Chrysanthemum morifolium Ramat.) cultivars, “Pearl Egg” and “Gaya Glory”, were subjected to a 10-h photoperiod with 600 μmol∙m−2·s−1 photosynthetic photon flux density (PPFD) provided by light-emitting diodes (LEDs) in each light-direction combination (top (1/1) (T), top (1/2) + side (1/2) (TS), top (1/2) + bottom (1/2) (TB), side (1/2) + bottom (1/2) (SB), and top (1/3) + side (1/3) + bottom (1/3) (TSB)). The TS lighting significantly enhanced the morphophysiological performance, compared to the other lighting direction combinations. Notably, the excellent branch formation and earlier flowering were induced by the TS lighting in both “Pearl Egg” and “Gaya Glory” plants.Light is a critical environmental factor that influences plant growth and development, ranging from seed germination to flowering and fruiting. This study was carried out to explore how the optimal combination of various lighting directions increases the light usage efficiency and influences the plant morphophysiology, by investigating the plant growth parameters, leaf anatomy, epidermal morphology, stomatal properties, chlorophyll content, key physiological changes, and correlated gene expressions. In closed-type plant growth chambers, rooted cuttings of two chrysanthemum ( Plant development and growth are influenced by both the quality and amount of the incident light. Furthermore, numerous photoreceptors are required for plants to appropriately process the light input [Since plants are sessile, external variables unavoidably have a significant impact on their physiology and development. Plants have evolved complex ways of sensing the environmental stimuli and converting them into internal signaling pathways in order to adapt to and survive in a changing environment. Light is one of the most important environmental cues, affecting practically every stage of a plant’s lifetime. After germination from the soil, etiolated growth causes the germinated seedling to grow toward the soil surface in search of light. When exposed to light, the seedling goes through photomorphogenesis, which includes de-etiolation, chlorophyll synthesis, and chloroplast growth, all of which help the seedling become an independent autotroph. Thus, light is used as a sophisticated signaling input to influence plant physiology and growth, in addition to being the only energy source for COht input ,2. For mht input .A controlled environment with artificial lighting (CEAL) is an airtight facility for growing plants indoors under artificially regulated circumstances, such as plant factories and growth chambers ,5,6. The2 concentrations which is lucrative for high photosynthetic efficiency is to have a dense, open, and smaller stoma [Variations in the light direction affect plant morphophysiology. The light interception is directly determined by leaf orientation. Phototropism-induced variations in the leaf angle and leaf movement (epinastic or hyponastic) have been proposed as a means of increasing the photosynthetic capacity, efficiency, and carbon gain in light competition conditions ,16,17. Ter stoma .Furthermore, measures of chlorophyll fluorescence have been recognized as a useful and informative indicator of photosynthetic light response efficiency. Under prevailing light and shadow conditions, chlorophyll fluorescence is principally and effectively used to evaluate the potential quantum yield of photosystem II and photoinhibition . Inappro2 fixation and RuBisCO-1, 5-bisphosphate (RuBP) regeneration, as well as the content and activity of light-capturing components, electron transport fragments, and energy transferring enzymes were used to assess the metabolic capability of photosynthesis in plants under diverse conditions [2 fixation in photosynthesis [3 crop plants [Moreover, plants have evolved complex acclimatization mechanisms to address adverse environments, such as the reactive oxygen species (ROS)-scavenging enzymatic antioxidant system . Under snditions ,31,32,33ynthesis , which ip plants . Previoup plants .Numerous studies have confirmed that the light intensity, quality, duration, and source type, such as the sun or artificial lighting systems, affect plant growth and development ,37,38. HThe chrysanthemum plants were kept in the growth chamber for 45 days. The morphology and plant growth measurements of “Pearl Egg” and “Gaya Glory” plants at the end of the experiment are shown in The light quality, quantity, intensity, and especially light direction noticeably affect leaf anatomy. In this study, significant differences in the thicknesses of leaves, palisade tissues, and spongy tissues were observed in response to all five lighting direction combinations in both “Pearl Egg” and “Gaya Glory” plants A–E. The Due to the positive phototaxis, the aboveground parts of plants will grow toward the light. Leaves grow downward to receive more light when the light comes from the side and bottom, which further influences the morphology of the epidermis in the leaf midribs A–F. In tIn addition, to further explore the epidermal cellular morphology response to various lighting direction combinations, we examined the stomatal characteristics in both “Pearl Egg” and “Gaya Glory” plants as affected by the different lighting direction combinations after 45 days . CompareThe chlorophyll (Chl) content is another important factor related to photosynthesis. The Chl content in chrysanthemum leaves was considerably affected by the different lighting direction combinations . In thisPn, Tr, Gs, and Ci in both “Pearl Egg” and “Gaya Glory” plants appeared in response to the TS and SB lighting, respectively. There were irregular differences in the Pn, Tr, Gs, and Ci values in response to the T, TB, and TSB lighting in both chrysanthemums, but these parameters were always higher than those in response to the SB lighting and lower than those in response to the TS lighting. This increase in the net photosynthetic rate indicates that TS lighting is highly effective for photosynthesis. These changes may be due to the enhancement of the stomatal properties and chlorophyll contents by the TS lighting.Fv/Fm, Fv’/Fm’, NPQ, and qP were notably changed in response to the different lighting direction combinations. Independent of the cultivar, the Fv/Fm, Fv’/Fm’, NPQ, and qP of chrysanthemum leaves were considerably higher in response to the TS lighting than in response to the SB lighting. These values were insignificantly different in response to the T, TB, and TSB lighting. Moreover, these parameters were not very different between “Pearl Egg” and “Gaya Glory” when they responded to the T, TB, and TSB lighting. For “Pearl Egg”, the values of Fv/Fm, Fv’/Fm’, NPQ, and qP were increased when the lighting changed from TB or TSB to T, while these values were enhanced when T or TB changed to TSB lighting in “Gaya Glory” plants, but these were always lower than those observed in response to the TS lighting. Overall, our results show that an appropriate lighting direction combination is critical for increasing the chlorophyll fluorescence characteristics and photosynthetic capacity in chrysanthemum plants.The absorbed radiation energy in both “Pearl Egg” and “Gaya Glory” plants was studied in response to the various light direction combinations . In thisFrom the previous results, it can be seen that the various lighting direction combinations induced differences in the photosynthetic efficiency . We deteIn this experiment, significant differences in the ROS scavenging enzymatic activities , guaiacol peroxidase (GPX), super-oxide peroxidase (SOD), and ascorbate peroxidase (APX)), sucrose synthesis enzymatic activities (sucrose synthase (SS) and phosphoenolpyruvate carboxykinase (PEPC)), starch synthesis enzymatic activities (soluble starch synthase (SSS)), and photosynthesis enzymatic activities (activated and non-activated activity of RuBisCO) were observed in response to the various lighting direction combinations . The CATCseSS-7), one gene related to starch synthesis (CseSSS-4), one gene related to photosynthesis (CsePsaA-7), and two genes related to flowering (Cse_sc015873.1_g020.1 and Cse_sc001459.1_g030.1) were investigated in our study. The relative expression levels of all 5 genes for sucrose synthesis, starch synthesis, photosynthesis, and flowering were up-regulated as the lighting direction changed from SB to TS in both “Pearl Egg” and “Gaya Glory”  scavenged enzymatic antioxidant system . ROS creMoreover, Chl a is more vulnerable to ROS than Chl b, and ROS induced direct degradation of Chl a and total chlorophyll concentrations under stress circumstances ,27. In oIn addition, results from this study demonstrated that the enzymatic activities of key enzymes related to sucrose synthesis (SS and PEPC), starch synthesis (SSS), and photosynthesis (RuBisCO) processes significantly changed with the variations in the lighting direction combinations. Higher activity levels of these enzymes were observed in response to the TS lighting E–H. ThesCseSS-7 and CseSSS-4, respectively involved in sucrose and starch synthesis were up-regulated by the TS lighting in both “Pearl Egg” and “Gaya Glory” plants  showed an extraordinary performance and followed the same pattern as that of flower induction  “Pearl Egg” and “Gaya Glory”, a qualitative SD plant) were obtained from the Flowers Breeding Research Institute, Gyeongnam Agricultural Research & Extension Services (GARES), Republic of Korea, in early September 2021. The cuttings were stuck in a commercial medium  in 21-cell zigzag trays . All cuttings were kept on a fogged propagation bench with an 80% relative humidity for 15 days and were subsequently acclimated for seven days on a greenhouse bench with an average light intensity of 350 μmol∙m−2∙s−1 PPFD of sunlight, and a natural photoperiod. The chrysanthemum plants with 8 ± 1 leaves per plant were transplanted into 10 cm plastic pots for the subsequent experiments.The non-rooted cuttings of chrysanthemum ) and transferred into three separate plant growth chambers  for use as three repetitions  at 25 °C temperature with an 80% relative humidity. Each chamber was divided equally into five compartments using plates according to the lighting