Desmonostoc muscorum CCALA125 strain extracts were enriched by polymeric resin treatment, and subjected to HPCCC affording three cyclic lipopeptides (1–3), which were further repurified by semi-preparative HPLC, affording 1, 2, and 3, with a purity of 86%, 92%, and 90%, respectively. The chemical identities of 2–3 were determined as muscotoxins A and B, respectively, by comparison with previously reported ESI-HRMS/MS data, whereas 1 was determined as a novel muscotoxin variant (muscotoxin C) using NMR and ESI-HRMS/MS data. Owing to the high yield (50 mg), compound 2 was broadly screened for its antimicrobial potential exhibiting a strong antifungal activity against Alternaria alternata, Monographella cucumerina, and Aspergillus fumigatus, with minimum inhibitory concentration (MIC) values of 0.58, 2.34, and 2.34 µg/mL; respectively, and weak antibacterial activity against Bacillus subtilis with a MIC value of 37.5 µg/mL. Compounds 1 and 3 were tested only against the plant pathogenic fungus Sclerotinia sclerotiorum due to their low yield, displaying a moderate antifungal activity. The developed chromatographic method proved to be an efficient tool for obtaining muscotoxins with potent antifungal properties.Muscotoxins are cyanobacterial cyclic lipopeptides with potential applications in biomedicine and biotechnology. In this study,  Cyanobacteria are Gram-negative photosynthetic microorganisms that have gained attention as a source of potential therapeutically useful compounds . One groβ-amino fatty acid [Desmonostoc muscorum CCALA125 (synonymous to Lukešová 1986/14 and NIVA-CYA 817). The structures of these compounds were determined by detailed HRMS/MS and NMR analysis, and Marfey’s reagent was used for chiral amino acid analysis after acid hydrolysis [Muscotoxins are cyanobacterial CLPs comprising 11 amino acid units including a tty acid . Our grodrolysis . Muscotodrolysis . HoweverCymbopogon citratus [Beta vulgaris [Siparuna glycycarpa [Microcystis aeruginosa [Chlorella zofingiensis [High-performance countercurrent chromatography (HPCCC) is a solid support-free chromatographic technique that uses two immiscible solvent phases. The equipment uses centrifugal force for retaining the stationary phase within the column, while the mobile phase is pumped through the column . Those ccitratus , Beta vuvulgaris , and Sipycycarpa , respectruginosa , and canngiensis , using cngiensis .As muscotoxins belong to the class of cyclic lipopeptides usually possessing antimicrobial activities, they could be interesting targets for addressing combinatorial biosynthesis strategies aimed at enhancing their potential pharmacological efficacy or mitigating toxicity issues. Herein, we report the separation of muscotoxins from cyanobacteria by the combined use of HPCCC, polymeric resins, and HPLC. The isolated compounds were tested for their antimicrobial activity against a panel of fungi, as well as Gram-positive and Gram-negative bacteria.2 and 3 from D. muscorum CCALA 125 extract were determined by comparing their ESI-HRMS/MS spectra with the literature data [2, the major CLP in the extract  for the target compound. Moreover, it should exhibit a short settling time (less than 30 s) and a suitable density difference between the upper and lower phases of the two-phase solvent system, in order to favor the retention of stationary phase inside the HPCCC column [K2/K1, K2 > K1) should be greater than 1.5 [K values show a poor resolution because they elute closer to the solvent front. On the contrary, those compounds with larger K value are better separated but elute as wider peaks and at later elution times [K values of the compounds 1–3 was investigated. The K value is frequently estimated by using HPLC with UV detection, which is calculated by dividing the peak area of the target compound in the upper phase by that of the peak area of the same target compound in the lower phase. However, given that UV chromatogram provided insufficient peak areas for minor muscotoxins, the MS detector was thus used to estimate the K values. By using this approach, the peak area of the molecular ions corresponding to the target compounds were selectively monitored from the base peak chromatogram. As presented in the n-hexane, ethyl acetate, ethanol, and water in a ratio of 1:5:1:5) gave suitable K values and a proper separation factor (α) for compounds 2 and 3, as well as a short settling time. However, the solvent system 10 gave a small K value (K ≤ 0.5) for compound 1, indicating that this compound would be poorly separated from other contaminants. Hence, to have a suitable K value, the solubility of compound 1 in the upper phase should be increased. The improvement of the K value of compound 1 could be achieved by finding out a new solvent system or by modifying the composition of the already selected solvent system. In the present study, the system 10 was acidified by adding acetic acid leading to the system 11 . This modification favored the solubility of compound 1 in the upper phase of the solvent system 11, giving rise to an appropriate partition coefficient (K = 1.09), a short settling time, and a suitable density difference. Although the partition coefficients of the target compounds in systems 10 and 11 are strongly correlated, the system 10 ensured a sufficient selectivity (α = 1.5) between the compounds 2 and 3. Overall, these results indicated that the separation of the compounds 1–3 would demand the application of a two-step HPCCC method. In this context, the solvent system 10 showed selectivity for the compounds 2 and 3, and solvent system 11 was selective for the compound 1. In HPCCC, it is very well established that when the retention of stationary phase is increased, a good chromatographic resolution is achieved [2 and 3 was achieved when injecting 100 mg of enriched extract.When working with HPCCC, the search for a proper two-phase solvent system has been informed to represent the major part of the work involved in the HPCCC separation . An apprC column ,26, whicthan 1.5 ,26. It ion times . In the achieved . A reducn-Hex–EtOAc–EtOH–H2O, 1:5:1:5) was used as mobile phase at a flow rate of 1 mL/min. This process was repeated 10 times to separate all enriched extract. The stationary phase retention during the HPCCC separation was 72%. Three peaks fractions that were eluted at the retention times from 68 to 88 min, from 110 to 135 min, and from 150 to 170 min, corresponded to the compound 1-containing fraction (Fr.1), compound 2, and compound 3, respectively , as well as the compounds 2  and 3 . Fr.1 was further subjected to a second HPCCC purification step using solvent system 11, with its lower phase as the mobile phase at a flow rate of 1 mL/min. The target compound 1 was eluted at the retention times from 170 to 190 min . The stationary phase retention during the HPCCC separation was 70%. In this study, the isolation of compound 1 is reported for the first time. The separation of this kind of CLPs with closely related chemical structures demonstrates the powerful selectivity of HPCCC.The optimized operating conditions were used to process 1000 mg of enriched extract using HPCCC, where the lower phase of the solvent system 10 , 2 , and 3  were obtained by HPLC when considering the stereoisomers of the target compounds as contaminants (The combined use of HPCCC and HPLC has proven to be efficient in the isolation of CLPs . This chaminants . The HPL2 and 3 was confirmed by ESI-HRMS/MS experiments. The fragment ions generated from the fragmentation of the molecular ions at m/z 1211.67 (2) and m/z 1225.68 (3) were in excellent agreement with those of the amino acid sequence of muscotoxins A and B; respectively [D. muscorum CCALA 125. Compounds 2 and 3 differ from each other only by a -CH3 group (methyl group) corresponding to the substitution of l-proline for γ-methylproline, as previously demonstrated by our team using 2D NMR methods [1 was determined by the combination of NMR and ESI-HRMS/MS. Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) experiments including COSY, TOCSY, and 1H-13C HSQC-TOCSY were performed to identify the amino acids composition of 1, as shown in 1, Gln2, Gly3, Pro4, Phe5, Val6, Ser7, dThr8, Ser9, Ile10, Pro11) (1) was assigned as muscotoxin C, which differs from the previously described muscotoxin A (2) in the substitution of Ile6 by Val6  . This pe, Pro11) . The nov by Val6 .1–3) were tested for their antifungal activity against common plant pathogenic fungi, S. sclerotiorum. Interestingly, despite the isolated variants differing only in one amino acid residue, their antifungal activity against S. sclerotiorum differed significantly . These data hint the possible role of the amino acid substitution in the peptidic core of the molecule, such as Pro by γ-MePro or Val by Ile enhancing the bioactivity. Owing to the high-yield isolation of compound 2 and the unavailability of sufficient amounts for minor variants isolated (1 and 3), the antimicrobial activity against five bacterial and eight fungal isolates was investigated only for compound 2. Among all the bacterial strains tested, 2 showed a weak antibacterial activity only against the Gram-positive pathogen Bacillus subtilis with a minimum inhibitory concentration (MIC) value of 37.5 µg/mL and MIC95 at ≤300 µg/mL. However, it exhibited a moderate to strong antifungal activity against all the fungal strains tested, except Bipolaris sorokiniana. The strongest activity was observed against Alternaria alternata, Monographella cucumerina, and Aspergillus fumigatus, with MIC values of 0.58, 2.34, and 2.34 µg/mL, respectively, and minimum fungicidal concentration (MFC) values of 75, 75, and 37.5 µg/mL, respectively  and Analytika . The methanol used for extraction was obtained from Analytika . Acetonitrile and water for HPLC-HRMS analyses were obtained either from Sigma-Aldrich  or Merck , and were of LC-MS grade purity. Deionized water was obtained using reverse-osmosis . Non-ionic polymeric adsorbents (Amberlite XAD16 and Amberlite XAD-7) were from Sigma-Aldrich .D. muscorum CCALA 125 was cultivated in a 15 L photobioreactor on Anabaena medium by bubbling CO2-enriched air (2%) at 28 °C for 10 days [g, 15 min), frozen at −40 °C, and then freeze-dried, affording 40 g of dried biomass. Sea sand was used for disintegration of the freeze-dried biomass and methanol (1 L) for extraction. The extraction operation was repeated three times on the same biomass. The resulting suspension (3 L) was centrifuged  and the supernatant was concentrated to total dryness using a rotary evaporator (at 40 °C). About 6200 mg of dried crude extract was obtained, which was stored at 2 °C for the subsequent enrichment process.The filamentous cyanobacterium  10 days . The celTwo non-ionic polymeric resins, that are commercially offered as XAD-16 and XAD-7 Amberlite resins, were consecutively used for enriching the dried crude extract that was obtained in the previous step. For that purpose, a suspension made of 6.200 g of dried crude extract and 500 mL of water was prepared and loaded into an Amberlite XAD-16 resin column . To remove the non-adsorbed components from the column, 1 L of water was passed thorough the column. The resulting aqueous solution (1.5 L), eluted from XAD-16 resin column, was further passed through the XAD-7 resin column . Again, the XAD-7 resin column was rinsed with water to remove the non-adsorbed components. Finally, the adsorbed compounds were released from each column using methanol as eluent (1.5 L). The resulting methanol eluates from each column were separately concentrated to total dryness using a rotary evaporator (at 40 °C). The dried enriched extracts were analyzed by HPLC-UV-ESI-HRMS.A HPCCC apparatus  was used for separating the target compounds from the enriched extract. It was used a semi-preparative HPCCC column (134 mL). A H50/H150 Smart Water Chiller  was used for adjusting the temperature of separation work. The stationary and mobile phases were pumped with a Q-Grad pump . The effluent from the column was continuously monitored with a Sapphire UV-VIS spectrophotometer  operating at 240 nm. The chromatographic run was processed using an EZChrom SI software platform .n-hexane, ethyl acetate, ethanol, water, and