total phenolic compounds in ethanol and water extracts of kınkor (oxidants . The quaoxidants . It has oxidants . Plants oxidants .Ferulago stellata) are rutin (14013.35 mg/kg), which is a naturally occurring flavonol glycoside in fruits, leafy vegetables, and several grains; chlorogenic acid as a polyphenolic compound that exhibits antioxidant, antibacterial, and antitumor activities (10103.18 mg/kg); and orientin that is a flavonoid from plant, derived often to use in various bioactivity studies (491.59 mg/kg). On the other hand, rutin that has wide variety of medicinal applications (156907.40 mg/kg); orientin (15329.03 mg/kg); and chlorogenic acid, as one of the natural products readily found in food, medicines, and cosmetics (44642.39 mg/kg), are the most plentiful phenolic compounds in 1 mg of ethanol extract of kınkor (Ferulago stellata)  . DiffereFerulago stellata) was collected from B9 Van: Çatak, Bilgi village, surroundings of Üçüzler district, 2200 m, in August 2019 .α-tocopherol, neocuproine, DPPH radical, ABTS, DMPD, and α-tocopherol were obtained from Sigma-Aldrich . The sources and purity of the standard compounds for LC-HRMS are given as follows: ascorbic acid , (-)-epigallocatechin , (−)-epigallocatechin gallate (>97% TRC Canada), chlorogenic acid (≥95% Sigma-Aldrich), fumaric acid (≥99% Sigma-Aldrich), verbascoside , orientin , caffeic acid , (+)-trans taxifolin , luteolin-7-rutinoside , naringin , luteolin 7-glucoside , rutin , rosmarinic acid , hyperoside , dihydrokaempferol , quercitrin , myricetin , quercetin , salicylic acid , naringenin , luteolin , nepetin , apigenin , hispidulin , isosakuranetin , CAPE (caffeic acid phenethyl ester) , chrysin , acacetin , and emodin . The other solvents used were of analytical grade and purchased from either Merck or Sigma-Aldrich. Kınkor (Ferulago stellata), a 50 g plant sample was cut into small pieces, ground into a fine powder using a mill and mixed with 0.5 L of ethyl alcohol, and then evaporated [Ferulago stellata) was transferred to an appropriate plastic bottle and kept at −20 °C until used in experiments.The used water and ethanol extractions methods were previously described . For detaporated . This prFerulago stellata), 50 g plant samples powdered and mixed with 500 mL water, boiled, and stirred for 20 min. Then extract was filtered and frozen at −87 °C in an ultra-low temperature freezer. Frozen extract was lyophilized at −50 °C at a pressure of 5 mm-Hg in a lyophilizator [Ferulago stellata) was kept in a plastic bottle and stored at −20 °C until used in experimental.For lyophilized water extraction shade-dried kınkor (ilizator . Prepare3+) reducing ability of ethanol and water extracts of kınkor (Ferulago stellata) were realized according to Oyaizu [Ferulago stellata) in distilled water or ethanol (10–50 µg/mL) were added to the same volume of phosphate buffers  and K3Fe(CN)6 solution . The mixtures were kept at 50 °C during 20 min and then, acidified with TCA . Finally, a portion of FeCl3  was transferred and their absorbances were spectrophotometrically measured at 700 nm.The ferric ions  were made according to spectrophotometric assay [2 solution , neocuproine solution (7.5 mM), and acetate buffer  were added to different concentrations of ethanol and water extracts of kınkor (Ferulago stellata) solutions (10–50 μg/mL) in test tubes. The volume of total mixture was adjusted to 2 mL with deionized water. Then, the tubes were closed and retained at 25 °C. Finally, their absorbances were spectrophotometrically recorded at 450 nm.The Cuic assay  as descric assay . For thiFerulago stellata) and standard solutions were transferred to the test tubes, which included several concentrations. A portion (2.25 mL) of TPTZ solution (10 mM TPTZ in 40 mM HCl) was freshly prepared and then transferred to 2.5 mL acetate buffer  and 2.25 mL of FeCl3 solution (20 mM) in water. Then, different concentrations of ethanol and water extracts of kınkor (Ferulago stellata) (10–30 μg/mL) were dissolved in 5 mL of appropriate buffer solvent, stirred, and incubated at 37 °C for 30 min. Finally, the absorbance of mixture was spectrophotometrically measured at 593 nm.FRAP reduction ability was realized according to our previous study . First, Ferulago stellata) was performed according to Blois method [Ferulago stellata) solution (1.5 mL) in ethanol or water (10–50 µg/mL) and incubated for 30 min in the dark. Finally, the absorbance of the mixtures was spectrophotometrically recorded at 517 nm.DPPH∙scavenging ability of ethanol and water extracts of kınkor  was realized according to the previous study [2S2O8 to an ABTS solution and their absorbances was set to 0.750 ± 0.025 nm diluted by buffer solution at 734 nm. Finally, 3.0 mL of ethanol and water extracts of kınkor (Ferulago stellata) at various concentrations (10–50 μg/mL) were mixed with 1.0 mL of ABTS•+ and the remaining absorbance was spectrophotometrically recorded at 734 nm.ABTSus study ,84. PrimFerulago stellata) were found as millimolar in the reaction medium. Both radicals (DPPH• and ABTS•+) scavenging effects were calculated as follows: Control and ASample are the absorbance values of the control and samples, respectively. The half maximal inhibitory concentration (IC50) was estimated by plotting percentages against the ethanol and water extracts of kınkor (Ferulago stellata) sample concentrations (μg/mL) [The radical removing capacities (RRC) of ethanol and water extracts of kınkor ( (μg/mL) ,86.Ferulago stellata) are used within the scope of anticholinergic studies. The AChE inhibitory effect of ethanol and water extracts of kınkor (Ferulago stellata) was realized according to Ellman’s method [Electrophorus electricus). For this, 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) and acetylthiocholine iodide (AChI) were used as substrate for cholinergic reaction [Ferulago stellata) solution were dissolved in ethanol and deionized water. Then, 50 μL of AChE (5.32 × 10−3 EU) solution was added and incubated for 10 min at 25 °C. After a short incubation period, 50 μL of DTNB (0.5 mM) was added. Finally, the reaction was started by the addition of 50 μL of acetylcholine iodate (AChI) (10 mM). The enzymatic hydrolysis of these substrates was spectrophotometrically determined by the formation of yellow 5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine at a wavelength of 412 nm.AChE and BChE inhibitions of ethanol and water extracts of kınkor  were studied within the scope of the antidiabetic study. α-glycosidase inhibition efficacy of ethanol and water extracts of kınkor (Ferulago stellata) was performed according to Tao et al. [d-glycopyranoside (p-NPG) substrate as described previously in details. The absorbances of samples were spectrophotometrically recorded at 405 nm [Ferulago stellata) sample dissolved in ethanol and deionized water, respectively. Then, it was pre-incubated at 35 °C for 10 min prior to the addition of p-NPG to the initiation of the reaction. In addition, 20 μL of p-NPG was added in phosphate buffer  after re-incubation at 35 °C. The absorbances were spectrophotometrically measured at 405 nm.Two digestive enzyme inhibitions of ethanol and water extracts of kınkor  sample dissolved in ethanol and deionized water were mixed and was preincubated at 35 °C for 30 min. Then, 20 μL of α-amylase solution was added to it and incubated for 30 min. The reaction was finished by addition of 50 μL of HCl (0.1 M). The absorbances were spectrophotometrically measured at 580 nm.α-Amylase activity, the second digestive enzyme, was determined according to the Xiao’s procedure . Starch 50 was obtained from activity (%) versus ethanol and water extracts of kınkor (Ferulago stellata) concentration plots. Furthermore, Lineweaver-Burk [i and other inhibition types [The ICver-Burk  graphs won types .Ferulago stellata) were calculated by Folin-Ciocalteu methods [Ferulago stellata) were calculated from the calibration curve. Total flavonoids in ethanol and water extracts of kınkor (Ferulago stellata) were determined according to our previous colorimetric method [3) colorimetric assay was used for the estimation of the total flavonoid content. The standard quercetin curve (0–100 μg/mL) was used to determine total flavonoids, and results are given as μg quercetin equivalents (QE) per g ethanol and water extracts of kınkor (Ferulago stellata).Total phenolics in ethanol and water extracts of kınkor  were dissolved in water in a 5 mL volumetric flask, which was kept in an ultrasonic bath until a clear solution was obtained. Then, 0.1 mL of dihydrocapsaicin solution, used as an internal standard, was added and diluted to the volume with mobile phase and stirred and heated to get clear solution. Then, the solution was filtered (0.45 µm Millipore Millex-HV filter). The concentration of final solution (1 mL) was added in a capped auto sampler vial, from which 2 µL of sample was injected to LC for each run. The prepared samples in the auto sampler were stored at 15 °C [The dried 100 mg of the ethanol and water extracts of kınkor  equipped with a Troyasil C18 column . The mobile phases A and B were composed of 1% formic acid–water and 1% formic acid–methanol, respectively. The gradient program of which was 0–1.00 min 50% A and 50% B, 1.01–6.00 min 100% B, and finally 6.01–10 min 50% A and 50% B. The flow rate of the mobile phase was 0.35 mL/min, and the column temperature was set to 22 °C. Environmental conditions were set as temperature of 22.0 ± 5.0 °C and relative humidity of (50 ± 15)% rh ,103.m/z 85–1500 were scanned in high-resolution mode of instrument [The best mobile phase was found to be an acidified methanol and water gradient in HPLC method. This mobile phase has also been found to be suitable for ionization abundance and separation of compounds. The best ionization of small and relatively polar compounds has been achieved with the ESI source. The ions between strument ,105. IdeFerulago stellata), an understudied endemic plant to Turkey, possessed effective antioxidant and some metabolic enzymes inhibitory properties. Evaluation of bioactivity and phytochemical screening of kınkor (Ferulago stellata) had great importance. Ethanol and water extracts of kınkor (Ferulago stellata) have been found to have efficient antioxidant properties when compared to BHA, BHT and ascorbic acid in various bioanalytical tests, including Fe3+ and Cu2+ reduction abilities, Fe2+ binding, as well as DPPH and ABTS radical scavenging activities. In addition, ethanol extract possessed higher antioxidant activity, phenolic contents, and demonstrated AChE, α-glycosidase and α-amylase inhibition effects. This study suggests that ethanol and water extracts of kınkor (Ferulago stellata) could be a promising potential source of beneficial phenolics. Data presented in this study demonstrated that kınkor (