direction combinations (top (1/1) (T), top (1/2) + side (1/2) (TS), top (1/2) + bottom (1/2) (TB), side (1/2) + bottom (1/2) (SB), and top (1/3) + side (1/3) + bottom (1/3) (TSB), respectively). The five lighting direction combinations were randomly distributed in each chamber to avoid the effects of the position. To prevent light from interacting with each other, all light-reflecting portions inside the chambers, as well as the plates of each layer, were enclosed in an opaque black curtain. Every plate (one lighting direction combination per plate) contained one group of plants with 10 cm between each plant. The tailor-made LED lamps  with a wide spectrum ranging from 400 to 720 nm and a distinct peak at 435 nm (blue) were employed from 08:00 to 18:00 (SD condition) by adjusting the timer. Moreover, the LED lamps were fixed 10 cm from the plants in various directions. The light intensity was measured with a quantum radiation probe  at the top-leaf level of the plant . In addi−1) 708.0 Ca(NO3)2∙4H2O, 246.0 MgSO4∙7H2O, 505.0 KNO3, 230.0 NH4H2PO4, 1.24 H3BO3, 0.12 CuSO4∙5H2O, 4.00 Fe-ethylene diamine tetraacetic acid, 2.20 MnSO4∙4H2O, 0.08 H2MoO4, and 1.15 ZnSO4∙7H2O. Additionally, our study was not only designed as a completely randomized layout but also had 18 biological replications with consistent growth to minimize external influences.The plants were watered every day at 09:00 from 23 September to 6 November 2021 with a nutrient solution composed of  without midribs were collected from fully expanded leaves at the same stage in the treated plants. These segments were fixed for three days at 4 °C in a formaldehyde solution containing 5% (v/v) formalin, 5% (v/v) acetic acid, and 90% (v/v) ethanol. The leaf samples were dehydrated three times in a graded series of ethanol solutions  ethanol for each treatment for 40 min before being sliced to an appropriate thickness using the freehand slice method. The slices were mounted on glass slides and observed without staining using an optical microscope . ImageJ was used to estimate the thicknesses of the leaves, palisades, and spongy tissues.For each lighting direction combination, six leaf segments , transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO2 concentration (Ci) were measured on the youngest mature leaf of each plant with a leaf porometer . For this purpose, a 3 cm2 area of a terminal leaflet was enclosed in a leaf chamber mounted horizontally. The lighting intensity in the plant growth chambers was 600 μmol∙m−2·s−1 (PPFD), relative humidity about 80%, and the CO2 concentration was the same as outside.The plants were kept in the plant growth chambers in which the net photosynthetic rate . The chlorophyll content was calculated in accordance with the method of Sim et al. . MoreoveFv/Fm), photochemical efficiency of PSII (Fv′/Fm′), non-photochemical quenching (NPQ), and coefficient of photochemical quenching (qP). All the parameters were calculated using the methods reported by Maxwell et al. [The miniaturized pulse-amplitude-modulated photosynthesis yield was used to detect the chlorophyll fluorescence. Each plant was moved to a dark chamber for 30 min to adapt before being measured with a photosystem . The measurements involved the maximal PSII quantum yield  triton-X, pH = 7.0). The resulting mixture was then centrifuged  to obtain the supernatant that would be used afterward for the total protein estimation and enzyme activity assay [For carbohydrate measurements, leaves at the same stage were taken at the end of the day or night. The Anthrone colorimetric method according to Vasseur and Ren et al. was used for the starch and soluble sugar measurements ,77. The ty assay . The totty assay ,80. The −2 s−1 for 15 min. Blanks and the control were run similarly, but without illumination and the enzyme extract, respectively. One unit of SOD was defined as the amount of enzyme which produced a 50% inhibition of NBT reduction by monitoring at A560 nm.Superoxide dismutase activity (SOD) was assayed as described by Becana et al. , monitor2O2 in A240 nm was directly measured in the homogenates, and the reaction medium contained a 50 mM potassium phosphate buffer (PH 7.0), 10 mM H2O2, and 0.1 mL enzyme extract in a final volume of 3 mL at 25 °C. The CAT activity was determined using the extinction coefficient (40 mM−1 cm−1) for H2O2.Catalase (CAT) activity was assayed according to Aeibi et al. . The ind290 nm (extinction coefficient 2.9 mM−1 cm−1) for 1 min. The reaction mixture contained a 50 mM sodium phosphate buffer (PH 7.0), 0.1 mM EDTA, 1 mM ascorbate acid, 2.5 mM H2O2, and 50 μL of the enzyme extract.Ascorbate peroxidase activity (APX) was analyzed according to Nakano  by monit2O2, a 50 mM phosphate buffer (PH 6.0), and 50 μL enzyme extract. The absorbance was recorded at A470 nm and was calculated using the extinction coefficient 26.6 mM−1 cm−1.Peroxidase activity (POD) was measured based upon the method described by Castillo et al. . The 3 mv/v) KOH was added to this mixture, followed by putting it in a water bath at 100 °C for 10 min and gradually cooling to room temperature. The mixture was subjected to incubation at 40 °C for 20 min after 200 μL 0.15% (v/v) anthrone-sulfuric acid solution was applied and the enhancement of A620 nm was monitored. The total RuBisCO activity was measured by injecting 100 μL of the supernatant into 400 μL of an assay mixture consisting of 50 mM Tris-HCl (pH 8.0), 5 mM DTT, 10 mM MgCl2, 0.1 mM EDTA, and 20 mM NaH14CO3 (2.0 GBq mmol−1) at 30 °C. After a 5-min activation period, the reaction was initiated by adding RuBP to 0.5 mmol L−1 and terminated after 30 s with 100 μL of 6 mol L−1 HCl. The PEPC was assayed in a 1 mL reaction mixture consisting of 50 mM Tris-HCl (pH 8.0), 5 mM MnCl2, 2 mM DTT, 10 mM NaHCO3, 0.2 mM NADH, 5 units of NAD-MDH, and 160 μL of the enzyme extract. The reaction was initiated by adding 2.5 mM phosphoenolpyruvate (PEP), and the increase in the A412 nm was monitored. The above description of enzymatic activities was as described in Feng et al. and Yang et al. [The enzymatic activities of the key enzymes related to sucrose synthesis (SS and PEPC), starch synthesis (SSS), and photosynthesis (RuBisCO) were measured using a UV spectrophotometer . The SS was determined in a 1 mL reaction mixture containing 500 μL enzyme extract at 34 °C for 1 h. A 300 μL 30% , starch synthesis (CseSS-7), photosynthesis (CsePsaA-7), and flowering (Cse_sc015873.1_g020.1 and Cse_sc001459.1_g030.1) were chosen from “https://plantgaden.jp/en/list/t1111766” and http://mum-garden.kazusa.or.jp/ (accessed on 27 November 2021). All the leaves were immediately frozen in liquid nitrogen. The total RNA was extracted using an Easy-Spin total RNA extraction kit , then used for first-stand cDNA synthesis with the GoScript Reverse Transcription System  according to the manufacturer’s protocols. Real-time quantitative PCR was conducted in a real-time PCR system . Reaction volumes (20 μL) contained 1 μL of cDNA, 1 μL of each amplification primer (10 μM), 10 μL of 2 × AMPIGENE qPCR Green Mix Lo-ROX , and 7 μL ddH2O (double distilled water). The 2−ΔΔCt method was used for the data analysis, and the ACTIN gene (Cse_sc001321.1_g010.1) was selected as the control. All the target gene primers are listed in The expression of key genes related to sucrose synthesis (p) < 0.05 with a statistical program . The Fisher’s least significant difference test was used for the F-test between the treatments. The experimental assays used to obtain all results were repeated six times and are presented as the mean ± standard error.Significant differences among the treatments were assessed by an analysis of variance (ANOVA) followed by Duncan’s multiple range test at a probability (Cse sc015873.1 g020.1 and Cse sc001459.1 g030.1) in both “Pearl Egg” and “Gaya Glory” plants. Particularly, when compared to the SB lighting, the TS lighting significantly enhanced leaf development, which helps improve the chlorophyll fluorescence and quantum yield of PSII, which in turn substantially promoted photosynthesis and increased chrysanthemum growth and development. Altogether, the TS lighting more properly changed the leaf orientation and adjusted the leaf angle to capture as much of the available light as possible, inducing better plant performances, especially splendid flowering and branching. Thus, the TS lighting provided the optimal lighting conditions in our experiment for chrysanthemums and obviously improved the light using efficiency. However, further investigation remains necessary to explore the involved internal signaling pathways .The significant effects of the lighting quality, quantity, and intensity on plants have been extensively investigated, but researchers have rarely studied the impacts of different lighting directions on chrysanthemum to understand the optimal combination of various lighting directions for better growth and development. In our study, differences in the lighting direction combinations induced epinastic or hyponastic leaf movements in chrysanthemums to efficiently capture the available light, resulting in variations in the leaf petiole angle, which is the threshold point that strongly regulates the morphophysiology. The TS lighting significantly improved the morphological characteristics, carbohydrate assimilation rate, key enzymatic activities, and other plant performances by up-regulating the correlated genes. Moreover, the excellent early flower induction occurred in response to the TS lighting by up-regulating the flowering-related genes (


The authors would like to draw the reader's attention to an error in the following article:J Diabetes Investig 2021; 12: 970–977.Seiichi T, Araki R, Motokura K, Tomobe Y, Yamauchi T, Hanaoka S, Tomotaki H, Iwanaga K, Niwa F, Takita J, Kawa M. Effects of passage through the digestive tract on incretin secretion: before and after birth. 2‐test to compare the proportions of categorical variables among the groups.” should be “Differences in the median values were compared using the Mann‐Whitney U‐test.”On page 971, the line “We used the χThe authors apologize for the errors and any inconvenience they may have caused.