acetic acid in different proportions (K) of the target compounds. In addition, given that the settling time as well as the density difference between the upper and lower phases of each two-phase solvent system are parameters associated with the retention of stationary phase in the column, they were thus estimated [K), which were calculated by dividing the peak area of the target compound in the upper phase by the peak area of compound in the lower phase.Different two-phase solvent systems were prepared using portions . The prestimated ,26 and uThe best two-phase solvent system selected from the previous step was prepared at a large scale for separating the target compounds by HPCCC. The components of the selected system were added into a separating funnel and shaken vigorously and were then left to equilibrate. After 20 min, the upper and lower phases were separated and degassed by applying sonication. The sample solution was prepared by dissolving the enriched extract in 3 mL of the lower phase of the selected solvent system.n-Hex–EtOAc–EtOH–H2O  was used in reverse elution mode for obtaining compounds 2–3, and the 1-containing fraction. In the HPCCC second step, the solvent system composed of n-Hex–EtOAc–EtOH–H2O–AcOH, 1:5:1:5:1 was used in reverse elution mode for obtaining the compound 1. In the reverse elution mode, the lower phase of the two-phase solvent system is used as the mobile phase and the upper phase as the stationary phase. At the beginning, the HPCCC column was filled with the upper phase (stationary phase), and it was rotated at 1200 rpm. After that, the lower phase (mobile phase) was pumped through the column at a flow rate of 1 mL/min. When reaching the hydrodynamic equilibrium, that is, when the volume of stationary phase that is eluted from the column is constant, the sample solution was injected. The separation was performed at 28 °C. The fractions that eluted from the HPCCC column were collected and analyzed offline by HPLC-ESI-HRMS. The retention of the stationary phase (Sf) during the HPCCC run was calculated as follows:The separation of the target compounds from the enriched extract was performed using a two-step HPCCC method. In the HPCCC first step, the solvent system composed of −1, and the column temperature was set at 28 °C.The HPCCC peak fractions corresponding to the target compounds were repurified using a semi-preparative Agilent 1100 HPLC system. The separation was performed on a reverse phase Reprosil 100 C18 column  using a mobile phase composed of acetonitrile (A) and water (B) using the following gradient: 0–2 min, 70% B; 2–6 min, 70–60% B; 6–15 min, 60–30% B; 15–16 min, 30–0% B; 16–20 min, 0–0% B; 20–21 min, 0–70% B. The peaks were detected with a diode array detector (DAD) at a monitoring wavelength of 240 nm. The mobile phase was pumped at a flow rate of 2 mL minm/z, operating in the positive ion mode. The peaks were recorded with a diode array detector (DAD) at a monitoring wavelength of 240 nm.The extracts and HPCCC fractions were analyzed using a Dionex UltiMate 3000 HPLC system  coupled with a diode array detector (DAD) and with electrospray ionization sourced-high resolution mass spectrometer . The separations were performed on a reversed phase column , held at a constant temperature of 30 °C. The mobile phase of the HPLC run consisted of the combination of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The following gradient was used: 0–1 min, 85% A; 1–20 min, 85–0% A; 20–25 min, 0% A; 25–30 min, 0–85% A, which was pumped at a constant flow rate of 0.6 mL/min. The source parameters were as follows: the spray needle voltage was set at 3.8 kV, nitrogen was used both as the nebulizing gas (3 bar) and the drying gas (12 L/min), and the temperature was 210 °C. The fragmentation of the molecular ions was induced by using nitrogen as a collision gas. The cyclic oligopeptide part of the target compounds was determined at collision energy of 60 eV. The scanning range was 50–2600 1–3 are presented in 1 was determined by NMR data as presented in 1H, 150.93 MHz for 13C, Bruker Biospin GmbH, Rheinstetten, Germany) in CD3OD, 303.2 K. The residual solvent signals were used as an internal standard (δH 3.330 ppm and δC 49.05 ppm). 1H-NMR, 13C-NMR, COSY, TOCSY, 1H-13C HSQC, 1H-13C HMBC, 1H-13C HSQC-TOCSY, and J-resolved spectra were measured using the standard manufacturer’s software TopSpin 3.5. The 1H-NMR spectrum was zero-filled to 2-fold data points and multiplied by a window function , before Fourier transformation to improve the resolution. The 13C-NMR spectrum was zero filled to 2-fold data points. Subsequently, the line broadening (1 Hz) was used to improve signal-to-noise ratio. Protons were assigned by COSY, TOCSY, and HSQC-TOCSY, and the assignment was transferred to carbons by HSQC. The chemical shifts are given on the δ scale (ppm), and coupling constants are given in Hz. The digital resolution allowed us to present the proton and carbon chemical shifts to three or two decimal places. The proton chemical shift readouts from HSQC are reported to two decimal places. The operating conditions of the ESI-HRMS instrument are indicated in the previous section.The chemical identity of the target compounds was determined by ESI-HRMS and NMR analysis in comparison with literature data . The MS 1–3) was initially tested only against plant pathogenic fungi Sclerotinia sclerotiorum by determining the minimum inhibitory concentration (MIC) using disc diffusion assay. Compounds (1–3) were dissolved in MeOH at different concentrations and further transferred (20 µL) on layer filter paper targets (size 5 mm) to get desired concentrations of 250–1.5625 µg/disk. Discs were placed at the border of Petri dish (average 35 mm) containing Czapek-Dox medium. The mycelial target of the plant pathogenic fungus S. sclerotiorum (0.5 cm in diameter) was inoculated on the opposite sites of dish. The fungicide RANCONA 15 ME  and methanol were used as positive and negative control, respectively. Each experiment was performed in triplicate. The growth of fungal mycelium after 4 days in the presence of the studied compounds was evaluated using digital image analysis. The macro-optic system, consisting of a digital camera DUS-1 (5Mpx) and objective lens type Cosmicar  were used for acquisition of images of the Petri dishes. The images (TIFF format) were converted by thresholds, and the area of binary objects corresponding to size of grown mycelium was measured and evaluated by software NIS-ELEMENTS AR ver. 3.2  and by a method published previously [Due to the unavailability of enough amounts of isolated compounds, the antifungal activity for all the isolated compounds , B. subtilis (CCM1999), S. sanguinis (CCM4047), P. aeruginosa (CCM1959), E. coli (CCM2024), and eight fungal isolates; C. friedrichii (BCC020_2879), A. fumigatus (BCC020_2845), F. oxysporum (BCC020_2866), T. harzianum (BCC020_0606), B. sorokiniana (BCC020_1571), M. cucumerina (BCC020_2872), C. globosum (BCC020_2527), and A. alternata (BCC020_0609) using broth two-fold microdilution method following the standard protocol [S. aureus and S. sanguinis, gentamycin (32–0.0625 µg/mL) was used as positive control for Bacillus subtilis, E. coli, and P. aeruginosa, whereas fluconazole (32–0.0625 µg/mL) was used as positive control for all the fungal isolates. After incubation, the well with the least concentration of the compound showing any inhibition in the growth was taken as the MIC value for the respective organisms. MIC95 was determined as the concentration required to inhibit or kill 95% of bacterial isolates spectroscopically at 600 nm. Minimum fungicidal concentration (MFCs) were determined after 48 h incubation by removing 10 µL of the contents from wells showing no visible growth and spreading them on to Sabouraud dextrose agar plates [Furthermore, compound protocol . Brieflyr plates . The plaDesmonostoc muscorum strain CCALA125 by the combined use of adsorption on polymeric resins, HPCCC, and HPLC methods. As these three chromatographic methods are recognized to be orthogonal against each other, therefore, their combined use rendered the separation of chemically similar compounds. Muscotoxin C was isolated for the first time and its chemical structure was established by MS and NMR spectroscopic data. The described isolation method is an efficient approach for obtaining CPLs from cyanobacterial biomass and represents a methodological reference than can be scaled up for obtaining these compounds in higher amounts. Muscotoxin A was found to exhibit a potent antifungal activity against plant pathogenic fungi, thus, it might have applications as an agricultural fungicide or be used as a potential chemical template for developing new fungicides.Three CLPs (muscotoxins A–C) were isolated and purified from the filamentous cyanobacterium


Nitrate (N) response is modulated by light, but not understood from a genome-wide perspective. Comparative transcriptomic analyses of nitrate response in light-grown and etiolated rice leaves revealed 303 and 249 differentially expressed genes (DEGs) respectively. A majority of them were exclusive to light (270) or dark (216) condition, whereas 33 DEGs were common. The latter may constitute response to N signaling regardless of light. Functional annotation and pathway enrichment analyses of the DEGs showed that nitrate primarily modulates conserved N signaling and metabolism in light, whereas oxidation–reduction processes, pentose-phosphate shunt, starch-, sucrose- and glycerolipid-metabolisms in the dark. Differential N-regulation of these pathways by light could be attributed to the involvement of distinctive sets of transporters, transcription factors, enriched cis-acting motifs in the promoters of DEGs as well as differential modulation of N-responsive transcriptional regulatory networks in light and dark. Sub-clustering of DEGs-associated protein–protein interaction network constructed using experimentally validated interactors revealed that nitrate regulates a molecular complex consisting of nitrite reductase, ferredoxin-NADP reductase and ferredoxin. This complex is associated with flowering time, revealing a meeting point for N-regulation of N-response and N-use efficiency. Together, our results provide novel insights into distinct pathways of N-signaling in light and dark conditions. Rice has the least NUE among cereals and therefore tops all other crops in N-fertilizer consumption in India2. The molecular aspects of nitrate transport, assimilation, signalling and crosstalk with water, hormone, and development are better understood than the biological determinants of crop nitrogen use efficiency14. Characterization of the phenotype for NUE will be crucial for progress in this regard15.A major challenge in improving crops for input use efficiency is to understand and optimize the inputs for various agroclimatic conditions including light and photoperiod, soil type, altitude, humidity etc. Nitrogen (N) is quantitatively the most important fertilizer input for intensive cropping, but globally, nitrogen use efficiency (NUE) is as low as 30–40% for various crops, which is a major cause for economic losses and environmental consequences of N pollutionNR) and nitrite reductase (NiR), followed by their assimilation into amino acids through the glutamine synthetase and glutamate synthase (GS-GOGAT) cycle. This requires 2-oxoglutarate (2-OG) from the carbon metabolism and hence coordination between C and N metabolism4. Transcriptomic studies have revealed thousands of nitrate-responsive genes in Arabidopsis18, rice23 and maize24. They include those involved in metabolism, redox balance, signaling, stress, hormones, development etc., indicating their possible role in NUE26. Heterotrimeric G-protein gamma subunit has been identified as a QTL for NUE in rice27, while the beta subunit has been shown to mediate nitrate-responsive root development28. G-proteins were implicated in light regulation of NR gene expression in our earlier studies in maize30 and rice31. Our transcriptome studies in G-protein mutants showed their role in other nitrogen and/or stress responses36.Nitrate is taken up into the cell by a family of transporters and converted into ammonium ions