Larotinib is a new first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. This open-label, phase 1b study is aimed to evaluate the efficacy, safety of larotinib in patients with advanced esophageal squamous cell carcinoma (ESCC) with EGFR overexpression or amplification pretreated with one or more system regimens, and to recommend an appropriate dose for its further study.Patients received larotinib orally at 3 doses , once daily. Clinical response was evaluated every 8 weeks according to RECIST v1.1 criteria by both investigators and independent radiology review (IRC).81 patients were enrolled. The investigator-assessed overall response rate (ORR) was 13.7% (10/73), all responses were observed in the 350 mg group of which ORR up to 20.0% (10/50), with 10 of them having EGFR overexpression and 4 having EGFR amplification. Per IRC assessment, ORR for all patients and 350 mg group were 13.9% (10/72) and 16.3% (8/50). In the 350 mg group, median overall survival (OS) and progression-free survival (PFS) were 8.0 (95% CI 4.9–10.2) months and 3.4 (95% CI 2.4–3.7) months, respectively. The most common treatment-related adverse events (TRAEs) were diarrhea, rash, and palmar-plantar erythrodysesthesia syndrome, elevated AST/ALT, vomiting, similarly with other EGFR TKIs.Larotinib demonstrated promising antitumor activity and manageable safety profiles in patients with pre-treated advanced ESCC with EGFR overexpression or amplification, especially at the dose of 350 mg, which showed better efficacy and acceptable safety. A phase 3 study is underway on 350 mg larotinib in ESCC patients with EGFR overexpression.https://clinicaltrials.gov/ct2/show/NCT03888092.This trial was retrospectively registered on 25/03/2019, NCT03888092. The online version contains supplementary material available at 10.1186/s12876-021-01982-4. The main exclusion criteria were history of other EGFR-targeted therapies, active infection, bleeding, unstable cardiovascular disease, and pregnancy or lactation.The status of EGFR in tumor specimens was tested in a central lab before enrolment. IHC assays were performed as per the manufacturer’s instructions using CONFIRM anti-EGFR rabbit monoclonal primary antibody . EGFR IHC staining scores were independently judged by two pathologists according to the evaluation criteria (definition of IHC 3+ = strong cytoplasmic or/and membranous reactivity in ≥ 10% of tumor cells). EGFR amplification was measured using a FISH assay  counting 50 cells. EGFR-amplified tumors were defined as an average copy number of EGFR ≥ 4, or ≥ 15.0% of all counted cells with EGFR copy number ≥ 6, or an average copy number of less than 4, but EGFR/CEP ratio ≥ 2.0.Eligible patients received larotinib  orally at 350 mg, 300 mg, or 250 mg daily (28 consecutive days as a treatment cycle) until disease progression, intolerable toxicity, or withdrawal of informed consent. A maximum of two dose reductions was permitted in 50 mg increments due to drug intolerance. Toxicity was assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) Version 4.03. Tumor responses were evaluated by both investigators and independent radiology review every 8 weeks according to the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1). Complete or partial responses were confirmed at a subsequent time point 4 weeks later. The primary efficacy endpoint was investigator-assessed objective response rate (ORR).At an early phase of the study, the evaluation of efficacy and safety was conducted in 3 dose groups, namely, 250 mg/d, 300 mg/d, and 350 mg/d. After the analysis of certain accumulated data, 350 mg was chosen to expand enrolment to determine its efficacy and safety. We calculated the sample size of the 350 mg group based on the primary efficacy endpoint, which is ORR. According to findings from previous studies, the ORR to second-line therapy was around 5% in ESCC , 25, we Subjects who met the selection criteria and received at least one dose of the treatment drug were included in the full analysis set and safety set. The response evaluable set referred to subjects who had a baseline and at least one post-treatment tumor assessment, and were used for the assessment of efficacy endpoints.For efficacy analysis, the ORR was calculated based on the observable number of cases achieving objective responses . The 95% confidence interval (95% CI) was calculated using the Closure-Pearson method. PFS and OS were calculated using the Kaplan–Meier method and the log-rank test to assess statistical significance. Descriptive statistics and MedDRA medical terminology was used to classify organ systems to summarize the incidence and severity of adverse events and serious adverse events. All analyses were conducted using SAS system version 9.2 .Between August 8, 2017, and March 01, 2021, a total of 173 patients were screened and 81 eligible subjects were enrolled  of them had confirmed tumor partial responses Fig. . All tum.7% of thAs of March 01, 2021, the median duration of follow-up was 20.6 months (IQR 16.2–23.6). The median PFS and OS for all patients were 2.90 (95% CI 2.1–3.6) months and 5.90 95% CI 4.6–7.7) months, respectively. Longer PFS and OS were both observed in patients treated with loratinib 350 mg, with a median OS 8.0 (95% CI 4.9–10.2) months and PFS 3.4 (95% CI 2.4–3.7) months  experienced at least one treatment-related adverse event (TRAE). The most common TRAEs were mostly mild (grade 1/2), including diarrhea (67.9%), rash (64.2%), palmar-plantar erythrodysesthesia syndrome 39.5%), oral ulcer (30.9%), and aneamia (30.9%)  or EGFR gene amplification (a positive fluorescence in situ hybridization [FISH] result), and the ORR increased to 16.7% 9/54); however, median PFS is only 1.7 months, with a median OS of 5.1 months. In this phase Ib clinical study in ESCC, the subject of EGFR biomarker requirement is almost the same as that of icotinib. More importantly, this study demonstrated a higher ORR and improved survival benefit, in the 350 mg group, the median OS is 8.0 months and median PFS is 3.4 months, with an ORR of up to 20.0% (10/50), which may be attributed to the difference in terms of drug property. This could be also explained in preclinical findings , suggesting that EGFR overexpression might be correlated with EGFR amplification, which corroborated previous findings , 23. ConLarotinib is well-tolerated in patients with ESCC. The toxicity profile in our study was similar to that of other first-generation EGFR-TKIs. Most of the TRAEs were grades 1 or 2, and most of the treatment-related SAEs could be resolved or became tolerable after therapeutic interventions. Although interstitial lung disease (ILD) was observed (five patients) in our study. Among them, two were recently pre-treated with anti-PD-1 monoclonal antibodies. The time intervals from the last dose of PD-1 inhibitor and the first dose of larotinib in the two subjects were 33 days and 34 days, respectively. Interestingly, no ILD was reported in the patients with intervals longer than 50-days, suggesting that the time interval between anti-PD-1 antibody administration and larotinib treatment may be related to the occurrence of ILD. A similar finding was observed in patients with NSCLC treated with sequential anti-PD-(L)1 monoclonal antibody followed by osimertinib , 28. TheDespite the encouraging results in this study, there remain several limitations. The study outcomes might be biased by the small sample size and the single-arm design. Furthermore, quality of life was not assessed in this study. Thus, a randomized, open-label, phase 3 clinical trial of larotinib in over 400 patients is ongoing .In conclusion, larotinib showed encouraging antitumor activity and a tolerable safety profile in pre-treated advanced ESCC patients with EGFR overexpression or amplification, especially at the dose of 350 mg. A phase 3 study is underway to further evaluate the efficacy and safety of 350 mg larotinib for the treatment of ESCC with EGFR overexpression .Additional file 1. Which is entitled with Preclinical studies on Xenografted Mice Models, is an in vivo assay reporting results regarding the evaluation of antitumor efficacy of larotinib in xenografted mice models, plasma, and tumor tissues.Additional file 2. Which is entitled with Supplementary Materials and Methods, is a description about protocol revision history for study PCD-DZ650-17-001 (NCT03888092).Additional file 3. Which is entitled with Overall Response Assessed by Independent Radiology Review, is a supplementary table describing confirmed overall response assessed by independent radiology review.Additional file 4. Which is entitled with Progression-free Survival and Overall Survival in Subgroups, is a supplementary table describing subgroup analyses of Progression-free Survival and Overall Survival at different dose levels.Additional file 5. Which is entitled with Preclinical Kinase Inhibition Assays, is a report describing a preclinical study designed to make comparison of inhibitory activity on EGFR family kinases in the same batch among different first-generation EGFR-TKIs.Additional file 6. Which is entitled with Preclinical Tissue Distribution Study, describes a preclinical tumor distribution study designed to confirm whether larotinib had a better distribution than other EGFR-TKIs in esophageal tumor tissues.Additional file 7. Which is entitled with Baseline characteristics, is Table Additional file 8. Which is entitled with Treatment related adverse events occurring in 10% or more patients, is Table


