Streptomycin and aminoglycoside derivatives are commonly used to treat tuberculosis and other stubborn infections; these drugs may alter auditory and/or vestibular function. Mutations in mitochondrial DNA have been associated with hypersensitivity to aminoglycosides; no studies have been conducted in Mexicans, which are very prone to such alterations because aminoglycosides have been prescribed carelessly for many years, irrespective of the ailment to be treated.We investigated “hot spot” mutations described previously as causing inner ear alterations.Hot spot mutations at the 12S rRNA gene and the tRNA Serine (UCN) gene were screened by PCR-RFLP and sequencing in 65 subjects undergoing audiological and vestibular testing.Experimental.32 individuals had healthy auditory and vestibular function, whereas 33 subjects had auditory affections. We found none of the previously reported mutations related to aminoglycoside hypersensitivity, or non-syndromic hearing loss. Two hearing-impaired patients that had been treated with streptomycin had the T1189C variant of the mitochondrial 12S rRNA region.Mutations related to hearing loss in other ethnic backgrounds were not found in Mexicans. However, the T1189C variant is possibly a putative mutation related to aminoglycoside hypersensitivity and was present in 2 patients. Deafness is one of the main diseases among the human world population. In Mexico, 5 of every 1,000 neonates are born deaf, contrasting with 1 of 1,000 deaf newborn children worldwideAminoglycosides affect mainly molecules involved in protein synthesis; they inhibit protein synthesis initiation by interacting with the site where the anticodon tRNA region  meets the A site of ribosomal RNA. This mechanism has been elucidated in bacterial 16S rRNA, and probably occurs in mitochondrial 12S rRNA(UCN) A7445G mutation gene has also been related with non-syndromic-deafnessThe non-syndromic deafness-associated 12S rRNA region has been studied in several ethnic groups, and putative mutations have been describedMitochondrial DNA is transmitted by the mother; its polymorphisms , when present in certain sites, seem to increase the risk or susceptibility to develop certain neurodegenerative conditions such as hearing loss(UCN) (A7445G) of mitochondrial DNA that could be associated with aminoglycoside hypersensitivity in patients previously screened for healthy or compromised auditory and/or vestibular function.Because aminoglycoside are used frequently (it is the treatment of choice for tuberculosis), we decided to investigate three different changes or hot spot mutations at the 3' end of the 12S rRNA region (T961+C(n)ins, A1555G, and C1494T), and tRNA SerineThe study sample comprised 65 Mexican subjects selected at the Instituto Nacional de Enfermedades Respiratorias, Mexico, who agreed to participate (Protocol B04-02). The clinical history was taken, focusing on past aminoglycoside treatments. Vestibular and auditory function was investigated in all selected individuals Peripheral blood samples were taken for DNA extraction  and screened for the presence of hot spot mitochondrial DNA mutations related to non-syndromic-deafness.The age of individuals ranged from 5 to 70 years (mean - 35 years). Otoscopy and pure tone audiometry at thresholds of 25, 250, 500, 1000, 2000, 4000, and 8000 Hz (Interacustic AC 40 audiometer) were carried out. Hearing above 25 dB was considered normal; hearing loss was mild (25 to 40 dB), moderate (45 to 60 dB), severe (65 to 80 dB), and profound (85 to 120 dB). Caloric tests were also run in all individuals to assess vestibular damage.Mnl), and A7445G . The procedure consisted of denaturation at 95°C for 5 min, followed by 35 cycles , and a final extension at 72°C for 7 min. Each PCR fragment was digested by enzymatic restriction with the abovementioned enzymes and separated by electrophoresis in agarose 3%.The technique was performed as in Hutchin T, et al for A1555GFor the C1494T mutation and other variants around the A1555G region, PCR amplification was done using the Phusion DNA polymerase (New England Biolabs) and oligonucleotides corresponding to the positions 1011-1134 and 1624-1644. The PCR conditions were: denaturation at 98°C for 5 min, followed by 35 cycles , and a final extension at 72°C for 5 min. Each fragment was column purified (Quiagen) and each sample was subsequently analyzed twice by direct bidirectional sequencing in an ABI PRISM 310 Genetic Analyzer Perkin-Elmer Applied Biosystems. The sequence alignment was performed with BLAST and DNAstar programs and compared with the consensus Cambridge sequence (GenBank Accession No. NC_012920), Human Mitochondrial Genome Database. Chromatograms were all manually checked for sequence analysis.The sequences of the following species were used for alignment to search for conservation of the sites where the variants were located in the 12S rRNA gene: human (NC_012920), cow (NC_006853), dog (NC_002008), rat (NC_001665), chimp (NC_001643), gorilla (NC_001645) and mouse (NC_005089).Clinical findingsAmong the 65 study subjects, audiological and vestibular evaluation revealed that 32 had healthy auditory and vestibular function, whereas the remaining 33 presented hearing loss . Of thesWhen the reported sites  were screened, they were not found in any of the samples of the study groups. However, we found a change (thymidine to cytosine) at the 1189 site in the 12S rRNA gene of 2 patients with auditory and vestibular diseases, who had been treated with streptomycin .The 12S rRNA region in mitochondrial DNA has been described as a hot spot for nucleotide variability related with non-syndromic deafness as a result of aminoglycoside hypersensitivity; several reports have confirmed this finding. In our molecular biological studies, we found no mutation associated with hearing loss and aminoglycoside hypersensitivity.However, we found a T to C change in the position 1189 in two patients, irrespective of alignment with the universal Cambridge database, which is the first report in the literature (to our knowledge) of such a nucleotide sequence change in the region close to the 3' end of the mitochondrial 12S rRNA gene, which could be correlated with auditory or vestibular alterations due to aminoglycosides .Figure 2Analysis of the position 1189 in different species has shown that it is a highly conserved site . WhetherOur results suggest that mutations associated with hearing loss and aminoglycoside toxicity, which are found in other ethnic groups, were not present in Mexicans. Furthermore, the T1189C variant, which had not been previously reported, is apparently associated with aminoglycoside treatment. Whether this putative mutation is present in other individuals is of significant importance, as it may be a valuable screening marker prior to therapy. Recommendations may be given to avoid damage due to these hazardous antibiotics.The authors wish to thank Doctor Robyn Hudson, of the Biomedical Institute (UNAM), for a critical review and English grammar corrections in the manuscript; also Doctor Ignacio Mora Magaña for some of the audiological measurements, and Mrs Cecilia Escalona Ochoa for much help in the laboratory. We thank Doctor Daniel Ortuño Sahagún for providing access to the reverse alignment DNAstar program, and the technical personnel of the Unit of Molecular Biology for significant support.