by the serial action of nitrate reductase , a positive regulator of light signalling, has been shown to enhance nitrate uptake in Arabidopsis40. Interestingly, N regulates flowering time by modulating a blue-light receptor cryptochrome 1 (CRY1) in Arabidopsis41. Sucrose mimics light-responses and its exogenous application induces NR activity possibly via hexose’s sensor independent N-signalling pathway in Arabidopsis42. Further, metabolite sensor SNRK1 regulates NR and sucrose phosphate synthase activity and therefore controls both N and C metabolism43.Light is an important regulator of plant N-responses, both through photosynthesis and C/N balance, as well as through light signalingMost of the above information on the role of light in N-response was based on Arabidopsis and not on crop plants. Therefore, to delineate the molecular basis of light-dependent and independent nitrate response, we analysed the nitrate-responsive leaf transcriptomes of light-grown and etiolated rice seedlings in this study.3 was found to be optimum in light and dark conditions  elongated mesocotyl and coleoptile length compared to light-grown condition   of ± 1 and p value ≤ 0.05 were used to define the differentially expressed genes (DEGs). Multiple testing corrections using Benjamini–Hochberg adjusted p value were tried but not considered, as they produced many false negatives by eliminating many well-known N-responsive genes obtained from qualifying our geomean cutoff (log2FC) of ± 1 45 to predict the subcellular distribution of proteins encoded by nitrate-responsive DEGs in light and dark, which revealed that they are predominantly targeted to the nucleus, followed by plasma membrane, chloroplast and others 48. The results were broadly similar to those from GO analysis, with nitrate-responsive changes to metabolism, cellular responses, regulation and hormone biosynthesis among others  pathways such as phenylpropanoid biosynthesis, nitrogen metabolism, phenylalanine metabolism, hormone signal transduction and photosynthesis in light, whereas in the dark, they were starch and sucrose metabolism, metabolic pathways, glycerolipid metabolism and N-Glycan biosynthesis Table .To validate the expression profile of nitrate-responsive genes involved in nutrient, stress and development, 6 DEGs out of 8 tested DEGs encoding transporter, transcription factors and others showed statistically significant differences in qRT-PCR, thus validating the microarray results Fig. .Figure 4bHLH120 (Os09g0455300/BGIOSGA030896), bHLH066 (Os03g0759700/BGIOSGA013600). However, universal stress domain containing protein (Os02g0707900/BGIOSGA005763), efflux transporter of nicotianamine 1 (Os11g0151500/BGIOSGA034485) and MYB family transcription factors  were up-regulated by nitrate in the dark but not detected in light condition. The SPX domain containing protein (Os02g0202200/BGIOSGA007749) was down-regulated in both light and dark. The reason for non-detected DEGs in microarray was their statistically non-significant expressions (Table The details of these DEGs and corresponding primer sequences used for qRT-PCR are provided in Table ns Table . The qRToryzabase database (https://shigen.nig.ac.jp/rice/oryzabase/) and found that N-responsive DEGs were assigned to culm, root, leaf, spikelet, seed, heading date and panicle  and LTP family protein (LOC_Os02g44310.1). Phytoene synthase (PSY) regulates carbon flux in carotenoid biosynthesis and is required to induce carotenogenesis-dependent ABA accumulation in root under different abiotic stress conditions50. The up-regulated expression of PSY in light may be associated with root inhibition by high nitrate in rice. Leaf is the primary organ for photosynthesis in rice and application of high N dose has been shown to reduce photosynthetic NUE51. Among leaf-associated N-responsive DEGs, we observed up-regulated expression of Zn transporter 3 (LOC_Os04g52310.1) and ABA stress-ripening (LOC_Os11g06720.1) in light, whereas plastid terminal oxidases 1 (LOC_Os04g57320.1) and YABBY domain containing protein (LOC_Os04g45330.1) were up-reregulated in dark , it was of interest to examine how many of the N-responsive genes identified here are expressed in other stages/tissues of the rice plant. This allows to separate those genes that are ubiquitously expressed in most of the organs from those that are unique to specific organs/stages for further validation of their nitrate response. This was particularly relevant in view of the incomplete characterization of the phenotype for N-response/NUE. We therefore searched the cle Fig. . The avaark Fig. .Figure 553, whereas YABBY domain containing proteins are known to regulate leaf morphology in plants54. A few DEGs were also associated with agronomically important traits , which revealed 270 and 216 N-responsive DEGs exclusive to light and dark conditions, respectively, with an additional 33 genes in common , RiceSRTFDB (https://www.nipgr.ac.in/RiceSRTFDB.html), STIFDB (https://caps.ncbs.res.in/stifdb/) and MapMan and classifying them into families using Rice SRTFDB  in rice. This was done by searching the for N-responsive DEGs in different transcription factor databases viz. RiceFREND  was used to identify the over-represented motifs in the 1 kb promoter sequences of the N-responsive DEGs identified in light and dark conditions.Transcription factors (TFs) act as master regulators by binding to cis-acting motifs in the promoter regions of genes to control diverse cell processes, including N signaling57. We further predicted the motif enrichment using 1 kb promoter sequence of all the DEGs identified in light and dark conditions. Examination of the 20 most significantly enriched motifs in DEGs identified under light and dark conditions revealed that they are entirely different sets, despite the fact that 33 DEGs were common to both conditions  networks built from yeast one-hybrid screens and validated by knockouts57, similar networks were developed in this study using their orthologues in rice. Prior to this, we verified the level of gene conservation between rice and Arabidopsis using Orthovenn 265. We observed 11,367 and 11,956 orthologous clusters in Arabidopsis and rice, respectively, of which 9,698 orthologous clusters were common . We identified 144 conserved orthologs in rice, with 91 in light and 53 in dark conditions, of which many, but not all were N-responsive in our microarray data  networks using N-responsive DEGs in light and dark conditions Figs. , S9. Theork Fig.  and noneork Fig. , suggestork Fig. . This cl24. Light regulation of nitrate assimilation was studied extensively in etiolated and green plants from the point of view of direct light signaling through photoreceptors or indirect signaling through C-metabolites. However, to the best of our knowledge, no study has comprehensively analyzed genome-wide nitrate signaling in light and dark conditions. Therefore, we exploited the availability of functional genomic tools to analyze the nitrate-responsive transcriptome in etiolated and green leaves of rice, a crop notorious for its poor NUE among cereals. This enabled us to understand light-dependent and light-independent nitrate responses, visualize the networks of underlying interactions and identify potential candidates to manipulate N-responses/NUE in rice.To date, a number of N-transcriptomic studies were carried out to identify the number of genes and associated pathways involved in N-uptake, -signaling, -metabolism and assimilation among others3, downstream metabolites, or the influence of root-shoot translocation parameters that may respond differently in light and dark29. These experimental factors can confound the interpretation of results, whereas we obtained consistent results when cut-leaves were treated with standardized concentrations of nitrate29 as was done in the present case. Our transcriptome analyses showed very distinctive sets of nitrate responsive genes in etiolated and green leaves, with only 33 common between them. The common up-regulated genes such as NR, FNR, ENOD20, BTBA4 were involved in N-metabolism while those commonly down-regulated such as peroxidase cysteine-rich repeat secretory protein 55, go35 NBS-LRR were involved in stresses  in dark condition ; its regulation by light offers a crucial meeting point in the mechanism of nitrate response and therefore a potential target to manipulate NUE. Moreover, flowering time is also an important determinant of crop duration in rice and we have already shown that long duration rice genotypes tend to have higher NUE15.Sub-clustering of the PPI networks using MCODE did not reveal any molecular complex in the dark, but a single molecular complex was identified in light, consisting of NiR, FNR and ferredoxin Fig. . FNR has72. Genes involved in the transport of chloride, amino acid, potassium, sulfate, and water channel were also up regulated in light  transporters were among the most abundant DEGs, though their members were different in light and dark conditions Table . They arht Table . It is k57. The constructed NTR networks using rice orthologs were similar to Arabidopsis NTR networks as expected, suggesting a core of nitrate-regulated genes and that their associated connections are highly conserved between rice and Arabidopsis. Mapping the nitrate-responsive gene expression data produced two distinct NTR networks for light and dark  or double distilled water (control) for 90 min. In case of dark experiments, similar treatments were given to excised leaves in dark condition. The control and nitrate-treated leaves were instantly frozen in liquid nitrogen and stored at -80 ºC till use.Seeds of rice https://imagej.nih.gov/ij/download.html). Statistical unpaired T test analyses were performed in the GraphPad Prism 6 software(https://www.graphpad.com/scientific-software/prism/).Ten days old seedlings grown in light and dark conditions were kept in horizontal position and digital images were captured. The lengths of mesocotyl and coleoptile were measured using ImageJ software  for 10 min at 4 °C. The supernatant was retrieved and 0.5 ml of it was mixed with 4.5 ml of aqueous 80% ice-cold acetone. The absorbance was measured at 663.2 and 646.8 nm and chlorophyll content was calculated using the formula: Ch-a = 12.25 × A663.2—279 × A646.8; Ch-b = 21.5 × A646.8—5.1 × A663.2. Statistical unpaired T test analyses were performed in the GraphPad Prism 6 software (https://www.graphpad.com/scientific-software/prism/).The chlorophyll content was estimated as described earlier38. Briefly, 100 µl of leaf crude extract was added to the reaction mixture containing 5 mM KNO3 and 5 mM EDTA in 0.1 M sodium phosphate buffer (pH 7.5) in a total reaction volume of 0.4 ml. The reaction was incubated for 20 min at 25 °C and terminated by adding 0.6 ml of freshly prepared stopping mixture [1:1 ratio of NED (0.1% w/v) and sulfanilamide (1% w/v in 3 N HCl)]. The pink colour was measured spectrophotometrically at 540 nm. NR activity was calculated as nmoles of nitrite/mg protein/min with the help of a standard curve generated using known concentration of nitrite. NR specific activity was expressed as enzyme activity per mg protein and mean data of three independent experiments with internal triplicates were used to plot the graph with standard errors.Nitrate reductase assay was performed after 6 h of treatment with potassium nitrate as described earlier78. The RNA pellet was washed with ethanol , dried at room temperature, dissolved in DEPC-treated autoclaved water and frozen at − 20 °C till further use. Microarray experiments were performed using RNA isolated from two independent biological replicates as flip-dye replicates at Genotypic Technology Pvt Ltd, Bengaluru, India. The quality and quantity of total RNA were analyzed using Agilent Bioanalyzer as per the manufacturer’s protocol.Total RNA was isolated from leaves frozen after 90 min of treatment with either water (control) or potassium nitrate (120 mM), using modified hot phenol method as described79. Labeling was performed using low RNA Input Fluorescent Linear Amplification Kit .Total RNA was used to synthesize the first and second strand cDNA. Reaction mixture containing 500 ng RNA and 1.2 µl of oligo dT-T7 Promoter Primer