Cryptococcus neoformans presents a worldwide threat to human health, especially in the context of immunocompromise, and current antifungal therapy is hindered by cost, limited availability, and inadequate efficacy. After the infectious particle is inhaled, C. neoformans initiates a complex transcriptional program that integrates cellular responses and enables adaptation to the host lung environment.The pathogenic yeast  Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills almost 200,000 people worldwide each year. It is acquired when mammalian hosts inhale the infectious propagules; these are deposited in the lung and, in the context of immunocompromise, may disseminate to the brain and cause lethal meningoencephalitis. Once inside the host, C. neoformans undergoes a variety of adaptive processes, including secretion of virulence factors, expansion of a polysaccharide capsule that impedes phagocytosis, and the production of giant (Titan) cells. The transcription factor Pdr802 is one regulator of these responses to the host environment. Expression of the corresponding gene is highly induced under host-like conditions in vitro and is critical for C. neoformans dissemination and virulence in a mouse model of infection. Direct targets of Pdr802 include the quorum sensing proteins Pqp1, Opt1, and Liv3; the transcription factors Stb4, Zfc3, and Bzp4, which regulate cryptococcal brain infectivity and capsule thickness; the calcineurin targets Had1 and Crz1, important for cell wall remodeling and C. neoformans virulence; and additional genes related to resistance to host temperature and oxidative stress, and to urease activity. Notably, cryptococci engineered to lack Pdr802 showed a dramatic increase in Titan cells, which are not phagocytosed and have diminished ability to directly cross biological barriers. This explains the limited dissemination of pdr802 mutant cells to the central nervous system and the consequently reduced virulence of this strain. The role of Pdr802 as a negative regulator of Titan cell formation is thus critical for cryptococcal pathogenicity. Cryptococcus neoformans and Cryptococcus gattii. C. neoformans is a ubiquitous opportunistic pathogen that infects mainly immunocompromised patients, while C. gattii is capable of infecting immunocompetent individuals  include transcellular migration, in which the yeast cells enter and exit vascular endothelial cells –9; parac–10–he brain . Cryptoche brain , 15.C. neoformans to survive and proliferate in the lung, and subsequently disseminate to the brain, depends on viability at mammalian body temperature and the expression of multiple virulence traits; these include secreted factors  greater than 10 or 15 μm or total cell diameter (including the capsule) that exceeds 30 μm , 43, 44.47–ze cells , 46. Somze cells . In contze cells , 46.C. neoformans experiences a dramatic change in conditions upon entering a host, including altered nutrient levels and pH. To adapt to the new environment, cryptococci activate a network of transcription factors (TFs) (rs (TFs) , 52. Forrs (TFs) , Cuf1 (5rs (TFs) , Pho4 (5rs (TFs) , Cir1 (5rs (TFs) , and Crzrs (TFs) . Alkalinrs (TFs) , 58.Overlapping TF circuits regulate cryptococcal virulence determinants, including polysaccharide capsule production and melanin synthesis. For example, Usv101, an important regulator of capsule thickness and polysaccharide shedding, also regulates three other TFs  and multiple polysaccharide-related enzymes . Gat201 C. neoformans virulence  (PDR802). To examine targets of Pdr802, we also constructed a strain that expresses the protein fused to mCherry at its N terminus   with theexpected , and PDR strains .10.1128/mBio.03457-20.1FIG S1C. neoformans strains in the KN99α background (middle) that either lack PDR802  or encode a tagged copy of the protein (mCherry-PDR802). (B) Qualitative analysis of gene expression in panel A strains and the complemented pdr802 mutant (PDR802). Cryptococcal mRNA isolated from cells grown in DMEM  was used to generate cDNA; from this, segments of the genes indicated at the left were amplified using the primers listed in PDR802 expression. Samples of RNA isolated as for panel B were analyzed for PDR802 expression by qRT-PCR. All results were normalized to ACT1 expression. Each symbol represents a biological replicate, with the mean and standard deviation also shown. ***, P < 0.001, compared to KN99α by one-way ANOVA with a post hoc Dunnett test. Download FIG S1, PDF file, 0.5 MB.Mutant strain construction and confirmation. (A) Scheme for generating Copyright © 2021 Reuwsaat et al.2021Reuwsaat et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pdr802), or the complemented mutant (PDR802). In this model, mice infected with the parent or complemented strains survived for roughly 3 weeks, while those infected with the deletion mutant showed a striking increase in survival: all animals survived for at least 65 days, and over half survived to the end of the study (100 days)  showed brain burdens similar to WT levels, while brain burdens in mice sacrificed at day 100 (blue symbols) ranged between zero fungal cells and the WT level.We next assessed the long-term survival of C57BL/6 mice infected with the parental wild-type (WT) strain (KN99α), the deletion mutant (00 days) . The lunfections , demonstfections , althoug10.1128/mBio.03457-20.2FIG S2pdr802 strain infections, red circles represent mice sacrificed at days 65 and 69, while blue circles represent mice sacrificed at the termination of the study (day 100). (B) Mean and SD of total CFU in the blood and brain at the indicated times post-infection. (C) Mean and SD of total CFU in the lung, blood, and brain 75 days after infection with the pdr802 strain. Each color represents one mouse. Download FIG S2, PDF file, 0.9 MB.Organ burdens. (A) Mean and SD values of total CFU in the indicated tissue of mice from the Copyright © 2021 Reuwsaat et al.2021Reuwsaat et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 5 times the original inoculum. Towards the end of this period, these cells were also detected in the blood and brain , we again noted some heterogeneity of fungal burden: one mouse had high lung burden with no dissemination, another had high lung burden with moderate brain burden, and the third had extremely low lung burden with no dissemination .We next examined the time course of fungal proliferation in the lungs. As expected, the burdens of WT and the complemented mutant strains increased steadily over an 18-day interval , eventuand brain . In contmination . No CFU pdr802 strain in vitro, including stress conditions that might be encountered in the host. We saw no differences in growth of the mutant compared to WT cells under conditions that challenge cell or cell wall integrity, including the presence of sorbitol, high salt, cell wall dyes, caffeine, sodium dodecyl sulfate (SDS), or ethanol  Tenfold serial dilutions of WT, pdr802, and PDR802 cells were plated on the media shown and incubated at 30°C (A), 37°C (B), or 37°C in the presence of 5% CO2 (C). Nitrosative (NaNO2) and oxidative (H2O2) stress plates were prepared with YNB medium, and melanization plates containing l-DOPA were prepared as described in Materials and Methods; all other plates were prepared with YPD medium. The lac1 control strain lacks the ability to melanize  Uree urease . DownloaCopyright © 2021 Reuwsaat et al.2021Reuwsaat et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pdr802 mutant under conditions more like those encountered inside the mammalian host, using tissue culture medium (Dulbecco’s modified Eagle medium [DMEM]) at 37°C in the presence of 5% CO2. We found that although the pdr802 mutant grew like WT in rich medium (yeast extract-peptone-dextrose [YPD]), it grew poorly in DMEM  (We next tested the growth of the  in DMEM A and B.  in DMEM . The pdrRNA-Seq) .10.1128/mBio.03457-20.4FIG S42 (B) was assessed by determining the optical density at 600 nm (OD600) at the times indicated. (C) Conditioned medium from the indicated strains was probed for the presence of GXM after growth in DMEM for 24 or 48 h. Equal volumes of culture supernatant were analyzed without normalization to cell density. Immunoblotting was performed using the anti-GXM monoclonal antibody 302. Download FIG S4, PDF file, 0.6 MB.Growth curves and capsule shedding. (A and B) Growth of the indicated strains in YPD at 30°C (A) or DMEM at 37°C and 5% COCopyright © 2021 Reuwsaat et al.2021Reuwsaat et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2 is the polysaccharide capsule, which we previously reported to be regulated by Pdr802, based on negative staining with India ink  for 24 h, followed by cell wall and capsule staining. Thousands of cells may be imaged per well and analyzed automatically with software that annotates and measures the capsule (annotated in dark blue) and cell wall (annotated in bright green). See Materials and Methods for details. (B) Capsule size distribution of WT cells after induction. Capsule thickness for each cell is the difference between the paired diameters of the cell wall and capsule, which is plotted with reference to the mean value. (C and D) Mean and SD (C) and cumulative percentage (D) analysis of WT compared to hyper- and hypocapsular control strains . Capsule thickness is in arbitrary units, related to the pixels measured. (E) Time required to analyze the capsule thickness of 1,000 cells by this method compared to manual assessment of India ink images. Download FIG S5, PDF file, 0.7 MB.Semi-automated assay for cryptococcal capsule imaging. (A) Schematic of applying this method to cryptococcal cells induced to form capsule by growth in DMEM  total cell diameter and (B) ratio of total cell to cell body diameters (diameter ratio), assessed by measuring at least 50 cells per strain with ImageJ. **, P < 0.01, and ****, P < 0.0001, compared to KN99α by one-way ANOVA with a post hoc Dunnett test for each day post-infection. (C) Percent of Titan cells in the indicated strain, evaluated using various published parameters: cell body diameter bigger than 10 or 15 μm  than in the WT and complemented cultures   . As expectively) .pdr802 mutant under both of these conditions, even though it showed normal engulfment when all strains were grown under control conditions (YPD). This is likely because the mutant culture is both hypercapsular and enriched in Titan cells.Titan cells are poorly engulfed by host phagocytes , 45, 73,2, as these conditions induce PDR802 expression dramatically compared to standard YPD growth conditions . We compared DNA sequences immunoprecipitated by anti-mCherry monoclonal antibody (MAb) from cells expressing mCherry-Pdr802, which grow similarly to the WT , and untnditions .10.1128/mBio.03457-20.7FIG S72 for the times shown, and samples were tested for their ability to form colonies on YPD medium. Plotted is the fold change in CFU relative to the initial culture. (B) Pdr802 self-regulation. The ratios (log2) of reads from immunoprecipitated (IP) DNA to reads from input DNA were calculated for 1,000 bp upstream of the first coding nucleotide (+1) of PDR802; shown is the difference in these values between tagged and untagged strains. Red triangles, complete Pdr802 DNA-binding motifs  The indicated strains were grown in DMEM at 37°C and 5% COg motifs ; blue trg motifs . PrimaryCopyright © 2021 Reuwsaat et al.2021Reuwsaat et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P values of <0.05, we identified 656 binding sites for mCherry-Pdr802 