The timing of pubertal development is strongly influenced by the genetic background, and clinical presentations of delayed puberty are often found within families with clear patterns of inheritance. The discovery of the underlying genetic regulators of such conditions, in recent years through next generation sequencing, has advanced the understanding of the pathogenesis of disorders of pubertal timing and the potential for genetic testing to assist diagnosis for patients with these conditions. This review covers the significant advances in the understanding of the biological mechanisms of delayed puberty that have occurred in the last two decades. It is broadly defined as puberty commencing more than two standard deviations later than the mean age for the population.DP can be caused by a variety of aetiologies, including self‐limited DP , hypogonadotropic hypogonadism caused by a permanent or functional gonadotropin‐releasing hormone (GnRH) deficit, and hypergonadotropic hypogonadism due to gonadal insufficiency.2Evidence from twin studies has demonstrated that the timing of puberty is strongly heritable, and that genetic regulation is an important element in determining when healthy individuals enter puberty.33.1Self‐limited DP and CHH can present with the same phenotype, that is, delay entering puberty; however, these two conditions are different in clinical course and requirement for treatment. CHH, or Kallmann syndrome (CHH with anosmia), are pathological conditions with failure to progress through puberty which usually need intensive hormonal therapy, whereas self‐limited DP is generally a more benign condition once puberty is established, either after a period of monitoring or a short treatment course of sex steroids.3.2ANOS1,HS6ST1,PROK2/PROKR2,SEMA3A,SEMA7A,SEMA3E,PLXNA1,CCDC141,FEZF1,CC/NTN1,AMH/AMHR2,NDNF,SOX10,TUBB3,GLCE,FGFR1,FGF17,FGF8,IL17RD,DUSP6,FLRT3,SPRY4,KLB,WDR11,NR0B1CHD7GNRH1/GNRHR,FSHBLHBIGSF1 deficiency, which results in a syndrome of X‐linked central hypothyroidism with DP and macro‐orchidism in male patients.To date, it has been shown that over 50 genes affecting the hypothalamic‐pituitary‐gonadal (HPG) axis contribute to the pathogenesis of CHH.3.3GNRHRTAC3 and its receptorHS6ST1, has been identified in a family segregating with pure self‐limited DP.n = 72) identified rare and predicted deleterious variants in CHH genes including AXL, FGFR1, HS6ST1, PROKR2, FEZF1 and TAC3, in patients with self‐limited DPAlthough self‐limited DP is the most common cause of DP, the underlying genetic basis of this condition remains incompletely understood. The first information about the genetic inheritance of isolated DP was from patients with CHH or Kallmann syndrome Figure , whose r3.4An increasing number of genes have been implicated in the pathogenesis of self‐limited DP over the last 5 years.To date, 14 genes have been identified as contributing to self‐limited DP, including those identified in relatives of CHH probands and others identified from large cohorts of familial self‐limited DP which have been extensively studied in vitro and in vivo. The majority of these genes have functions related to GnRH biology, including regulation of GnRH neuronal development and migration, GnRH upstream control, GnRH downstream action, and energy metabolism  analysis in a study which found that deleterious variants in this gene impaired its transcriptional activity on the GnRH promoter, resulting in reduced GnRH transcription and secretion.TAC3 and TACR3 have also been reported to cause self‐limited DP. These genes code for neurokinin B and its receptor, an important element of the KNDy neuronal complex which controls GnRH pulsatility.TAC3/TACR3 have been identified in self‐limited DP patients using WES,Other important upstream regulators of GnRH transcription have been implicated in the timing of human puberty,3.7GNRHR mutations which lead to the phenotype of CHH. Additionally, heterozygous mutations of GNRHR have been identified in patients who manifest only with self‐limited DP.GnRH needs to bind to its receptor, GNRHR, to stimulate pituitary gonadotrophs to prompt gonadotrophin secretion. Abnormal GnRH‐GNRHR signalling has been demonstrated with 3.8A wide range of environmental factors have been found to influence the timing of puberty. The effect of endocrine‐disrupting chemicals (EDCs) on pubertal timing have been widely studied. EDCs are environmental compounds that have a potential contribution to the observed shift towards an earlier onset of puberty in the developed world, Figure MKRN3 and DLK1 identified in pedigrees with central precocious puberty.Epigenetic mechanisms have been implicated in the regulation of the timing of puberty. Experimental data from rats and goats give evidence for changes in DNA methylation and histone acetylation leading to altered gene expression during puberty.3.9Fat mass and obesity‐associated protein (FTO) had been implicated by GWAS to have role in the timing of puberty, and to impact on BMI and risk of obesity.FTO were identified in 3 Finnish families from a self‐limited DP cohort.Fto‐knockdown mice showed delayed pubertal onset. Although the mechanism by which FTO might influence pubertal timing is unclear, this may involve energy homoeostasis. FTO might act directly via the mTORC1 signalling pathway, which has role in energy balance and expression of kisspeptin in the hypothalamus, or it might affect BMI, thus influencing pubertal timing indirectly, or potentially exert an effect via both mechanisms.LEP and LEPR respectively, have an important role in mediating the relationship between energy metabolism and pubertal timing. Loss of function mutations in LEP or LEPR