In addition to its well-established immunosuppressive actions, tryptophan 2,3-dioxygenase (TDO) appears to elicit direct effects on tumor cell function. Although TDO has been associated with cancer stemness, its involvement in melanoma stem cell biology remains largely unknown. Since we showed that by upregulating TDO, dexamethasone (dex) promotes proliferation and migration of SK-Mel-28 human melanoma cells, we sought to investigate dex effects on melanoma spherogenesis and stemness, and whether these events are mediated by TDO. We demonstrate here that dex significantly upregulates TDO in A375, a more aggressive melanoma cell line, confirming that dex effects are not limited to SK-Mel-28 cells. Moreover, dex stimulates spherogenesis of both cell lines, which is mediated by TDO, evident by its suppression with 680C91, a TDO inhibitor. The formed melanospheres appear to be enriched with embryonic stem cell marker mRNAs, the expression of which is potentiated by dex. Expression of cancer stem cell markers  was significantly increased in A375 spheres, as detected by flow cytometry. Taken together, our results suggest that TDO could represent a promising target in the management of melanoma and that dex, routinely used as a co-medication also in advanced melanoma, may stimulate melanoma cell function/tumor-supporting properties, a rather debilitating and undesired side effect. Degradation of tryptophan (Trp) into kynurenine (kyn) via the kynurenine pathway (KP) is known to play an important role in cancer progression . Indeed,TDO is constitutively expressed in malignant glioma and triple-negative breast carcinoma, and its upregulation has been associated with immune escape . RecentlThe use of GCs for the treatment and management of several cancers has been well established, particularly in selected combination therapy regimens. However, the ability of GCs to modulate cancer progression is quite controversial. While GCs are indeed routinely used to induce apoptotic cell death in hematological malignancies  or to alRecently, it was reported that dex may decrease the efficacy of chemotherapy by virtue of its ability to induce a stem cell phenotype in ovarian cancer cells, thus promoting cancer progression . In thisd-glucose DMEM, with 10% (v/v) heat inactivated fetal bovine serum , 100 U/mL penicillin, 100 μg/ml streptomycin and 2 mmol/L glutamine  in a humidified chamber with 5% CO2 in air. The culture medium was changed every 2 days.SK-Mel-28 and A375 , human metastatic melanoma cell lines, were grown in high 2) for SK-Mel-28 and 7600/well (2000 cells x cm2) for A375 in selective serum-free Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium supplemented with 1X B27 supplement , human recombinant epidermal growth factor  and basic fibroblast growth factor  . The number of melanospheres was followed up to 5 days, and the sphere-forming efficiency (SFE) was calculated after 5 days as percentage (%) of spheres formed, by counting the number of spheres in the entire well using an inverted microscope with a 10x objective and dividing them by the total number of seeded cells and multiplied by 100.Melanospheres were generated from A375 and SK-Mel-28 human melanoma cells as previously reported with few modifications . Cell cuParental melanoma cells and melanospheres were labelled and analyzed by direct immunofluorescence staining with a panel of monoclonal antibodies: CD133-phycoerythrin (PE) , CD44-allophycocyanin (APC), , anti-hGD2  . Isotype-matched antibodies were used as negative control and dead cells were excluded by 7-amino-actinomycin D  . Results® kit with the Agilent 2100 Bioanalyzer . Two hundred ng of total RNA from samples obtained as described above were retro-transcribed using iScript  in 20 µL of total volume, and amplified with specific primers listed in ® Green Supermix  according to manufacturer’s instructions using a RotorGene 3000  instrument. In the present analysis, 18s rRNA was confirmed to be stable and was used as the internal control. RT-qPCR was performed using the following procedure: 98°C for 2 min, 40 cycles of 98°C for 5 s, 60°C for 10 s. The program was set to reveal the melting curve of each amplicon from 60 to 95°C with a read every 0.5°C.Total RNA was isolated from SK-Mel-28 and A375 melanoma cell lines by means of NucleoSpin Total RNA kit according to the manufacturer’s protocol . RNA was quantified by Qubit™ 3.0 Fluorometer . After quantification, total RNA was analysed to determine the quality of samples by the Agilent RNA 6000 Nano LabChip® Green PCR kit and specific miRCURY LNA miRNA PCR Assay for miR-200c and miR-155-3p. The cycle was set at 9°C for 15 s, 55°C for 30 s and 70°C for 30 s, repeated 40 times. RNU-6B was used as an endogenous control. Relative expressions between miRNAs and RNU-6B were calculated by using a 2−ΔΔCt formula.For miRNA-specific cDNA synthesis, total RNA was reverse transcribed by using the miRCURY LNA RT kit (Qiagen). Quantitative real-time PCR amplification and analysis were performed using the Rotor-Gene Q thermal cycler (Qiagen), the miRCURY LNA SYBR4 cells) in high d-glucose DMEM with 10% FBS into LabTek Slides Chamber and incubated in a 5% CO2 atmosphere at 37°C for 24 h. Fresh medium with low serum concentration (1% FBS) was then added, and cells were stimulated with dex every 3 h up to 12 h. Double immunofluorescence analyses were performed on cells after fixation with cold acetone for 5 min. Non-specific binding sites were blocked with 10 mg/ml bovine serum albumin in PBS for 1 h at room temperature, with 0.2% triton X-100 (Sigma-Aldrich). A primary antibody , polyclonal rabbit anti-human IDO1  or monoclonal anti human-TDO ) was then added overnight at 4°C, followed by washing. Then a 2 h incubation at room temperature with a secondary goat anti-rabbit or anti-mouse antibodies, conjugated with FITC AF488 (green fluorescence) . The signal was amplified with anti-FITC Fluorescein/Oregon green antibody for 1 h 30 min  at room temperature. Nuclei were labelled with Hoechst 33342 . Omission of primary antibodies was used as negative controls. Slides were mounted with Fluoromount  and examined with Leica DC200 microscope digital color camera and Leica DC Viewer software.A375 were seeded . Parametric data were reported as means ± SEM and differences between groups were tested with ANOVA test (followed by Bonferroni’s and Dunnett’s Multiple Comparison Test). For comparisons between two groups, we used Student’s unpaired p < 0.001)  . Convers< 0.001) .This increase in mRNA expression was concomitant with increased protein expression. Quantitative analysis showed significant increase in the immunofluorescent signal of TDO but not AhR or IDO1 . Dex-indMelanoma progression is associated with the expression profile of miRNA-200 family . Interesin vitro (Cancer stem cells (CSCs) are pivotal for cancer initiation and recurrence, and several studies have demonstrated that melanoma stem cells are crucial for both initiation of the disease and metastasis. Since sphere formation assay (SFA) is the gold standard assay to evaluate self-renewal and differentiation of stem cells at the single-cell level in vitro , we invein vitro , while Ain vitro .p < 0.05). Interestingly, TDO inhibition did not affect sphere formation of unstimulated cells, but it significantly abolished the effect of dex in both melanoma cell lines , and a significant increase of Klf4  compared to parental cells. However, spheres derived from parental cells that had been stimulated with dex exhibited a higher increase in all stem cell markers. Indeed, the mean fold increase was 7.18 ± 3.4, 16.4 ± 6.1, 7.25 ± 4.0, 7.06 ± 2.1, 13.91 ± 7.7, 22.8 ± 5.6 for Ap-1, NANOG, Klf4, c-Myc, Sox2, Oct3/4, E2f1, respectively  . In unstectively . Importaectively . InteresIn parental A375, basal expression of stem cell markers mRNAs was relatively undetectable. However, treatment with dex causes a significant increase in all stem cell markers, except Klf4. Indeed, there was a 19.6 ± 1.7, 75376 ± 884, 84.07 ± 2.6, 33.76 ± 2.6, 2505 ± 242, and 170.2 ± 5.31-fold increase in AP-1, NANOG, c-Myc, Sox2, Oct3/4 and E2f1, respectively . Like inIt has been reported that the presence of CD133 or the ganglioside GD2 is associated with increased stemness in many cancers . HoweverAfter 5 days in low attachment and serum-free medium conditions, melanoma stem-like subsets  showed high levels of CD44 expression, similar to that of the parental cells . While iIt was not until recently that the role of TDO in cancer was appreciated, when emerging evidence showed that TDO, but not IDO1, is the central Trp-degrading enzyme in human glioma cells, and that the TDO–AhR pathway was associated with malignant progression and poor survival for this tumor . Owing tMore attention has been given to TDO’s role in tumor progression as a result of the interesting finding that glucocorticoids (GCs) increase its expression. GCs, particularly dexamethasone, are routinely used to induce cancer cell death, or to mitigate the undesirable side effects of chemotherapy . It was We previously demonstrated that dex stimulates SK-Mel-28 growth and migration via TDO upregulation. Here, we wished to investigate the possible involvement of dex in spherogenesis and stemness of SK-Mel-28, and the more aggressive A375 melanoma cell line. We also sought to determine whether TDO plays a role in dex-modulated spherogenesis. This becomes increasingly important since cancer stem subpopulations play critical roles in malignancy, making it imperative to better understand the role of dex in melanoma.Here, we showed that dex upregulates mRNA and protein levels of TDO in A375 cells, demonstrating that dex effects are not only restricted to SK-Mel-28 cells. Furthermore, this dex-mediated effect appears to involve repression of miRNA-200c, a player known to target TDO2 in breast carcinoma cells, resulting in reduced production of the immune-suppressive metabolite kynurenine . By suppOur present data could suggest a possible connection between dex treatment and melanoma cell survival or aggressiveness. The pharmacology of glucocorticoids is rather complex and is the subject of ongoing studies that seek to better understand their role in tumorigenesis. Apparently, glucocorticoids may control cell proliferation, migration, and survival by influencing miRNA expression. Unfortunately, however, the effects of dex on cancer cell treatment are still controversial, partly because the effects of dex on the expression of certain miRNAs and downstream targets is itself inconsistent . Thus, fCancer stem cells play increasing roles in promoting tumorigenesis, and thus, targeting them may prove essential in the fight against cancer. To this end, we assessed the involvement of dex and TDO in cancer stemness. Our results demonstrate, for the first time, that dex stimulates the formation of melanospheres in a TDO-dependent mechanism. In Pham’s study , TDO wasTo the best of our knowledge, this is the first report to show that dex can promote melanoma stemness. This underscores the potential involvement of dex in growth, survival and heterogeneity of melanoma cell. Pluripotency genes of stem cells are known to initiate tumorigenesis by promoting a highly proliferative and aggressively metastatic phenotype.A key player in melanoma is E2f1, a member of the E2f family of transcription factors involved in cell