in genomic DNA. Of these, 540 occurred within 1,000 bp upstream of transcription start sites  data. Sheet 1, annotated peaks that occur in gene promoter regions that showed ≥2-fold enrichment when Chip-Seq was performed on strains expressing tagged versus untagged Pdr802. Sheet 2, ChIP-Seq primary data of Pdr802-specific peaks. Sheet 3, RNA-Seq data. Sheet 4, DTO filtered data. Sheet 5, DTO primary data. Sheet 6, ChIP-Seq data of all peaks in mCherry-Pdr802 samples. Sheet 7, ChIP-Seq data of all peaks in untagged (WT) control samples. Download Copyright © 2021 Reuwsaat et al.2021Reuwsaat et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pdr802 cells after growth for 24 h in DMEM at 37°C and 5% CO2 . We then used dual-threshold optimization (DTO) to analyze the RNA-Seq and ChIP-Seq data sets together. This statistical method allowed us to combine the evidence from binding and expression studies to converge on a set of direct and functional TF targets  or proteins that mediate its maturation and import . We found that Pdr802 positively regulates PQP1 and OPT1 gene expression  is induced. Our analysis showed that Pdr802 positively regulates expression of LIV3 and STB4, while it negatively regulates ZFC3 ..pdr802 cC. neoformans  values throughout are for the DTO results. Download Data Set S2, XLSX file, 0.02 MB.Pdr802 target analysis and primers used in this study. Sheet 1, Pdr802 regulated genes that are down-regulated during Titan cell formation in vitro . Sheet 2in vitro . Sheet 3in vitro . Sheet 4in vitro . Sheet 5Copyright © 2021 Reuwsaat et al.2021Reuwsaat et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ concentrations through the regulation of calcium transporters , which reinforces our hypothesis that Pdr802 acts primarily under host-like conditions. To further explore the relationship of Pdr802 and calcineurin, we compared published gene expression profiles of a calcineurin mutant  that negatively regulate capsule size  in the KN99α strain background (PDR802) and construction of a strain that expresses Pdr802 with N-terminal mCherry (mCherry-Pdr802) are detailed in the supplemental methods. For all studies, C. neoformans strains were inoculated from single colonies into YPD medium  and grown overnight at 30°C with shaking at 230 rpm before further handling as detailed below. To assess viability during growth in tissue culture medium, overnight cultures were washed with phosphate-buffered saline (PBS), diluted to 106 cells/ml in DMEM , plated (1 ml/well) in triplicate in 24-well plates, and incubated at 37°C and 5% CO2. At the indicated times, cells were mixed thoroughly, diluted in PBS, and plated on YPD agar  for assessment of CFU. To assess viability during growth in mouse serum, mouse blood was collected as described below in “Animal experiments”; YPD-grown cryptococcal cells were incubated in 100 μl of serum in 96-well plates for 24 h at 37°C and 5% CO2, and CFU were counted as described above.We previously reported the ckground  that wasckground . Complem4 cryptococcal cells. The mice were monitored and humanely sacrificed when their weight decreased to below 80% of initial weight or if they showed signs of disease, at which point organ burden was assessed. The lungs and brains were harvested, homogenized, diluted, and plated on YPD agar. The resulting CFU were enumerated, and survival differences were assessed by Kaplan-Meier analysis.All animal protocols were approved by the Washington University Institutional Animal Care and Use Committee (reference 20170131) or Comissão de Ética no Uso de Animais (CEUA) (reference 30936), and care was taken to minimize handling and discomfort. For survival studies, groups of five 4- to 6-week-old female C57BL/6 mice (The Jackson Laboratory) were anesthetized by subcutaneous injection of 1.20 mg ketamine and 0.24 mg xylazine in 120 μl sterile water and intranasally infected with 5 × 10C. neoformans overnight cultures were centrifuged , washed with sterile PBS, and resuspended in PBS to 1 × 106 cells/ml. Groups of three 4- to 6-week-old female C57BL/6 mice (Centro Multidisciplinar para Investigação Biológica na Área da Ciência em Animais de Laboratório [CEMIB]) were anesthetized as described above, intranasally infected with 5 × 104 cryptococcal cells, and monitored as described above. At set time points post-infection (see above), mice were sacrificed and fungal burden was assessed from organs (as described above) or blood (obtained by cardiac puncture). Organ burden was analyzed by Kruskal-Wallis test with Dunn’s multiple comparison post hoc test for each day post-infection.For timed organ burden studies, g for 15 min and then heat inactivated at 56°C for 30 min.To assess cryptococcal viability in mouse serum, 6 BALB/c mice were anesthetized with isoflurane and blood was collected from the retro-orbital space using a sterile capillary tube. Collected blood was incubated at 37°C for 30 min, and serum was isolated by centrifugation at 1,000 × 3 cells were incubated in mouse serum for 24 h at 37°C and 5% CO2. After incubation, cells were fixed in 4% paraformaldehyde and washed three times with PBS. C. neoformans cells were placed on glass slides and mixed with similar volumes of India ink, and the capsule was measured as previously described  were then plated in quadruplicate in the middle 32 wells of a poly-l-lysine-coated 96-well plate  and incubated at 37°C and 5% CO2. After 24 h, the cells were washed with PBS and incubated with 150 μl of a staining mixture  for 30 min at room temperature in the dark. The cells were washed again with PBS, fixed with 4% formaldehyde for 10 min at room temperature, and washed with PBS, and each well was refilled with 150 μl PBS. The cells were imaged using a BioTek Cytation 3 imager, which automatically collected 100 images per well in a grid pattern at the well center. Image files were prepared for analysis with GE Healthcare IN Cell Translator and assembled into .xdce image stacks for analysis with GE Healthcare IN Cell Developer Toolbox 1.9. Cell wall and capsule images were first filtered to remove background noise and border objects, and then cells were identified using shape-based object segmentation  followed by watershed clump breaking to prevent apparent connectivity caused by incomplete segmentation. Target linking was performed to assign each cell wall object to one capsule object based on known 1:1 pairing and location, generating a target set. Capsule and cell wall object diameters were calculated for each target set (hundreds to thousands per well), and the difference between measurements in each pair was defined as the capsule thickness. Data were normalized by the difference in capsule thickness between uninduced and induced WT cells, which were included in each experiment, and compared to data for hypercapsular (pkr1) (ada2)   and hypo) (ada2)  control PDR802 cells from mouse lungs after 18 days of infection, the tissue was treated with 50 μg/ml DNase I for 30 min at 37°C.To measure capsule thickness of cryptococcal cells grown in the lungs of infected mice, lung homogenates were filtered through a cell strainer with 40-μm pores using a syringe plunger, fixed in 3.7% formaldehyde, and used for India ink staining and measurement as described above. For the visualization of KN99α and 6 cells/ml were grown in DMEM for 24 and 48 h. Culture supernatant fractions were then resolved by gel electrophoresis on 0.6% agarose, transferred onto nylon membranes, and probed with 1 μg/ml anti-GXM antibody 302.GXM immunoblotting was conducted as previously described . Briefly7 cells/ml in PBS. Aliquots (3 μl) of 10-fold serial dilutions were spotted on YPD or YNB agar supplemented with various stressors  at the concentrations indicated in the figures. Melanization was tested on plates made by mixing 10 ml of 2× minimal medium  with 10 ml of 2% agar-water per plate. A control strain lacking the ability to melanize was used as a control (lac1) . Plates were incubated at 30°C or 37°C.For stress plates, cryptococcal cells were grown overnight in YPD, washed with PBS, and diluted to 10l (lac1) . For the2 as recently described  for 72 h at 37°C and 5% COescribed  and quanescribed , 48. Forescribed  with a fescribed .5 cells/well) in a 96-well plate and incubating in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2 for 24 h. C. neoformans cells were prepared for uptake experiments by inoculating an overnight culture in YPD into either DMEM or Titan cell induction medium  for 1 h at 37°C, while macrophages were activated with 50 nM phorbol myristate acetate (PMA) for 1 h at 37°C and 5% CO2; 106 cryptococcal cells were then incubated with the macrophages for 2 h at 37°C and 5% CO2. The wells were then washed three times with warm PBS, and the macrophages were lysed with 0.1% Triton in PBS and plated for CFU enumeration as described above. Fold change in numbers of CFU was assessed by comparison to the CFU of opsonized cells. One-way ANOVA with Dunnett’s multiple-comparison post hoc test was used to compare phagocytosis of pdr802 and PDR802 strains with that of KN99α.J774.16 cells were prepared for uptake experiments by seeding  tethered to protein A Sepharose (IP) or Sepharose alone overnight at 4°C. The beads were then washed, incubated at 65°C to reverse DNA-DNA and DNA-protein cross-links, and the DNA was recovered by phenol-chloroform-isoamyl alcohol (25:24:1) extraction, ethanol precipitation, and resuspension in nuclease-free water.ChIP studies were performed as previously described , 105. BrP value of <0.05, and read coverage of each peak was obtained using SAMtools  was isolated and sequenced as previously described  less than or equal to 20% at 80% sensitivity. In addition to requiring a statistically significant overlap between the ChIP-Seq and RNA-Seq gene sets, we filtered out any genes for which traditional differential expression analysis yielded an adjusted P value of ≤0.15 or absolute fold change of ≥0.3, leaving 380 bound targets.RNA from wild-type and escribed . Brieflyescribed . Fastq fescribed  using Noescribed , SAM filescribed . The numescribed , and difescribed , using tP values . Dual-thP values . This is2+, yeast cells were cultured overnight in YPD at 30°C with shaking, washed three times with deionized water, diluted to 106 cells/ml in DMEM , plated (1 ml/well) in triplicate in 24-well plates, and incubated at 37°C and 5% CO2 for 24 h. At the indicated times, cells were mixed thoroughly, diluted in PBS containing 2 μM Fluo4-AM (Thermo Fisher), incubated at 30°C for 30 min, and analyzed using flow cytometry. The overnight culture was used as a control and treated as above.To measure intracellular free CaChIP-Seq and RNA-Seq data files are available at the NCBI Gene Expression Omnibus under accession numbers GSE153134 and GSE162851, respectively.10.1128/mBio.03457-20.10TEXT S1Text S1, PDF file, 0.04 MB.Supplemental methods. Download Copyright © 2021 Reuwsaat et al.2021Reuwsaat et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