cause monogenic obesity.LEP.Kiss1 promoter.Murine models have provided further data linking metabolic inputs with pubertal timing. Overexpression of SIRT1 in a rodent model decreases Kiss1 mRNA expression and leads to DP.4BMP15, GDF9, FIGLA, FSHR, POLR3H, NOTCH2, FOXL2, AHM/R, FMR1, POF1B and DIAPH2.Patients with primary gonadal disorders may present with delayed or absent pubertal development. Turner syndrome is the most common form of hypergonadotropic hypogonadism in females, and puberty is usually absent, or otherwise delayed and followed by progressive ovarian insufficiency.In males, the commonest condition is Klinefelter syndrome , where individuals enter puberty spontaneously at a normal age, but testosterone levels become increasingly deficient by Tanner stages 4‐5.LHCGR gene present in females usually with primary amenorrhoea rather than DP.LHCGR mutations lie along a phenotypic spectrum from disorders of sexual differentiation to undermasculinisation and infertility due to lack of testosterone secretion.FSHR are extremely rare, affecting mostly females with variable degree of pubertal development and complete ovarian failure. Point mutations in the extracellular domain of the FSHR, most frequently seen in the Finnish population, lead to subsequent inactivation of the receptor function resulting in raised FSH levels.Mutations in the gonadotropin receptors leading to hypergonadotropic hypogonadism are not a common cause of delayed or absent puberty. Loss‐of‐function mutations in the 5While a variety of clinical and biochemical investigations are available to assist with diagnosis of individuals with central DP, none of these can reliably distinguish CHH from self‐limited DP in adolescence.6Accumulation of knowledge relating to the genetic basis of pubertal delay has greatly accelerated over the last two decades with the improvements in sequencing technologies. Identification of genetic defects underlying hypogonadotropic and hypergonadotropic hypogonadism have led to a greater understanding of the pathophysiology of these disorders. The genetic control of self‐limited DP is still mostly undiscovered, but from the initial findings it appears that the pathogenic mechanisms are related to GnRH neuronal development and biology, starting from neuronal development in the embryo to transcription and secretion of GnRH in the pubertal brain. The main pathophysiology of this condition is thus likely to be due to changes in responsiveness of the GnRH neuroendocrine system, resulting from defects of development of GnRH neurons and GnRH network functionality. Further developments will led to greater clarity on the biology of these conditions and the factors that determine timing of puberty in the healthy population, and can provide the opportunity for improved diagnostics and therapies for patients with disorders of puberty.


High VPD decreased stomatal conductance (sg) but increased transpiration rate (rT). High VPD increased the threshold of soil water for rT began to decrease, while the soil water threshold for sg depended on temperature due to the varied ABA response to temperature. High VPD decreased leaf water potential, leaf area, and shootDM, which exacerbated the inhibition of soil drying to plant growth. Across the well-watered plants in both experiments, negative linear relationships of sg and WUE to VPD and positive linear relations between rT and VPD were found. The results provide some novel information for developing mechanistic models simulating crop WUE and improving irrigation scheduling in future arid climates.Drought stress often occurs concurrently with heat stress, yet the interacting effect of high vapor pressure deficit (VPD) and soil drying on the physiology of potato plants remains poorly understood. This study aimed to investigate the physiological and growth responses of potatoes to progressive soil drying under varied VPDs. Potato plants were grown either in four separate climate-controlled greenhouse cells with different VPD levels  or under a rainout shelter in the field. The VPD of each greenhouse cell was caused by two air temperature levels (23 and 30 °C) combined with two relative humidity levels (50 and 70%), and the VPD of the field was natural conditions. Irrigation treatments were commenced three or four weeks after planting in greenhouse cells or fields, respectively. The results indicated that soil water deficits limited leaf gas exchange and shoot dry matter ( Regardless of irrigation regimes, plants grown at normal temperature showed a significant difference in leaf[ABA] between high and low relative humidity under soil drying, in contrast to plants at high temperature with no differences  of DS plants was lower than WW plants across all VPDs. The lψ of DS plants under high temperature and low relative humidity (VPD4) was the lowest (−0.57 MPa) among the treatments (SD) than those grown at normal temperatures (SA) than DS plants at each VPD level. Regardless of irrigation, plants developed under high humidity had a higher SA as compared to those grown under low humidity at a given temperature  of WW plants was higher than DS plants regardless of VPD regimes. Moreover, plants grown under low relative humidity had a lower LA as compared to those under high relative humidity at normal temperature; the LA of plants grown at normal temperature was higher than those grown at a high temperature (SLA) of WW plants was higher across all VPD levels as compared to the DS plants, especially at high temperatures: SLA of DS plants was 40 and 36% lower than WW plants under high and low relative humidity, respectively  of WW plants was higher than DS plants across all the VPD