in nuclease-free water was incubated at 65 °C for 10 min. Then 4.0 µl of 5 × First strand buffer, 1 µl of 10 mM dNTP mix, 2 µl of 0.1 M DTT, 1 µl of 200 U/µl MMLV-RT, and 0.5 µl of 40U/µl RNaseOUT were added and incubated at 40 °C for 2 h. After CDNA synthesis, 8 µl of NTP mixture, 2.4 µl of 10 mM Cyanine-5-CTP or Cyanine-3-CTP , 6 µl of 0.1 M DTT, 20 µl of 4 × Transcription buffer, 0.6 µl of inorganic pyrophosphatase, 0.5 µl of RNaseOUT, 0.8 µl of T7 RNA polymerase, and 15.3 µl of nuclease-free water were added to reaction mixture and incubated at 40 °C for 2 h. Qiagen'sRNeasy mini spin columns were used to purify the amplified samples. The cRNA quantity and specific activity were determined using NanoDrop ND-1000 (v 3.2.1.) and samples with specific activity > 8 were used for hybridization. Reaction mixture containing 1,650 ng of each Cyanine labeled cRNA (41.8 µl), 10 × Blocking agent (11 µl) and 25 × Fragmentation buffer (2.2 µl) was incubated at 60 °C for 30 min in dark. The fragmented cRNA were mixed with 2 × Hybridization Buffer (55 µl) and resulting 110 µl mixtures was hybridized at 65 °C for 17 h in an Agilent Microarray Hybridization Chamber with Hybridization Oven. After hybridization, slides were washed with Agilent Gene Expression Wash Buffer I and incubated them for 1 min at room temperature and 37 °C. Slides were washed again with Wash buffer II in similar condition, cleaned and dried by rinsing with acetonitrile and then scanned using Agilent scanner (G2565B) set at 100% laser power. Agilent Feature Extraction software (version 9.1) was used to extract the data, which was normalized as per the recommended Per Spot and Per Chip protocol. This is a 2-color default normalization, (Per Spot and Per Chip: Intensity dependent (Lowess) normalization) where each raw intensity value corresponding to the control channel is adjusted using a locally-weighted regression method called Lowess. Each value in the signal channel was divided by the adjusted control value, resulting in the final normalized value. The raw data were analyzed using GeneSpring 9 GX software and submitted at NCBI GEO database (Accession number: GSE12940).Microarray data analyses were performed as described earlier47 using default parameters. We have considered only statistically significant  GO terms for further analyses. TreeMap (https://www.treemap.com/) software was used to graphically represent the GO enrichments obtained from Expath analysis. Expath 2.0 tool47 was also used to perform the comparative pathway enrichment analyses of DEGs in light and dark. Mapping of DEGs onto various biological pathways was done using MapMan version 3.5.1 (https://mapman.gabipd.org/mapman-download)48. The fold change value (log2FC) and corresponding p value were used for significant enrichment of DEGs associated pathways in PageMan80. Over-represented and under-represented pathways are depicted with red and blue coloured boxes, respectively.Gene ontology based functional annotations of the DEGs were performed by Expath 2.0 toolhttps://rice.plantbiology.msu.edu/) and then analyzed by CELLO program (https://cello.life.nctu.edu.tw/) using default parameters for eukaryotes. Subcellular predictions were also made using TargetP 2.0 (https://www.cbs.dtu.dk/services/TargetP/)81.To predict the subcellular localization, the amino acid sequences of DEGs were retrieved from RGAP database (https://quantprime.mpimp-golm.mpg.de/?page=about). DEGs were selected based on their up- and down-regulated expression for described biological pathways. Approximately 2 µg of total RNA isolated from the control and nitrate-treated leaves was reverse transcribed into cDNA (20 μl volume) using cDNA synthesis kit . Its amplification reaction was carried out in 10 μl volume containing 1 μl of cDNA, 0.5 μl of forward and reverse gene specific primers (10 µM) and 5 μl of KAPA SYBR FAST Master Mix (2X) Universal . The reactions were performed in Aria Mx real-time PCR system . The relative accumulation of transcripts was analyzed by the comparative C(T) method using actin (BGIOSGA013463) as an internal control. Melting curve analyses of the amplicons were used to determine the specificity of qPCR reactions. Statistical unpaired T test analyses were performed using GraphPad Prism 6 software (https://www.graphpad.com/scientific-software/prism/).To confirm the expression pattern of the DEGs, qRT-PCR was performed in three independent biological replicates, with three technical replicates using gene-specific primers Table . All thehttps://string-db.org/), MCDRP (https://www.genomeindia.org/biocuration/), BioGRID (https://thebiogrid.org/) and PRIN (https://bis.zju.edu.cn/prin/). We mapped the DEGs to the protein–protein interaction (PPI) networks based on experimental score, using Cytoscape version 6.066. To detect the molecular complexes, we used molecular complex detection (MCODE) plugin in Cytoscape. We also downloaded all the Arabidopsis orthologs from PlantGDB database (https://www.plantgdb.org/), which were used to generate and annotate DEGs-associated PPI and nitrate-responsive transcriptional regulatory networks in rice.The lists of experimentally validated interacting proteins for the DEGs analyzed in this study were retrieved from the databases STRING (https://rapdb.dna.affrc.go.jp/tools/dump). The motif discovery oligo analysis tool of Regulatory Sequence Analysis Tools (RSAT) software82 was used to identify enriched motifs (6–8 bases oligonucleotides) in the promoter sequences using default parameters. Rice (Oryza sativa IRGSP-1.0.42) whole genome was used as the background and motifs were predicted in both the DNA strands.One kb promoter regions upstream of the translational start site of the DEGs were downloaded from RAPDB database (Supplementary information 1Supplementary information 2


Hordeum brevisubulatum evolves better strategies to retain K+ to improve high-salt tolerance. Hence, uncovering K+-efficient uptake under salt stress is vital for understanding K+ homeostasis. HAK/KUP/KT transporters play important roles in promoting K+ uptake during multiple stresses. Here, we obtained nine salt-induced HAK/KUP/KT members in H. brevisubulatum with different expression patterns compared with H. vulgare through transcriptomic analysis. One member HbHAK1 showed high-affinity K+ transporter activity in athak5 to cope with low-K+ or salt stresses. The expression of HbHAK1 in yeast Cy162 strains exhibited strong activities in K+ uptake under extremely low external K+ conditions and reducing Na+ toxicity to maintain the survival of yeast cells under high-salt-stress. Comparing with the sequence of barley HvHAK1, we found that C170 and R342 in a conserved domain played pivotal roles in K+ selectivity under extremely low-K+ conditions (10 μM) and that A13 was responsible for the salt tolerance. Our findings revealed the mechanism of HbHAK1 for K+ accumulation and the significant natural adaptive sites for HAK1 activity, highlighting the potential value for crops to promote K+-uptake under stresses.Potassium retention under saline conditions has emerged as an important determinant for salt tolerance in plants. Halophytic  Potassium represents 2.6% of the weight of the Earth’s crust. However, dissolved K in soil—as the only fraction directly available to plant—is deficient, especially in saline–alkali land and arid land . Retaini+/K+ uptake proteins/K+ transporter) proteins function as K+-transporters and the major contributor for K+ nutrition in K+ depleted soil [Arabidopsis), which indicates the important physiological roles of such transporters in crops. Their proteins possess 10–15 transmembrane (TM) domains and are divided into 5 clusters [AtHAK5, HvHAK1, OsHAK1, OsHAK5, ThHAK1, SlHAK5, CcHAK1 and so on [+ transport feature that allows plant to thrive under low-K+ (<1 mM) conditions. In fact, K+ uptake in plants also demands these HAK-type transporters under multiple stresses such as salinity, Cs+ -polluted or drought soils [+, Cs+ or drought indirectly reduces soluble K+ concentration in soil that leads to K+ deficiency [+ or Cs+ in cells causes ion imbalances with a reduced K+/Na+ or K+/Cs+ ratio that inhibits plant growth [OsHAK1 in rice leads to about 50–55% lower K+ uptake than wild types in K-starved environments, and causes sensitivity to drought and salt [+/K+ homeostasis and the membrane potential of root cells under salt conditions [Zea mays L. showed ZmHAK4-mediated shoot K+/Na+ homeostasis for improving salt tolerance, conferring the natural variation of this gene [+ nutrition largely depends on HAK/KUP/KT system, affecting agriculture worldwide.As ion fluxes control ion concentration, the HAK/KUP/KT , and A13 directly affect the K+ uptake under high external Na+ condition. Our study significantly revealed the natural adaptive sites of HbHAK1 were the key residues for improving HAK1-type activity, highlighting the potential value for crops to promote K+ uptake under stresses.Saline land occupies more than 20% of irrigated soil. It reduces crop productivity because most crops are glycophytes . As barllophytes ,23. Hordth China . Our preh barley ,26. AlthH. brevisubulatum plants could maintain a relatively stable level of K+ content under saline conditions and the numbers of upregulated genes of potassium transporters greatly exceeded that in H. vulgare, which showed obvious K+ loss under saline conditions [H. brevisubulatum and H. vulgare plants under 350 mM NaCl treatment. We searched the potential HAK/KUP/KT transporters in PacBio isoform sequencing (Iso-seq) data. Finally, 13 genes with intact coding DNA sequences were obtained and named following the homologous genes in rice and barley. These proteins contained about 708–945 amino acids and were predicted to contain 11–14 trans-membrane regions with a long tail in the C-terminal. All these proteins were localized in the plasma membrane, which were analyzed using TargetP tool (http://www.cbs.dtu.dk/services/TargetP/)  . Based ostructed . HAK/KUP+ uptake in H. brevisubulatum, we extracted the expression information of 13 identified HAK/KUP/KT proteins from transcriptomic data of root tissues. The homologous genes coding these 13 HAK/KUP/KT proteins in H. vulgare from transcriptomic data were also extracted. Comparing these genes in H. brevisubulatum and H. vulgare, 9 members with different expression patterns were identified , however, C170A or R342K single mutant transformed strains became unable to grow  via H+/K+ symports at low (<0.2 mM) external K+ content and a low-affinity system (LATS) via ion channels at high (>0.5 mM) external K+ content. High-salt soil frequently causes K+ deficiency, therefore, the HATS in roots dominates K+ uptake from soil under saline conditions to maintain a stable cytosol K+ concentration (100–150 mM) [Arabidopsis [+ transport in K+/Na+ homeostasis under salt [+ acquisition at high external Na+ content is crucial for K+/Na+ homeostasis. The halophytic H. brevisubulatum could grow well under heavy saline-alkali land and be used as a saline-tolerant grass for soil improvement in North China [H. brevisubulatum plant may evolve some distinctive strategies for K+ acquisition improvement under saline conditions. The hypothesis was supported by the salt-treated root transcriptome data which showed abundant genes related to potassium ion transport were upregulated [+ uptake under saline conditions.K–150 mM) ,29. Sinc–150 mM)  and AtHAbidopsis , HATS isbidopsis . AtHAK5 bidopsis , while dder salt ,8,9,19. th China ,32, implegulated . Among tH. brevisubulatum and H. vulgare, the expression of HbHAK1 was induced by salt while transcripts of HvHAK1 was greatly downregulated , as the same experiments confirm HvHAK1, OsHAK5, CcHAK1 and CaHAK1 transformants recover the growth at 0.1-mM K+ added [+ content [+ transport activity and extended the minimum threshold of K+ uptake operated by HAK-type transporters.Through comparing the expression patterns of HAK/KUP/KT proteins between egulated . We furtolerance . HAK/KUPK+ added ,33,34 an content ,10. The H. brevisubulatum and barley, HbHAK1 and HvHAK1 have only 15 different amino acid residues. We used site-directed mutations in cDNA of HbHAK1 and identified the transport activity of mutants HbHAK1 in Cy162 system. C170A and R342K mutant transformants critically reduced the capacity of strains growth under 10 μM K+ conditions which showed even weaker growth compared with HvHAK1 at 0.1 mM K+ and 0.3+ mM K+ added, while A13T mutants dramatically decrease Na+ tolerance  were plated on MP (Medium lacking Potassium using for plant culture) medium with 0.5% sucrose and 0.8% agar for a week at 21–22 °C with a 16 h/8 h (day/night) photoperiod and 60–70% relative humidity.Seeds of d in lab  and ColuH. brevisubulatum and H. vulgare via an RNAprep Pure Plant Kit . Deep sequencing was performed by Beijing Ori-Gene Science and Technology Corp., Ltd. . The transcriptome library construction and sequencing data analysis was described in previous research [http://www.ncbi.nlm.nih.gov/sra/; the accession number is SRP161471.Total RNA was isolated from the root of research . The rawOryza sativa were obtained from the NCBI website. These sequences were used as a BLAST query in the transcriptomic data to identify putative homologs. 