cycle regulation . IncreasOct4, NANOG and Sox2 are transcription factors that work in concert to maintain ESC pluripotency and self-renewal . Oct4 ex+ fraction in SK-Mel-28 spheres, a significant increase was observed in A375-derived spheres since they represented 35.7% of cell population. This data could highlight the heterogeneity of melanoma cell lines and may account for the more aggressiveness of A375 compared to SK-Mel-28 (+ isolated cancer stem cells exhibit increased stem cell markers, such as NANOG and Oct3/4, and possess tumor-initiating potential, compared to CD133- cells (A role for CD133 expression in cancer has been suggested. In this study, we showed that CD133 was expressed in less than 1% of parental A375 and SK-Mel-28 cells, as reported in previous studies . While tK-Mel-28 . Indeed,3- cells .Gangioside GD2 is a sialic acid-containing glycosphingolipid, expressed on different types of tumor cells including melanoma cells . GD2 wasTaken together, our results highlight a direct role of TDO in cancer cell property, besides its well-known immune-mediated capacity. As such, targeting TDO could have a double impact on cancer growth inhibition: a direct effect on cancer cells and an indirect one on immune escape. Our data support the notion that TDO’s expression is regulated in an autocrine-paracrine way by dex stimulation. Dex, indeed, upregulates TDO mRNA and protein, the latter required for the acquisition of stem cell features by SK-Mel-28 and A375 cells, that are prevented by 680C91 treatment . Our datOur results provide evidence that TDO may have an important role in melanoma progression. Moreover, dex modulates growth and stemness of melanoma cells, which indeed corroborates clinical evidence suggesting a detrimental effect of systemic steroids during immunotherapy . Several


Obtained ceramic materials crystallize in scheelite-type structure with space group I41/a. Morphological features and grain sizes of powders under study were investigated by SEM technique. Spectroscopic studies within the UV-vis spectral range were carried out to estimate the direct band gap (Eg) and Urbach energy (EU) of obtained powders. EPR studies confirmed the existence of two types of magnetic objects, i.e., Mn2+ and Gd3+ ions, and significant antiferromagnetic (AFM) interactions among them.New Gd D,8. D4 fo3+)-doped molybdates and tungstates have been used extensively mainly in optoelectronics as efficient phosphors, lasers, and scintillators [Rare earth  has tremendous importance, because of its high spin magnetic moment due to seven unpaired electrons (8S7/2). Therefore, materials doped with Gd3+ ions can be applied in magnetic resonance imaging [3+ is responsible for the application of these materials in X-ray computed tomography [Gadolinium, one of the rare earth metal families, plays an important role in chemistry and biomedicine. It can be applied simultaneously to magnetic resonance imaging (MRI), X-ray computed tomography (CT), and neutron capture therapy for cancers. Gadolinium ion Mo(W)O4 by trivalent RE3+ ones leads to the formation of vacant sites in these matrices (denoted as ▯) what is very well observed by many spectroscopic techniques. Above mentioned RE3+-doped scheelite materials revealed very strong luminescence both in visible and NIR spectral ranges that could be suitable for ultra-short pulses lasers and efficient phosphors [3+-doped scheelite-type molybdates and molybdato-tungstates provided much important information on their properties and potential applications [n-type conduction not extending 2·10−9 S/m and a direct (or indirect) band gap energy higher than 3 eV [3+-doped micro- and nanomaterials with metallic aluminium and silver as intrinsic electrodes make them suitable for LTCC (Low-Temperature Co-fired Ceramic) technology [x1−3▯xGdx2(MoO4)x1−3(WO4)x3 where 0 < x ≤ 0.1774 exhibit electrical relaxation processes [Our research group has been conducting studies on new doped micro- and nanomaterials of scheelite-type structure for many years ,20,21. Whosphors ,23. Dielications ,23. Theshan 3 eV ,25,26,27Doping is a common procedure to optimize materials for specific industrial applications. The combination of several dopants  is a very promising way of enhancing many properties of materials such as photoluminescence, and magnetic and dielectric properties.3+- and Mn2+-co-doped scheelite-type calcium molybdato-tungstates using two different synthesis methods. Their structure, morphological features, and thermal stability were systematically investigated and analyzed. The optical and magnetic properties of these materials were compared as a function of both dopant’s concentration.For this reason, in this work, we have obtained new Gd4 and CaMnGdMoWO samples with different Gd3+ ions content, i.e., when 0 < x ≤ 0.2500 and y = 0.0200 as well as with different Mn2+ concentration, i.e., when 0 < y ≤ 0.0667 and x = 0.1667 obtained by solid-state reaction method and combustion route are shown in 2Θ angle with increasing Gd3+ content (2Θ angle with increasing Mn2+ concentration (2Θ with the (112) and (103) diffraction lines are presented in I41/a . 4 and doped samples. To observe the changes of unit cell parameters clearly, cell constants (a and c), lattice parameter ratio c/a as well as the volume of unit cell (V) of CaMnGdMoWO materials obtained by solid-state reaction method and combustion route as a function of Gd3+ concentration are shown in a and c lattice parameters are not identical  Å  or 5.2374740(8) Å  and then, its value decreases and then increases again with further increasing content of gadolinium ions. The c parameter shows a different relationship, i.e., for low Gd3+ ions concentrations its value decreases to 11.4173(7) Å  or 11.4188(9) Å . With a further increase of Gd3+-doping, the value of c parameter increased and then decreased (high-temperature sintering) or only increased (combustion route). Generally, the unit cell volume tends to increase with increasing Gd3+ ion concentration, i.e., practically within the whole homogeneity range of CaMnGdMoWO solid solution an expansion of crystal lattice is observed  > ionic radius of Gd3+ (1.053 Å) > ionic radius of Mn2+ (0.96 Å) for CN = 8) [4  Mo6+ and W6+ ions are tetrahedrally coordinated by oxygen ones and their ionic radii are very similar, i.e., they are 0.41 and 0.42 Å, respectively [6+ ions with W6+ ones in scheelite-type structure does not cause significant changes in both lattice parameters of materials. The crystal lattice expansion observed for CaMnGdMoWO materials when Gd3+ ions concentration was increasing is probably caused by a strong defect of their structure, i.e., when three crystallographic positions of calcium ions are occupied by two Gd3+ ones only and an appearance of cationic vacants takes place. An analogous phenomenon in other doped materials, e.g., RE3+-doped CdMoO4  [3+ and Mn2+-co-doped calcium molybdato-tungstates [c/a was also calculated. This parameter evidently changes nonlinearly with increasing of Gd3+ ions concentration  . In scheectively . Therefo Eu, Dy) , Eu3+ anngstates  have alrntration . This mea and c) as well as the volume of a tetragonal unit cell of CaMnGdMoWO ceramics decrease with increasing Mn2+ concentration when Gd3+ ions content in these samples is constant  and variable Gd3+ ions content (0 < x ≤ 0.2500), calculated lattice parameters and volume of unit cell obey the Vegard’s law, i.e., they are nearly linear functions of y concentration parameter.Moreover, we have found that the lattice parameters  and CaWO4 (scheelite) melt congruently at 1480 and 1590 °C, respectively [3+ and Mn2+-co-doped calcium molybdato-tungstates are lower than the melting point of calcium molybdate and they gradually decreased with both increasing of Gd3+ and Mn2+ ions contents. These temperatures were determined as: 1430 °C (x = 0.0050 and y = 0.0200), 1290 °C (x = 0.1667 and y = 0.0200), 1260 °C (x = 0.2500 and y = 0.0200), and 1260 °C (x = 0.1667 and y = 0.0667).Our earlier studies showed that both calcium scheelites, i.e., CaMoOectively . The melx = 0.1667 and y = 0.0200) obtained by two different routes, i.e., high-temperature sintering  exhibit an optical absorption spectrum governed by a direct transition, i.e., an electron located in a maximum-energy state in a valence band achieves a minimum-energy state in a conduction band under the same point in the Brillouin zone [Generally, there are two types of optical transition that can occur at the fundamental edge of crystalline materials: direct and indirect transitions ,32,33,34uin zone ,32,33,34g) of CaMoO4 and CaMnGdMoWO materials was determined by the method proposed by Kubelka and Munk [g values. The UV-vis absorption spectra of CaMoO4 and CaMnGdMoWO ceramics obtained by solid-state reaction as well as combustion methods are shown in 2– → W6+, O2– → Mo6+ as well as O2– → Mn2+ charge transfer bands [3+ has half-filled 4f orbitals ; therefore, it is expected that the intensity of absorption bands due to 4f-4f transitions would be very weak. All of these bands should be observed in the very high-energy part of the spectrum, i.e., within the UV region (195–310 nm) [Optical energy gap  ,26. So, h is the Plank constant, ν is the light frequency, A is a proportionally coefficient characteristic of each material, and n is a constant associated with electron transition type [n = ½ [αhν)2 curve of each material to the photon energy axis as it is shown in g valuesE are given in g valuesE with Gd3+ ions concentration (when y = 0.0200) of materials under study. It was observed that the optical band gap of CaMnGdMoWO samples decreased when Gd3+ concentration increased up to x = 0.2222 (solid state reaction method) or x = 0.1667 (combustion synthesis) and it reached the lowest values, i.e., 3.45 and 3.98 eV, respectively, whereby, the g valuesE values determined for the materials with smaller grain size were clearly higher. We have already observed an analogous relationship for other nano- and micromaterials, i.e., Cax1−MnxMoO4 and Cax1−Mnx(MoO4)0.50(WO4)0.50 solid solutions [0.95Mn0.05MoO4 and Ca0.95Mn0.05(MoO4)0.50(WO4)0.50 microcrystals was 3.76 and 3.85 eV, respectively [g valuesE value, i.e., Mn2+-doped calcium molybdate—4.18 eV and Mn2+-doped calcium molybdato-tungstate—4.07 eV [When a structure of a band gap is parabolic, the absorption coefficient and optical band of materials can be determined using the Tauc relation ,38,39: and x > 0.1667 , direct band gap systematically increases. This means that defects in the CaMnGdMoWO crystal lattice are ordered. This structure is rearranged so as to be stable and regular. On the other hand, the band gap values determined for CaMnGdMoWO samples, in which the Mn2+ ions concentration changed, non-linearly decreased with increasing concentration of these ions  [α is an experimentally determined absorption coefficient and α0 is a constant characteristic of the material. Due to the correlation of the band gap to the tailing of band edges, gE values reduce as UE values increase. Hence, these are complementary to each other. The Urbach energy is estimated by plotting ln(α) vs. hν and fitting the linear portion of the curve with a straight line [UE value. The Urbach energy values determined for CaMoO4 and CaMnGdMoWO samples are collected in UE variation as a function of Gd3+ (x) or Mn2+ (y) concentration. As is clearly seen from these