On the contrary, downregulation of PIK3R1 decreased levels of osteoblast-related genes and increased levels of osteoclast-related genes. Besides, in vitro experiments revealed that PIK3R1 facilitated proliferation and repressed apoptosis of osteoblasts but had an opposite impact on osteoclasts. In summary, PIK3R1 exhibits an osteoprotective effect via regulating osteoblast differentiation, which can be represented as a promising therapeutic target for osteoporosis.In an effort to bolster our understanding of regulation of bone formation in the context of osteoporosis, we screened out differentially expressed genes in osteoporosis patients with high and low bone mineral density by bioinformatics analysis. PIK3R1 is increasingly being nominated as a pivotal mediator in the differentiation of osteoblasts and osteoclasts that is closely related to bone formation. However, the specific mechanisms underlying the way that PIK3R1 affects bone metabolism are not fully elucidated. We intended to examine the potential mechanism by which PIK3R1 regulates osteoblast differentiation. Enrichment analysis was therefore carried out for differentially expressed genes. We noted that the estrogen signaling pathway, TNF signaling pathway, and osteoclast differentiation were markedly associated with ossification, and they displayed enrichment in PIK3R1. Based on western blot, qRT-PCR, and differentiation analysis  Osteoporosis is featured by low bone mineral density (BMD). Escalating global epidemic of osteoporosis, as a systemic skeletal disorder, because of its association with a serious structural deterioration of bone tissue correlated with risk of (fragility) fractures , in oldeOsteoclasts are specialized cells that resorb bone and thus are essential for normal skeletal development and remodeling . Unlike α, PIK3R1 accounts for 65-75% of intracellular regulatory subunits [PI3K, a heterodimer, consists of a regulatory subunit (p85) and a catalytic subunit (p110), and both of them are expressed in multiple isoforms . In the subunits . PIK3R1 subunits . Currenthttp://www.ncbi.nlm.nih.gov/geo). GSE2208 included 19 women   was utilized to fill the missing expression data, followed by logbase2 scaling. Finally, the limma packet  was normalized. Regarding several probes that only match one gene symbol, the average expression value of probes was computed and regarded as the conclusive gene expression value. For subsequent analysis, differentially expressed genes (DEGs) were obtained.GSE2208 (Affymetrix Human Genome U133A Array) was downloaded from the Gene Expression Omnibus  annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Thresholds for significant enrichment were Murine monocyte macrophages, RAW264.7, were obtained from the American Type Culture Collection . The culture condition was DMEM  with 10% fetal bovine serum , 100 U/ml penicillin, and 100 mg/ml streptomycin . For osteoclastogenesis, RAW 264.7 cells were treated with M-CSF (50 ng/ml) and RANKL (50 ng/ml) for 7 d. The medium was renewed 3 times (less than 48 h). Cells were cultured in a standard environment.α-MEM . All media were added with 10% FBS and 1% penicillin/streptomycin . Cells were incubated under routine conditions. To induce osteoblast differentiation, MC3T3-E1 cells were cluttered in an osteogenic induction medium containing the standard growth medium, supplemented with 50 mg/l ascorbic acid, 10 mmol/l β-glycerophosphate, and 10 nmol/l dexamethasone .Murine preosteoblast MC3T3-E1 cells were accessed from the Cell Bank of the Chinese Academy of Sciences  and maintained in For ALP staining, after formalin fixation (10%) and rinsing twice with PBS (pH 7.4), osteoblasts were maintained with ALP substrate solution, naphthol AS-MX , and fast violet B salt  in 0.1 M Tris-HCl (pH 8.5). In addition, ALP activity was assayed by a LabAssay ALP kit  after osteoblasts being rinsed twice with PBS and lysed in Mammalian Protein Extraction Reagent  per the specification. Besides, protein quantification using the BCA protein assay kit  was implemented under the manufacturer's protocol.For ARS staining, cells were rinsed twice with PBS and then fixed for 10 min in 2% formaldehyde, followed by 30 min of cell staining with 0.2% ARS solution at 37°C. Each well was rinsed 3 times with distilled water and pictured. The ARS red staining indicated calcium deposition.β-actin as an internal reference. Primer sequences are listed in The RNeasy Mini Kit  was utilized for RNA isolation. Complementary DNA (cDNA) was generated by reversely transcribing RNA on the SuperScript III First-Strand Synthesis System . qRT-PCR was conducted on the Step One Plus Real-Time PCR System  and Fast SYBR Green Master Mix , with μg total proteins in each sample were resolved by 10% SDS-PAGE, followed by being electrotransferred onto a PVDF membrane with a semidry blotter . Then, the membrane was sealed for 1 h with 5% nonfat dry milk and then incubated overnight with primary antibodies at 4°C, followed by incubating with a secondary antibody. Antibodies used for western blotting are listed in Proteins were extracted from osteoblasts and osteoclasts of mice. Homogenates were extracted in radioimmunoprecipitation assay buffer ; then, ultrasonic apparatus was used to complete cell lysis. Lysates were subject to 12000 rpm centrifugation at 4°C for 5 min. Subsequently. 50 μg envelope plasmid, 9 μg packaging plasmid, and 12 μg targeting plasmids into cells. 48 h later, lentivirus was obtained and filtered. The sh-NC and oe-NC were controls for sh-PIK3R1 and oe-PIK3R1, respectively.Stable knockdown (pLKO.1) PIK3R1 vectors were obtained from Vigene Biosciences . Lipofectamine 2000  was implemented to transfect short hairpin-PIK3R1 (sh-PIK3R1). 24 h later, cells were harvested and subjected to qRT-PCR and western blotting, so as to evaluate transfection efficacy. To generate overexpression-PIK3R1 (oe-PIK3R1) lentivirus, Lipofectamine 2000 was used to transfect 3.6 4 cells were seeded into a 96-well plate and 10 μl cell counting kit-8 (CCK-8) reagent  was added at 24, 48, 72, 96, and 120 h. A microplate reader  was utilized for quantification at 450 nm.After transfection, 1 × 10Cells were placed into 48-well plates after transfection. EdU reagent  was added to each well at 24 h. 2 h later, cells were subjected to 15 min of fixing with 4% formaldehyde and were treated with 0.5% Triton X-100 for permeabilization. Next, Apollo reaction mixture was dropped into each well and Hoechst 33258 was used for cell staining.4 cells were rinsed twice in PBS after transfection, followed by resuspension in 100 μl binding buffer. Next, 5 μl Annexin V-FITC and 10 μl PI were used for 15 min of staining with 50 μg/ml RNase A , followed by 1 h of cell incubation in an incubator in the dark. A flow cytometer  was used for cell assay. FlowJo software  was utilized for data analysis. The number of viable apoptotic cells was computed by calculating the percentage of Annexin V+/PI- cells. The number of nonviable apoptotic cells was computed by calculating the ratio of Annexin V+/PI+ cells. The number of necrotic cells was computed by calculating the proportion of Annexin V-/PI+ cells. Annexin V-/PI- cells are considered as viable cells [First, 2 × 10le cells .t-test or one-way analysis of variance (ANOVA) with Tukey's post hoc test was performed accordingly. The significant difference was considered when p < 0.05.All data were expressed as mean ± SD and processed by GraphPad Prism software v.8.0 (USA). All experiments were repeated 3 times independently, including 3 biological replicates and 3 technical replicates. The Student In an effort to probe DEGs in osteoporosis patients with high/low BMD, we analyzed data in the GSE2208 dataset. GSE2208 included 19 women . Through microarray data processing, 756 DEGs were identified from high and low BMD groups .Subsequently, DEGs were subjected to enrichment analysis. The results of GO annotation on 756 DEGs are depicted in Figures DEGs were then subjected to KEGG signaling enrichment analysis to reveal pathways for these DEGs. As presented in sh-PIK3R1 was transfected into osteoblasts to decrease PIK3R1 level Figures . ALP andIn addition, a specific lentivirus was used to upregulate PIK3R1 in osteoblasts, so as to validate its role in osteoblast differentiation. qRT-PCR and western blotting were performed to evaluate the efficacy of lentivirus Figures . ALP andCollectively, PIK3R1 stimulated the expression of osteoblast-related genes and had a promoting impact on osteoblast proliferation and differentiation.As such, PIK3R1 expression was silenced to investigate its role in osteoclast differentiation Figures . The mRNIn addition, PIK3R1 expression was upregulated to verify its role in osteoclast differentiation. Western blotting and qRT-PCR analyses were performed to examine lentivirus efficacy Figures . As depiBone is metabolically active and constantly undergoes remodeling via repetitive cycles of bone formation and bone resorption , 21. Funα suppressed osteoblast differentiation of mesenchymal stem cells and ossification in estrogen-deficiency-induced osteoporosis [α can induce apoptosis of osteoblasts [Currently, modern high-throughput technology identified a new candidate gene expression profile related to osteoporosis and bone formation , 24, 25.oporosis , 28. Apooporosis . The TNFoporosis . TNF-α ceoblasts  and syneeoblasts . FinallyPIK3R1 expression in osteoblasts and osteoclasts was explored, and ALP and ARS assays were conducted as proof of osteoblastic differentiation. The results showed that downregulation of PIK3R1 in osteoblasts noticeably decreased ALP and ARS activities while increased osteoclast-related protein levels. These findings demonstrated that PIK3R1 promoted differentiation of osteoblasts and hampered the formation of osteoclasts, consistent with previous studies. For example, Zhu et al.  proposedOverall, PIK3R1 acts as a regulator in bone formation through modulating osteoblast differentiation and osteoclast formation to maintain bone homeostasis, which could be represented as an encouraging therapeutic target for osteoporosis. Nonetheless, this study failed to probe into the mechanism by which PIK3R1 promoted bone formation. Further investigations are ongoing.