13 HAK/KUP/KT proteins with full length of CDS were obtained and their expression data were also extracted. A phylogenetic tree was constructed in MEGA5.0 software (http://www.megasoftware.net/) using protein sequences of these 13 genes and 27 protein sequences of HAK/KUP/KT family in Oryza sativa. The protein sequences of 13 HAK/KUP/KT transporters were list in Protein sequences of HAK/KUP/KT family genes in HbHAK1 was constructed into the vector pBASTA, with green fluorescent protein (GFP) expression driven by the CaMV 35S promoter present in the parent plasmid pBI121 [Arabidopsis athak5 plants using the floral dip method. Transgenic T2 seeds were used for the phenotype analysis. Seeds from the wild type, athak5 and two different transgenic lines overexpressing HbHAK1 on an athak5 background (HbHAK1-1 and HbHAK1-2) were planted in a seed germination pouch with liquid MP medium containing different concentrations of potassium chloride (KCl) [d pBI121  using gade (KCl) . The SeeHbHAK1 overexpression transgenic T1 generation seeds of athak5 were germinated and grown for 4 days at 21–22 °C under dark condition. The leaf epidermal cells of the etiolated seedlings were used for microscopic observation. FM4–64 was used to stain the cell plasma membrane. These 4-day seedlings were immersed in 20 μg/mL FM4–64 buffer for 1 min and then washed with water twice. Confocal images were captured using confocal macroscope equipment . The fluorescence signals were excited at 488 nm for GFP and 561 for the FM4–64 dye.The cDNA sequences of HbHAK1 and HbHAK2 were amplified with special primers and digested with the SpeI/XhoI or EcorⅠ/XhoI restriction enzymes, respectively, and ligated into the yeast expression vector p424 [HbHAK1, p424-HbHAK2, p424-HvHAK1, p424-AtHAK5, and p424-OsHAK1 were transformed into yeast (Saccharomyces cerevisiae) strain CY162 trk1△trk2△ [ena1–4△ nha1△ [tor p424 . P424, pk1△trk2△  and B31 4△ nha1△ . The pri+ were performed in solid AP-T (Arginine Phosphate medium lacking Trp) medium, as described previously [+ ranging from 0.001 to 10 mM, and in the absence or presence of various concentrations of NaCl (50 to 750 mM). For the growth curve, the yeast strains  were grown in liquid SD-T  medium at 30 ℃ overnight and then transferred to liquid AP-T medium supplemented with different concentration of K+ (0.05 or 2 mM) with the same initial OD600 of about 0.1. The shaker was adjusted to 200 rpm, and the OD600 of the strains was measured every 3 h for three consecutive days of growth. This experiment was repeated in triplicate.Yeast complementation assays at low Keviously  and supp+ depletion experiments, yeast cells were grown overnight at 30 °C in liquid SD-T medium and then then transferred to liquid AP-T medium for about 4 h for K starvation. The cells were then suspended in 10 mM 2-(N-morpholino) ethane sulfonic acid (MES) supplemented with 2% glucose and adjusted to pH 6 with Ca(OH)2. At time zero, 0.03 mM KCl were added to the medium and the samples were collected at intervals over a 2-h period. K+ contents were identified and quantified by atomic emission spectrophotometry using a PerkinElmer Model 2380 spectrophotometer  [For Kass, US) .For the changes, the different amino acids of HbHAK1 with HvHAK1, we mutated 15 amino acid residues of HbHAK1 according to the right sequence of HvHAK1 by using the Quick change method with site-directed mutagenesis kit . The protocol was followed as recommended. The primers used was listed in H. brevisubulatum were exhibited in H. vulgare can be found in the http://plants.ensembl.org/Hordeum_vulgare/Info/Index, as HvHAK1 (HORVU2Hr1G071570), HvHAK2 (HORVU2Hr1G020220), HvHAK7 (HORVU3Hr1G098670), HvHAK9(HORVU2Hr1G018190), HvHAK11 (HORVU2Hr1G098940), HvHAK15 (HORVU2Hr1G099810), HvHAK18 (HORVU5Hr1G090010), HvHAK23 (HORVU5Hr1G059200) and HvHAK25(HORVU6Hr1G073030).The selected 13 genes for the HAK/KUP/KT in


The COVID-19 pandemic has hit almost all countries around the globe, seriously affecting the welfare of populations. Spain is especially hard-hit. In this context, the purpose of the present study is to analyze social, demographic, and economic correlates of mental health during the COVID-19 pandemic in the population residing in Spain.The sample of this cross-sectional study was comprised of 801 participants aged 18 or older and residing in Spain. Data collection was carried out during March and April 2020. Data of mental health (GHQ12) and well-being (Positive and Negative Affect Schedule) indicators, and those of a wide number of social, demographic, and economic variables were recorded. Linear regression models were built to value associations between mental health and social, demographic, and economic indicators.Mental health morbidity was higher in women, younger people, individuals with medium studies, people with fewer children, singles, students, and unemployed individuals. Positive affect was higher among women, people with a high level of studies, those not co-living with dependent seniors, the self-employed, the employed, and those working outside home. Negative affect was negatively associated with age and number of children and was higher among women, people with basic studies, singles, individuals co-living with dependent seniors, homemakers, and students.The most vulnerable populations were found to be women, younger people, people with basic or medium studies, students and individuals with no remunerated activities, single populations, and those co-living with dependent seniors as well as those with a reduced number of children. The entire world is now struggling to overcome one of the most devastating pandemics of the XXI century, until now . COVID-1In order to inhibit the spread of the virus, most countries have established some form of a state of emergency including quarantine periods in which citizens are under strict lockdown and isolation . While tIn periods of uncertainty such as natural disasters, economic crises, and serious health threats, a great variety of studies have found significant changes in people’s mental health and well-being . The exiSimilar studies carried out in United Kingdom have reported higher self-harm behaviors and thoughts of suicide among people experiencing socioeconomic disadvantage, unemployment, disability, chronic physical illnesses, mental disorders, and COVID-19 diagnosis . PreexisMost existing long-term studies on global pandemics were carried out in China and other Asian countries during the SARS pandemic or during the Ebola and influenza pandemics . AccordiThe impact of a pandemic on mental health does not seem to affect everyone at the same level. A study carried out in Australia during the 2007 influenza pandemic found that younger age , lower eLastly, post-quarantine effects may also be taken into account. Both the economy and individuals—particularly the most vulnerable—suffer from the impact of financial loss when people are unable to work. Considerable socioeconomic distress and symptoms of psychological disorders may materialize . Social Mental health is defined by the World Health Organization as “a state of well-being in which the individual realizes his or her own abilities, can cope with the normal stresses of life, can work productively and fruitfully, and is able to make a contribution to his or her community” . Mental Well-being is a key aspect of mental health . The hedFor all these reasons, the purpose of the present study is to analyze social, demographic, and economic correlates of mental health during the COVID-19 pandemic in the population residing in Spain. We aim to evince the factors capable of predicting improvement or exacerbation of psychological distress.This cross-sectional study was designed to assess the associations between social, demographic, and economic factors and mental health indicators during the COVID-19 pandemic in Spain. Snowball technique and convenience sampling were followed to recruit participants as follows: (1) Students enrolled in the nursing degree at the Autonomous University of Madrid were contacted by email and through academic platforms. All potential participants contacted were invited to share the study information with other people within their environment. (2) Professors and researchers directly involved in the present research informed their personal and professional contacts of the study by email and invited them to participate and disseminate the information. (3) Social networks  were used to recruit additional participants. The advert of the study was published on behalf of participation by the European University of Madrid which was accessible to the general public. Similarly, the proposal to participate in the study was published in the professional and personal profiles of each of the researchers involved in the present research. Participants as well as those who decided not to participate in the study were able to share the information of the study with their social and professional networks.After potential participants were informed of the objectives and relevant information of the study, they could indicate consent to participate in the study or not. Upon a positive response, the anonymous questionnaire was deployed. All participants who met the inclusion criteria were recruited. Inclusion criteria were to currently reside in Spain, be aged 18 or over, be able to read, understand, and complete the questionnaire in Spanish, be interested in participating in the study, and provide informed and written consent. Data was collected between March 19th and April 26th, 2020, the most critical periods of the COVID-19 pandemic registered in Spain.Post hoc statistical power calculations were also carried out, for an alpha error of 0.05 and according to the effect size range obtained in the models (considering the two predictors used), showing a statistical power higher than 0.95 in all cases.A total of 37 participants were excluded from the study because they did not meet the inclusion criteria of age (they were under 18 years old). Further, participants in the study were asked if they were active health professionals. Those who met this condition (117 participants) were not included in the present analysis, since their status as health workers has important implications for both risk of infection and mental health and well-being status. As a result, 801 participants provided valid data of mental health indicators and were considered for the analysis. The sample size was calculated using the G-Power tool, for a linear multiple regression, considering an Alpha error of 0.05 and a 0.95 statistical power. Three mental health and well-being indicators were considered in the present study: psychological health status and positive and negative affect.Have you been able to concentrate on whatever you are doing?”The Goldberg General Health Questionnaire (GHQ-12) was used to assess mental health, employing its short version. This questionnaire is a widely used instrument designed to discriminate whether or not psychological morbidity is present. The validation study of the Spanish version revealed an adequate internal consistency, which ranges between 0.82 and 0.90, a sensitivity between 76 and 100, and a Cronbach’s alpha