figures, the optical band gap values change opposite to the degree of disorder in the structure of CaMnGdMoWO materials. As a result, both a decrease in the optical band gap and the broadening of the Urbach tail occurred. The lowest value of gE (3.45 eV) was observed when the Urbach energy reached its highest value, i.e., 541 meV for CaMnGdMoWO sample when x = 0.2222 and y = 0.0200.Hybridization properties of doping materials cause lattice strain and its defects, e.g., Mn rgy (EU) ,44,45,46ght line ,44,45,46-2+ contribution, i.e., y = 0.0200),obtained using solid-state reaction method  and,obtained using solid sta-te reaction method .obtained using combustion synthesis  experiment. Considering the doping metallic ions and the used synthesis method, CaMnGdMoWO samples were divided into the following three groups:2+ doping and varied gadolinium ions content, denoted by the x parameter. As can be seen, EPR spectra revealed the existence of multi-line agreement of manganese signal centered at c.a. 340 mT. This signal is clearly visible for samples with lower gadolinium ions content (x = 0.0050), where only six narrow lines of Mn2+ paramagnetic ions appear  only a wide resonance line of Gd3+ ions is observed, as a result of the crystal field (fine) interaction of S = 7/2 electronic spin of gadolinium.Changes in the observed EPR signal is caused by the coexistence of magnetic Mn3+ ions on the Mn2+ magnetic system originates not only from the paramagnetic nature of three-valent gadolinium ions. As we mentioned in our previous work, where comparable scheelites Ca1−3xy−Mny▯xEu2x(MoO4)1−3x(WO4)3x were studied [2+ is visible also under coexistence with non-magnetic Eu3+ ions. This mechanism was explained by charge non-equilibrium between di- and three-valent ions where the creation of additional electronic vacancies (▯x) takes place. An increasing number of electronic vacancies have an influence on nominally isolated manganese ions, leading to the creation of complex magnetic arrangements. Disturbance of the magnetic system is reflected in the evolution of the Mn2+ EPR spectra where, at a significantly high x value, only a wide overleaped resonance line is observed. Thus, as we believe, a similar mechanism is responsible for the evolution of manganese resonance signal in present materials. Overleaping of the manganese line leads to circumstances, where the gadolinium EPR signal dominates in the overall spectrum at a high x value.But influence of Gd studied , disturbI= C/(T-θ), where I is the integral intensity of the resonance signal. Calculated values of the θ parameter are negative in all cases, indicating an antiferromagnetic (AFM) interaction among responsible magnetic centers: Mn2+ and Gd3+. The negative sign of magnetic interactions could be visible in a graph with the relation of I·T product as a function of temperature  power of magnetic interactions in CaMnGdMoWO materials.Detailed calculations of 3+ ions. Such a situation was clearly observed in the second group of GdMnGdMoWO materials, i.e., samples with fixed gadolinium ions content when x = 0.1667. The spectra shown in 2+-doping. Only for the sample with the smallest manganese concentration, i.e., when y = 0.0066, the trace of the Mn2+ signal is hardly visible in the spectrum  and varied gadolinium ions contribution (x). EPR spectra of two samples with: x = 0.0455 ((3) line) and x = 0.2000 ((1) line) are presented in 3+ content, the magnetic structure of present materials is modified, where the EPR signal of Mn2+ ions is significantly overleaped by a wide gadolinium line. It could be explained by the increasing role of exchange interactions between two magnetic centers: tetrahedral Mn2+ and dodecahedral Gd3+ ions taking place via an oxygen bridge. Thus, at higher Gd3+ concentrations, the manganese ions are affected by the environment, and could not be treated as isolated particles anymore.The last part of our investigations concerned the group of samples obtained by combustion method, with fixed Mnx parameters one can conclude that EPR signals are almost unchanged, in respect to the synthesis method. As we reported in earlier work [x1−MnxMoO4 nanomaterials, combusted at 450 °C we observed relatively well-resolved manganese EPR lines. According to us, it was connected with a low size of grains in these materials, where the skin effect prevent the development of the magnetic interaction along the structure, allowing the maintenance Mn2+ ions as isolated centers with visible fine structure. On the other hand, EPR signal of combusted samples: Cax1-Mnx(MoO4)0.50(WO4)0.50 was significantly overleaped, compared to a similar one obtained by solid–solid reaction. As these samples were synthesized at a combustion temperature 900 °C, we proposed an explanation with a mechanism, where a higher temperature process prefers the creation of manganese-enriched areas embedded in the matrix deprived of these ions. Thus, we observed that the EPR signal originates from a dense Mn2+ system, being far from an isolated ion model.ier work , synthes2+ ions distribution in low grain materials, as in higher grains CaMnGdMoWO obtained by solid–solid reaction at a lower temperature. A similar weak influence of the thermal process on the EPR spectra wasobserved in our previous studies on Pbx1−3▯xGdx2(MoO4)x1−3(WO4)x3 (0 < x ≤ 0.1774) scheelite structures [If we suppose, that the shape of EPR spectra is caused by competition between grain size and temperature of the thermal process, in the present case both mentioned factors lead to a specific equilibrium, where no changes in EPR spectra are observed b. It meaructures .3), manganese oxide (MnO), molybdenum oxide (MoO3), gadolinium oxide (Gd2O3), and tungsten oxide (WO3) . Calcium molybdate (CaMoO4), manganese molybdate (MnMoO4), and gadolinium tungstate (Gd2(WO4)3) were obtained analogously to our previous studies [4, MnMoO4, and Gd2(WO4)3 were prepared, i.e., when MnMoO4 concentration was constant and equaled 3.00 mol% (y = 0.0200) and gadolinium tungstate was variable and ranged from 0.50 (x = 0.0050) to 50.00 mol% (x = 0.2500). The initial MnMoO4/Gd2(WO4)3/CaMoO4 mixtures of the second series contained constant amount of Gd2(WO4)3, i.e., 25.00 mol% (x = 0.1667), and the content of MnMoO4 varied from 1.00 (y = 0.0066) to 10.00 mol% (y = 0.0667). The composition of all prepared mixtures is given in Microcrystalline samples of the new solid solution were obtained by two-step synthesis. In both steps, a high-temperature solid-state reaction between appropriate reactants was applied. The following initial materials were used in the first step of synthesis: calcium carbonate 6Mo7O24·1.36 H2O, Alfa Aeasar), ammonium tungstate (NH4)10W12H2O42·3.45 H2O, Alfa Aeasar) and citric acid  [Some samples of CaMnGdMoWO solid solution were synthesized via the combustion method . As prec Aeasar) . The Gd, Aeasar) .2Θ range with the scanning step 0.013° on an EMPYREAN II diffractometer  using CuKα1,2 radiation (λ = 1.5418 Å). Next, XRD patterns were analyzed by HighScore Plus 4.0 software, and lattice parameters were calculated using POWDER 2.0 software [Powder X-ray diffraction patterns of all samples were recorded within the 10–100° software ,49. Dens−1, and in the temperature range from 25 to 700 °C (the air flow 110 mL h−1). The mass of each sample for DTA-TG measurements was ~30 mg. The mass losses recorded on TG curves and connected with the dehydration processes of both hydrates allowed a precise determination of the number of water molecules in Mo and W precursors.Simultaneous TG and DTA measurements (the results not presented here) of ammonium molybdate and tungstate were carried out on a TA Instruments thermoanalyzer  at the heating rate of 10 deg minx = 0.0050; 0.1667; 0.2500 and y = 0.0200 as well as x = 0.1667 and y = 0.0667, previously pressed into pellets using a hydraulic press at 15 MPa, were heated in a resistance furnace. Their image and temperature were continuously recorded by a pyrometer Raytek (model RAYMM1MHSF2V) during the gradual rise of temperature in a furnace. The melting point of each sample was determined at this temperature when its image of pellets disappeared in a camera. This moment indicated that the pellet had melted.Melting point of some CaMnGdMoWO samples was determined using a pyrometric method. The samples of the solid solution when UV-vis diffuse reflectance spectra were recorded within the range of 200–1000 nm using a JASCO-V670 spectrophotometer  equipped with an integrating sphere.Grain size and morphology of some CaMnGdMoWO materials as well as their elemental composition were examined using field emission scanning electron microscopy (FE-SEM) Hitachi SU–70  microscope and NORAN™ System 7 of Thermo Fisher Scientific  equipped with UltraDry energy dispersive X-ray detector), respectively. SEM analysis was performed at an accelerating voltage of 10kV and secondary electron images were acquired. The samples under study were coated with a palladium–gold alloy thin film using the thermal evaporation PVD method  to provide electric conductivity.EPR spectra of doped materials were recorded on a conventional X-band Bruker ELEXSYS E 500 CW-spectrometer operating at 9.5 GHz with 100 kHz magnetic field modulation. The first derivative of the absorption spectra has been recorded as a function of the applied magnetic field. Temperature dependence of the EPR spectra of solid solutions under study in the 78–300 K temperature range was recorded using Oxford Instruments ESP helium-flow cryostat.x−y1−3Mny▯xGdx2(MoO4)x1−3(WO4)x3 solid solution  was synthesized using solid state reaction method and combustion route. XRD analysis identified that new ceramic materials tend to crystallize in tetragonal scheelite-type structure (space group I41/a) within the whole homogeneity of solid solution. Unexpectedly, both lattice parameters (a and c) changed nonlinearly  with increasing Gd3+-doping when Mn2+ ions content in the samples was constant. Expansion of the unit cell volume was observed practically within a whole homogeneity concentration range of solid solution. New ceramics melt in air and their melting point decreased as Gd3+ as well as Mn2+ ions concentrations increased. Applying of combustion method made it possible to obtain CaMnGdMoWO powders with a submicro grain size, i.e., from ~500 nm to max. a few micrometers. All materials are insulators and their direct band gap nonlinearly changed with the increase of both doping ions’ concentrations. The UV-vis studies revealed a structural disorder in CaMnGdMoWO samples that was confirmed by an increase of Urbach energy with Gd3+ and Mn2+-substitution.In this study, new Ca2+ and Gd3+ ions. The fine structure of six well-resolved manganese ions was observed only in samples with very low gadolinium ions contents. Under these circumstances manganese ions occupy a central position in MoO4 tetrahedra. Increasing concentration of Gd3+ ions in the dodecahedral position disturbs the Mn2+ arrangement, which means that manganese should not be treated as well-isolated ions, and MoO4 tetrahedra are significantly distorted. The fine structure of Mn2+ starts to be overleaped by complex dipolar and exchange interactions thus, as a result, only a wide gadolinium ions resonance line is visible in EPR spectra at higher Gd3+ contents.EPR spectra of CaMnGdMoWO materials confirmed the existence of two types of magnetic objects, i.e., Mn2+and Gd3+ ions in this case. But the general domination of AFM interactions is proved and similar results were reported earlier, where Mn2+ ions were embedded in comparable scheelite materials.The temperature dependence of the EPR signal intensity fulfills the Curie–Weiss relation, indicating significant antiferromagnetic (AFM) interactions among responsible ions. Detailed analysis is difficult due to the combined coexistence of two different magnetic Mn2+ ions as being better isolated in structure, compared to samples obtained by solid–solid reactions. On the other hand, a high temperature of combustion could be the reason for the non-uniform distribution of Mn2+ ions, where mutual interactions are conducive to creating complex magnetic arrangements.A significantly lower grain size of samples obtained by combustion route is the reason of maintaining Mn