levels, especially under relative humidity at normal temperature. Moreover, the shootDM of WW and DS plants developed at high temperature and low relative humidity (VPD4) was 34 and 25% higher than those grown at normal temperature and high relative humidity  of DS plants was lower than WW plants across all the VPD levels. Meanwhile, the WU of the WW plants grown at high temperature and relative humidity (VPD2) was lower than that for the other treatments  on the fourth day than on the third day, probably due to the increased VPD , which could reduce sg even for WW plants leaf . This reW plants ,35. In re curves  fall in humidity c. The laand leaf . Thus, tor to Tr c, but deon (ETo) . Howeversg compared to those grown under low relative humidity due to modified stomata morphology, i.e., larger stomata length and pore aperture [sg to VPD [leaf[ABA] of DS plants increased with increasing VPD under normal temperatures, suggesting ABA involvement in the regulations of gas exchange by VPD  and soybean (Glycine max L.) also pointed to soil water content as the key factor determining the response of transpiration to soil water deficit, regardless of the size of plants or pots ..gs of WWeriments . Such a  unclear  and meriSolanum tuberosum L. cv. Oleva (40–55 mm in diameter) were planted in a depth of 3 cm in 2.5 kg peat substrate  filled into 108 cylindrical pots . Fertilizer was applied to each pot with 1 g N (NH4NO3), 0.8 g P, and 1 g K (H2KPO4). Prior to planting, potato tubers were pre-sprouted for two weeks at 12–14 °C with dim overhead light. Only one sprout was left to emerge during the germination phase. The tubers were planted in four greenhouse cells, and the VPD treatments in each cell are shown in −2 s−1 photosynthetic active radiation provided by sunlight plus LED lamps in each greenhouse cell. The greenhouse experiment was conducted at the Faculty of Science, Copenhagen University, Taastrup, Denmark. Tubers of −3. The soil had a volumetric soil water content of 21 and 5% at field capacity (FC) and permanent wilting point, respectively. Each pot was fertilized with three granular compound (NPK) fertilizers: 2.1 g N, 0.5 g P, and 2 g K. The treatment before planting and at the germination of the potato tubers was analogous to that in the greenhouse experiment. All pots were placed under a rainout shelter to prevent rainfall. The field experiment followed the greenhouse experiment and was carried out from May to June 2019 at the experiment station of Copenhagen University, Taastrup, Denmark. On 21 May, two potato tubers were sown in 25 pots (24 L) containing 21.5 kg of air-dried sandy soil with a bulk density of 1.44 g cmFTSW) ). Xylem sap was collected by pressurizing the roots of the potted plants in a pressure chamber. The whole pot was inserted into the pressure chamber and sealed, leaving only the shoot, which was then cut at 3–5 cm height from the stem base, and the cut surface was dried with blotting paper. Pressure was applied slowly until the xylem sap outflowed from the cut. Pipettes were used to collect the xylem sap (0.5 mL) from the cutting surface into an Eppendorf vial wrapped with aluminum. All sap samples were frozen immediately and stored at −80 °C for ABA analysis.The concentrations of ABA in leaf and xylem were determined only in the greenhouse experiment. After leaf gas exchange measurement, three leaves (one leaf per plant) per treatment were cut and immediately packed in aluminum foils and frozen in liquid nitrogen, then stored at −80 °C for the measurement of leaf ABA concentration  were determined by enzyme-linked immunosorbent assay (ELISA)  and xylem[ABA] at each VPD level. High VPD significantly affected sg, rT, plant ABA concentration, lψ, SA, SD, LA, shoot,DM and WUE, while sg and WUE were negatively correlated with VPD and rT was positively correlated with VPD. Furthermore, the results demonstrated that the plant water relation and dry matter of potatoes developed well under low VPD, while the combined effects of high VPD and soil water deficits had significantly on leaf[ABA], xylem[ABA],LA and SLA exacerbated the adverse effects on plant growth and development. With respect to sg, the FTSW threshold depended on temperature due to a differential ABA response to temperature, apparently affecting the ABA concentration. This is a novel and important finding for ecosystem studies and modelling actual rT. In summary, the results provide essential and detailed insights into plant mechanisms of stomatal control and water status under varied VPD and soil moisture conditions, which are essential for developing mechanistic models predicting crop WUE in a future arid climate.In conclusion, soil water deficits significantly limited leaf gas exchange rates,


P = 0.025). Moreover, P50 demonstrated a significant positive association with extrapulmonary organ dysfunction score  and risk of mortality , irrespective of initial PARDS severity. The relationship between P50 and mortality was largely mediated by extrapulmonary organ dysfunction. A high P50 value at the time of PARDS diagnosis may be associated with mortality via dysfunctional extrapulmonary organs. Future studies should consider P50 as a potential candidate index for risk stratification of PARDS patients.Hypoxemia and multiple organ dysfunction are significant contributors to mortality in patients with pediatric acute respiratory distress syndrome (PARDS). P50, the oxygen tension at which hemoglobin is 50% saturated, is a measure of hemoglobin-oxygen affinity, and its alteration might have implications for tissue hypoxia and organ dysfunction. The purpose of this single-center, retrospective study was to evaluate P50 levels in PARDS and