of 0.76 . The scoIndicate the extent to which you have felt distressed over the past week.”The Positive and Negative Affect Schedule (PANAS) was employed to measure well-being. This questionnaire is formed by two independent scales, each consisting of 10 items. The positive affect scale measures feelings such as joy or pleasure, and the negative affect scale includes feelings such as anxiety and sadness. Higher scores indicate higher levels of positive and negative affect. The instrument consists of a Likert scale that ranges from very slightly or not at all, to extremely . This qu2) were reported as a number by the participants.Age, number of children, and dwelling size .Country of origin was indicated after the question: what is your country of origin? (Spain/other).Level of education was identified by participants as basic level of studies (primary and secondary school), medium level of studies , and high level of studies (completed university studies).Marital status was identified by each participant from the possible answers: married, single, unmarried partner, separated/divorced, and widowed.Current employment status, during the COVID-19 pandemic was defined as self-employment, employment, unemployment, homemaker, retired, or student.Living with dependent seniors, current reduced income due to the COVID-19 pandemic, and working outside home  were indicated as yes or no.Length of confinement was calculated from the date of completion of the questionnaire, considering March 14th as the first day of confinement .Self-referred current medical diagnosis of COVID-19 (yes/no) was included as covariate for the analysis, given its potential influence on mental health indicators.The protocol for the present study obtained approval from the Ethics Committee of the Faculty of Biomedical and Health Science of the European University of Madrid (No CIPI/20/135). All participants were informed of the purpose and intent of the study and provided written consent. Similarly, anonymity of each of the participants was ensured.All statistical analysis was conducted using the Statistical Package for the Social Sciences software version 21.0  and STATA/SE 14.1 software (Stata Corp LP).p < 0.001).Descriptive statistics  were calculated to describe participant characteristics. Differences between categorical variables and mental health indicators were addressed using Student’s t test for dichotomous variables and ANOVA test for variables with more than 2 categories. The Spearman correlation test was employed to value associations between quantitative variables and mental health indicators after assessing the distribution of each variable using the Kolmogorov–Smirnov test  values of the mental health indicators are presented in p < 0.001, r = −0.23), women , people with a medium level of studies , those with a lower number of children , single people , and students . The positive affect score was higher in women , people with a high level of studies , those not living with dependent seniors , self-employees , those with a shorter length of confinement , and those working outside home . Finally, the negative affect score was higher in younger people , women , people with a basic level of studies , those with a lower number of children , single and widowed people , people living with dependent seniors , and homemakers .Characteristics of the participants are also presented in p < 0.001) and in number of children , p < 0.001) was associated with decreased mental health scores. Similarly, a high level of studies , p = 0.001), being married , p < 0.001), being self-employed , p = 0.025), and being retired , p = 0.004) were linked to lower mental health scores. On the other hand, referring gender as female , p < 0.001), reporting a medium level of studies , p < 0.001), being single , p < 0.001), being unemployed , p = 0.029), and being a student , p < 0.001) were associated with a higher mental health score.Linear regression models for the mental health score are presented in p < 0.001), living with dependent seniors , p = 0.006), being unemployed , p = 0.007), being retired , p = 0.005), and being a student , p < 0.001) were linked to decreased positive affect scores. On the other hand, referring gender as female , p < 0.001), a high level of studies , p < 0.001), being self-employed , p = 0.008), being employed , p < 0.001), and working outside home , p = 0.012) were linked to a higher positive affect score.Linear regression models for positive affect scores are presented in p < 0.001) and in number of children , p = 0.004) was associated with lower negative affect scores. Also, a high level of studies , p = 0.004), being married , p = 0.028), being separated or divorced , p = 0.004), being and retired , p = 0.002) are linked to lower negative affect scores. However, reporting gender as female , p < 0.001), basic studies , p = 0.027), being single , p = 0.001), living with dependent seniors , p = 0.006), being a homemaker , p = 0.049), and being a student , p = 0.033) were related to higher negative affect scores.Finally, linear regression models for negative affect scores are presented in According to our results, the most vulnerable populations in terms of mental health morbidity were women, younger people, individuals with a medium level of studies, those with fewer children, single individuals, students, and the unemployed. In contrast, positive affect was higher among women, those with a high level of studies, those not co-living with dependent seniors, the self-employed, the employed, and those working outside home due to the COVID-19 pandemic. Lastly, negative affect was negatively associated with age and number of children and was higher among females, people with a basic level of studies, single individuals and those with unmarried partners, individuals co-living with dependent seniors, homemakers, and students.As expected, and in line with prior studies, more vulnerable individuals in terms of socioeconomic status were more likely to report symptoms of psychological distress . EducatiUnexpectedly, no significant associations were found between length of confinement, mental health morbidity, and negative/positive affect. Prior studies have found contradictory results. While some pointed out negative effects of quarantine duration on psychological health , not allStressors during quarantine should also be considered. Number of children, co-living with a dependent senior, and being alone were found to be related to mental health or well-being. It was also unexpected to find that an increased number of children at home was associated with better mental health status and lower levels of negative affect. Prior studies have identified similar tendencies, suggesting the protective effects of having two or more children at home . HoweverDependent seniors co-living at home were found to be a stressor during quarantine, with lower levels of positive affect and higher levels of negative affect among caregivers, a finding validated by the existing literature on caregiving and its damaging effects on mental health and well-being . This asLastly, although previous literature suggests that having more space at home might be related to increased well-being , particuFinally, sociodemographic factors should also be considered. Age, gender, and marital status have been found to be related to mental health and well-being. In congruence with prior studies carried out in Spain during the COVID-19, women showed higher mental morbidity  and highThis study is not without its limitations, as follows: (1) Perhaps the most relevant limitation we must point out is that the use of social networks to recruit participants for this study may associate a sample selection bias. However, the need to assume this limitation was due to the confinement of the entire Spanish population for the full duration of data collection. There were scarce possibilities of reaching potential participants by other means. In spite of the limitations associated with the use of social networks for data collection, the decision to proceed was supported by some scientific works that have pointed out that social media data maintain the capacity for addressing broad social questions while upholding methodological integrity . Other sHowever, this study provides information about social, demographic, and economic factors able to influence the mental health of a population unable to exert their basic freedoms in the unique instance of a health emergency.In conclusion, the most vulnerable populations in terms of mental health morbidity and well-being were women, younger people, people with basic or medium level of studies, students and individuals with no remunerated activities, singles, and those with unmarried partners. Stressors during confinement were co-living with dependent seniors and having few children. These results highlight the need to consider psychosocial predictors of mental health and well-being in order to design and implement future intervention programs to monitor mental health and well-being outcomes among the most vulnerable individuals in the highly probable context of future pandemics.The raw data supporting the conclusions of this article will be made available by the authors, under limited conditions.The protocol for the present study obtained approval from the Ethics Committee of the Faculty of Biomedical and Health Science of the European University of Madrid. The patients/participants provided their written informed consent to participate in this study.SE-G was the coordinator, main investigator of the study, and oriented and revised the article. LE-G was the coordinator and main investigator of the study, and analyzed the data. JG-P was an investigator of the study and revised the article. MC-G was an investigator of the study and revised the article. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MTNR1B gene and year‐round oestrus or the litter size in Small Tail Han sheep. To better understand the effects of single nucleotide polymorphism (SNP) rs400827589 in MTNR1B, a population polymorphism analysis was conducted using genotyping data in 45 sheep breeds around the world. The results indicated that TT was the dominant genotype in all sheep breeds. The associations of this SNP with reproductive seasonality and litter size in Small Tail Han sheep showed rs400827589 was correlated with fecundity as assessed by reproductive seasonality and litter size (p < .05). Bioinformatics analysis indicated the change in amino acid from Ile to Leu may affect the function of the MTNR1B protein by impacting the secondary and tertiary protein structures. The present results demonstrate that rs400827589 could be used in the marker‐assisted selection of the litter size in Small Tail Han sheep.In mammals, the melatonin receptor gene has been widely studied since it has a great influence on reproductive traits. However, little is known about the association between polymorphism of the coding region of the  p < .05). Bioinformatics analysis indicated the change of amino acid from Ile to Leu may affect the function of the MTNR1B protein by impacting the secondary and tertiary protein structuresrs400827589 was correlated with fecundity as assessed by reproductive seasonality and litter size ( In addition, there was ethics approval by the animal ethics committee of IAS‐CAAS (No. IAS2018‐3).Jugular vein blood samples were collected from 737 ewes in six sheep breeds for DNA isolation using the phenol‐chloroform method. The information of sheep breeds included in the study was shown in Table 2.2MTNR1B sequences available in the Ensembl (Accession No. ENSOARG00000002933), the primer sequences for genotyping were 5′‐TGG ATG AAC AAC CCC TCT GGG ATC CG‐3′ (Forward), 5′‐ACG TTG GAT GTT GTG ATC TTC GCC ATC TGC‐3′ (Reverse) and 5′‐TTG TGA GCC ACT TCT TCG GGG TCA/A3′ (Extension reaction), which amplify a region of 120bp. The MassARRAY®SNP analysis (http://www.sequenom.com) was subsequently applied for genotyping all 737 sheep. The polymerase chain reactions system and temperature were described in detail in a previous study  (SAS Institute Inc.). p values less than .05 were considered to be significant. The model was described in the previous study ; Gj is the fixed effect of the jth genotype ; IPG is the interaction effect of parity and genotype and ijne is the random residual.The calculations of allele frequencies and genotype frequencies and the Hardy‐Weinberg equilibrium tests were performed by using Popgene (version 1.31) , and amino acid sequences were subsequently obtained from NCBI (https://www.ncbi.nlm.nih.gov/protein/195972821). The transmembrane domains before and after mutation in MTNR1B were predicted using TMHMM (http://www.cbs.dtu.dk/services/TMHMM‐2.0/). Prediction of the secondary structure of MTNR1B and its mutants occurred using Predict Protein (https://www.predictprotein.org/). The MTNR1B protein three‐dimensional (3D) structure in sheep was predicted by Iterative Threading ASSEmbly Refinement (I‐TASSER) (http://zhanglab.ccmb.med.umich.edu/I‐TASSER/), the protein‐ligand binding site prediction was performed by a meta‐server approach (COACH) (http://zhanglab.ccmb.med.umich.edu/COACH/).The coding sequences of the 33.1MTNR1B exon 2 was selected for genotyping, the different alleles resulted in amino acid changes. The genotype results in 737 samples with a >95% success rate and samples with successful genotyping were included in the population polymorphism analysis (Table p > .05).In this study, rs400827589 in http://asia.ensembl.org/Ovis_aries/Variation/Population?db=core;g=ENSOARG00000002933;r=21:1373657‐1392132;t=ENSOART00000003171;v=rs400827589;vdb=variation;vf=33224018). The three genotypes were found in different sheep breeds from various countries in the world. The distribution of genotypes was similar to native breeds in China, the dominant genotype was TT and a few sheep breeds had the GG genotype.A population polymorphism analysis was also conducted in another 39 sheep breeds, the results are listed in Table p < .01).We classified six breeds into two categories, year‐round oestrus and seasonal oestrus, based on the oestrous characters. The results of the population polymorphism analysis shown in Table 3.2p < .05).The association analysis between rs400827589 with litter size in Small Tail Han sheep was performed using data from our previous study  sequence. The results indicated that there were seven transmembrane domains, a mutation from T to G led to no change in the transmembrane domains Figure . In addi4Melatonin is a highly lipophilic circulating hormone. In addition to regulating insulin secretion and glucose levels, it controls circadian rhythms and reproductive processes . Compared with the distribution of this locus in sheep breeds around the world  in SNP rs400827589 (Table MTNR1B in rs400827589 also affected ovine reproductive seasonality and litter size differently, with litter size of the mutant homozygote individuals decreased significantly. This phenomenon indicated that the rs400827589 may be an adverse mutation for litter size in sheep. Further study is required to confirm the mechanism of the effect of rs400827589 on the litter size in sheep.As a key link in the melatonin signalling pathway, polymorphisms of 89 Table . The res89 Table , 2008, p5MTNR1B rs400827589 was the dominant genotype in sheep around the world. Polymorphism of MTNR1B in rs400827589 can affect reproductive seasonality and was associated with litter size in some Chinese native sheep. In addition, bioinformatics analysis indicated the change of amino acid from Ile to Leu may affect the function of the MTNR1B protein by impacting the secondary and tertiary protein structures. Findings in the present study also indicate that MTNR1B might use to select for litter size in sheep.The TT genotype in We certify that there is no conflict of interest with any organization regarding the material discussed in the manuscript.Xiaoyun He: Visualization; Writing‐original draft. Zhuangbiao Zhang: Software. Ming‐Xing Chu: Formal analysis; Funding acquisition; Project administration.


Pediatric-adolescent or developmental gynecology has been separated from general gynecology because of the unique issues that affect the development and anatomy of growing girls and young women. It deals with patients from the neonatal period until maturity. There are not many gynecological problems that can be diagnosed in newborns; however, some are typical of the neonatal period. This paper aims to discuss the most frequent gynecological issues in the neonatal period. Gynecology (from the Greek word ‘gyne’ = woman) is the area of medicine that specializes in the diagnosis and treatment of diseases affecting female reproductive organs (“woman’s diseases”). In a broader sense, this medical specialty covers the entire woman’s health, including preventive actions, and represents the specificity of anatomical and physiological distinctness of sex. Pediatric-adolescent gynecology or developmental gynecology is separated from general gynecology because of the unique issues that affect the development and anatomy of growing girls and young women. It deals with patients from the neonatal period until maturity. Girls with gynecological disorders constitute 10% of all gynecological patients. That is why they should be included in specialistic gynecological care, particularly in its preventive aspect.There are not many gynecological problems leading to abnormalities in later development that can be diagnosed in newborns. There are, however, some disorders that are typical of the neonatal period. As far as health promotion and prevention are concerned, it is essential to take care of a baby girl’s health in relation to her future maternity since birth. In the neonatal period, it is crucial to instruct mothers of the proper hygienic procedures for girls’ genitals and of how to prevent diaper dermatitis. Adequate feeding is also important with regard to development.The neonatologist or the pediatrician is usually the first specialist who detects genital abnormalities in a little girl. In most cases, the related changes are physiological. However, if these abnormalities are disturbing or unusual, the baby should be referred to a pediatric gynecologist for a proper gynecological examination and to a pediatric endocrinologist or pediatric surgeon for further examination and treatment.Knowledge of normal prepubertal anatomy and the use of an accurate terminology are essential for describing and documenting anatomic findings. The gynecologist assesses the appearance of the vulva, the size and color of labia minora and majora, the introitus of the vagina, the color of the mucous membrane, and the size of the clitoris. It is also essential to evaluate the hymen and the presence of a discharge from the vagina . This examination is mostly performed visually, without the use of instruments. Normally, the labia majora are full, and the labia minora are thickened. The hymenal folds appear thick and redundant. The vaginal mucosa is pink and moist, with an acidic pH. As the maternal hormone levels decrease, the labia majora lose their fullness, and the labia minora and hymen become thinner and flatter. Visualization of the mid and upper vagina may be accomplished while the baby is in the knee-chest position but often requires a sedated examination and instruments. When performing an anesthesia examination, a lighted Killian nasal speculum and a fiberoptic scope  are useful for examining the prepubertal vagina. A liquid distention medium can be used for vaginoscopy to visualize the whole vagina and cervix. Vaginal cultures should be obtained before the vaginoscopy. The cervix, uterus, and adnexa in the child can be evaluated through an examination, using a finger placed rectally and the other hand abdominally, with the patient lying supine. An ultrasonographic exam allows for assessing the uterine proportions and the appearance of the ovaries. For newborns and small girls, the cooperation between the gynecologist and the mother is obligatory. Educating the mother before this examination is essential both for her reassurance and for gaining her trust.In utero, the vaginal epithelium of the neonate is stimulated by maternal hormones that cross the placenta into the fetal circulation. After delivery, these hormone levels fall rapidly, and a thick, grayish-white, mucoid discharge from the neonate’s vagina can be observed. The secretion usually resolves in 10 days.In some baby girls, the discharge from the vagina is blood-tinged or even grossly bloody. This is usually treated as physiological menstrual-like neonatal bleeding, and it is assumed that no treatment is needed. However, in some studies, it has been shown that such bleeding might be a marker of intrauterine distress in late pregnancy. It is more often observed in infants with intrauterine growth restriction or in those whose mothers suffered from preeclampsia. Moreover, neonatal menstrual-like bleeding may represent a sign of increased risk of developing endometriosis during adolescence and plays a role in the transgenerational evolution of major reproductive disorders ,2,3. PreA foreign body is a common cause of prepubertal vaginal bleeding. Most commonly, the object is toilet paper, a small plastic toy, a cap, or other small objects. Bleeding in such a situation might be accompanied by pelvic pain and a foul-smelling discharge. The unidentified foreign body may lead to urinary tract infection or dermatosis and, in serious cases, to perforation into the peritoneal cavity or fistula formation .A trauma leading to vaginal bleeding in infants can be either accidental or nonaccidental. The most common injury is the straddle injury following a fall on the edge of some object . It rareStreptococcus, S. pyogenes), enteric pathogens , or Candida spp. in infants wearing diapers [Vulvovaginitis  is anoth diapers ,5.Other skin conditions predisposing to vaginal bleeding are atopic dermatitis (eczema and psoriasis) and lichen sclerosis . Trauma Vaginal bleeding may also be a sign of a gynecologic tumor, such as ovarian, vaginal, or vulvar tumor. The most common malignant tumor in young girls is rhabdomyosarcoma, specifically, sarcoma botryoides. The peak age for the development of this tumor is two years, and over 90% of them are detected before five years of age. Patients may present with an intralabial mass that is grape-like in appearance and vesicular . EndoderIn newborns, the hymen is redundant, estrogenized, thick, and elastic, often with a prominent ridge at six o’clock. As the estrogen subsides, the hymen becomes unestrogenized, thin, and significantly more vascular. Assessment of the hymen includes the observation of its shape, variations in the hymenal rim, the degree of estrogen effect, and any signs of trauma or scar tissue. The examination might reveal anatomic variants that sometimes are mistaken for signs of sexual abuse, such as midline sparing (linea vestibularis), failure of midline fusion, urethral prolapse, or labial adhesion. Midline sparing refers to a symmetric, flat avascular area of the posterior vestibule observed in 10% of healthy newborns. It can sometimes be confused with scarring. Congenital abnormalities of the hymen include imperforate, microperforated, cribriform, septated, and annular hymens. They occur in 3–4% of the female population. Redundant or fimbriated hymens that appear to be sleeve-like or ruffled are common in newborns because of maternal estrogen effects. The crescentic shape becomes the most common shape around the age of 4 years in the prepubertal female . TypicalIn the presence of congenital malformations such as vaginal atresia or an imperforate hymen, the physiological discharge of the vaginal epithelium stays inside or releases in drops only when the baby cries. The incidence of imperforate hymen is estimated at 0.014–0.1%, and its presentation is often late, with amenorrhea in adolescence ,7. HowevPrenatal diagnosis of imperforate hymen with hydrometrocolpos has been reported as early as 25 weeks of gestation, although most cases are described during late pregnancy or after birth ,8,9,10. Labial adhesions are identified when the labia minora are partly or entirely agglutinated. The incidence is reported to be around 1.8%, and the diagnosis is done most frequently between 13 and 23 months of age when the estrogen level is physiologically low . The symInterlabial cysts