Leishmania infantum. Several ammonium, methylimidazolium, pyridinium and phosphonium organic cations combined with amphotericin B as anion were synthesized in moderate to high yields and high purities by the water-reduced buffer neutralization method. All prepared compounds were characterized to confirm the desired chemical structure and the specific optical rotation ([α]D25) was also determined. The biological assays performed on L. infantum promastigotes showed increased activity against this parasitic disease when compared with the starting chloride forms and amphotericin B alone, highlighting [AmB] as the most promising formulation. Possible synergism in the antiprotozoal activity was also evaluated for [AmB], since it was proven to be the compound with the highest toxicity. This work reported a simple synthetic method, which can be applied to prepare other organic salts based on molecules containing fragile chemical groups, demonstrating the potential of these OSILs-AmB as possible agents against leishmaniasis.Nowadays, organic salts and ionic liquids (OSILs) containing active pharmaceutical ingredients (APIs) are being explored as drug delivery systems in modern therapies (OSILs-API). In that sense, this work is focused on the development of novel OSILs-API based on amphotericin B through an innovative procedure and the evaluation of the respective biological activity against  Moreover, the biological properties of these new compounds were evaluated against Leishmania infantum, aiming to understand their potential as a therapeutically antiprotozoal drug. In this context, to quantify the improvement in activity, the relative decrease in inhibitory concentration (RDIC) [AmB], [Ch][AmB], [AmB]:D25: 162.0° ± 0.6 (c = 1 mg mL−1 in methanol); 1H-NMR 2SO, J = 7.5 Hz), 3.09−3.01 , 2.92 , 2.33−2.15  1.86−1.71 , 1.59 , 1.25 , 1.15 , 1.11 , 1.03 , 0.91 , 0.86 ; FTIR  was converted into hydroxide and, afterward, added to amphotericin B  predissolved in 1 M dried triethylamine methanolic solution. The desired product was obtained as an orange solid . [α]TIR KBr, : υ = 344Synthesis of choline amphotericin B, [Ch][AmB]:D25: 50.0° ± 5.8 (c = 1 mg mL−1 in methanol); 1H-NMR 2SO, J = 5.8 Hz), 1.91−1.70 , 1.65−1.31 , 1.23 , 1.14 , 1.10 , 1.03 , 0.91 , 0.83  ppm; FTIR  was converted into hydroxide and, afterward, added to amphotericin B  predissolved in 1 M dried triethylamine methanolic solution. The desired product was obtained as an orange solid . [α]TIR KBr, : υ = 3392OHMIM][AmB]:Synthesis of 1-(2-hydroxyethyl)-3-methylimidazolium amphotericin B, TIR KBr, : υ = 3433OMIM][AmB]:Synthesis of 1-(2-methoxyethyl)-3-methylimidazolium amphotericin B, TIR KBr, : υ = 34316Pyr][AmB]:Synthesis of cetylpyridinium amphotericin B, TIR KBr, : υ = 3436,6,6,14][AmB]:Synthesis of trihexyltetradecylphosphonium amphotericin B, TIR KBr, : υ = 343Leishmania infantum promastigotes were selected as a biologically relevant model to test the effectiveness of newly prepared compounds. In this context, a cloned line of virulent L. infantum (MHOM/MA/67/ITMAP-263) was maintained at 27 °C in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U mL−1 penicillin, 100 mg mL−1 streptomycin and 20 mM HEPES buffer, all acquired from Lonza. The promastigotes were subcultured each week with dilutions of 1 × 106 parasites/mL. After 5 days, they became stationary, reaching a density of around 2 × 107 parasites/mL. A maximum of 10 passages is recommended. After 10 passages, a new culture was started with parasites recovered from infected mice.To evaluate the biological activity of amphotericin B organic salts, 7 parasites/mL parasite solution was added for a final density of 2 × 106 parasites/mL in the well. After 72 h of incubation at 27 °C, 20 μL of a 0.5 mM resazurin solution was added and plates were incubated for further 4 h under the same conditions. Then, fluorescence was measured in excitation (540 nm) and emission (620 nm) wavelengths using a Synergy 2 Multi-Mode Reader (Biotek). For each individual assay, the Z factor was calculated through Zf = 1 − [3 × (σp + σn)/|μp − μn|], where μ and σ are, respectively, the means and the standard deviations of both positive and negative controls and Zf is the Z-Factor. In addition, the IC50 value was determined to evaluate the antiparasitic effect. A non-linear regression analysis was conducted on Prism 9 for Windows (Version 9.4.0), corresponding to the averages of the results obtained in at least two independent experiments. Lastly, the investigation of the potential synergism in the context of [AmB] activity − molecular peak (m/z 922 in negative mode) was absent in MALDI-TOF-MS spectra, as only one fragment was detected with m/z 420. One possible explanation is the occurrence of Grob fragmentation  of the prepared compounds was also measured and is depicted in D24 of neat amphotericin B is 333° in acidic N,N-dimethylformamide conditions [AmB] and [Ch][AmB] are substituted tetralkylammonium derivatives, the magnitude of optical rotations is significantly different, being 162° for the first and 50° for the latter, which was also registered for analogues derivatives by Carreira et al.  anion . Moreovenditions . When coa et al. . FurtherLeishmania infantum promastigotes of the prepared compounds, the half maximal inhibitory concentration (IC50) of both OSILs-AmB and the starting organic cations in their chloride form was determined. Moreover, the relative decrease in IC50 (RDIC) of each formulation containing the API was also calculated. The results collected are displayed in Aiming to evaluate the biological activity against 50 obtained by this biological assay, [AmB] (61.4 nM) exhibited the lowest value, indicating the highest growth inhibition capacity when compared to the original AmBH (86.6 nM). This implies that is required less quantity to achieve the same biological outcome and, therefore, this formulation is the most promising one. The remaining OSILs-AmB were classified according to the decrease in antiparasitic activity: [Aliquat][AmB] > [C3OMIM][AmB] > [C16Pyr][AmB] > [Ch][AmB] > [C2OHMIM][AmB]. For the organic cations in their chloride form, only [Cl], [Aliquat][Cl] and [C16Pyr][Cl] present anti-Leishmania activity in the tested concentrations range. Nonetheless, the obtained values are not statistically relevant since they are much higher than the IC50 value of AmBH. All compounds were tested in order to demonstrate if the starting materials exhibit any activity against this protozoan disease, allowing the conclusion that the improvements concerning the biological activity of OSILs-AmB are associated with the incorporation of this pharmaceutical drug. Regarding the ICLeishmania activity when compared with API, the relative decreases in inhibitory concentrations (RDIC) were calculated. Higher RDIC values are desirable as they indicate increased activity of the OSILs-AmB in respect to the starting agent. These values, displayed in 6,6,6,14][AmB] is more active against L. infantum promastigotes than AmBH alone. Moreover, the other OSILs-AmB exhibited values below 1, with the exception of [Aliquat][AmB] and [C3OMIM][AmB], which registered very similar biological activity to AmBH.In order to fully understand the impact of each formulation in the anti-16Pyr][AmB] is 0.84, while the same formulation exhibit a positive effect against fungi). On the contrary, for polar cations, as choline, an opposite phenomenon can occur, probably, due to ion trapping of AmBH in solution, as previously reported for tests conducted on fungi [AmB] is the most promising formulation, revealing higher antiparasitic activity. Therefore, the possible synergism in the antiprotozoal activity was evaluated, since other combinatorial therapies with AmBH have already demonstrated significant synergistic effects responsible for the improvement in their therapeutic index + is sufficient to partially overlap the [AmB]− potency, leading to a small incremental potency  was also measured, revealing values that are in agreement with the ones already reported for other organic salts combined with chiral molecules.This work emphasizes the use of a simple buffer neutralization method to synthesize organic salts and ionic liquids based on complex antimicrobial agents, such as amphotericin B or other molecules containing fragile chemical groups. The crucial step to successfully prepare the desired products is dissolving the API in 1 M dry triethylamine buffer methanolic solution, which impaired the occurrence of Grob fragmentation and the consequent degradation of AmBH. Furthermore, this innovative synthetic procedure allowed OSILs-AmB to be obtained in high yields and purity levels through an easier and faster process. The resulting compounds were characterized by NMR, FTIR and MALDI-TOF-MS, which confirmed the desired chemical structure as well as the suitable proportion between cation and anion. The specific optical rotation ([α]50 and RDIC assays were performed, which suggested that only [AmB] has higher activity than AmBH alone. The potential synergism in the antiprotozoal activity was also evaluated, showing the enhancing effect of the starting chloride organic salt form, leading to an increase in the activity of this prepared OSIL-AmB. This is quite a promising result that prompts the way for the development of new bioactive formulations with AmBH.Since AmBH has proven itself effective against leishmaniasis, bioactivity studies were conducted to evaluate the therapeutic potential of the prepared formulations. In this context, IC