to determine the association between P50 and clinical outcomes. The study included 212 children diagnosed with PARDS according to the Pediatric Acute Lung Injury Consensus Conference definition who required invasive mechanical ventilation and had arterial blood gas results of hemoglobin oxygen saturation < 97% at the time of diagnosis. P50 levels were calculated using Doyle’s method, and organ dysfunction was assessed using the Pediatric Logistic Organ Dysfunction-2 score. Most patients exhibited more than one dysfunctional extrapulmonary organ at PARDS onset. P50 increased with increasing PARDS severity (mild (26.6 [24.9–29.6]), moderate (26.8 [25.0–29.5]), and severe PARDS (29.1 [26.1–32.4] mmHg;  Although improvements in pediatric critical care support have led to a decrease in mortality rates, pediatric ARDS (PARDS) remains a significant cause of childhood morbidity and mortality worldwide5.Acute respiratory distress syndrome (ARDS) refers to hypoxic respiratory failure due to diffuse pulmonary inflammation. ARDS results in increased capillary endothelial permeability following a variety of direct and indirect injuries to the lung7. PARDS is a severe lung disease exhibiting complex pathophysiology that involves not only the respiratory system but also the extrapulmonary end-organs. Therefore, extrapulmonary organ dysfunction is common in both adults and children10. It generally occurs concurrently with respiratory dysfunction; however, additional organ dysfunction may develop after the onset of respiratory failure11. Previous studies have consistently demonstrated that extrapulmonary organ dysfunction is a strong risk factor for mortality that is independent of hypoxemia degree, or other respiratory criteria12. The Pediatric Acute Lung Injury Consensus Conference (PALICC) sub-committee has stated that multiple organ dysfunction is ‘the single most important independent clinical risk factor for mortality at the onset of ARDS’13. However, the pathophysiological mechanisms linking multiple organ dysfunction to PARDS remain unclear.Of note, the predominant cause of death in PARDS does not appear to be severe hypoxemia, which is one of the defining criteria in PARDS, but multiple organ dysfunction14. Nearly 98% of circulating oxygen is bound to hemoglobin, and the affinity of hemoglobin for oxygen in relation to the partial pressure of oxygen (pO2) is demonstrated by the hemoglobin-oxygen dissociation curve. The oxygen tension at which hemoglobin is 50% saturated (P50) is a measure of the affinity of hemoglobin for oxygen that determines the release of oxygen from microcirculation into tissues15. Therefore, alterations in P50 might have implications for tissue hypoxia and subsequent organ dysfunction via altered oxygen extraction16.At the tissue level, oxygen supply and demand are dependent on various factors including peripheral oxygen delivery, diffusion from microcirculation to tissue mitochondria, and local tissue oxygen consumption. Abnormalities of systemic gas exchange, such as imbalances between oxygen delivery and uptake, have been suggested as potential mechanisms underlying extrapulmonary organ dysfunction in ARDS with a low level of oxygen uptake below the critical thresholdFew studies have investigated P50 in ARDS. In addition, no investigation has reported P50 in relation to multiple organ dysfunction in PARDS. Thus, we aimed to evaluate the level of P50 in PARDS patients, investigate the associations between P50 and various organ dysfunctions, and assess the possible link between P50, extrapulmonary organ dysfunction, and clinical outcomes in patients with PARDS.17, which requires invasive mechanical ventilation; and (2) at least one documented arterial blood gas result at the time of PARDS diagnosis. Patients with congenital cyanotic heart disease were excluded because these patients were cared for in a separate cardiothoracic intensive care unit.First, we extracted data from the Severance Hospital electronic database and retrospectively reviewed the records of patients aged 1 month to 19 years who were admitted to the intensive care unit (ICU) from January 1, 2010 to January 30, 2017. A total of 1,101 patients were then screened for inclusion in this study. Next, we identified patients with respiratory difficulty requiring invasive mechanical ventilation. Finally, we reviewed medical records to identify if the patient met all the diagnostic criteria of PALICC definition consistent with the timing of onset, etiology for ARDS, chest radiographic images for the presence of pulmonary infiltrates, the origin of edema not fully explained by cardiac failure or fluid overload, and oxygenation parameters. The eligibility criteria were as follows: (1) diagnosis of PARDS according to the PALICC criteria18 based on Hill’s equation192 and hemoglobin oxygen saturation (sO2) from a single-point measurement of arterial gases. All arterial gas results were the nearest approximation to the time of PARDS diagnosis and within a maximum of 4 h after diagnosis. Only results with measured sO2 < 97% were utilized because sO2 values ≥ 97% might lead to erroneous calculations at the extremes of the hemoglobin-oxygenation equilibrium curve20. In addition, patients younger than 1 year were excluded from the analysis because fetal to adult hemoglobin switching might not yet fully occurred in this population, and fetal hemoglobin has a higher affinity for oxygen than adult hemoglobin21.In vivo P50 values were calculated using the method described by DoyleThis study was approved, and a waiver was obtained from the Institutional Review Board of Yonsei University Health System, Severance Hospital for informed consent (IRB No. 