Citrus reticulata Blanco or its cultivated varieties, is a national geographical indicated product that has the concomitant function of both medicine and foodstuff. The primary source of Citri Reticulatae Pericarpium is Citrus reticulata “Chachi,” called “Guang chenpi,” while it differs in variety, propagation, grafting rootstock, and tree age, and the hereditary stability of its biological information between intraspecific plants is worthy of our attention. Homologous analysis result of 4 DNA barcodings in the ribosome or the chloroplast showed that the homology of them  of 22 samples was 100.00%, 99.97%, 99.99%, and 99.81%, respectively, which indicated that 4 DNA barcodes maintained a high degree of genetic stability in Citrus reticulata “Chachi.” Also, ITS2 was considered to identify Citrus reticulata “Chachi” from other varieties because it presented not only low variability within a certain taxon but also a high level of interspecies variability. Simultaneously, variant site detection of Citrus reticulata “Chachi” was analyzed by comparing with the reference Citrus reticulata genome, and 2652697 SNP sites and 533906 InDel sites were detected from whole-genome resequencing data of 22 samples, providing the data resources and theoretical foundation for the future study about the relevant molecular makers of “Guang chenpi.”Citri Reticulatae Pericarpium, the desiccative mature peel of  Citrus reticulata Blanco or its cultivated varieties. Thereinto, Citrus reticulata “Chachi,” the main cultivated variety of Citrus reticulata Blanco, is the primary source of genuine Chinese medicinal materials “Guang Chenpi” [Citri Reticulatae Pericarpium (CRP), a traditional Chinese medicine that has the concomitant function of both medicine and foodstuff, is the desiccated mature peel of  Chenpi” . As a na Chenpi” –4, and m Chenpi” –7.Citrus reticulata “Chachi.” As an emerging method of identification of food and natural medicinal materials, molecular marker  has considerable untapped potential in the quality control and origin identification of food and medicinal materials. DNA barcoding, an important tool for ecological research, has been widely used in species identification [Citrus reticulata “Chachi” was less studied. Previous studies have reported that the ITS2 region was selected for discrimination of the four CRP cultivars; however, this study did not take the intraspecific variation of Citrus reticulata “Chachi” into consideration [Citrus reticulata “Chachi,” they showed some difference in the tree age and the variety including big-leaf species small-leaf species. Besides, propagations of Citrus reticulata “Chachi” include layerage on its maternal plant or graftage on different rootstocks such as Citrus limonia Osbeck, Citrus reticulata Blanco, and Poncirus trifoliata (L.) Raf.Up to now, a large number of studies have focused on the chemical compounds in CRP by morphological identification, microscopic identification, TLC, UV, HPLC, GC-MS, and LC-MS –10, whilfication –14. A nufication –17. Nevederation . BetweenCitrus reticulata “Chachi” of different propagation methods, different tree ages, different varieties, and different rootstocks. Among them, ITS2 is a segment of DNA in the ribosome [Herein, 4 DNA barcodings including ITS2, rbcl, matK, and psbA-trnH were chosen for biological evolutionary information analysis about ribosome , and rbcCitrus reticulata “Chachi” was carried out thought whole-genome resequencing technology with DNBSEQ-T7, compared with the reference published genomic data of Citrus reticulata from the NCBI (GenBank accession number ASM325862v1) [Citrus reticulata “Chachi” samples.Except for a study on genetic stability of 4 barcodes, genetic diversity analysis of 25862v1) , furtherCitrus reticulata “Chachi” plants, which can provide screening indicator of DNA barcoding to distinguish Citrus reticulata “Chachi” and other varieties of CRP. Also, variant type detection based on whole-genome resequencing data provides more potential molecular markers to distinguish Citrus reticulata “Chachi” between intraspecific plants or other cultivars, laying a foundation for the further development of “Guang chenpi.”The objective of this work was to research the hereditary stability of 4 DNA barcodings  in different Citrus reticulata “Chachi” samples were different in variety, plant propagation, rootstock, and tree age.Twenty-two batches of biological materials were collected from the Germplasm Source and Seedling Breeding Center of “Guang chenpi” . 22 CitrGenomic DNA was extracted using the plant DNA extraction kit (TSP101-200) of Tsingke. The quality of the extracted genomic DNA was checked by 1% agarose gel electrophoresis with DL2000 DNA marker, and the concentration of them was carried out through the NanoDrop 1000 .μl−1 and then was amplified by performing polymerase chain reaction (PCR) using 4 pair of universal primers of DNA barcodings listed in Genomic DNA was diluted to 15 ng·Genomic DNA will be randomly interrupted, the end will be repaired, “A” will be added, and the unique connector of DNBSEQ-T7 sequencer will be added. Then, DNA libraries will be constructed by PCR enrichment. Finally, the DNA library was denatured, cycled, and digested to obtain single-stranded circular DNA. Single-stranded circular DNA was amplified by rolling circle amplification (RCA), further producing DNA nanoball (DNB). Illumina sequencing was performed on DNBSEQ-T7 sequencer after the DNA libraries were qualified.Q30 was over than 85%. The clean reads were used for subsequent bioinformatics analysis. For further analysis, we downloaded previously published genomic data of Citrus reticulata from the NCBI (GenBank accession number ASM325862v1). We mapped high-quality data per individual to the reference Citrus reticulata genome using Burrows–Wheeler Aligner (BWA) software [To exclude bias from low-quality reads that arise from the process of base-calling or adapter contamination, the quality of the raw data obtained by whole-genome resequencing was evaluated until the value of software , then thSNPs and InDels can be called by mapping the unitigs against a reference genome. The main calling procedures are as follows: (1) for the results of BWA comparison, Mark Duplicate tool of Picard software is used to remove the duplication and shield the influence of PCR-duplication; (2) the Genome Analysis Toolkit (GATK) software  is used Citrus reticulata “Chachi” samples by using the plant DNA extraction kit, and OD value (A260/280) and DNA concentration are shown in In this work, Genomic DNA was extracted from tender leaves of 22 According to the agarose gel electrophoresis result of PCR amplification products , ITS2, rFour barcodes  were analyzed by DNAMAN software for the length and base composition of each sequence fragment and further identified by BLAST in Genebank. The success rates of PCR amplification and sequencing of 3 DNA barcodings  of 22 samples were 100%. However, due to the large number of fragments missing in the sequencing of two samples (A16 and A21) of the psbA-trnH barcode, 20 psbA-trnH sequences were actually obtained in the experiment. PCR success rate, barcoding length, GC content, variable site, and BLAST rate of 4 DNA barcodings are listed in Citrus reticulata “Chachi” samples was carried out by DNAMAN software. The homologous analysis result showed that the homology of ITS2 of 22 samples was 100.00%, which indicated that ITS2 maintained a high degree of genetic stability in Citrus reticulata “Chachi” of different propagation methods, different tree ages, different varieties, and different rootstocks. Also, the homology of rbcl, matK, and psbA-trnH of 22 samples was 99.97%, 99.99%, and 99.81%, respectively.Moreover, homologous analysis of 4 DNA barcodings about 22 batches of Citrus reticulata “Chachi,” making us realize molecular breeding of Citrus reticulata “Chachi” and the distinction of Citrus reticulata “Chachi” and related species need more valuable molecular markers. Therefore, whole-genome resequencing was also performed on 22 Citrus reticulata “Chachi” samples of different propagation methods, different tree ages, different varieties, and different rootstocks, which provided more scientific basis for molecular breeding.In addition, among the 22 batches of CRP samples, 2 SNP sites were identified in the matK barcode and 1 Indel site was identified in the psbA-trnH barcode. The results showed that there were still some variations within the species of Citrus reticulata “Chachi,” Citrus reticulata “Dahongpao,” Citrus reticulata “Unshiu,” and Citrus reticulata “Tangerina,” while ITS, trnH-psbA, and rbcL could not distinguish these CRP samples [Citrus reticulata “Chachi” with different varieties, propagation methods, grafting rootstocks, and tree ages. Because of DNA degradation in moderately or highly processed products with time, PCR amplification of standard-length (around 650 bp) barcodings is a huge challenge [Citrus reticulata “Chachi” from other varieties, which presented not only low variability within a certain taxa but also a high level of interspecies variability. Also, this work indicated that matK was not considered because of its long length and variable sites within taxa, while rbcl and psbA-trnH had the potential to distinguish Citrus reticulata “Chachi” from other varieties. Actually, combining DNA barcodes in the ribosome and in the chloroplast makes it more convincing in species identification of plants [Previous studies have reported that the ITS2 region was selected for discrimination of the four CRP cultivars including  samples . Differehallenge . Combinef plants , 26.Citrus reticulata “Chachi” sample genomes were sequenced, which generated 158 Gb raw data. Base coverage depth distribution curve and coverage distribution curve indicated that the coverage depth of the bases on the genome was evenly distributed. The statistical results of insert fragment distribution with a single peak show that insert fragment distribution fits the normal distribution and the construction of DNA libraries was reliable. The chromosome coverage depth map showed that the genome was evenly covered, indicating good randomness of sequencing.A total of 22 Citrus reticulata “Chachi” samples is given in Citrus reticulata is 344.27 Mb (assembly level: scaffold). In this work, the average coverage depth was 16X, and the value of Q30 reached 88.94%. The average mapped ratio and genome coverage of all the samples were 98.97% and 93.55%, respectively. The average GC content of Citrus reticulata “Chachi” was 38.68% in line with reference genome Citrus reticulata.Summary of clean sequencing data results about 22 Citrus reticulata “Chachi” for the national geographical indicated product CRP was firstly analyzed by SNP and InDel calling from whole-genome resequencing data , 902182 SNP sites were transversion (Tv), and 9008 SNP sites were transition or transversion. These SNP sites were with a Ti/Tv ratio of 1.93, which is in line with general rules of base mutation in natural organisms [Citrus reticulata “Chachi,” transition happens much more frequently than transversion, which means that evolution of Citrus reticulata “Chachi” tends to accept the substitution between purines and purines or the substitution between pyrimidines and purines, the substitution between purines and pyrimidines causes bad things to happen, and that substitution has mostly been eliminated by evolution.A total of 2652697 SNP sites were excavated between 22 rganisms . In the Citrus reticulata “Chachi” samples, among which 275380 InDel sites were insertion, 241768 InDel sites were deletion, and 9008 InDel sites were insertion or deletion. Unlike SNP sites, insertions and deletions of InDel sites are equally likely to occur.In addition, InDel sites, as codominant molecular markers, are widely distributed in the genome with high density, which are suitable for genome-wide molecular marker exploration. A total of 533906 InDel sites were detected between 22 Citrus reticulata “Chachi” of different propagation methods, different tree ages, different varieties, and different rootstocks. Because ITS2 presented not only low variability within a certain taxa but also a high level of interspecies variability, it was considered to be an useful DNA barcoding to identify Citrus reticulata “Chachi” from other varieties. Moreover, 2652697 SNP sites and 533906 InDel sites were detected from whole-genome resequencing data of 22 Citrus reticulata “Chachi” samples, fully reflecting the genetic diversity of Citrus reticulata “Chachi” with different varieties or propagation methods. To excavate more useful molecular markers for distinguishing Citrus reticulata “Chachi” between intraspecific plants or other cultivars, DNA barcoding analysis and variant type detection of the Citrus reticulata “Chachi” were studied for the first time in this investigation, which laid a special foundation for the biological information analysis of Citrus reticulata “Chachi” for the national geographical indicated product Citri Reticulatae Pericarpium.Overall, our work indicated that 4 DNA barcodes  maintained a high degree of genetic stability in


Scientific Reports 10.1038/s41598-020-73579-y, published online 19 October 2020Correction to: The original version of this Article contained an error.In the Discussion section, subheading “Large-scale climate influences on foraging effort, success and efficiency”,94) and the foraging behaviour and success of marine top predators (e.g.95).”“Sustained elevated positive SOI conditions are indicative of El Niño events, which are known to have strong influences on the distribution and abundance of fish species (e.g.now reads:94) and the foraging behaviour and success of marine top predators (e.g.95).”“Sustained elevated negative SOI conditions are indicative of El Niño events, which are known to have strong influences on the distribution and abundance of fish species (e.g.The original Article has been corrected.