Pou5f1 (EcPou5f1) and Nanog (EcNanog) were cloned from orange-spotted grouper, Epinephelus coioides. The full-length cDNAs of EcPou5f1 and EcNanog were 2790 and 1820 bp, and encoded 475 and 432 amino acids, respectively. EcPou5f1 exhibited a specific expression in gonads, whereas EcNanog was expressed highly in gonads and weakly in some somatic tissues. In situ hybridization analyses showed that the mRNA signals of EcNanog and EcPou5f1 were exclusively restricted to germ cells in gonads. Likewise, immunohistofluorescence staining revealed that EcNanog protein was limited to germ cells. Moreover, both EcPou5f1 and EcNanog mRNAs were discovered to be co-localized with Vasa mRNA, a well-known germ cell maker, in male and female germ cells. These results implied that EcPou5f1 and EcNanog could be also regarded as reliable germ cell marker genes. Therefore, the findings of this study would pave the way for elucidating the mechanism whereby EcPou5f1 and EcNanog regulate germ cell development and gametogenesis in grouper fish, and even in other protogynous hermaphroditic species.Pluripotency markers Pou5f1 and Nanog are core transcription factors regulating early embryonic development and maintaining the pluripotency and self-renewal of stem cells. Pou5f1 and Nanog also play important roles in germ cell development and gametogenesis. In this study,  Nanos3 [Ly75 [Plzf [Slbp2 [Vasa, Dazl and Piwi [Germ cell development is indispensable for animal reproduction and fertility. The complex process from the formation of primordial germ cells (PGCs) to the differentiation into gametes is strictly regulated by many factors, such as hormones and reproduction-related genes ,2,3. GerNanos3 , mitotic75 [Plzf ,7, oocytf [Slbp2 , as welland Piwi ,10.Pou5f1, Sox2, Klf4, and C-myc are capable of reprogramming somatic cells into induced pluripotent stem cells (IPSCs) [Nanog promoter indicates that Pou5f1 and Sox2 are direct modulators of Nanog transcript [Homo sapiens) and mouse (Mus musculus), Pou5f1 and Nanog are mainly expressed in multipotent stem cells, such as embryonic stem cells (ESCs), IPSCs, PGCs, spermatogonial stem cells, and oogonial stem cells [Danio rerio), medaka (Oryzias latipes), and Japanese flounder , Pou5f1 and Nanog are also detected in the above-mentioned stem cells [Pou5f1 and Nanog participate in the differentiation and development of germ cells [Pou5f1 results in the developmental arrest of oocytes [Sus scrofa) testis [Cxcr4b which is the guider of PGCs migration [Pou5f1 and Nanog are restricted to oogonia, oocytes, and spermatogonia [Larimichthys crocea), Pou5f1 is expressed in spermatogonia and primary spermatocytes, as well as all female germ cells [Megalobrama amblycephala), Nanog is detected in all male germ cells and most female germ cells [Carassius auratus), the Nanog promoter is in a state of hypermethylation in oocytes, while hypomethylation in sperm [Pou5f1 and Nanog in germ cells indicate that they are functionally important in sustaining germ cell development and gametogenesis in mammals and fish.Pou5f1  and Nanog are closely related to each other and characterized as core transcription factors essential for embryogenesis and the maintenance of cell pluripotency ,12,13. P (IPSCs) . Nanog p (IPSCs) . The exianscript . Moreoveanscript . In humaem cells ,20,21. Iem cells ,23,24,25rm cells , and the oocytes . Intrigu) testis . In medaigration . In gonaatogonia ,31. Neverm cells . In blunrm cells . In goldin sperm . The expE. coioides) is a protogynous hermaphroditic fish whose gonad first develops as matured ovary and then reverses into testis, and therefore is regarded as an ideal model for studying the mechanism of sex reversal [Pou5f1 and Nanog have been characterized as germ cell-specific genes in some dioecious fish, but their expression patterns present a species difference to a variable extent [Pou5f1 (EcPou5f1) and Nanog (EcNanog) were cloned and characterized in orange-spotted grouper, and their expression patterns were analyzed during gametogenesis. These results would contribute to further studies on the mechanisms underlying PGCs formation and migration, germ cell development, and sex reversal in hermaphroditic fish.Orange-spotted grouper  before being sacrificed. Tissue samples were as follows: brain, pituitary, gill filament, heart, head kidney, kidney, muscle, liver, stomach, intestine, spleen, testis, and ovary. These tissues were collected from groupers for the subsequent experiments, including RNA extraction, protein extraction, and frozen section.EcPou5f1 and EcNanog were cloned from testis using SMARTer RACE cDNA Amplification Kit . Based on the cDNA fragments of EcPou5f1 and EcNanog in E. coioides genome data (unpublished data), specific primers were designed and listed in EcPou5f1 and EcNanog were spliced into full-length cDNA sequences. Amino acid sequences of EcPou5f1 and EcNanog were predicted and performed for multiple sequence alignments using DNAMAN version 7.0 . Phylogenetic trees were generated with MEGA version 5.0 .Full-length cDNA sequences of EcNanog was performed with KOD One PCR Master Mix-Blue (Toyobo), and PCR procedure was as follows: initial denaturation at 94 °C for 2 min, followed by 35 cycles of 5 s at 98 °C, 5 s at 60 °C, and 5 s at 68 °C, finally 68 °C for 5 min. After gel electrophoresis, bands were photographed with a Tanon 1600 image system . The β-Actin was used as an internal control. Genetic expression quantifications were normalized to β-Actin. Primers were listed in Total RNA of tissue was extracted with TRIzol reagent . RNA quality was evaluated by agarose gel electrophoresis. The cDNA was synthesized with 1 µg total RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover . Real-time quantitative PCR (RT-qPCR) was performed on a Roche LightCycler 480 System  with SYBR Green Realtime PCR Master Mix (Toyobo). Produce was as follows: 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C, 5 s at 58 °C, and 15 s at 72 °C, with final step for 15 s at 95 °C and 30 s at 60 °C. Semi-quantitative PCRs for about 240 bp DNA fragments were performed with Taq PCR StarMix , and PCR procedure was as follows: initial denaturation at 94 °C for 2 min, followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 15 s at 72 °C, finally 72 °C for 5 min. Semi-quantitative PCR for the 1299 bp open reading frame (ORF) of The monoclonal anti-Nanog antibody from medaka was provided by Professor Hongyan Xu . The totw/v) sucrose at 4 °C overnight, and embedded in OCT compound . These gonad samples were cryosectioned at 4 µm using a Leica RM2135 Microtome . Probes were synthesized using DIG RNA labeling kit . Lengths of EcPou5f1 and EcNanog probes were 1037 bp and 1299 bp, respectively. Ingredients of hybridization buffer were as follows: 50% deionized formamide, 5 × saline sodium citrate (SSC), 0.5 mg/mL salmon sperm RNA, 1 × Denhart’s solution, and 5% dextran sulphate. Gonadal sections were pre-hybridized with hybridization buffer for 2 h and then hybridized with 1 µg/mL DIG probes at 65 °C for 15 h. Subsequently, sections were washed with SSC and blocked with Blocking Reagent (Roche) for at least 1 h. Sections were incubated with anti-Digoxigenin-AP (Roche), then colored with NBT/BCIP (Roche). Photographs were imaged by a Leica DMI8 microscope (Leica).ISH protocol was described in our previous study . BrieflyVasa probe was 1040 bp and synthesized using Fluorescein RNA labeling kit (Roche). Gonadal sections were pre-hybridized for 2 h, then hybridized by 1 µg/mL Vasa probe and one of EcPou5f1 and EcNanog probes at the same time at 65 °C for 15 h. Subsequently, sections were washed with SSC and blocked with Blocking Reagent for at least 1 h. The Vasa probe was incubated with anti-Fluorescein-POD (Roche), then stained red fluorescence for 10 min with TSATM PLUS Fluorescein system . After being washed, the EcPou5f1 or EcNanog probes were incubated with anti-Digoxigenin-POD (Roche) and were stained green fluorescence for 10 min. Finally, gonadal sections were counterstained by DAPI (Solarbio) for cell nuclei staining. Sections were imaged with a Zeiss LSM 800 microscope  or a Leica TCS SP5 microscope (Leica).Dual-label ISH protocol referred to our previous study . The VasTM Plus Fluorescence System. The nucleus was colored by PI (Solarbio). Photographs were imaged by a Leica DMI8 microscope.Gonadal sections were dried in a drying oven and washed with PBS. After being blocked with 5% goat serum (Sangon) for 1 h, sections were incubated with the anti-Nanog antibody for 2 h (1:200 dilution in PBS containing 2% goat serum). After being washed with PBS, sections were incubated with HRP-conjugated goat anti-rabbit IgG (1:3000 dilution in PBS) for 1 h. Signals were developed using the TSAt-test. A probability level less than 0.05 (p < 0.05) was considered statistically significant. All statistics were carried out using GraphPad Prism version 5.0 .All data were shown as the mean values ± SEM. Statistical analysis was implemented by one-way ANOVA and Student’s EcPou5f1 was 2790 bp with a 1428 bp ORF, a 269 bp 5′-untranslated region (UTR), and a 1093 bp 3′-UTR, as well as encoded a predicted protein of 475 amino acids containing POUs and POUhD , for example, brain, can