4-2013-0207). The study was carried out according to the Declaration of Helsinki.22. The total score was subdivided into five types of organ dysfunction  by relevant variables. Similarly, extrapulmonary organ dysfunction was assessed using the non-respiratory PELOD-223, which was calculated by simply removing the corresponding respiratory variables.During the ICU stay, demographics and clinical data were assessed, including PARDS etiology, comorbidity, clinical progress, and outcomes. The Pediatric Index of Mortality 3 scores at ICU admission, and Pediatric Logistic Organ Dysfunction (PELOD)-2 score at the time of PARDS diagnosis were collected. ICU mortality and degree of multiple organ dysfunction were selected as the main outcomes. The PELOD-2 score is generally adopted as a clinically meaningful descriptor for the number and severity of organ dysfunctions in critically ill childrenClinical outcomes of PARDS, such as ICU mortality, duration of invasive mechanical ventilation, and ventilator-free days at 28 days post-diagnosis were also collected. Ventilator-free days were determined for survivors by subtracting the total duration of invasive ventilation from 28 days. In patients who required ≥ 28 ventilator days or ICU non-survivors, the number of ventilator-free days was 0.24. All statistical analyses were conducted using software SPSS Statistics  and R . Two-tailed P value < 0.05 was considered to indicate statistical significance.Categorical data were presented as counts and percentages. Continuous data were tested for normality using the Kolmogorov–Smirnov test and reported as the median with interquartile range. Categorical variables were analyzed using the Chi-square test or Fisher’s exact test, and continuous variables were compared using the Kruskal–Wallis H test with Dunn’s method for multiple comparisons. Correlation between the P50 level and PARDS severity was analyzed with the Spearman’s rank correlation test. Multivariate linear regression analyses controlling for age, sex, weight, comorbidity, and initial PALICC grade were performed to assess the associations between P50 and degree of various organ dysfunctions. Cox’s regression model was employed for survival analysis by adjusting for the aforementioned variables. To investigate the possible mechanism underlying P50 and mortality in PARDS patients, mediation analysis was performed for extrapulmonary organ dysfunction as a potential mediator using the method proposed by Lange et al.This study was approved by the Severance Hospital’s Institutional Review Board, and the requirement for informed consent was waived (IRB No. 4-2013-0207).2 < 97%. Among them, 45 patients were aged younger than 1 year. The remaining 212 patients were included in the final analysis  in patients with severe PARDS (29.1 [26.1–32.4] mmHg) than in those with mild (26.6 [24.9–29.6] mmHg) or moderate (26.8 [25.0–29.5] mmHg) PARDS. The correlation analysis showed a weak but significant positive correlation between the P50 level and PARDS severity .A trend of increasing P50 levels with increasing PARDS severity was observed Fig. . P50 wasMost patients 92.0%) exhibited at least one extrapulmonary organ dysfunction at PARDS onset. The number of extrapulmonary organs with dysfunction increased with increasing hypoxemia severity Table . Further.0% exhibP = 0.007), extrapulmonary , and overall organ dysfunction  scores. Extrapulmonary organ dysfunction was further divided into neurological, cardiovascular, renal, and hematologic dysfunction, which revealed significant associations between cardiovascular and renal dysfunction and P50  . The same association was found after adjustment for potential covariates such as age, sex, weight, comorbidities, and initial PALICC grade . In contrast, no significant associations were observed between P50 and ICU length of stay, invasive mechanical ventilation duration, and ventilator-free days (data not shown).Univariate analysis using Cox’s regression revealed that the mortality risk was significantly increased with increasing P50 levels . The analysis confirmed a significant direct effect  and indirect effect  of extrapulmonary organ dysfunction consistent with the presence of mediation. Extrapulmonary organ dysfunction was estimated to mediate 81.5% of the effect of P50 in PARDS mortality.The mediation analysis produced models demonstrating that extrapulmonary organ dysfunction partially mediated the relationship between P50 and mortality in PARDS Fig. . After cIn this study, P50 levels increased with increasing PARDS severity, with a notable difference in children with severe PARDS. P50 exhibited a significant correlation with the degree of extrapulmonary organ dysfunction as well as respiratory dysfunction. The mortality risk was significantly associated with the P50 level, even after adjusting the initial PARDS severity criteria. Moreover, we demonstrated that extrapulmonary organ dysfunction partially mediated the relationship between P50 and mortality in PARDS patients.11 reported a prevalence of 63% of concurrent multiple organ dysfunction within a day of the onset of acute respiratory failure, using a multi-centered, prospectively collected dataset of 2449 children. In addition, Kallet et al.8 conducted an analysis on 15 years of data from 1747 adults with ARDS and demonstrated that at least one dysfunctional extrapulmonary organ system was present in 80–90% of patients. This pervasive early-onset extrapulmonary organ dysfunction further supports the concept of PARDS as a pulmonary manifestation of a systemic syndrome25.In PARDS patients, extrapulmonary organ dysfunction occurred frequently, with an incidence above 90% at the time of