Nowadays, in clinics, there is a lack of reliable biomarkers that could serve as tools allowing for early cancer detection, prediction of therapy response, tumor recurrence, and TNBC course. In this review, we summarized the most recent findings on the applicability of unique blood circulating ncRNAs for management of TNBC. This review was supplemented by bioinformatics analysis for better understanding of molecular processes in which ncRNAs are involved, to promote individual TNBC phenotype and tumor action.Triple negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer, and is related to unfavorable prognosis and limited treatment strategies. Currently, there is a lack of reliable biomarkers allowing for the clinical management of TNBC. This is probably caused by a complex molecular background, leading to the development and establishment of a unique tumor phenotype. Recent studies have reported non-coding RNAs (ncRNAs) not only as the most promising class of molecular agents with a high applicability to manage human cancers, including TNBC, but also as robust and non-invasive biomarkers that are able to be monitored in blood circulation, with the application of liquid biopsy. There is a lack of papers discussing the role of blood-circulating ncRNAs as diagnostic, predictive, and prognostic biomarkers for TNBC. In this paper, we summarized the available literature reports on the utility of blood-circulating ncRNAs for TNBC management. Additionally, we supplemented this review by bioinformatics analysis, for better understanding of the role of ncRNAs’ machinery in the development of a unique TNBC phenotype. Despite the advances in diagnosis and implementation of adequate treatment options, including tailored targeted therapies, breast cancer (BC) is the second most common cause of cancer-related deaths in women worldwide ,2. The rAmong the BC histological subtypes, the triple-negative breast cancer (TNBC) differs from others by its substantial aggressiveness, limited therapy options, and the poorest prognosis ,8. It isNowadays, in clinics, there is a lack of reliable biomarkers that could serve as tools allowing for early cancer detection, prediction of therapy response, tumor recurrence, and TNBC course. Recent efforts made to adapt novel techniques to reveal such biomarkers have proven futile to some extent. Hitherto, clinical decisions are made exclusively upon either histopathologic analysis or analysis of a small number of genes, including their coding proteins in the tumor tissue, which are also distinctly limited ,17,18. MThe majority of transcripts in the human genome are non-coding sequences, that represent a regulatory role in the whole molecular processes of the cell. ncRNAs represent the largest family of RNAs that are not coding for proteins, and form a significant proportion of the genome. According to the recent findings, the three of the most important players in the regulation of gene expression were identified in the ncRNAs family, as followed by microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA) ,23. TheiOne of the major pros for the analysis of ncRNAs as TNBC biomarkers is their remarkable stability in body fluids and significantly altered expression under the cancer conditions. They are both released into blood circulation from cancerous tissues (directly or within the exosomes) or from host tissues affected by tumor occurrence . MoreoveWe conducted a literature search until October, 2021 using the databases: PubMed, EMBASE, Scopus, and by manual searching with the use of keywords (Google) for papers written in the English language. The following approach of literature searching was used with the application of keywords: “triple-negative breast cancer”, “TNBC”, “breast cancer”, “blood”, “serum”, “plasma”, then supplemented by “miRNA”, “microRNA”, “miR”, “long non-coding RNA”, “lncRNA”, “circular RNA”, and “circRNA”. Also, the following search builder was used: (“triple-negative breast cancer” OR “TNBC” OR “breast cancer”) AND (“blood” OR “serum” OR “plasma”) AND (“miRNA” OR “microRNA” OR “miR) AND (“long non-coding RNA” OR “lncRNA”) AND “circular RNA” OR “circRNA”). In Notably, circulating miRNAs were widely examined as prospective diagnostic biomarkers of human malignancies, including BC. However, only a few papers focused on their diagnostic accuracy exclusively for TNBC. In One of the first papers that has begun a series of works analyzing the utility of miRNAs for TNBC detection was the study of Shin et al. Using microarray (MA) followed by qRT-PCR validation, they selected miRNA-16,21 and 199a as promising TNBC biomarkers, and achieved quite satisfactory diagnostic accuracy for TNBC detection with the AUC ranging from 0.798 to 0.884. Interestingly, authors also found that expression of studied miRNAs differs between pre- and post-operative patients, suggesting miRNAs as non-invasive markers for TNBC monitoring . Some of−8), pathways in cancer (3.94 × 10−8), p53 signaling (4.19 × 10−7), ErbB signaling (0.0002), and Wnt signaling (0.0013).Using the bioinformatics tool, we analyzed all of TNBC-related, circulating miRNAs in order to predict their regulatory role in molecular pathways (WikiPathways) . Most of−8), small cell lung cancer (1.3 × 10−9), and melanoma (7.5 × 10−7), and for the breast cancer pathway, FDR was 8 × 10−6 (−10), migration (7.4 × 10−10), and proliferation (4.25 × 10−9). Disease-related predicted analysis (Lnc2Cancer2.0 and MNDR2.0 tools) suggested their key involvement in BC-related carcinogenesis .Circulating lncRNAs demonstrate similar diagnostic accuracy as blood miRNAs, and their combination into diagnostic signatures improves test sensitivity and specificity  66,67,6,70,71,728 × 10−6 C. The GONFIB, promotes cell proliferation and tumor migration to distant organs. This satisfactory diagnostic accuracy was achieved by distinguishing between TNBC and healthy individuals (AUC of 0.897) [−6), p53 signaling pathway (3.2 × 10−5), cell cycle (1.1 × 10−4), breast cancer (1.6 × 10−4), and mTOR signaling (9.6 × 10−3). The top GO terms are summarized in circRNAs are the most recently discovered ncRNA, thus their role as tumor circulating biomarkers is limited. We found only three papers related to blood circRNAs and TNBC. Therefore, it is hard to assess their reliability and tissue-specificity. Li et el. found significantly lower expression of circ0104824 in the circulation of TNBC patients in contrast to those suffering from other BC histological subtypes. However, the expression of circRNA was higher in all BC subtypes in contrast to healthy controls (AUC = 0.849). Authors also noticed that circ0104824 was clinically correlated with tumor size, estrogen, and progesterone receptor status . Two othf 0.897) . Using bDiagnostic accuracy of the discussed blood-circulating ncRNAs for the non-invasive detection of TNBC is summarized in The disease course can be monitored unrestrictedly and ina minimally invasive manner, thanks to the application of liquid biopsy. On the one hand, it is believed that the level of expression of circulating ncRNAs reflects the tumor aggressiveness affecting patients’ survival. On the other hand, changes in ncRNA expression after the therapy can be a valuable precursor of tumor recurrence. In the Prognostic and predictive value of miRNAs for TNBC has been proven by numerous studies analyzing tissue expression of the molecules ,87,88. TData regarding the utility of lncRNAs for prediction and prognosis is still limited and only few papers have focused on this idea ,83,84,85p values)  D. AnalysTargeted treatment strategies for TNBC, including immunotherapy, will obviously require clinically useful biomarkers for therapy enrollment, monitoring, and prediction. ncRNAs are a promising group of biomarkers that can deal with the above-mentioned expectations, which seem to confirm recent in vitro studies made on pembrolizumab—a monoclonal antibody targeting PD-1 that was approved for immunotherapy of locally recurrent unresectable/metastatic TNBC . ncRNAs Nevertheless, a regulatory network of miRNA–lncRNA–circRNA–mRNA interactions demonstrates complex molecular machinery, in which each independent participant seems to be crucial for proper regulation of cell cycle and biological pathways. Even in the case of deregulation of single mode in this highly expanded machinery, it can lead to the failure of the entire anti-oncogenic and tumor suppressing mechanisms. To underline the complexity of ncRNA machinery, and thus the difficulty in their analysis as TNBC biomarkers, we introduced all of the discussed ncRNAs to bioinformatics interaction analysis to create a regulatory network, as well as KEGG and GO enrichment analysis . Noticea