P < 0.05). These genes and metabolites were significantly enriched in lipid metabolism pathways, including arachidonic acid metabolism and PPAR signaling. Several candidate genes were associated with muscle growth, such as MYLK3, MYL10, FIGN, MYH8, MYOM3, LMCD1, and FLRT1. Among these, MYH8 and MYL10 participated in regulating muscle growth and development and were correlated with meat quality-related fatty acid levels (|r| > 0.5 and p < 0.05). We selected mRNA from four of these genes to verify the accuracy of the sequencing data via qRT-PCR. Our findings provide further insight into the key genes and metabolites involved in muscle growth and meat quality in hybrid sheep populations.Crossbreeding improves and enhances meat quality and is widely used in sheep production; however, the molecular mechanisms underlying the meat quality of various crossbred sheep remain unknown. In this study, male Southdown, Suffolk and Hu sheep were crossbred with female Hu sheep, and the transcriptomes and metabolomes of the longissimus dorsi muscle of the F1 generation were sequenced to explore how different sire breeds affect meat quality. The results showed that 631 differentially expressed genes and 119 significantly altered metabolites contributed to muscle development characteristics and meat quality-related diversity ( As consumers increasingly focus on how nutrition, safety, and high-quality meat products affect health, demand for high-quality sheep meat is growing in most areas of China. Studies have shown that crossbreeding can improve and enhance meat quality. Crossbreeding between Duroc and Polish Landrace sheep can improve tenderness , and hybACLY, ADIPOQ, ELOVL6, LEP and ME1, as main gene factors defining the processes influencing meat composition and quality , Suffolk × Hu (SH), and Hu × Hu (HH), those animals were obtained from Gansu Qinghuan Sheep Breeder Co., Ltd., . We used 18 rams (1 year old) in this experiment, and all rams received the same feeding management and dietary levels. Sheep weights were similar among the groups . The animals were slaughtered after 24 h with food deprived and had free access to water. Subsequently, the longissimus dorsi between the 12 and 13th rib was excised from all 18 sheep on the left side of each carcass within 30 min after exsanguination. Any visible external fat and connective tissues were trimmed before the following analyses.via agarose gel electrophoresis, PCR amplified, and sequenced by Gene Denovo Biotechnology Co.,  using an Illumina NovaSeq6000. The raw reads were filtered, and the clean reads were mapped to the reference sequences using HISAT2.2.4 software . Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the DEGs were performed using R based on hypergeometric distribution. GO terms and KEGG pathways with P < 0.05 were considered significantly enriched.Muscle tissues from the sheep were used to extract the total RNA using a TRIzol reagent kit  per the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer  and checked using RNase-free agarose gel electrophoresis. After extracting the total RNA, eukaryotic mRNA was enriched with Oligo (dT) beads, and prokaryotic mRNA was enriched by removing the rRNA using a Ribo-Zero™ magnetic kit . The enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcribed into cDNA with random primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTP and buffer. The cDNA fragments were purified using a QiaQuick PCR extraction kit , end-repaired, A-base-added, and ligated to Illumina sequencing adapters. The ligation products were size selected software . Gene exsoftware . Genes/t® HSS T3  column maintained at 40°C. The autosampler temperature was 8°C. The analytes were gradient eluted with 0.1% formic acid in water (C) and 0.1% formic acid (D) in acetonitrile or 5 mM ammonium formate in water (A) and acetonitrile (B) at a flow rate of 0.25 mL/min. After equilibration, 2 μL of each sample was injected, and the following increasing linear gradient of solvent B (v/v) was used: 0–1 min, 2% B/D; 1–9 min, 2%–50% B/D; 9–12 min, 50%–98% B/D; 12–13.5 min, 98% B/D; 13.5–14 min, 98%–2% B/D; and 14–20 min, 2% D + the positive control. The ESI-MSn experiments were performed on the Thermo Q Exactive mass spectrometer with spray voltages of 3.8 and −2.5 kV in positive and negative modes , respectively. Sheath and auxiliary gases were set at 30 and 10 arbitrary units, respectively. The capillary temperature was 325°C. The analyzer scanned over a mass range of 81–1,000 m/z for a full scan at a mass resolution of 70,000. Data-dependent acquisition (DDA) MS/MS experiments were performed with an HCD scan. The normalized collision energy was 30 eV.To extract the metabolites, the freeze-dried samples were dissolved in methanol and concentrated to dry in a vacuum after centrifugation. The samples were then dissolved with 80% 2-chlorobenzalanine and filtered [20 μL of each sample was used for quality control (QC)]. Chromatographic separation was performed in a Thermo Ultimate 3000 system equipped with an ACQUITY UPLCTo extract the metabolites, 50 mg (± 5%) of the samples were weighed and transferred into 2-mL Eppendorf tubes. Next, 0.5 mL of an acetonitrile: isopropanol: water  mixed solution (−20°C) and 3–42-mm zirconium beads were added and placed in a high-flux tissue grinder, shocked at 30 Hz for 20 s, allowed to stand for 10 s, cycled eight times, and sonicated in an ice water bath for 5 min. Another 0.5 mL acetonitrile: isopropanol: water  solution was added on an ice water bath, sonicated for 5 min, and centrifuged at 12,000 rpm for 2 min. Next, 500 μL of supernatant solution was added to a new 2-mL Eppendorf tube, the vacuum concentrator was concentrated to dryness (8–10 h), and the remaining supernatant was placed in a −80°C freezer for backup. Next, 80 μL of 20 mg/mL MEOX solution was added for redissolution, vortexed for 30 s, and incubated at 60°C for 60 min. Finally, 100 μL BSTFA-TMCS (99:1) reagent was added, then reacted at 70°C for 90 min and centrifuged at 14,000 rpm for 3 min, then 90–100 μL of supernatant was added to the detection bottle. Samples were placed in sealed cuvettes to be tested and processed for GC-TOF upper detection within 24 h.Gas chromatography was performed on a DB-5MS capillary column  to separate the derivatives at a constant flow of 1 mL/min helium. Next, 1 μL of sample was injected in split mode at a 1:10 split ratio by the autosampler. The injection temperature was 280°C, and the transfer-line ion source temperatures were 320°C and 230°C. From an initial temperature of 50°C, the temperature was increased at 15°C/min to 320°C for 0.5 min and then held at 320°C for 9 min. Mass spectrometry was performed using a full scan method with a scan rate of 10 spec/s, electron energy of −70 V, and a solvent delay of 3 min , 20.Denovo Biotechnology Co., Ltd., Metabolic alterations among the groups were analyzed via principal component analysis (PCA) and  partial least-squares discriminant analysis (OPLS-DA) after data preprocessing by mean centering (Ctr) and Pareto variance (Par) scaling, respectively. Metabolites were identified with P < 0.05 by t-test, and metabolites with a VIP ≥ 1 were considered differential between the groups. Identified differential metabolites were subjected to metabolic pathway analysis through the KEGG database.Metabolites were distinguished by comparing the m/z values of the precursor ions, retention time, and fragmentation patterns with the standards in a database compiled by Gene via pairwise comparison using the Hmisc package in R1. DEGs and SCMs with a threshold of |r| > 0.5 and P < 0.05 were considered significantly correlated and were subjected to conjoint biological annotation using the KEGG database. Here, only the pathways containing DEGs and their highly related metabolites were selected. The networks were visualized using Cytoscape2.Pearson correlation coefficients were calculated between the SCMs and DEGs Ct–ΔΔ method. Statistical analyses were performed using GraphPad Prism 8. Quantitative data are presented as the mean ± standard error of the mean. The significance level was calculated using one-way analysis of variance and Tukey’s post hoc test. P < 0.05 was considered statistically significant.qRT-PCR was performed per the manufacturer’s protocol . The reactions were performed in 20 μL volumes on a Bio-Rad C1000 Thermal Cycler. The qRT-PCR procedure was 94°C for 30 s, 40 cycles of 94°C for 5 s, 60°C for 15 s and 72°C for 10 s. β-actin was used as a reference gene to normalize gene expression. 3). We obtained 631 DEGs in total for the HH vs SH, HH vs NH, and SH vs NH comparisons , MYL10 (myosin light chain kinase 10), FIGN (Fidgetin), MYH8 , MYOM3 (Myomesin-3), LMCD1 (Homo sapiens LIM and cysteine-rich domains 1), FLRT1 (Fibronectin leucine rich transmembrane) and other genes (P < 0.05). KEGG enrichment results showed that the DEGs for all three groups were mainly enriched in pathways related to fatty acid metabolism, including arachidonic acid metabolism, fatty acid biosynthesis and regulation of lipolysis in adipocytes.To further understand the functions of the DEGs, we performed functional enrichment analysis , which yer genes . Figure via OPLS-DA showed clear differences between the meat quality of the three sheep breeds 1 × PC2 score plot . Furtherp breeds , thus dep breeds , which wP < 0.05). In the HH vs SH comparison, 38 differential metabolites were significantly annotated into eight pathways, and most were involved in lipid metabolism-related pathways, such as glycerophospholipid metabolism, protein digestion and absorption, arginine and proline metabolism and ABC transporters (P < 0.05), most of which were involved in glycerophospholipid metabolism, protein digestion and absorption, arginine and proline metabolism. In the SH vs NH comparison, two metabolic pathways were significantly enriched in fat metabolism, cysteine and methionine metabolism and arginine and proline metabolism (P < 0.05).KEGG enrichment analysis was used to determine the biological mechanisms associated with phenotypic changes. All differential metabolites were co-enriched in 131 biological pathways  with seven meat quality-related fatty acids . Among them, MYH8 and MYL10 participate in regulating muscle growth and development   . In the elopment , includielopment . Compareelopment , indicatTM7SF2 expression was significantly higher in the SH sheep than in the HH sheep , myristic (r = 0.71) and 9-octadecenoic acid levels (r = 0.67)  and is involved in lipid and lipoprotein metabolism  . TM7SF2 tabolism , 37. TheFatty acids are important chemical substances constituting fats and are important aromatic substances or precursors of aromatic substances. Studies have shown that fatty acid metabolism levels and composition affect meat quality and its nutritional value; for example, dodecanoic C12:0), myristic (C14:0), palmitic C16:0), stearic (C18:0), linoleic (C18:1), alpha-linolenic (C18:2) and linolenic fatty acids (C18:3) can enhance meat flavors –41. Dode:0, stear2:0, myriIn this study, a great number of DEGs and differential metabolites were identified based on the integrative analysis of transcriptomics and metabolomics in hybrid sheep’ muscle. Different hybrid male parents caused differential expression of meat quality-related genes, metabolites, and related pathways, in different hybrid populations, such as fatty acid metabolism  and lipid metabolism-related pathway. But most importantly, fatty acid metabolism played a key role in the meat quality of the three hybrid sheep populations. Overall, these results will provide effective information and more evidence to support further insight into the key genes and metabolites involved in muscle growth and meat quality in hybrid sheep populations.PRJNA859945, and our data have been released.The data presented in this study are deposited in the SRA repository, accession number: The studies involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Lanzhou Institute of Husbandry and Pharmaceutical Science of Chinese Academy of Agricultural Sciences .ZL, BC, and BY conceived and designed the study. YY, JBL, DZ, and JYL collected the samples. BC, CY, and TG performed the experiments and analyzed the data. BC wrote the manuscript. BC and ZL contributed to revisions of the manuscript. All authors read and approved the manuscript.