PARDS onset. These findings are consistent with previous studies in children with acute respiratory failure or ARDS. Weiss et al.9. In ARDS, the redistribution of cardiac output or a loss of recruitable capillary reserve may compromise perfusion within individual organs. Direct endothelial injury and the ensuing alveolar edema increase the diffusion distance for oxygen from the alveoli to the capillaries, and parenchymal damage may impair oxygen utilization14. Furthermore, the concept of systemic microcirculatory injury has been supported by the presence of globally activated intravascular inflammatory mediators in ARDS28. Other than the lung, a primary target organ, the inflammatory process may spill over into the systemic circulation, resulting in widespread endothelial and parenchymal damage. This may cause altered gas exchange in distal organs in an analogous manner as pulmonary injury and dysfunction30.The exact nature of the association between lung injury and extrapulmonary organ failure in ARDS remains largely unknown. ARDS could be both the cause and the consequence of multiple organ dysfunction32. The pathology of multiple organ dysfunction includes the primary insult, which evokes the host response of the systemic inflammatory process, and the resultant phenomenon of 'organ cross-talk' among individual systems25. Our findings support this reciprocal interaction between the lung and other organs, as well as the contribution of extrapulmonary organ dysfunction to mortality in PARDS, which reinforces the results of previous studies34.Our results have demonstrated that the degree of extrapulmonary organ dysfunction and the number of dysfunctional organ systems increased as the PARDS severity increased. All extrapulmonary organ dysfunctions were closely related to the mortality risk, and the greater the number of dysfunctional organs, the higher the mortality rate. Indeed, mortality in critically ill children results from the interactions among multiple failing organs35. Factors that increase P50 include decreased pH, high levels of erythrocyte 2,3-diphosphoglycerate , and fever36. The complicated interplay among these various factors makes it difficult to determine the final P50. For instance, acidemia may increase P50 via the Bohr effect; however, it also reduces 2,3-DPG production, which may decrease P50. Chronic hypoxemia and anemia increase 2,3-DPG, whereas hypophosphatemia decreases 2,3-DPG.Increased P50, which is equivalent to a rightward shift of the hemoglobin-oxygen dissociation curve, is indicative of a decreased hemoglobin-oxygen binding affinity, which promotes oxygen unloading15. Myburgh et al. reported reduced P50 in critically ill patients37, whereas two recent investigations39 revealed no difference in P50 compared to the healthy population. Focusing on ARDS, an Australian group demonstrated leftward shifts of the oxyhemoglobin dissociation curves40. In contrast, a group of Belgium ARDS patients demonstrated a rightward shifted curve41, which is consistent with our findings.Although this area of clinical research remains rather small, several previous studies that have investigated hemoglobin-oxygen affinity in critical illness have demonstrated conflicting resultsIn addition, our study revealed that P50 was associated with mortality regardless of initial PARDS severity, and extrapulmonary organ dysfunction largely mediated this relationship. P50 is ultimately thought to be a comprehensive reflection of increased systemic oxygen demand following increased consumption. Moreover, P50 showed a significant association with the dysfunction of several types of extrapulmonary organs. Extrapulmonary organ dysfunction was a strong risk factor for mortality in PARDS, and the mediation analysis confirmed the link between P50 and death with concurrent extrapulmonary organ dysfunction being a significant mediator independent of potential covariates, including the degree of hypoxemia. Our findings support the role of extrapulmonary organ dysfunction as a potential risk stratification system, and P50 as a likely explanation for this organ cross-talk.2 < 97% were included. We utilized Doyle’s method to determine P50. This method is a single-point analysis using a pair of oxygen tension and saturation measurements, which is thought to be suitable in daily practice, even for the pediatric population. Although this method had been validated using the data provided by Severinghous42, the calculated P50 involves fundamental limitations, as the values are estimates at best. Finally, our findings primarily focused on P50 and organ dysfunction at the time of PARDS onset, but we did not consider subsequent changes or newly developing organ dysfunctions. Further prospective validations with a larger sample size are needed to overcome the inherent limitations of this study design.The strength of this study was that it investigated P50 in association with clinical outcomes in PARDS patients. It builds upon the findings of previous studies and demonstrates causal inferences with explanatory variables, including validated criteria for organ dysfunctions. Nevertheless, we acknowledge several limitations that should be considered when interpreting the results. First, our study was conducted using a limited number of patients as this was a single-center study. Only patients who were older than 1 year and had arterial gas results of sOIn conclusion, P50, a measure of hemoglobin-oxygen affinity, may reflect the degree of extrapulmonary organ dysfunction in PARDS and may be associated with mortality. Future studies should consider P50 as a potential candidate index for risk-stratification in PARDS.Supplementary Information.