Xanthomonas campestris pv. campestris (Xcc), which causes black rot disease in crucifers, is a representative species of Gram-negative phytopathogenic bacteria, relatively little is known regarding the factors influencing the stringent response in this species. However, previous studies in other Gram-negative bacteria have indicated that RelA and SpoT play a critical role in ppGpp synthesis. The current study found that these proteins also had an important role in Xcc, with a ΔrelAΔspoT double mutant being unable to produce ppGpp, resulting in changes to phenotype including reduced production of exopolysaccharides (EPS), exoenzymes, and biofilm, as well the loss of swarming motility and pathogenicity. The ppGpp-deficient mutant also exhibited greater sensitivity to environment stress, being almost incapable of growth on modified minimal medium (mMM) and having a much greater propensity to enter the viable but nonculturable (VBNC) state in response to oligotrophic conditions (0.85% NaCl). These findings much advance our understanding of the role of ppGpp in the biology of Xcc and could have important implications for more effective management of this important pathogen.The alarmone ppGpp plays an important role in the survival of bacteria by triggering the stringent response when exposed to environmental stress. Although IMPORTANCEXanthomonas campestris pv. campestris (Xcc) is a typical seedborne phytopathogenic bacterium that causes large economic losses worldwide, and this is the first original research article to investigate the role of ppGpp in this important species. Here, we revealed the function of RelA and SpoT in ppGpp production, physiology, pathogenicity, and stress resistance in Xcc. Most intriguingly, we found that ppGpp levels and downstream ppGpp-dependent phenotypes were mediated predominantly by SpoT, with RelA having only a supplementary role. Taken together, the results of the current study provide new insight into the role of ppGpp in the biology of Xcc, which could also have important implications for the role of ppGpp in the survival and pathogenicity of other pathogenic bacteria. Escherichia coli in 1969 , Pseudomonas syringae, Xanthomonas citri subsp. citri, and Erwinia amylovora , type 2 secretion system (T2SS), and other genes related to virulence from transcriptome sequencing (RNA-Seq) data  secretion in Ps. syringae, and swarming motility in Agrobacterium tumefaciens ppGpp, a global regulator in bacteria, was first reported in  in 1969 , 3. For ze ppGpp , 5. Howeze ppGpp . After sze ppGpp . In the ze ppGpp , 6, recemylovora –11. For efaciens , 12–15.Beta- and Gammaproteobacteria generally utilize two homologous RSH enzymes, RelA and SpoT, and possibly contain SAS to synthesize (p)ppGpp (such as fpRel in Ps. syringae pv. tomato and RelV in Vibrio cholerae)  and RelQ  (–The enzymes responsible for ppGpp metabolism in bacteria can be divided into three main categories: long RelA/SpoT homologue (RSH) proteins, which contain both a synthetase and hydrolase domain, small alarmone synthetases (SAS), which contain only a synthetase domain, and small alarmone hydrolases (SAH), which contain only a hydrolase domain , althougholerae) , 16, 18,1, YjbM) –23.Xanthomonas campestris pv. campestris (Xcc), a representative species of Gram-negative plant-pathogenic bacteria, is an important seed-borne pathogen  broth, tryptic soy broth (TSB), amino acid, and sodium pyruvate in preliminary experiments, the current study went on to examine the role of RelA and SpoT in ppGpp production and its effect on stress resistance, biofilm production, and exoenzyme secretion, as well as its effect on pathogenicity and the VBNC state in a series of RelA/SpoT mutants and complementation strains. To the best of our knowledge, this constitutes the first report regarding the role of ppGpp in Xanthomonas campestris pv. campestris and provides important insight into the role of ppGpp in the VBNC state and survival of this important plant-pathogenic bacterium.The current study was initiated to investigate the hypothesis that ppGpp also plays a key role in both the stringent response and induction and maintenance of the VBNC state in Xcc. Having established that Xcc could be reproducibly induced into the VBNC state by CuSOBioinformatic analysis of the Xcc 8004 genome identified two putative RSH proteins, XC_RS05900 and XC_RS04795, which corresponded to RelA and SpoT, respectively. Further analysis indicated that both proteins contained the ppGpp synthetase domain, as well as other characteristic motifs, such as a hydrolase domain . The ppGpp synthetase domains of the Xcc RelA and SpoT proteins were then used to search for further homologous proteins in the Xcc 8004 genome. Although nine possible candidates were identified, none exhibited significant homology (Table S1) or contained the characteristic synthetase domain.relA and spoT genes on the stringent response, VBNC state, and pathogenicity of Xcc. Although ΔrelA and ΔrelAΔspoT mutants were successfully generated using the triparental mating method, attempts to produce a single gene knockout of the spoT gene were unsuccessful. Similar results were found in a previous study of E. coli  compared with those in the wild type, which confirmed that neither the ΔrelA nor the ΔrelAΔspoT mutant had any detectable relA expression  and that the ΔrelAΔspoT mutant had no spoT expression . The qPCR also confirmed that relA expression could be restored to the deletion mutants by the reintroduction of a functional relA gene in the complementation strains, and it was interesting to note that relA expression in the complementation strains was significantly higher than that of the wild-type isolate and that expression in the ΔrelAΔspoT(relA) complementation strain was higher than that of ΔrelA(relA) . Similar results were observed in the ΔrelAΔspoT(spoT) mutant, which had significantly higher levels of spoT expression compared to either the wild-type isolate or the ΔrelA(relA) complementation strain, which itself had slightly (though not significantly) increased levels of spoT expression . These results indicated that both the complementation genes were under the control of their native promoters but do seem consistent with the hypothesis that SpoT has a more important role in the regulation of ppGpp accumulation than RelA. The lack of any detectable relA or spoT expression in the ΔrelAΔspoT mutant was also evidence that this strain lacked the capacity to produce ppGpp, a fact that was later confirmed in the response to stress experiments described below.The expression levels of relA and ΔrelAΔspoT (ppGpp-deficient) mutants differed when they were grown on the NYGA, mMMA, LBA media  × 108 CFU/mL and (4.10 ± 0.77) × 108 CFU/mL in the wild type and ΔrelA mutant, respectively. However, no differences were found between the cell titers of ΔrelAΔspoT mutant on LBA and NYGA , which indicates that ppGpp deficiency restricts the capacity of Xcc to survive under nutrient-limiting conditions. Further investigation revealed that ΔrelAΔspoT as well as the single gene ΔrelA mutant also produced smaller colonies when grown on NYGA plates amended with 2% glucose, a medium used as an indicator of EPS production, with colony diameters of 11.5 ± 0.0 and 13.3 ± 0.3 mm, respectively, compared to 17.0 ± 0.5 mm for the wild type  values of 0.043 ± 0.005 and 0.011 ± 0.004, respectively, compared to 0.058 ± 0.006 in the wild type  or ΔrelAΔspoT(relA), which had OD630 values of 0.043 ± 0.003 and 0.010 ± 0.003, respectively, and the functional spoT gene only partially restoring phenotype in the ΔrelAΔspoT(spoT) complementation strain (OD630 = 0.035 ± 0.007). In this case, the secretion of extracellular protease, amylase, and cellulase was greatly reduced in the ΔrelAΔspoT mutant, resulting in a reduction in the zone of activity from 15.67 ± 0.76, 13.17 ± 0.29, and 15.67 ± 0.29 mm in the wild type to 12.33 ± 0.29, 9.17 ± 0.29, and 13.33 ± 0.29 mm, respectively, in ΔrelAΔspoT  complementation strain, indicating that enzyme secretion was largely mediated by the activity of SpoT. Taken together, these results indicate that ppGpp plays an important role in the response of Xcc to oligotrophic conditions, EPS production, biofilm formation, and secretion of extracellular enzymes.The growth of the wild type and the ΔBA media . Examinaild type  and C. Sild type . HoweverelAΔspoT  to J. It2 in ΔrelA and ΔrelAΔspoT, respectively, compared to 6.55 ± 0.30 cm2 in the wild type (relA mutant in the complementation strains ΔrelA(relA) (6.58 ± 0.21 cm2) and ΔrelAΔspoT(spoT) (4.78 ± 0.75 cm2), respectively, it was noted that relA could also partially restore phenotype in the complementation strain ΔrelAΔspoT(relA) (3.84 ± 0.25 cm2). This result indicates that, while the swarming motility phenotype is predominantly mediated by SpoT, it can also be influenced by RelA.The swarming motility of the deletion mutants was also found to be significantly altered, but in this case the effect was more clearly defined, with the phenotype seeming to be mediated by both RelA and SpoT. Similar to the EPS, biofilm, and extracellular enzymes assays, swarming motility was reduced in both mutants but to a higher degree in the double mutant, which almost completely lacked the ability to swarm on semisolid NYGA media . This reild type . HoweverTaken together, these results indicate that the interaction between RelA and SpoT and their effect on ppGpp production and downstream biological processes in Xcc could be more complicated than previously thought.Raphanus sativus L.). Visual inspection at 7 days postinfection (dpi) revealed that all strains, with the exception of the SpoT-deficient strains [ΔrelAΔspoT and ΔrelAΔspoT(relA)], caused typical chlorotic and necrotic symptoms around the site of injection  complementation strain yielded a bacterial titer of 2.04 × 106 CFU/cm2, which was intermediate between the values for the ΔrelAΔspoT mutant (2.12 × 104 CFU/cm2) and the wild-type isolate (6.51 × 106 CFU/cm2)  . Taken t2+, and this was considered a good starting point for the experiments investigating the effect of environmental stress. Expression analysis using qPCR revealed that the expression of relA and spoT changed quite dramatically when log-phase cells of the wild-type Xcc strain 8004 (OD600 = 0.18) were exposed to the copper stress . However, no evidence of any additional SASs was detected (Table S1). Single (ΔrelA) and double (ΔrelAΔspoT) mutants were successfully generated using the triparental method, but it was not possible to produce a single gene ΔspoT mutant. Similar difficulty in generating a ΔspoT mutant has also been observed in a previous study of E. coli, a phenomenon the authors attributed to the essential hydrolyze activity of SpoT, which prevents ppGpp from accumulating to lethal levels in the bacterial cell  was constructed using a shuttle plasmid that could