also express Pou5f1 [Nanog transcript [EcPou5f1 was not detected in those nongonadal tissues expressing the short DNA fragment or protein of EcNanog. We guess that other regulators, such as Sox2 and Klf4, and the auto-regulatory of Nanog might account for the absence of EcPou5f1 in nongonadal tissues [Pou5f1 and Nanog show species differences to a variable extent in diverse fish, and suggest that they play multifunctional roles in gonads and somatic tissues.n gonads . In zebrnd heart . On the d testis . We deduem cells ,43,44. S [Pou5f1 , EcPou5fs Pou5f1 ,45. Pou5anscript . Intrigu tissues ,47. In sEcPou5f1 and EcNanog are dependable germ cell-specific genes in orange-spotted grouper. EcPou5f1 was restricted to germ cells, but scarce in vitellogenic stage oocytes. Using Tyramide Signal Amplification system, EcPou5f1 signal was faintly observed in spermatids, suggesting that a few EcPou5f1 transcripts existed in spermatids. These ISH results indicate that EcPou5f1 exists exclusively in germ cells of gonads and would downregulate dramatically from spermatocyte to spermatid. In medaka and Japanese flounder, Pou5f1 is limited to spermatogonia, oogonia, and most oocytes, but absent in spermatocytes and spermatids [Pou5f1 is detected in spermatogonia and primary spermatocytes [EcNanog was detected in all male germ cells and the female germ cells from oogonia to early vitellogenic stage oocytes. A similar expression pattern is also observed for Nanog in gonads of blunt-snout bream [Nanog is only expressed in spermatogonia [EcPou5f1 and EcNanog may participate in spermatogenesis, whereas the Pou5f1 and/or Nanog of medaka, zebrafish, Japanese flounder, and Nile tilapia is mainly responsible for the pluripotency and self-renewal of spermatogonia. In accordance with the EcNanog ISH, EcNanog protein specifically existed in all male and female germ cells, including vitellogenic stage oocyte in which EcNanog mRNA was difficultly detected. EcNanog contained a motif of YKQVKTWFQN that had been identified as a nuclear localization motif in human Nanog [Vasa is a widely accepted germline-specific marker [Vasa is exclusively expressed in germ cells in gonads of orange-spotted grouper [Macaca mulatta), Nanog is restrictively expressed in all spermatogenic cells, most of which are Vasa-positive [Vasa was co-localized with EcPou5f1 and EcNanog in gonads. Furthermore, in ovary, the small oval cells expressing EcPou5f1 or EcNanog would be marked by Vasa signal, suggesting that these cells exactly were oogonia or oogonial stem cells. On the basis of these results, we conclude that EcPou5f1 and EcNanog are reliable germ cell-specific markers in orange-spotted grouper.ermatids ,30. In termatids ,49. Howeatocytes . EcNanogut bream . Howeveratogonia ,31,42. Tan Nanog . As expean Nanog ,42. Intean Nanog . The traan Nanog . Vasa isc marker ,52, and c marker  and mousc marker . Our prepositive . In humapositive . In thisPou5f1 and Nanog in differentiated germ cells in mammals [Pou5f1 and Nanog are mainly responsible for the pluripotency of germline stem cells [Nanog and Pou5f1 are expressed in spermatogonia, oogonia, and a large proportion of oocytes, as well as have different expression patterns in the differentiated male germ cells in diverse fish [EcPou5f1 and EcNanog can be detected in all spermatogonia and oogonia, most oocytes, spermatocytes, and spermatid. The high expressions of teleost Nanog and Pou5f1 during oocyte development may be related to maternal inheritance, i.e., mRNA or protein delivery to the next generation [Pou5f1 and Nanog may have some differences in promoting spermatogenesis in different fish. Moreover, the differentials among diverse fish would provide a new perspective for understanding the evolution and conserved nature of teleost Pou5f1 and Nanog.Although there have been a few studies about the functions of  mammals ,27,28, iem cells ,21,56,57rse fish ,33,42,49neration ,25,33. TEcPou5f1 and EcNanog are germ cell-specific marker genes and play important roles in gametogenesis in a protogynous hermaphroditic fish, orange-spotted grouper. Our findings would contribute to further studies on the molecular mechanism underlying germ cell development, gametogenesis, and sex reversal in hermaphroditic fish.In summary,


The objectives of the present work were sequencing of matK and rbcL, the in vitro establishment of recalcitrant rice embryogenic calli, the determination of their sensitivity to gamma radiation, and optimization of the generation procedure. All sequenced genes matched perfectly with previously reported matK and rbcL O. sativa genes. Embryogenic calli induction improved using MS medium containing 2 mg L−1 2,4-D, and regeneration was achieved with MS medium with 3 mg L−1 BA and 0.5 mg L−1 NAA. The optimized radiation condition was 60 Gy, (LD20 = 64 Gy) with 83% regeneration. An immersion system  of either 60 or 120 s every 8 h allowed systematic and homogeneous total regeneration of the recalcitrant line. Other well-known recalcitrant cultivars, CR1821 and CR1113, also had improved regeneration in the immersion system. To our knowledge, this is the first study reporting the use of an immersion system to allow for the regeneration of gamma-ray mutants from recalcitrant indica rice materials.The development of gamma ray-mutated rice lines is a solution for introducing genetic variability in indica rice varieties already being used by farmers. In vitro gamma ray ( Oryza genus consists of 22 species, but only two are commonly planted, namely, O. sativa and O. glaberrima [Rice is an important cereal that provides 20% of the world’s energy, particularly in Asia, Africa, and Latin America . The Oryaberrima ,4,5,6. Faberrima ,10,11,12Plant-tissue culture represents an opportunity to overcome time limitations, land requirements, and the selection of the desired variability that results when using gamma-ray mutagenesis in seeds. Once irradiated, the seeds must be planted several times from M0 = seeds prior to mutagenic treatment; M1 = plants produced from material treated with the mutagen, to subsequent generations termed M2, M3, M4, to avoid chimeras and heterogenicity until exposed to stress-selection conditions, as has been done in the past ,8. In co−1 2,4-D, resulting in pro-embryos and somatic embryos [−1 2,4-D [The latter is possible because plant cells are totipotent, which means that whole plants can develop from single cells . We prev embryos ,18,19. C−1 2,4-D .matK) and ribulose-1,5-bisphosphate carboxylase small/large subunit (rbcL). Second, the improvement of our plant-tissue culture methods. Third, determining the radiosensitivity of embryogenic calli and further plant regeneration. We present a simple method to induce mutations using gamma rays in embryogenic calli of a recalcitrant cultivar, with an alternative immersion method that allowed our material to generate homogeneous and predictable in vitro plants after irradiation.Here, we faced three challenges. First, the identification of Lazarroz FL rice line with molecular markers, the chloroplast maturase gene K (matK) and ribulose-1,5-bisphosphate carboxylase small/large subunit (rbcL), MZ558335 and MZ558334 sequences of Lazarroz FL showed perfect matches with the already published NCBI Oryza sativa indica demonstrated the identity of the non-irradiated material as expected. We detected three synonymous mutations in the rbcL sequence that are important for the characterization of the variety  and although it is a small value, for the next steps of regeneration represented a challenge and particularly in our final irradiation goal, which also triggers browning  was achieved with 2 mg L-D alone . In our browning .−1 were produced after 15 days of culture from the scutellum of mature zygotic embryos  gamma radiation on embryogenic indica rice calli was evaluated. A lethal dose (LD50) of the embryogenic calli was found to be 110 Gy, while the 20% lethal dose was 64 Gy, resulting in a 0 to 120 gray gradient exposure, with 200 calli per exposure  decreased significantly (p < 0.05) occurred at doses higher than 60 Gy (Compared to the non-irradiated control (0 Gy), a significant decrease in the sprouting of embryogenic calli was observed, as the dose increased from 0 to 80 Gy. Sprouting of embryogenic calli irradiated with gamma rays  provided predictable and homogeneous regeneration  in 60 s (100%) compared to regeneration on the best semi-solid medium (70%). Similarly, the sprouting rate was higher using the RITA® system independently of the immersion time compared to that obtained using the selected semi-solid medium  was evaluated. Our results showed no significant differences between the immersion time (60 vs. 120 s) on the regeneration capacity, sprouting and browning . Neverthd medium .Although, the immersion time (60 vs. 120 s) did not significantly affect the browning rate, and it was higher using an immersion of 120 s (97.56%) compared to 60 s (60.00%) and a semi-solid medium (9.52%) .The potential for a temporary immersion system to regenerate recalcitrant materials was also validated for the well-known recalcitrant cultivars CR-5272, CR-1821 and CR-1113 .60Co) mutagenesis in an embryogenic calli of a recalcitrant Costa Rican Lazarroz FL cultivar. The cultivar seems to be related to Southeast Asian rice cultivars based on its rbcL sequence pattern.The present work optimized in vitro gamma ray  with other cultivars, such as MR220, GNY-53, and JP-5 [The best calli induction was observed for MS with 2mg of 2,4-D and regeneration with MS with 0.5 mg ANA + 3 mg BA. The calli induction step with a 2 mg Liors [−1 . On the and JP-5 ,24.