Solute carrier family 17 member 9 (SLC17A9) encodes a member of a family of transmembrane proteins that are involved in the transport of small molecules. SLC17A9 is involved in the occurrence and development of various cancers, but its biological role in liver hepatocellular carcinoma (LIHC) is unclear.The expression level of SLC17A9 was assessed using The Cancer Genome Atlas (TCGA) database and immunohistochemistry of tumor tissues and adjacent normal liver tissues. The receiver operating characteristic (ROC) and R software package performed diagnosis and prognosis. Gene Ontology/Kyoto Encyclopedia of Genes and Genomes functional enrichment and co-expression of SLC17A9, gene–gene interaction (GGI), and protein–protein interaction (PPI) networks were performed using R, GeneMANIA, and STRING. Western blot, real-time quantitative PCR (RT-qPCR), immunofluorescence, colony formation, wound scratch assay, ATP production assays, and high connotation were applied to determine the effect of SLC17A9 knockdown on HEPG2 (hepatocellular liver carcinoma) cells. TIMER, GEPIA, and TCGA analyzed the relationship between SLC17A9 expression and immune cells, m6A modification, and ferroptosis.+ T cells , and macrophages ; m6A modification: HNRNPC , METTL3 , and WTAP ; and ferroptosis: HSPA5 , SLC7A11 , and FANCD2 .SLC17A9 expression in LIHC tissues was higher than in normal liver tissues (p < 0.001), and SLC17A9 was related to sex, DSS , and PFI  . SLC17A9 expression has diagnostic  and prognostic potential (p = 0.015) in LIHC. Gene Ontology/Kyoto Encyclopedia of Genes and Genomes (GO/KEGG) functional enrichment analysis showed that SLC17A9 was closely related to neuronal cell body, presynapse, axonogenesis, PI3K/Akt signaling pathway. GGI showed that SLC17A9 was closely related to MYO5A. PPI showed that SLC17A9 was closely related to SLC18A3. SLC17A9 silencing inhibited HepG2 cells proliferation, migration, colony formation, and reduced their ATP level. SLC17A9 expression level was related to immune cells: B cells , CD4Our data show that SLC17A9 may influence LIHC progression. SLC17A9 expression correlates with tumor immune infiltration, m6A modification, and ferroptosis in LIHC and may have diagnostic and prognostic value in LIHC. Liver hepatocellular carcinoma (LIHC) is one of the most common malignant tumors in the world the most commonly diagnosed cancer in 13 countries and the leading cause of cancer death in 20 countries (SLC17A9 (solute carrier family 17 member 9), a vesicular nucleotide transporter (VUNT), is most abundantly expressed in the stomach, intestines, skeletal muscles, and liver . SLC17A9https://portal.gdc.cancer.gov/) (www.linkedomics.org/login.php) (www.genemania.org) and STRING (www.string-db.org) were used to construct gene–gene interaction (GGI) and protein–protein interaction (PPI) networks (https://cistrome.shinyapps.io/timer) (gepia.cancer-pku.cn) (www.rndsystems.com/cn/resources/cell-markers/immune-cells). Correlation between SLC17A9 and m6A and ferroptosis was analyzed using TCGA LIHC datasets.Datasets on SLC17A9 expression in pan-cancer, LIHC, and normal liver tissues were downloaded from The Cancer Genome Atlas (TCGA) (er.gov/) . Correlagin.php)  was usednetworks , 22, reso/timer)  was used-pku.cn)  was usedWe recruited 28 patients with liver cancer who underwent surgery in Taihe Hospital, Shiyan City, Hubei province. Ethical approval for this study was granted by Taihe Hospital’s medical ethics committee. The study adhered to the Helsinki Declaration and its subsequent amendments.5 cells per well and cultured for 24 h. Transfection was done using Lipofectamine 8000 reagent (Beyotime) following the manufacturer’s guides. For each transfected well, 125 μl of serum-free OptiMEM, 2.5μg of plasmid DNA, and 4 μl of Lipo8000™ transfection reagent were mixed in a sterile centrifuge tube. The transfection mixture was applied to the cells. The oligonucleotide sequences of the small interfering RNA (siRNA) were displayed as follows: SLC17A9: ; and GAPDH: .HepG2 cells were seeded on six-well plates at 6 × 10At 48 h after transfection, cells were homogenized in RIPA lysis buffer  on ice for 30 min, and then, protein was measured by the Bicinchoninic Acid (BCA) assay . Protein was separated on 10% sodium dodecyl sulfate–polyacrylamide gels and transferred onto a polyvinylidene difluoride membrane using a semiwet method; next, we blocked the membrane with 5% dried skimmed milk for 1 h and incubated overnight with a SLC17A9 rabbit anti-human monoclonal antibody  in a 4°C refrigerator. Next day, horseradish peroxidase (HRP)–conjugated secondary antibodies  for 1 h at room temperature. Images were obtained using a gel imaging system.Total RNA was extracted from cells 48 h after transfection using Trizol reagent (Invitrogen). Trizol Reagen (600 μl) was added, and the samples were placed on ice for 20 min to lyse the cells. Subsequently, the cell lysate in Tirol was phase separated by addition of 120 μl of chloroform, and the RNA was precipitated by addition of 300 μl of isopropanol, and then, cDNA was synthesized by utilizing the TaqMan Reverse Transcription Reagents kit (TaKaRa). Real-time quantitative PCR (RT-qPCR) analysis was performed with different primer sequences using the SYBR Green Master Mix (TaKaRa). The primers used for RT-PCR were in Cells were fixed with 4% formaldehyde for 1 h and permeabilized with 0.5% Triton X-100 at room temperature. They were then blocked with 1% Bovine Serum Albumin (BSA) for 2 h then incubated with primary antibody at 4°C, overnight. Next, they were incubated with secondary antibody 2 h and nuclei counterstained 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI). They were then examined and imaged fluorescent confocal microscopy.The cells were seeded onto 12-well plates at 100 cells per well and cultured for 2 weeks. They were then fixed with 4% Paraformaldehyde (PFA) for 10 min and rinsed with Phosphate Buffered Saline (PBS) before staining with crystal violet. They were then examined by microscopy and imaged.6 per well) were seeded onto 12-well plates and cultured until confluent. A white gun was used to make vertical scratch along the ruler to ensure uniform scratches in each well. The image was taken at 0, 12, 24, and 48 h.HepG2 cells .4/ml and seeded onto 96-well plates in six replicate wells. After 24 h of culture, the 96-well plates were moved into a high-connotation instrument. High-connotation cell images were then taken using a 10× objective. Multiple visual fields were selected per well for monitoring and cell growth recording.Control and siSLC17A9 groups were suspended at 1 × 10Gene expression analysis was done using Pearson correlation analysis. Other experimental results were analyzed using two-tailed t-test. Data were expressed as mean ± SD. P < 0.05 indicated statistically significant differences.We analyzed the expression level of SLC17A9 in TCGA pan-cancer dataset Table 2.Analysis of genes’ co-expression with SLC17A9 in LIHC on LinkedOmics found that SLC17A9 expression positively correlated with 4,084 genes and negatively correlated with 3,702 genes , macrophages , neutrophils , and CD8+ T cells  , CD4 = 6.05) Table 3.m6A modification is a potential target for designing drugs for tumor treatment. Therefore, the correlation between expression level of SLC17A9 and levels of m6A-related genes was explored using TCGA LIHC datasets , F. RT-Further, the correlation between SLC17A9 and ferroptosis gene was explored using TCGA LIHC dataset –F.in vitro experiments. We find that SLC17A9 silencing HepG2 markedly suppressed ATP production as well as cell proliferation, migration, and colony formation. In LIHC, SLC17A9 expression significantly correlated with sex, DSS, and PFI, indicating that it may have prognostic value in LIHC. ROC curve analysis highlighted the diagnostic value of SLC17A9 expression in LIHC. Second, we find that patients with LIHC expressing high SLC17A9 levels had worse prognosis than those with low SLC17A9 levels, indicating that SLC17A9 has potential diagnostic and prognostic value in LIHC.SLC17A9 is a vesicle nucleotide transporter involved in ATP transport and is highly localized on lysosomes . Reducedvia MAPK/ERK signaling , and monocytes differed significantly with SLC17A9 expression. Together, these results confirmed that SLC17A9 was associated with tumor infiltration by immune cells in LIHC, especially B cells, CD4+ T cells, and macrophages. Macrophages have been associated with immunosuppression and angiogenesis, which provides sufficient nutrition for tumor proliferation , the general project of Hubei Provincial Department of Education (No. B2021160), the HuBei Provincial Department of Science and Technology Innovation Group Programme (grant no. 2019CFA034), Innovative Research Program for Graduates of Hubei University of Medicine (grant nos. YC2020011 and YC2021018), and the Key Discipline Project of Hubei University of Medicine.The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The agricultural irrigation and fertigation systems have a non-negligible impact on the soil microenvironment in arid and semi-arid areas. Therefore, studying the processes and changes of soil microenvironment under different plastic mulch drip irrigation systems can reveal the “soil-microbe” mechanism and provide a theoretical support for the optimal irrigation and nutrition management of maize in the semi-arid area of Northeast China. Three treatments were used for this study in the semi-arid area of northeast China, namely; mulched fertigation system (MF), drip irrigation system (DI), and farmers’ practices system (FP). We used high-throughput sequencing to study the soil bacterial community structure targeting the 16S rRNA gene. The agricultural irrigation and fertigation systems significantly affected soil properties. MF significantly increased bacterial abundance and bacterial diversity and richness. Moreover, MF and DI markedly increased some relative abundance of beneficial bacterial. The bacterial network in MF was more conducive to the health and stability of the agroecosystem and the relationships among species in MF bacterial network were more complex. The agricultural irrigation and fertigation systems had indirect effects on community composition and bacterial diversity through soil organic carbon (SOC), ammonium nitrogen ( The semi-arid area in China is influenced by natural environmental factors like drought and soil nutrient depletion; due to the rainfall and crop water requirements mismatch, soil water supply is often one of the important limiting factor of crop productivity. However, the cultivation of maize is known to be largely dependent on water and soil nutrients. The traditional methods for managing water and fertilizer, like flooding irrigation and the single basal fertilizer application, are known to result in lower nutrient use efficiency compared with high nutrient use efficiency and severely restrict crop yield . SeveralThe MF system combines plastic film mulch and fertigation technology, where the plastic film can increase soil temperature through the conductive transfer of solar energy during the day. Moreover, the plastic mulch reduce evaporation because of their water-impermeable nature. This process makes the soil environment conducive for microbial activities thereby improving decomposition and the release of nutrients into the soil . The MF Soil bacteria are important in agricultural ecosystems and soil function . BacteriSoil microbial community is implicated in numerous processes such as the biochemical transformation of carbon and nitrogen and the development of soil physical structure, sustainable crop productivity, and soil ecosystem health. Being a source and sink of soil nutrients, structural alterations in the microbial communities also influence the rate of nitrogen fixation, nitrification, and de-nitrification, processes of nutrient cycling, and thus crop production . Soil miNitrosospira and Nitrosomonas were dominant in soils irrigated with FAW, and effluent-irrigated soils. Therefore, studying the process of soil microenvironment changes under different plastic mulch drip irrigation systems can provide a better understanding to determine the best irrigation and fertilization management method, so as to optimize the crops yield. Some investigations have reported MF system had a significant positive impact on the characteristics of soil water and heat migration, crop yield, nutrient efficiency and water productivity , while i−1 soil organic matter, 105.83 mg kg−1 available N, 12.79 mg kg−1 available P, 104.52 mg kg−1 available K and pH 7.6. The long-term field experiment was established in 2015 and had since been subjected to continuous maize cropping. Maize was sown in early May and harvested in early October. The maize cultivar used was FuMin-985, planting density was 70,000 plants ha−1, and sowing depth was two cm. The area size of each treatment was 300 m2 (15 by 20 m). After the maize entered the maturity period, the maize harvester was used for harvesting. After harvest, used a 1JHY-200 straw chopper to crush maize straw (length < 10 cm), and evenly scatter it in the field. The 1LYFT-450 fence