not reintroduce the relA gene in situ and that it therefore lacked some of the surrounding regulatory elements, leading to unpredictable changes in gene expression. It was interesting to note that the reintroduction of spoT had a much more significant impact on phenotypes that were affected by both genes, with ΔrelAΔspoT(spoT) exhibiting a higher degree of phenotype recovery than ΔrelAΔspoT(relA) with regard to the secretion of extracellular enzymes, biofilm formation, and swarming motility, as well as pathogenicity ppGpp in the virulence traits of X. citri subsp. citri were reported and the mechanism by RNA-seq was analyzed . When required, solid medium was prepared by the addition of 12.8 g/L agar, and selective antibiotics were added at the following concentrations: rifampicin, 50 μg/mL, kanamycin, 50 μg/mL, chloramphenicol, 20 μg/mL, and tetracycline, 5 μg/mL for Xcc and 15 μg/mL for E. coli.The bacterial strains and plasmids used in the current study have been listed in https://www.ebi.ac.uk/interpro/search/sequence/), and the amino acid sequences of both the RelA and SpoT synthetase domains were used as a query to search the Xcc 8004 genome for other homologous proteins.The putative Xcc RelA and SpoT sequences identified in a previous survey of the RelA/SpoT superfamily  were retrelA single mutant and ΔrelAΔspoT double mutant were generated from the parental Xcc 8004 strain using the triparental mating methods. Five hundred base pair upstream and downstream fragments of the Xcc relA and spoT genes were amplified using the XccRelAF8-F/R, XccRelAF10-F/R, XccSpoTF8-F/R, and XccSpoTF10-F/R primer sets, respectively, to produce amplification products that contained a fusion sequence compatible with the p2P24Km vector (relA and EcoRI/HindIII for spoT) to yield the two donor vectors p2P24KmrelA and p2P24KmspoT, respectively.The Δm vector . The resE. coli donors containing either the p2P24KmrelA or the p2P24KmspoT plasmid in conjunction with an E. coli helper strain containing pRK600 (Xanthomonas-like colonies (yellow in color) were picked and verified by PCR using the Xcc-specific primers DLH120/125 detailed in a previous study  and cloned into the pLAFR3  Xcc receptor strains.Complementation strains reintroducing functional e pLAFR3  vector trelA and spoT was initially assessed in wild-type cells exposed to copper, which were prepared in a manner identical to that described in the ppGpp extraction experiment detailed below (OD600 = 2.0), and sampled at 6 time points . A second expression experiment was then used to assess the expression of the two genes in the wild-type isolate, as well as the deletion mutants and complementation strains without copper treatment, during log-phase growth (OD600 = 1.0). In both cases, the cells were harvested by centrifugation at 12,000 rpm for 3 min at 4°C and total RNA was extracted using the SV total RNA isolation system  according to the protocol of the manufacturer. The quality and quantity of the resulting RNA were determined using NanoDrop 2000  before being stored at −80°C until required. First-strand cDNA was synthesized using the PrimeScript RT reagent kit with gDNA Eraser  and stored at –20°C.The relative expression of relA and spoT genes was determined by quantitative PCR (qPCR), which was performed using the Applied Biosystems 7500 fast real-time PCR system  in conjunction with SYBR Premix DimerEraser . Each reaction mixture (20 μL) contained 2 μL diluted cDNA, 10 μL SYBR Premix DimerEraser, each primer at a final concentration of 500 nM, 0.4 μL Rox Reference Dye II, and 5.6 μL of sterile distilled water and processed using the following PCR program: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 56°C for 30 s, and 72°C for 30 s. The standard curve was generated using 4-fold dilutions of the template cDNA, and the PCR efficiency (E) was calculated using a linear regression model: E (%) = (10−1/slope − 1) × 100%  values of each gene were normalized to tC values of the housekeeping genes ugpC and pbpA. Each treatment was represented by three biological replicates and the entire experiment was conducted three times. All the primers used for qPCR analysis are listed in Table S3.The relative expression of the Xcc ) × 100% . The 2−Δ4Cl, 0.5 mmol/L MgSO4 · 7H2O, 0.4% glucose, and 1.6% agar, which was included to investigate the effect of oligotrophic stress  containing 40 mmol/L MOPS (morpholinepropanesulfonic acid) and 4 mmol/L tricine (adjusted to pH 7.2 with KOH), 50 mmol/L KCl, 10 mmol/L NHc stress . Each pl600 = 0.3) were carefully pipetted onto separate plates and cultured at 28°C for 4 days before the size and morphology of the resulting colonies were assessed.The production of EPS was investigated using NYGA amended with 2% glucose, as described in a previous study . Two-mic600 = 0.3) were inoculated onto each plate, and the degree of enzyme activity was quantified by measuring the diameter of transparency (zone of activity) around each colony after 24 h of incubation at 28°C.Similar plate-based assays were also used to assess the enzyme activity of the different Xcc strains. In this case, NYGA amended with 1% (wt/vol) skimmed milk, 0.1 (wt/vol) soluble starch, or 0.5% (wt/vol) carboxymethyl cellulose was used to estimate the extracellular protease, amylase, and cellulase activities, respectively, in accordance with the protocols of previous studies –79. Agai–600 = 1). After 5 days of incubation at 28°C, the bacterial cultures were discarded from the tubes, replaced with an equivalent volume (10 mL) of 0.1% (wt/vol) crystal violet (CV), and allowed to stain for 30 min. The CV was then removed, and the tube was carefully washed twice with sterilized water before the pigment bound to the inside of the tube was eluted in absolute ethanol and quantified by spectrophotometry at 630 nm in accordance with the protocol of a previous study (The biofilm assay was conducted using 9-mL liquid cultures (LB) inoculated with 1-mL bacterial suspensions (ODus study .600 = 0.3) were inoculated onto semisolid NYGA plates (3 g/L Noble agar), and the diameters of the resulting growth zones were measured after 4 days of incubation at 28°C in order to calculate the total colony area.The swarming motility of the different Xcc strains was assessed using the plate assay described in a previous study . In thisIn all cases, each treatment was represented by three biological replicates, and the entire experiment was conducted three times.Raphanus sativus L. cv. Japan 501) leaves. Twenty microliters of the inoculum, which was prepared from overnight cultures by centrifugation at 12,000 rpm for 3 min and resuspension in 0.85% NaCl (OD600 = 0.3), was injected into the main vein on the back of randomly selected leaves of radish seedlings at the 5-true-leaf stage. Identical negative-control samples were prepared by injecting an equivalent volume of sterile 0.85% NaCl. Symptoms of disease were observed and evaluated at 7 days postinoculation (dpi), when 3 leaf disks (9 mm) were collected from each inoculated leaf using a hole puncher and processed for bacterial isolation and counting. The leaf disks were first disinfected by immersion in a 1% NaOCl solution for 1 min before being washed twice in sterilized water and frozen in liquid nitrogen. The samples were then homogenized using a Retsch MM400 ball-milling machine at 60 Hz for 1 min. The resulting leaf powder was resuspended in 0.85% NaCl and a 10-fold dilution series plated on LBA. The number of resulting colonies was counted after 2 days of incubation at 28°C. Each treatment was represented by 6 individual seedlings, and the entire experiment was conducted three times.The pathogenicity of the deletion mutants, complementation strains, and wild-type Xcc 8004 strain were assessed on radish  was assessed in both the wild type and the deletion mutants. In preparation, each strain was cultured on solid medium (LBA) for 48 h at 28°C, before a single colony was picked and inoculated into 10 mL LB broth and incubated at 28°C with shaking (120 rpm) for 12 h, after which the cells were harvested by centrifugation.600 of 0.18 in either 0.85% (wt/vol) NaCl solution amended with CuSO4 to a final concentration of 50 μM or 0.85% (wt/vol) NaCl alone. The cells were then kept at 28°C and samples were taken at intervals  for ppGpp extraction as described below.In the case of the copper and oligotrophic stress, the cells were resuspended to an OD4 · 7H2O, 0.1 M CaCl2, and 15 g/L glucose; OD600 = 2) amended with 0.8 g/L serine hydroxamate (SHX) to induce amino acid starvation  (The ppGpp extraction itself was conducted using a cold methanol method developed in a previous study . The bacm/z 152)  in compaThe cells used to assess the effect of oligotrophic condition (0.85% NaCl) on the induction of the VBNC state were prepared in a manner identical to that described above.The flow cytometry (FCM), itself, was performed using the FACSCalibur system  in conjunction with SYTO 9 (Invitrogen) and propidium iodide  to measure the total and viable population of Xcc cells in the induction mixture, respectively, as described in a previous study . Each trP < 0.05) between treatments being determined by Dunnett’s multiple-comparison test. However, the cell counts on the media  were assessed using a two-way ANOVA (GraphPad Prism 5) in comparison to the wild-type sample, while the ppGpp accumulation data were analyzed by a one-way ANOVA (DPS software) with statistical differences (P < 0.05) determined by the least significant difference (LSD) test.The enzyme activity, biofilm formation, swarming motility, and pathogenicity data collected in the current study were subjected to a one-way analysis of variance (ANOVA) using GraphPad Prism 5 software with significant differences (The data and material can be available from the authors.