®, Saint-Mathieu-de-Tréviers, France) with either 60 or 120 s of immersion every 8 h, which achieved a predictable and more homogeneous regeneration response. The radiation dose with which to start mutagenesis was proposed as a lethal dose 20 at 60 Gy, as regeneration was not affected but remained as high as 80%. At 80 Gy, the regeneration fell to 30%, consistent with the oxidative damage provoked by radiation and corresponding to the lethal dose 30 of the embryogenic calli. We believe 60 Gy is an excellent condition to start mutagenesis, considering that in other in vitro plants, such as pineapple, potato, and banana, a 5–40 Gy dose of gamma irradiation was shown to be sufficient to produce variability [Regeneration was improved using a temporal immersion system  [2−), hydroxyl (OH−) radicals, and hydrogen peroxide (H2O2) [Optimization of embryogenic calli in our recalcitrant Lazarroz FL cultivar became a challenge while at the same time an opportunity compared to our previous results from CR-5272 for the following reasons First, the alternative immersion method allowed our material to generate homogeneous and predictable in vitro plants after irradiation. The technique enabled regeneration for our Lazarroz FL cultivar and other recalcitrant rice lines CR-1821 and CR-1113, and consequently, is a great potential tool for regenerating gamma radiation mutated materials. The constant liquid and airflow can dilute oxidative compounds and facilitate more homogeneous exposure to nutrients. We validated the results with recalcitrant cultivars CR-1821 and CR-1113, which showed a total regeneration rate in contrast to the null regeneration in the conventional semisolid method. Plant cultivars’ nonhomogeneous in vitro behavior is not fully understood, but recent discoveries provide insights into the genetic basis of the sucrose metabolism and calli browning. External phytohormones seem to trigger rice sucrose metabolism required for regeneration. The system appears to rely on the expression of endogenous cytokinin, auxin, and ABA signaling genes: ORYZA SATIVA RESPONSE REGULATOR 1 (ORR1), PIN-formed 1 (PIN1), and 1(LEA-1) . The expe (H2O2) . Tissue e (H2O2) .Second, we can develop the desired mutations in a cultivar that farmers are already using, leading to faster breeding and adoption of a derived improved cultivar. Rice traits associated with specific genes are well known, which paves the way for producing novel cultivars based on mutation of desired or required conditions, such as biotic and abiotic stress tolerance . We foreTM Tissue Kit Macherey-Nagel  was used for DNA extraction from 1 mg of on non-irradiated lyophilized leaf tissue of Lazarroz FL. Thermo Fisher K1071  was used for the subsequent PCR following the recommendations of the manufacturer. The primers used in this study are as follows: for rbcL, rbcLaf 5′ATGTCACCACAAACAGAGACTAAAGC3′ and rbcLar 5′GTAAAATCAAGTCCACCRCG-3′ or rbcLaf 5′ATGTCACCACAAACAGAGACTAAAGC3′ and rbcLr590 5′AGTCCACCGCGTAGACATTCAT-3′; for matK, matK-xf 5′TAATTTACGATCAATTCATTC-3′ and matKr 5′ACAAGAAAGTCGAAGTAT-3′. Briefly, the PCR master mix consisted of a mixture (50 μL) containing 1X Dream Taq Master mix Thermo Fisher , 20 μM of each primer, and 5 μL of DNA (50 ng/uL). The thermocycling program was 95 °C for 5 min, 40 cycles at 95 °C for 45 s, 55 °C for 45 s and 72 °C for 1 min, and a final cycle of 72 °C for 7 min.A NucleoSpinv/v) NaOCl for 10 min each with constant agitation, using 10 mL of disinfectant solution for every gram of seeds. After the first and final incubation, the seeds were washed six to seven times with distilled sterilized water and were cultured in media composed of mineral salts and vitamins as described by Murashige and Skoog (MS), with 20 g L−1 sucrose and 0.1 g L−1 hydrolyzed casein. Treatments consisted of supplementing the basal medium with one of the following combinations of plant growth regulators: (i) 2.5 mg L−1 2,4-dichlorophenoxyacetic acid  was used as a control, (ii) 2.0 mg L−1 2,4-D, (iii) 1.0 mg L−1 2,4-D + 1.0 mg L−1 6- benzyladenine (6-BA), (iv) 2.0 mg L−1 2,4-D + 1 mg L−1 6-BA, and (v) 2.0 mg L−1 2,4-D + 0.25 mg L−1 1-phenyl-3-urea   were added as a gelling agent. All previously mentioned chemicals were supplied by Phytotechnology Laboratories® . An autoclave (1.2 ATM. cm−2 and 121 °C for 30 min) was used for the sterilizing medium and further dispensed on 94 × 16 mm vented polystyrene Petri dishes  in a laminar flow chamber. For each treatment, at least 900 seeds were cultured after surface sterilization. Cultures were maintained in the dark at 26 ± 2 °C. Calli induction and browning rates  were recorded as response variables and analyzed in a completely randomized design with a generalized linear model with a Poisson distribution and logit link function. Post hoc analysis consisted of an Honest Significant Difference test on IBM SPSS version 27 [For initial assays, the palea and lemma of rice caryopsis of a commercial local indica cultivar were removed with No. 80 grit sandpaper. The obtained seeds were surface-sterilized as previously reported  through on, TDZ) . After a−1 sucrose and 0.3 g L−1 hydrolyzed casein. Treatments consisting of supplementing the basal medium with variations of growth regulators, and the control was supplemented with 0.5 mg L−1 α-Naphthaleneacetic Acid (NAA) + 3 mg L−1 6-BA, while the second treatment was supplemented with 0.5 mg L−1 NAA + 0.5 mg L−1 6-BA and the third treatment was supplemented with 0.5 mg L−1 NAA + 1.5 mg L−1 Kinetin (KIN) and a fourth treatment consisted of supplementing basal medium with 0.5 mg L−1 NAA + 0.5 mg L−1 TDZ. After adding growth regulators, pH was adjusted to 5.8 with 1 N NaOH or 1 N HCl and 5.4 g L−1 Gelzan ®  were added as gelling agent. All previously mentioned reagents were supplied by Phytotechnology Laboratories® . After a properly dissolving of gelling agent, 60 mL of media were dispensed on 475 mL polypropylene WNA Deli Containers, and afterwards, media were sterilized at 1.2 ATM.cm−2 and 121 °C for 30 min. For each treatment, 6 replicates of 7 calli were cultured on a factorial design. Factor 1 consisted of induction media and factor 2 was regeneration media. Cultures were maintained at an irradiance of 72 µmol s−1m−2, a 16 h light/8 h dark photoperiod was used and 26 ± 2 °C for a period of 4 weeks. Calli showing mature coleoptilar germinated embryos were determined as regenerated, calli with completely differentiated plantlets greater than 1 cm were categorized as sprouted, and necrotic calli with a dark coloration were identified as browning calli. These response variables were analyzed with a generalized linear model with a Poisson distribution and logit link function. Post hoc analysis consisted of an Honest Significant Difference test on IBM SPSS version 27 [Embryogenic calli obtained from each induction medium were transferred to different regeneration treatments. Basal regeneration medium was similar to that described by Sudhakar et al. , and wasrsion 27  and diff−1 NAA + 3 mg L−1 6-BA, selected after previous experiments were analyzed. Culture conditions were the same indicated above and after 4 weeks lethal doses were calculated using probit analysis on IBM SPSS version 27 [Embryogenic calli irradiation was achieved with a gamma irradiator Ob-Servo Ignis type with 24 cobalt 60 source pencils . To determine radiosensitivity and the median lethal dose (LD50), calli were irradiated at 0, 40, 60, 80, 100, 120 Gy. Ten repetitions, and 20 embryogenic calli per exposure were used. The survival rate was recorded after calli were transferred to the regeneration medium composed by basal regeneration medium supplemented with 0.5 mg Lrsion 27  based on®  temporary immersion system regeneration of the embryogenic calli consisted of 200 mL regeneration media previously described in 4.3, with 23 four-week-old calli per unit, different rice cultivars . Light and temperature culture conditions remained unchanged, with 60 or 120 s immersion used as treatments, every eight hours. After four weeks of culture, the variables evaluated were regeneration, sprouting, and browning, green area calculations with ImageJ version 1.52p [The RITAon 1.52p .Rice tissue culture is a tool for conventional and modern breeding, but is limited to the genotype response, particularly during the regeneration of recalcitrant varieties. Our results collected using an immersion system helped to overcome such difficulties and allowed for the induction of gamma-ray mutants. A temporary immersion system seems to help overcome calli browning while allowing the tissue to recover and consequently presenting a more efficient method. We foresee that having access to such methods could diminish the time to trigger innovation and focus on selecting mutants with desired traits in commercially used varieties.