type hydraulic-overturning plough was used to plough the straw into the soil , and turn the straw to a depth of 20–30 cm soil layer. The combined land leveler was used to spin and rake to reach the sowing state. When the soil temperature of five cm was stable and passed 8 °C, adopted wide and narrow rows for planting. After heavy suppression (the suppression strength was 400–800 g/cm2), we used the 2BJHM-2 multifunctional water-saving mulching planter to complete fertilization, sowing, drip irrigation pipes, film mulching, and soil covering at the same time. After the experiment set up, the crop type, cropping density, fertilizer application rate, crop straw management are consistent.This study was conducted in Minle Village , Jilin Province, Northeast China. The soil at the experiment site is chernozem sandy loam, with 17.68 g kg−1. Maize was sown in broad-narrow ridges pattern, with broad and narrow rows’ spaces of 80 and 40 cm, respectively, both ridges were covered with the plastic film. Each plot was supplied with an independent unit of the gravity drip irrigation system, which was a dripline placed in the middle of each narrow row, and the emitter spacing was 30 cm. A fertilizer tank was installed upstream of the pressure regulating valve to apply fertilizer. The nominal flow rate of the dripper was 2.0 L h. In treatment DI, the shallow-buried drip irrigation system was applied with no plastic mulch. In treatment FP, the fertilizer and water management were controlled by local farmers’ management practices, without plastic mulch and irrigation by flooding with water pipe. The irrigation amount of the three treatments was consistent. The total irrigation amount of three treatments in maize growth period were 220 mm, of which the irrigation amount at before sowing (BS) and eighth leaf stage were 20 mm, and the irrigation amount in twelfth leaf, tasseling stage (VT), and milk stage (R3) were 60 mm. Each treatment was irrigated at the same time, and the same irrigation amount was controlled by water meter. The test water source was groundwater. Each irrigation system received equal amounts of 210 kg N ha−1, 90 kg P2O5 ha−1, and 90 kg K2O ha−1. Under MF and DI systems, the total N was split into five applications of 30, 30, 20, 10, and 10% BS, eighth leaf, twelfth leaf, VT, and R3, respectively. The total P2O5 and K2O were divided into five applications of 50, 20, 15, 10, and 5% at the five growth stages. In addition, urea, triple superphosphate, and potassium chloride were dissolved in the irrigation water and applied by drip fertigation. However, under treatment FP the total N was applied once (100%) before sowing while the total P2O5 and K2O were applied with the same proportion as DI and FP at the five growth stages.The experiment included three treatments: mulched fertigation system (MF), drip irrigation system (DI), and farmers’ practices system (FP). The three treatments were arranged in a completely random block design with three repetitions. Under treatment MF, the non-degradable plastic film that a conventional white polyethylene (PE) plastic mulch for maize with a width of 110 cm and a thickness of 0.008 mm was selected, and the amount was 66.45 kg haSoil samples for this study were collected at BS (5.5), sixth leaf (V6) (6.26), VT (7.24) and harvest stages (R6) (9.27) in 2019. Soil samples were taken on the 7th day after irrigation fertilization at BS and VT. Under each treatment, eradicated the vegetation and weeds on the soil surface carefully, five soil samples were randomly collected from the tillage layers (0–20 cm) with a soil auger and mixed into them to form a composite sample. Each composite sample was about 1 kg. Each treatment was composed of four replicated composite samples. The soil samples were placed into respective sterile bags in an icebox and carried back to the laboratory. The samples were sieved with a 2-mm mesh to remove roots and stones and a part of the soil samples were stored at −80 °C for soil DNA extraction. Other soil samples were air-dried at room temperature to determine soil physicochemical properties.3BO3 indicator solution in the inner chamber of the diffusion dish. Quickly add 10 ml of NaOH solution, and then put it into a 40 °C incubator. After alkali hydrolysis and diffusion for 24 h, remove it. Titrate NH3 in the absorption solution in the inner chamber with a sulfuric acid standard solution. The solution changes from blue to reddish as the titration end point. Soil available phosphorus (AP) was measured with the molybdenum blue method. Weigh 5.00 g of air-dried soil, add a small spoonful of phosphorus-free activated carbon, 100 ml of 0.5 mol/l NaHCO3 extraction solution, shake for 30 min, remove for filtration, suck 10 ml of the filtrate, add 5 ml of molybdenum antimony anti developer, and compare the color on the spectrophotometer after 30 min. Available potassium (AK) was measured with flame photometry . Weigh 5.00 g of air-dried soil into a plastic bottle, add 50 ml of 1 m ammonium acetate solution, shake for 30 min, and remove it for filtration. The filtrate was measured directly on the flame photometer. Three points were randomly selected in the harvest period, the harvest area of each yield measuring plot was 30 m2. The maize aboveground part of 30 m2 was harvested, threshed, and the seeds were dried to calculate the maize grain yield (water content 14%).The soil physicochemical properties were determined according to the methods of A total of 0.5 g fresh soil sample was weighed, using PowerSoil DNA Isolation Kit  and the total soil DNA was extracted according to the instructions. DNA quality and concentration were detected by NanoDrop spectrophotometer 2000  and checked in 1% agarose gel electrophoresis.2+ (25 mM), 0.5 µL of (10 µM) each forward and reverse primers, 2.0 µL of template DNA, 0.5 µL of dyestuff and 9.5 µL of sterile double-distilled water (ddH2O). The PCR reaction procedure was performed as follows: initial activation at 95 °C for 3 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and elongation at 72 °C for 30 s. The PCR products were checked in a 2% agarose gel and purified using AxyPrep DNA Gel Extraction Kit  and quantified by QuantiFluor™ _ST  according to the manufacturer’s instructions.The bacterial 16S rRNA gene was amplified using the primer set F515 (5′-GTG CCA GCM GCC GCG G-3′) and R907 (5′-CCG TCA ATT CMT TTR AGT TT-3′) . Q-PCR wThe bacterial 16S rRNA was amplified using the primer set F515/R907 in a thermocycler PCR system  . The PCRhttp://qiime.org/tutorials/tutorial.html) and merged by FLASH. An average quality score < 20 and < 200 bp in length were removed. The Uchime algorithm was used to eliminate chimera from noisy sequences (http://drive5.com/uparse/) at 97% similarity level (http://www.arb-silva.de) against the 16S rRNA database using confidence threshold of 0.80  were conducted with a Fisher’s protected least significant difference (LSD) using SPSS software version 20. The student’s t-test was used to assess differences in the relative abundances at phylum and genera levels with 95% confidence intervals in STAMP 2.1.3   . A heatme/STAMP) . The logent}X¯2) , where Xent}X¯2) . The Shawhere Sobs is the number of observed OTUs, n1 is the number of individuals in the OTU, and Nis is the total number of individuals in the community . The Chahttp://huttenhower.sph.harvard.edu/galaxy/). The LDA was considered to be an important contributor with an LDA score ≥ 2.0. The Pearson correlation analysis between soil bacterial communities and soil physicochemical properties was performed in the PerformanceAnalytics package in the R environment (version 3.5.0) (χ2 tests (P > 0.05), goodness-of-fit index (GFI > 0.9), Akaike Information Criteria (AIC), and root square mean errors of approximation (RMSEA < 0.08). Co-occurrence network visualization was performed in Gephi software (0.9.2) with the robust Spearman correlations coefficient ρ > 0.7 and P < 0.05.where Sobs is the observed number of OTUs, n1 is the number of OTUs with only one sequence, and n2 is the number of OTUs with only two sequences . Principn 3.5.0) . StructuP < 0.05). The ANOVA results showed that P < 0.05). Compared with FP, DI and MF increased the maize yield by 16 and 20%, respectively , while Shannon had a significantly negative correlation with P < 0.01). PCoA1 was significantly positively correlated with P < 0.05).Pearson correlation analysis manifested that there was a significant correlation between soil bacterial communities and soil physicochemical properties . The bacχ2 = 91.65, df = 33, P = 0.001, RMSEA = 0.2, GFI = 0.63, AIC = 157). This model explained 92.1%, 98.9%, 90.4% and 98.2% of the variation in yield, bacterial community composition, diversity, and abundance, respectively. λ = 241, P < 0.01). Moisture showed direct effect on bacterial diversity . SOC and pH showed direct effect on bacterial abundance . The results showed that irrigation systems had indirect effects on community composition and bacterial diversity through SOC, SEM was used to evaluate the direct and indirect effects of irrigation on soil bacterial community composition, diversity, abundance, and yield . The finThe co-occurrence network was constructed based on the sequencing results of bacterial 16S rRNA gene in three agricultural irrigation and fertigation systems, and the top 100 OTUs of abundance were selected to construct the network. The interaction results in the community structure being affected by species. The co-occurrence network analysis revealed that the significantly different structure characteristics among the three agricultural irrigation and fertigation systems were from each other . The co-P < 0.05) than that of FP , V6 (b), VT (c) and R6 (d). MF, mulched fertigation system, drip irrigation under plastic mulch; DI, drip irrigation system, drip irrigation without plastic mulch; FP, farmers’ practices system; BS, before sowing stage; V6, sixth leaf stage; VT, flowering stage; R6, harvest stage.Click here for additional data file.10.7717/peerj.14044/supp-3Figure S3MF, mulched fertigation system, drip irrigation under plastic mulch; DI, drip irrigation system, drip irrigation without plastic mulch; FP, farmers’ practices system; BS, before sowing stage; V6, sixth leaf stage; VT, flowering stage; R6, harvest stage.Click here for additional data file.10.7717/peerj.14044/supp-4Figure S4P < 0.05, P < 0.01. MF, mulched fertigation system, drip irrigation under plastic mulch; DI, drip irrigation system, drip irrigation without plastic mulch; FP, farmers’ practices system; SOC, soil organic carbon; *, ** indicate Click here for additional data file.10.7717/peerj.14044/supp-5Table S1Click here for additional data file.10.7717/peerj.14044/supp-6Table S2Click here for additional data file.10.7717/peerj.14044/supp-7Table S3Click here for additional data file.10.7717/peerj.14044/supp-8Table S4Click here for additional data file.10.7717/peerj.14044/supp-9Table S5Click here for additional data file.10.7717/peerj.14044/supp-10Table S6Click here for additional data file.10.7717/peerj.14044/supp-11Table S7Click here for additional data file.


In the original publication , there wFigure 5. VdKin2 affects septa development and vacuole morphology in Verticillium dahliae. (A) Hyphae of wild type (WT), ΔVdKin2, or ΔVdKin2comp strains were stained with Calcofluor White and the septa distance was observed. Scale bar = 10 μm. Measurement of septa using ImageJ software. The error bars represent standard deviations. Asterisks (****) indicate significance at p < 0.001. The experiments were performed three times independently. (B) Vacuoles were dyed with FM4-64 and photographs were acquired by fluorescence microscopy with DIC and RFP filters. Scale bar = 5 μm.2.8. Hyphal Septa, Vacuoles, and Lipid Droplet Staining, Paragraph 3, regarding the staining method of the septum, the “Congo red” should be replaced by “Calcofluor white (CFW)”. The correct paragraph appears below:1. In Section To observe the septa, the mycelium was washed with sterile water, and then the mycelium was strained with freshly prepared Calcofluor white (CFW) dye with a working concentration of 1 mg/mL, strained for 10 min at 25 °C in dark, and washed with PBS buffer.3.1. Identification of Vdkin2, Paragraph 1, “an open reading frame of 822 codons” should be modified to “an open reading frame of 928 codons”, and “86.6 kDa” should be “102.6 kDa”. The correct paragraph appears below:2. In Section V. dahliae genome, a single-copy Kin2-type gene (VDAG_09024) was identified as homologous to U. maydis Kin2 . Here, VDAG_09024 was designated as VdKin2. The VdKin2 sequence includes three exons and two introns with an open reading frame of 928 codons, and the deduced molecular weight of VdKin2 was 102.6 kDa . The VdKin2 sequence harbors a motor domain and Smc superfamily domain (chromosome segregation ATPase). Multiple sequence alignment confirmed that the kinesin motor domains of Kin2 orthologs from different fungi are highly conserved .Phylogenetic analysis suggested that fungal motor enzyme genes were divided into two subfamilies: Kin1-type and Kin2-type . In the The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated.