Nanocrystalline materials exhibit many unique physical and chemical properties with respect to their coarse-grained counterparts due to the high volume fraction of grain boundaries. Research interests on nanocrystalline materials around the world have been lasting over the past decades. In this study, we explored the room temperature strain rate sensitivity and creep behavior of the nanocrystalline Mg–Gd–Y–Zr alloy by using a nanoindentation technique. Results showed that the hardness and creep displacements of the nanocrystalline Mg–Gd–Y–Zr alloy decreased with increasing loading strain rate. That is, the nanocrystalline Mg–Gd–Y–Zr alloy showed negative strain rate sensitivity and its creep behavior also exhibited negative rate dependence. It was revealed that the enhanced twinning activities at higher loading strain rates resulted in reduced hardness and creep displacements. The dominant creep mechanism of the nanocrystalline Mg–Gd–Y–Zr alloy is discussed based on a work-of-indentation theory in this paper. Mg alloys are promising metallic structural materials and regarded as ideal candidates for lightweight applications in automotive and aerospace industries owing to their low density, high specific strength and rich mineral resources on earth ,2. HowevPrevious studies have demonstrated that when grain sizes are refined to nanoscale, most metals and alloys can obtain double or even much higher strength with respect to their coarse-grained (CG) counterparts ,19,20. Wm, is a crucial parameter which reflects the underlying deformation mechanisms of metallic materials during plastic deformation. Extensive investigations have been conducted on the SRS of NC materials and the relationship between m and grain sizes of crystalline materials g et al.  and Wei or CG Fe  to ~0.00Creep is a crucial in-service deformation mode in engineering materials. Considering practical engineering applications, creep performance is an important factor that needs to be evaluated. Due to the enhancement of GB-related activities in NC materials, their creep behaviors may significantly differ from those in their CG counterparts. Studies on the creep behaviors of NC materials, such as Cu ,33, Al ,35, Ni ,37, etc.A nanoindentation test is an effective and high-efficiency method to characterize the SRS and creep behaviors of NC materials ,42. HereInitial alloy ingot used in the present work was produced using semicontinuous casting, with a measured chemical composition of Mg-6.0Gd-3.5Y-0.5Zr (wt%). After solid solution treatment at 510 °C for 16 h, the billets with dimensions of Φ115 mm × 200 mm were extruded to Φ18.2 mm rods at 400 °C. Subsequently, the extruded rods were rotary swaged to Φ14.7 mm by four passes at room temperature, with a total area reduction of 34.8% [2 60-300 transmission electron microscope (TEM)  operated at 300 kV. EBSD samples were prepared by electropolishing in ethanol solution containing 5 vol% perchloric acid at −40 ℃. EBSD data were analyzed using HKL Channel 5 software . TEM samples were mechanically ground to ~40 μm and then ion milled to perforation using a Gatan 691 precision ion polishing system . X-ray diffraction (XRD) was conducted on a Bruker D8 Advance diffractometer  using CuKα radiation (λ = 0.154 nm) with a scanning step size of 0.02°, a counting time of 3 s, and a 2θ range of 10–80°.Microstructural observations were performed on a FEI Helios NanoLab 600i dual beam scanning electron microscope (SEM)  equipped with an Oxford electron backscatter diffraction (EBSD) system  and FEI Titan G−3 s−1 to 1 s−1, loaded to a predetermined depth of 2000 nm and held at corresponding maximum loads for 1500 s. hcr represents the creep displacement during the holding stage  with a load and displacement resolution of 0.01 μN and 0.01 nm, respectively. Nanoindentation tests were conducted at loading strain rates (LSRs) ranging from 5 × 10ng stage c. For ean, can be defined as [σ. For indentation creep, the displacement time (h-t) curves during the holding stage can be fitted using an empirical equation [h0, a, t0, b and k are fitting parameters. Based on Equation (2), creep strain rate h is an instantaneous indentation depth, and t is creep time. Creep stress σ during the holding stage can be obtained via Tabor relation, σ = H/3 [H can be calculated by:F is real time load during the holding stage, hc is contact depth, and c = 24.56 for the Berkovich indenter [n can be obtained by plotting H in a double logarithmic coordinate system. The fitting slop of the linear segment of the logH curve equals the creep stress exponent n.According to the classical power-law creep theory, creep stress exponent fined as :(1)n=∂loequation :(2)h=h0+fined as :(3)ε˙=1h σ = H/3 , and theindenter . CombiniFmax, decreased monotonously. That is, it exhibited negative strain rate dependence. Fmax corresponding to all the tested LSRs. Fmax decreased from 136.7 mN at 5 × 10−3 s−1 to 83.2 mN at 1 s−1. Plotted in m. For comparison, SRS of the extruded CG alloy is also shown in m, the NC Mg alloys show unexpected negative m values, which is different from those of most CG and NC materials [−1 s−1 in the SRS of NC–RS sample existed. Its m value decreased abruptly from −0.030 to −0.254. However, the saltation disappeared in NC–RS+A sample. Moreover, the m value increased to −0.016 after ageing treatment.aterials ,29,30,31−2 s−1 the m value of the longitudinal section is positive and when 3 × 10−2 s−1 < LSR < 3 × 10−1 s−1, both sections possess similar m values; when LSR > 3 × 10−1 s−1, m of longitudinal section maintains −0.029, while that of cross section decreases suddenly to −0.254. Abnormal SRS undoubtedly stems from specific deformation mechanisms. The variation of m with LSR should be correlated to the transition of dominant deformation modes, which will be discussed in detail in For materials with specific texture, the change of loading direction may influence their dominant deformation modes, which would therefore result in the variation of SRS. m, here we define the strain rate dependence of indentation creep displacement as ω using Equation (5):hcr is the indentation creep displacement, and ω of both RS and the RS+A NC alloys are negative. That is, not only the hardness, but the creep displacements of the NC Mg alloys exhibit negative strain rate dependence as well.Fmax, decreases with increasing LSR in the present work, as shown in The aforementioned results were obtained based on the mode that the indenter was loaded to a fixed depth, 2000 nm, at a constant strain rate followed by holding at the corresponding maximum load (designated as CSR-depth mode hereafter). Under such a loading mode, the holding load, n is an important parameter in describing the creep process, and its value reflects the creep mechanisms of materials. Generally, n = 1 corresponds to diffusion creep, n = 2 corresponds to GB sliding, and n = 3–7 corresponds to dislocation creep [n based on conventional theory. Taking LSR = 5 × 10−3 s−1 as an example, fitting using Equation (2) gives an excellent agreement with the experimental result, as shown in t curve corresponding to H-t curve corresponding to n can be obtained by plotting versus H in a double logarithmic coordinate system. H curves for RS and the RS+A NC Mg alloys, in which the n values of RS and the RS+A NC Mg alloys are 88.9 and 32.4, respectively. Similar to the results of most indentation creep studies on metals and alloys, the n values obtained via conventional data processing procedures were considerably larger than the range of 1–7 corresponding to the classical power-law creep theory. It is impossible to accurately analyze creep mechanisms based on such abnormally large n values.Creep stress exponent on creep . Figure HWI is hardness based on work-of-indentation theory, Wp is plastic work conducted by the indenter, and Vp is plastically deformed volume. Referring to the definition of HWI, indentation creep hardness, Hcr, can be defined as:Wcr is the plastic work conducted by the indenter during the creep stage, and Wcr and Fm is holding load, h0 and hm are displacements at the start and end of holding stage, respectively, and c = 24.56 for the Berkovich indenter [Vp, it is reasonable to treat Vp as constant during the holding stage. Therefore, the creep strain rate based on work-of-indentation theory, can be formulated as:t is the holding duration. According to the hemisphere hypothesis, the plastically deformed volume Vp can be obtained as follows [r can be expressed as [σs is yield strength and can be obtained via Tabor relation, σs = Hp/3. Based on the work-of-indentation theory, Tuck et al. [Hp to holding load Fm and plastic work Wp using Equation (13):n in Equation (1), the creep stress exponent based on the work-of-indentation theory ncr can be expressed as:To overcome this difficulty, a redefined creep strain rate and indentation creep hardness were put forward based on the concept of work of indentation ,48,49. Aindenter . Since c follows :(11)Vp=1Hcr and ncr by plotting Hcr obtained above in a double logarithmic coordinate system, as shown in ncr suggest that room temperature indentation creep mechanisms of RS and RS+A NC Mg alloys are a dislocation creep. For the former, owing to the existence of supersaturated solute atoms inside grains, the dislocation glide is dragged by the solute atmosphere, resulting in a dislocation viscous glide creep mechanism (ncr = 2.9) [α-Mg matrix and therefore weakened the solute drag effect. Hence, the dislocation climb becomes the rate-controlling step during the room temperature creep of RS+A NC Mg alloy (ncr = 7.7) [Combining Equations 7)–(13), r = 2.9) . For the–(13), r r = 7.7) .Generally, metals and alloys become harder at higher strain rates due to enhanced dislocation accumulations, known as usual positive SRS (PSRS). However, under some specific circumstances, materials can soften at higher strain rates, showing a phenomenon of negative SRS (NSRS). By far, there are four mechanisms that can explain the NSRS phenomenon: dynamic strain ageing (DSA) ,53,54,55m value for the cross section remains negative under all tested strain rates, and not only does it not increase gradually with increasing strain rate, but it decreases abruptly when LSR > 3 × 10−1 s−1 (see DSA works via solute-dislocation interactions . At rela s−1 see . Meanwhi s−1 see , which iβ-Ta [SIPT commonly lead to NSRS in materials that may experience phase transformation under applied stress, such as β-Ta , 304 ausβ-Ta , and Ti–β-Ta . Obviousβ-Ta , there iRecently, studies on metallic multilayers suggests that the propensity of crack formation increases at higher strain rates, and this can result in NSRS . This sum increases gradually and turns out to be positive when the true strain exceeds 