are another abnormality that may cause anxiety to the parents and pediatricians. They occur in 1:1000 to 1:7000 newborn girls. Most commonly, they are simple hymenal cysts  and paraurethral gland cysts  and present as single small, bulging, golden-colored, or whitish interlabial protrusion adjacent to the urethral meatus and slightly distorting it. The cysts usually resolve spontaneously over 2–3 months, and their presence is related to exposure to maternal estrogens. The differential diagnosis of interlabial masses includes anomalies of the ureter or urethra, ectopic tissue, prolapse , or neoplasia (botryoid rhabdomyosarcoma) .An essential part of the physical examination of newborns is an assessment of mammary glands. Breast development occurs during fetal life and is well described as proceeding in female infants for several months after birth. It ceases at 3–6 months of age, and the breast bud may regress after that .Neonatal breast enlargement is defined as the benign proliferation of glandular tissue due to estrogens crossing the placenta into the fetal circulation. It occurs in approximately 70% of newborns, both girls and boys, and can be unilateral or bilateral. Usually, the diameter of a breast bud is 1 to 2 cm in the first few weeks of life. Mammary glands in newborns are often hard, hyperemic, and tender. In some newborns, the breasts are excessively big, and the reason for such an exaggerated response to maternal hormones is unclear ,15.Postnatally, falling levels of maternal estrogens are thought to trigger prolactin secretion in the pituitary of the newborn. Prolactinemia stimulates neonatal breasts and causes milk secretion in 5 to 20% of newborns ,15,16. MStaphylococcus aureus (83–88%). Typical presentation includes unilateral swelling, erythema, warmth, tenderness, and induration in the absence of systemic signs of infection. If left untreated, it can lead to breast abscess and, rarely, to generalized sepsis [When the mammary glands are enlarged and produce milk, the infant may be more susceptible to breast inflammation and infection. Inadequate secretion of milk due to improper canalization of the lactiferous ducts may lead to stagnation of milk . In such a situation, the infection may result in complications such as mastitis and breast abscess. Neonatal mastitis usually occurs under five weeks of age, with a peak incidence at three weeks of age, and is mostly unilateral. The most common causative agent is d sepsis ,15,21.Supernumerary nipples are a common finding. They might extend along the nipple line on either side of the chest wall, down the abdomen, and may occur in the labia. Bilateral ectopic breast tissue has been described in the vulva .Atypical-appearing genitals pose significant neonatological, gynecological, genetic, surgical, and psychological problems. The examination of the external genitals should include precise measurements of anatomic features, in reference to the normative values ,24,25. TDrs. Charmian Quigley and Frank French proposed a scale to grade the appearance of external genitalia in babies with androgen insensitivity syndrome (AIS), as shown in The diagnosis and treatment of a child with disorders of sex development (DSD) require a multispecialty team experienced in the long-term management of such patients and generally available only in tertiary-care centers. An integrated approach is essential to convey consistent information and an appropriate plan to the parents. They should be given a complete biological background concerning sex determination and the potential causes of DSD in their child, presented in a manner that ensures their understanding of the issues. Parents should be kept fully informed regarding planned diagnostic tests and the interpretation of results. Psychological counseling should be offered to the parents on how to optimally share critical information with more distant members of their family. If surgical intervention is recommended, indications, timing, and access to appropriate services should be discussed with the parents.While our understanding of the genetics of sex determination has advanced rapidly (more than 30 genes are known to contribute to the etiopathogenesis of DSD), the specific molecular disorder may not be defined in many affected children. Existing algorithms are of limited value, due to the broad spectrum of phenotypes and genetic polymorphisms as well as the often long time needed to complete genotyping in highly specialized, but rare, laboratories with appropriate expertise ,26,28. PPhysical examination includes inspection of the neonate for potential dysmorphic features, including head or facial dysmorphisms, orthopedic or heart defects, as DSD may be part of congenital syndromes. The presence of a micropenis due to hypogonadotropic hypogonadism may suggest a coexistent pituitary dysfunction with secondary hypocortisolemia and hypothyroidism. The examination of the external genitalia includes the precise measurement of anatomical structures, in relation to normative values. Besides, the analysis includes the detecton of the presence of gonads, hyperpigmentation of the external genitalia, breast areola and ear lobes, crease of the labioscrotal folds, length, width, and symmetry of the corpora, ventral flexion of the penis, presence and degree of hypospadias, degree of labioscrotal fusion. A well-formed penis and significant labioscrotal rugae may suggest prenatal exposure to elevated testosterone and dihydrotestosterone (DHT) levels. Asymmetric virilization indicates that only one gonad secretes testosterone. A contralateral dysgenetic testis or ovary is present (in the case of ovotesticular DSD) ,32,33.Ovarian cysts usually arise from mature follicles and are the most common abdominal cysts in female fetuses and newborn girls ,35. TheyTorsion is the most common complication of ovarian cysts (50–78%). It appears antenatal in most cases and is more common in large cysts. This complication may cause inflammatory adhesions, intracystic bleeding, and rupture. In large cysts, pulmonary hypoplasia or polyhydramnios have been described due to the pressure of the mass on the small intestine, interfering with the fetal swallowing mechanism .The management of congenital ovarian cysts is controversial. It depends on cyst size and complications that might occur during gestation or after birth. Early surgical intervention used to be recommended to prevent torsion and cyst hemorrhage or rupture that may lead to intestinal obstruction. The current trend favors watchful waiting and close follow-up with serial ultrasounds until cyst involution, especially for cysts smaller than 4 cm, which are less likely to be associated with torsion ,39. ThesSymptomatic cysts and those that do not regress after several months should be resected. Complex masses that can lead to such problems as cyst recurrence and small bowel obstruction also require surgical management . ConcernIn preterm babies, multiple ovarian cysts may result from excessive ovarian stimulation syndrome . Other sGynecological problems in neonatal life are unusual and rare. Many conditions that are thought to be pathological are, in fact, anatomical variants or physiological situations. In some cases, however, it is necessary to monitor the newborns with gynecological problems over a longer time to promote fertility and supervise the prepubertal phase. Therefore, knowledge of these abnormalities is essential so that explanations and prognosis can be reassuringly offered to parents.


Toxoplasma gondii infections. This review was performed to obtain a better understanding of the in vivo and in vitro effects of chitosan on T. gondii strains.The preferred treatment for management of toxoplasmosis is the combined use of pyrimethamine and sulfadiazine. However, there are a wide number of adverse side effects with these medications. Recent research has focused on the use of chitosan for the treatment of Toxoplasma gondii”, “Chitosan”, “nanoparticles” and “anti-toxoplasmosis” with AND or OR.The current study was carried out according to the PRISMA guideline and registered in the CAMARADES-NC3Rs Preclinical Systematic Review and Meta-analysis Facility (SyRF) database. The search was performed in five scientific databases, including Scopus, PubMed, Web of Science, EMBASE, and Google Scholar, with date limits of 1992 to December 2019. The search was restricted to articles published in the English language. The words and terms searched were “T. gondii, with Me49 and PRU each included in one study. Five studies (56%) were performed in vivo, one study in vitro and 3 studies included in vivo and in vitro tests.Of 2500 manuscripts, 9 met the eligibility criteria for review. All studies used the RH strain of T. gondii, chitosan nanoparticles show potential as an alternative treatment for T. gondii infections.Considering the low toxicity and the high inhibitory potency of chitosan against  The sea2.2After removing duplicate publications, three authors independently reviewed the titles and abstracts of the studies, and the approved studies were selected for further analysis. With scientific documentation and at this stage, studies with inadequate information, with lack of evidence, noncompliance with the methods and misinterpretation of the results were excluded from the present study. A flowchart depicts the study design process . Moreove3in vitro, 5 in vivo, 3 in vitro and in vivo) from 2003 to 2019, met the inclusion criteria for discussion in this systematic review with the data extracted are presented in in vitro studies were performed with the RH strain while two of the in vivo studies included type-2 strains (Me49 and PRU).Nine papers  resulted in morphological changes of the tachyzoites. Also, altered induction of cellular apoptosis in the tachyzoite surface and their structure degradation  encapsulated with chitosan nanospheres or with complete Freund's adjuvant accomplished. And the end challenge of mice with RH and Me49 strains increased the survival time of these groups for 80 and 57 days, respectively. The life span of these vaccinated groups was significantly increased compared to the control group. Also, the level of INF-γ was significantly increased in the CTLV-encapsulated groups with chitosan and adjuvant Freund compared to the control group and in the vaccinated group. With CTLV encapsulated chitosan nanospheres (CTLVECNS) and then challenged with Me49 strain, the number of smaller cysts formed by traverse inflammatory cells in the brain of this group was not seen . This study indicated that BALB/c mice injected intramuscularly (G10E-CS) with RH and PRU strains for two weeks and after these injections, were treated with survival time in the RH challenge group for 21 days. That this significantly higher than the other groups.in vitro and elevated INF-γ cytokines. It also decreased IL-4 and IL-10 cytokines. The results of this study could be lead to the effectiveness of chitosan microspheres as an effective system for delivering antigenic peptides to dendritic cells in research and development of new vaccines preventing T. gondii infections , and an increase in IgM against parasitoid tachyzoites. Results implied improvement and enhancement of the immune system in mice treated with spiramycin-loaded chitosan nanoparticles .T. gondii.Chitosan nanoparticles have been studied as a natural polymer for the stabilization of metallic nanoparticles. Chitosan-coated zinc oxide by binding to IgG antibodies can evaluate the low level of this antibody using a microfluidic LIF immunosensor device. This method develops chitosan-coated zinc oxide nanoparticles as an efficient and effective analytical tool in the detection and monitoring of toxoplasmosis clinical . There a5T. gondii, chitosan nanoparticles show potential as an alternative treatment for T. gondii infections. Chitosan nanoparticles in combination with other therapies also could provide a higher level of efficacy. However, extensive in vivo and clinical trials on human subjects are needed to approve the impact of chitosan nanoparticles for treatment of toxoplasmosis.Considering the low toxicity and the high inhibitory potency of chitosan against None.The authors declare no conflict of interest.