VEGF mRNA and protein production by senescent pericytes and other retinal cells. Collectively, these results confirm that TGFβ1 is strongly implicated in the vascular complications of DR.Diabetic retinopathy (DR) is a microvascular disease of the retina and a serious complication of type I and type II diabetes mellitus. DR affects working-age populations and can cause permanent vision loss if left untreated. The standard of care for proliferative DR is inhibiting VEGF. However, the mechanisms that induce excessive VEGF production in the retina remain elusive, although some evidence links elevated VEGF in the diabetic retina with local and systemic TGFβ1 upexpression. Here, we present evidence from animal models of disease suggesting that excessive TGFβ1 production in the early DR is correlated with  Diabetic retinopathy is a complication of diabetes mellitus characterized by alterations in the retinal vasculature . The retThere are two blood–retinal barriers (BRBs) in the retina. The inner blood–retinal barrier (iBRB) supports photoreceptors and interneurons, and is the vasculature most impacted by diabetic retinopathy ,8. TightLoss of vascular capillaries leads to retinal hypoxia and release of vascular endothelial growth factor (VEGF). VEGF has a central role in the pathophysiology of diabetic retinopathy and is identified as both a predictive marker of disease progression and the primary driver of diabetes-induced vasculopathy ,15. IL6 Another key effector factor in diabetic retinopathy may be TGFβ1. TGFβ1 is elevated in total serum of diabetic patients  and its TGFβ1 is a pleiotropic and highly conserved growth factor that plays a role in various cellular functions during development and homeostasis ,30,31. DFurthermore, TGFβ1 is implicated in the process of cellular aging, where it induces senescence in various cells and tissues  via exprSenescence was first reported in 1961 by Hayflick et al. , and wasBesides alterations in TGFβ1 and VEGF, senescence in retinal cells may contribute to diabetic retinopathy pathogenesis. Furthermore, elevated TGFβ1 levels may be responsible, at least in part, for both microvascular and senescent phenotypes in diabetic retinopathy. Moreover, TGFβ is elevated in DR patients. Together, elevated VEGF expression and pericyte dropout are key characteristics of DR. Based on these observations, we set out to determine if/how TGFβ could affect pericyte homeostasis and VEGF levels in vitro and in an animal model of disease.In this study, we report that exogenous TGFβ1 can induce VEGF expression in primary retinal pericytes and in mouse retinas. In addition, TGFβ1 can induce senescence in retinal pericytes in vitro and increased expression of VEGF, IL6, and an SASP potentially via p15 and PAI1 pathways. Collectively, these findings suggest that TGFβ1, which is compensatory upregulated in aging populations, could be an important causative and disease-propagating factor in diabetic retinopathy pathogenesis.2.Primary human retinal pericyte cells  were maintained in complete medium  and cultured at 37 °C in a humidified incubator with an atmosphere of 95% O2 and 5% CORecombinant human TGFβ1 protein (240-B-002/CF), human TGFβ2 protein (302-B2-002/CF), human TGFβ3 protein (243-B3-002/CF), recombinant mouse TGFβ1 protein (7666-MB-005/CF), and mouse TGFβ2 protein (7346-B2-005/CF) were obtained from R&D and reconstituted as recommended by the manufacturer. Recombinant mouse TGFβ3 protein (SRP6552-5UG) was purchased from Sigma .5 cells per well) in a 24-well plate and incubated for 24 h, and then treated with TGFβ1 and incubated for 24, 48, and 72 h. After the treatment, the media was removed and replaced with fresh media containing 1.5 µL of senescence dye  in 500 µL of fresh media. After incubation for 1 h at 37 °C, 5% CO2, the cells were washed two times with 500 µL wash buffer . Cells were collected by trypsinization and resuspended in 500 µL wash buffer and analyzed for Alexa488 signal immediately using flow cytometry .An assay kit from Abcam (ab228562) was used to identify senescent cells. Pericytes were seeded .MTT cell proliferation assay kit from Abcam (ab211091) was used to quantify cell proliferation. Cells were plated in a 96-well plate and treated with TGFβ1 for 24, 48, and 72 h. After the treatment, the media was replaced with 50 µL of serum-free media and 50 µL of MTT reagent  per well. After incubation at 37 °C for 3 h, MTT solvent  was added to each well. The plate was protected from light and shaken on an orbital shaker for 15 min. The absorbance at 590 nm was recorded using a plate reader  (11644793001) was utilized according to the manufacturer’s instructions. Briefly, 100 µL of supernatant from treated cells and 100 µL of freshly prepared reaction mixture were mixed in a 96-well plate and incubated for up to 30 min at room temperature protected from light. The absorbance was measured at 490 nm.The supernatants from TGFβ1-treated pericytes were analyzed with Luminex multiplex technology. We custom selected cytokines from R&D and utilized Luminex200 to detect levels of TNFα, IL6, IL8, GM-CSF, EGF, VEGF, bFGF, PLGF, ANGPTL4, ICAM1, Collagen I, Collagen IV, Fibronectin, MMP1, MMP3, and TIMP1, and the results were analyzed with the software Bio-Plex Manager (Version 6.2.0175). Luminex technology was also used to assess protein levels of cytokines in ocular fluid collected from TGFβ1-, TGFβ2-, and TGFβ3-injected mice. For these studies, a customized Luminex multiplex immunoassay panel for mice cytokines was generated and purchased from R&D.Total RNA was isolated with RNeasy Mini Kit  and measured with nanodrop . IScript™ cDNA Synthesis Kit  was used for cDNA synthesis. Primers for VEGF (Hs00900055_m1), IL6 (Hs00174131_m1), PAI1 (Hs00167155_m1), and p15Ink4b (Hs00793225_m1) were obtained from TaqMan , and β-actin primers were obtained from Sigma (05046165001). Quantitative PCR was performed using TaqMan Fast Advanced Master Mix  and The Applied Biosystems QuantStudio 12K Flex Real-Time PCR System. Further, the following protocol was utilized: denature for 2 min at 95 °C, followed by 40 cycles, each consisting of 95 °C denaturation for 1 s, and 60 °C annealing for 20 s. The relative expression level of the transcripts was analyzed using the 2-△△Ct approach.® M1000 PRO) was utilized for absorbance detection.Supernatant from TGFβ1-treated cells were used to detect cytokines of interest. The kits from Invitrogen  for the detection of human VEGF, IL6, and PAI1, respectively, were used. A Tecan , program one, for dehydration of mice eyes. In addition, eyes were embedded in paraffin.The eyes were fixed in 10% NFB  overnight and washed in PBS. We used the dehydration machine . Ventana system, ACD probes, and ACD kits were used for automated ISH. ACD probe used: RNAscope® 2.5 VS Probe- Mm-Vegfa-O1 Cat No. 436969.Glass slides  were dried and washed in xylene  in advance. Eyes were cut into 5 µm sections with a Leica microtome (RM2255) and mounted onto the glass slides in mounting media  and the signal was quantified with HALO software (V3.2).t-test, one-way ANOVA, or two-way ANOVA. Furthermore, applicable post hoc correction tests  were executed. For all graphs, data are presented as mean ± SD. p values < 0.05 were considered significant.Each experiment was repeated at least three times unless stated otherwise. Prism 7 (GraphPad) was used to perform statistical analyses and generate charts. Statistical analysis included unpaired, Student’s TGFβ1 could induce defects in retinal pericytes, we performed FACS-based senescence assays. We cultured primary retinal pericytes and treated them with 50 ng/mL of TGFβ1 for 24, 48, and 72 h. After these timepoints, we set out to quantify production of senescence-associated β-galactosidase , which is detectable only in senescent cells , a pleiotropic factor under the regulation of TGFβ, reportedly regulates cell cycling. Therefore, we analyzed the expression patterns of cell cycling factors and found that p15 and PAI1 are upregulated upon TGFβ1 stimulation c, whereaCollectively, our data suggest that TGFβ can control multiple molecular pathways of senescence in pericytes. Furthermore, we speculate that this could be an early step in diabetic retinopathy disease progression that contributes to pericyte dropout.Since local and systemic TGFβ1 increases are detectable in diabetic patients, understanding the local effect of TGFβ1 in the retina is essential. To determine the impact of TGFβ1 protein in vivo, animals were intravitreally injected with 50 ng/mL of TGFβ1, and eyes were collected after 48 h for ocular fluid analysis with Luminex, and tissue was paraffin-embedded for ISH analysis. VEGF protein levels detected via Luminex were increased in TGFβ1-injected animals after 48 h a. In addLocal expression of VEGF within retinal neurovascular units could be a major contributing driver of microvascular complications in disease progression. The prevalence of diabetes has been steadily increasing over the past few decades ,45. DiabThe mechanism by which high blood glucose levels induce progressive damage of the retinal vessels remains unclear. Here, we propose an early role of TGFβ in the cascade of structural and functional alterations in the endothelium that eventually leads to retinal dysfunction. Glucose increases activation of TGFβ ,46 and, In the retina, TGFβ signaling plays an essential part in retinal vasculature development, verified in the conditional knock-out TβRIIΔeye mice. The authors detected impaired retinal capillaries exhibiting torturous, leaky vessels, microaneurysms, and hemorrhages . Beyond TGFβ is implicated in pathological angiogenesis in the retinal vasculopathies; however, the mechanism is not fully understood. Contrasting published data support both antiangiogenic and proangiogenic roles of TGFβ1 ,59. Here, we have shown that TGFβ1 stimulates VEGF expression in vitro and in vivo. Further, we hypothesize that a mechanism by which TGFβ1 induces the changes in the diabetic retina could be cellular senescence. In cultured retinal pericytes, TGFβ1 induces characteristics of cellular senescence, including cell cycle arrest, SASP production, and morphological changes. Moreover, we show that this effect possibly occurs via the regulation of p15 and PAI1 genes. This is in agreement with the reported role of TGFβ in cell cycle arrest where TGFβ signaling stimulates p21, p15, and p57 expression in cancer cells .We have also demonstrated that TGFβ1 induces senescence in retinal pericytes for the first time. Therefore, we propose that cellular senescence plays a role in early events of diabetic retinopathy, such as pericyte dropout, angiogenesis, and immune cell infiltration. Furthermore, we show that TGFβ1 induces, besides VEGF, the release of many essential cytokines that play a role in the progression of diabetic retinopathy. For example, IL-6 is recognized as an inducer of retinal vascular inflammation and permeability . ANGPTL-Our data suggest that TGFβ1 induces VEGF production and release in vitro and in vivo. In retinal pericytes, this effect is via the mechanism of cellular senescence, and it further elevates other cytokines that play a role in the development of diabetic retinopathy. Therefore, we have proposed that TGFβ1-induced senescence may play a role in the disease progression.We have conducted this pilot study that will be extended in the future; nevertheless, our results strongly suggest that TGFβ should be further studied as an early player in diabetic retinopathy. Furthermore, identifying other early players in the disease should be elucidated in future studies, as early underlying processes remain elusive. Finally, the contribution of senescence in DR phenotypes has become more evident with recent studies; therefore, we believe that studying the involvement of this process in diabetic retinopathy might shed new light on overlooked drivers and mechanisms of this devastating disease.