0.08. Microstructural examination indicates that twinning dominates plastic deformation when the strain is lower than 0.08, while the dislocation slip takes over when the strain is higher than 0.08, where twinning is saturated. Accordingly, it can be concluded that materials exhibit NSRS when plastic strain is dominantly mediated by deformation twinning. Karimpoor et al. [m from −0.030 to −0.016 (see −2 s−1, twinning is activated and takes over plastic deformation, causing NSRS (see m of the cross section at 3 × 10−1 s−1 might result from the activation of less favored twin variants in addition to the most favored ones, which further enhances twinning activities at higher strain rates (see It is well-known that twinning propensity is enhanced at higher strain rates . Therefor et al.  also four et al. , which cr et al. ,69. Thus.016 see . Second,.016 see . When LSNSRS see . Third, ates see .During the plastic deformation of NC metallic materials, dislocations are emitted from GBs, traverse through the grains, and are eventually accumulated at or absorbed by the opposite GBs. The dislocation absorption model established by Carlton et al.  suggestsNanocrystalline Mg–Gd–Y–Zr alloy exhibits negative strain rate sensitivity. Enhanced twinning activities at higher loading strain rates lead to the decrease in hardness, that is, the negative strain rate sensitivity.Initial unaged nanocrystalline Mg–Gd–Y–Zr alloy creeps via a dislocation viscous glide mechanism due to the solute drag effect, while the creep mechanism of aged alloy turns to a dislocation climb owing to the depletion of solute atoms by segregation and clustering.Indentation creep displacements of nanocrystalline Mg–Gd–Y–Zr alloy exhibit a negative strain rate dependence. Enhanced twinning activities at higher loading strain rates generate more twin boundaries in the alloy, which impede dislocation motion, shorten their mean glide distances, and therefore reduce the creep displacements. Suppressed twinning activities in aged alloy result in larger creep displacements than those in unaged alloy.The strain rate sensitivity and indentation creep behavior of a bulk nanocrystalline Mg–Gd–Y–Zr alloy were investigated. The main findings were as follows:


Conclusions. During “Phase 1”, many people faced several negative changes in lifestyle/eating behaviours with potential negative impact on health. These findings highlight that pandemic exacerbates nutritional issues and most efforts need to be done to provide nutrition counselling and public health services to support general population needs.Background. COVID-19 pandemic has imposed a period of contingency measures, including total or partial lockdowns all over the world leading to several changes in lifestyle/eating behaviours. This retrospective cohort study aimed at investigating Italian adult population lifestyle changes during COVID-19 pandemic “Phase 1” lockdown (8 March–4 May 2020) and discriminate between positive and negative changes and BMI (body mass index) variations (ΔBMI). Methods. A multiple-choice web-form survey was used to collect retrospective data regarding lifestyle/eating behaviours during “Phase 1” in the Italian adult population. According to changes in lifestyle/eating behaviours, the sample was divided into three classes of changes: “negative change”, “no change”, “positive change”. For each class, correlations with ΔBMI were investigated. Results. Data were collected from 1304 subjects (973F/331M). Mean ΔBMI differed significantly ( COVID-19 pandemic has imposed a period of contingency measures, including total or partial lockdowns all over the world, with different modalities according to the countries. In Italy the first lockdown, “Phase 1” , occurred March 8. This phase has greatly impacted everyday life of all citizens, leading to different adaptive strategies.Emerging studies ,2,3,4 haThis pandemic has more than ever mastered the notion that there are inevitable repercussions of the general malaise on mental wellbeing. A large multicenter Italian study  exploredTherefore, based on these previous observations ,2,3,4, tA 38 multiple-choice web-form survey in Google Forms was used to collect retrospective demographic and anthropometric data as well as lifestyle habits and eating behaviors during the first lockdown phase of COVID-19 pandemic  in Italy. The survey was launched on 30 April 2020, through WhatsApp, institutional social networks channels  and our Lab  mailing list. Adults (>18 years) residing in Italy were eligible to participate. Data collection ended on 10 May 2020.This web-form administration did not allow a probability sampling procedure; however, it was effective for the research objectives since it facilitated wide dissemination of the survey.The web-survey was conducted in agreement with the national and international regulations and the Declaration of Helsinki. All participants were fully informed about the study objectives and requirements and were asked to provide informed consent accepting the data sharing and privacy policy, before accessing the survey. Participants completed the survey by connecting directly to Google Forms, a web-based app used to create forms for data collection purposes and which is included as part of the free Google Docs Editors suite offered by Google . All the0) and during “Phase 1” . For this purpose, in the present research, we considered 10 multiple-choice items, out of the 38 ones, exploring (i) physical activity; (ii) daily water consumption; (iii) daily caffeine consumption; (iv) daily alcohol consumption; (v) daily breakfast consumption; (vi) habitual sandwich/pizza consumption at lunch; (vii) habitual sweets/dessert consumption at lunch; (viii) habitual fruit consumption at lunch; (ix) habitual vegetable consumption at lunch; (x) “craving or eating between meals” habit. To investigate changes experienced during “Phase 1” COVID-19 pandemic home confinement, participants were asked to describe their lifestyle habits and eating behaviors, before “Phase 1”  both before and during COVID-19 pandemic “Phase 1”. The survey also collected demographic information (such as gender and residency) as well as anthropometric data (such as weight and height) useful to calculate BMI  ; 1 = no The total score was then divided into tertiles. According to the tertiles, subjects were classified into three different “classes of change” that occurred during “Phase 1”: (i) subjects with negative change,    were significantly related to BMI change (dependent variable), adjusting for sex. Basic description of data and statistical analyses were performed using STATA 16.1 . BMI mean values at Tn = 1078) resided in the northern regions of Italy while 17.3% (n = 226) in the Central and Southern ones. We received 1360 questionnaires and selected 1304 subjects (973F/331M) after removing potential duplicates by comparing their IDs. Most of the sample , with a total of 1241/1304 responses (4.8% missingness).1 (p < 0.0001). The median value of the lifestyle habits and eating behaviors total score showed that most of the subjects belonged to the 2nd tertile, meaning substantially that no changes occurred during “Phase 1”; no significant differences were recorded between residents in different regions (Northern vs. Centre + Southern) nor between gender (As reported in n gender . Changes2) as dependent variable and lifestyle habits and eating behaviors  as independent ones were simultaneously assessed. The analysis showed that BMI increase (ΔBMI > 0) during “Phase 1” was significantly and negatively related to the following behaviors: (i) inadequate water consumption ; (ii) excessive alcohol consumption ; (iii) decreased physical activity ; (iv) increased frequency of “craving or eating between meals” ; (v) habitual consumption of dessert/sweets at lunch .Multiple regression analysis  was condp = 0.737). On the contrary, mean (± SD) ΔBMI differed significantly (p < 0.001) between subjects in the “negative change” class (0.4 ± 10.8), subjects in the “no change” class (0.12 ± 0.74), and subjects in the “positive change” class (−0.18 ± 0.9), as reported in In the three classes of change, mean (± SD) BMI value did not differ significantly . The groups experiencing negative lifestyle changes and the one experiencing positive ones reported an increased and decreased BMI respectively, while the one experiencing no lifestyle changes reported no substantial BMI modifications. In particular, those subjects who reported negative changes of their behaviors, namely “adequate daily water consumption”, “alcohol consumption”, “physical activity”, “craving or eating between meals”, “habitual sweets/dessert consumption at lunch”, were also those who displayed a significant increase in BMI during “Phase 1”.Our sample population was characterized overall by a slight but significant BMI increase during COVID-19 pandemic “Phase 1”. After classifying lifestyle changes into three classes , we observed a significant variation of BMI (ΔBMI) between the three groups , the authors reported that the “sensitive” one, ate more and often and snacked more between meals, influenced by a lower mood due to social isolation.In a survey conducted in Spain during home confinement, where the sample was grouped in three clusters based on food and cooking habits , age was not registered, and the analysis was not age-adjusted. Finally, the authors investigated lifestyle habits and eating behavior changes to simply discriminate between positive and negative changes and their relationship to BMI during COVID-19 pandemic, unable to exclude pre-existing eating or mood disorders.This study showed that during “Phase 1” COVID-19 pandemic home confinement, several changes in lifestyle habits and eating behaviors occurred, with individual differences probably depending on personal resilience. However, the short duration of “Phase 1” did not allow to highlight the impact of these changes on long term outcomes Nevertheless, we underline the need to increase public health actions to meet emergency needs and reduce vulnerability over the long term through expanded social protection, health care and education, especially for the most vulnerable groups including seniors, children and women, and for those with poor access to essential goods such as food, education and health care.This is not going to be the last pandemic we face; therefore, we need to build resiliency in our population and nutrition is a key factor. Public health interventions should consider the need to decrease levels of inequity and protect people from further health threats and disease. Further research to understand if and to what extent these changes have regressed, or have remained stable, in the long term and what impact they have had on health is recommended.