Proteobacteria and Bacteroidetes, whereas these bacteria started to increase only when QS was subjected to drought. When drought stress was applied, AH was enriched with more drought-tolerant bacteria, mainly Acidobacteria, while the enrichment was weaker in QS rhizosphere. Therefore, the increase in drought tolerance of the drought-tolerant variety AH was greater than that of the drought-sensitive variety QS. Overall, this study confirmed the key role of drought-induced rhizosphere bacteria in improving the adaptation of alfalfa to drought stress, and clarified that this process is significantly related to the variety (genotype). The results of this study provide a basis for improving drought tolerance in alfalfa by regulating the rhizosphere microbiome.Drought is a major abiotic stress that threatens crop production. Soil microbiomes are thought to play a role in enhancing plant adaptation to various stresses. However, it remains unclear whether soil microbiomes play a key role when plants are challenged by drought and whether different varieties are enriched with specific bacteria at the rhizosphere. In this study, we measured changes in growth phenotypes, physiological and biochemical characteristics of drought-tolerant alfalfa (AH) and drought-sensitive (QS) under sterilized and unsterilized soil conditions with adequate watering and with drought stress, and analyzed the rhizosphere bacterial community composition and changes using 16S rRNA high-throughput sequencing. We observed that the unsterilized treatment significantly improved the growth, and physiological and biochemical characteristics of alfalfa seedlings under drought stress compared to the sterilized treatment. Under drought stress, the fresh and dry weight of seedlings increased by 35.24, 29.04, and 11.64%, 2.74% for unsterilized AH and QS, respectively, compared to sterilized treatments. The improvement was greater for AH than for QS. AH and QS recruited different rhizosphere bacteria when challenged by drought. Interestingly, under well-watered conditions, the AH rhizosphere was already rich in drought-tolerant bacterial communities, mainly  Global climate change exacerbates the frequency and duration of drought stress, which severely impacts crop yield and quality. Drought can reduce yields of major crops by 50–80% . Below gPlants employ a variety of strategies to overcome drought stress, including a combination of stress avoidance and regulation of drought tolerance, depending on their genotype. The interaction between plants and the soil microbiomes that colonize the rhizosphere and root system is considered a key factor in the rapid adaptation of plants to soil environmental stress . HoweverMedicago sativa L.) is a globally important, widely cultivated leguminous forage crop due to its high biomass production and nutritional value for livestock  , which had not been planted or fertilized, and transported it back to the laboratory for air drying. The air-dried soil was sieved through a 2 mm mesh to remove the debris and large clods, and stored at 4°C.AH and QS seeds were first sandpapered and then rinsed with sterile water to break up the seed coat. Surface sterilization was then carried out, by treating seed surface with 4% NaClO for 5 min, and washing it with sterile water six times. Sterilized seeds were placed in a 9 cm Petri dish (two layers of sterile filter paper on the bottom and 5 ml of sterile water). The dish was placed in dark culture at 4°C for 2d, followed by dark culture at 25°C for 2d, then removed and placed at room temperature for 1d. The soil substrates were divided into sterilized (M) and unsterilized (W) treatments. Specifically, vermiculite, volcanic stone, and some field soil were sterilized twice by autoclaving for 30 min. 150 g of volcanic stone and 75 g of vermiculite were weighed separately in 10 × 10 cm pots and the sterilized and unsterilized treatments were defined by adding 25 g of sterilized or unsterilized soil and mixing thoroughly. We then add 1/2 Hoagland nutrient solution to 100% soil water content (250 ml of saturated water content). Seedlings of AH and QS were selected at the same germination status and transplanted into pots containing sterilized and unsterilized soil, with five plants per pot, and 16 pots per treatment. Also, we used pots without transplanted seedlings as bulk soil (BS) controls. The seedlings were grown in an artificial climate chamber under 16/8 h light/dark and 25°C/22°C day/night with approximately 60% relative humidity, and watered with sterilized water every 2 d to 70% saturated water content.2PO4.H2O, 16.5 g Na2HPO4.7H2O, and 200 μl Silwet L-77 in 1 l ddH2O, pH = 7.0) and vortexed briefly. After removing the roots, the suspension was filtered through a 100 μm cell strainer and spun down for 10 min at 4000 x g. The supernatant was discarded and the remaining pellet was the rhizosphere. All samples were frozen in liquid nitrogen and stored at −80°C. Leaves from the remaining replicate pots were collected for immediate determination of relative conductivity and chlorophyll content, and the remainder were snap-frozen in liquid nitrogen and placed at −80°C for physiological index determination. The roots were also removed to determine root length, fresh weight, and dry weight.Healthy 4-week-old alfalfa seedlings of approximately uniform growth were selected for the experiment. The sterilized and unsterilized treatments of AH and QS were divided into two groups, one with normal watering  and one with continuous drought , with eight pots per treatment as replicates. During the growing period, we used the weighing method to irrigate with sterile water every day. The well-watered group was maintained at 70% saturated water content during the growth period by daily irrigation, while drought stress was applied to the treated group by withholding irrigation until the end of the experiment. Every 2–3 days the pots were randomly rearranged to eliminate the effect of location on the experiment. We measured the soil water content of each treatment every day, and measured the plant height of each alfalfa seedling. During the drought period, plants gradually consumed water and nutrient reserves in the matrix, which simulated field conditions, and no visual symptoms of nutrient deficiency were observed. Sampling was carried out when the plants were observed to show signs of wilting. The chlorophyll fluorescence parameter (Fv/Fm) was measured using a Handy PEA . Three pots were selected to measure the height, leaf length and width of each plant, then the aboveground part of each plant was cut off from the base and immediately weighed for fresh weight, after which it was dried in an oven at 65°C for 48 h and the dry weight was measured. At the same time, rhizosphere soil was collected and the rhizosphere soil was separated from the roots of the alfalfa as previously described . In brie2O and gently shanked for 6 h at room temperature. Then, the leaves were boiled at 100°C for 20 min. When the leaves were cooled to room temperature, measured by the formula REC (%) = (Ci /Cmax) × 100, where Ci and Cmax, respectively, represent the conductivity before and after boiling of the detached leaves. Chlorophyll fluorescence parameters of leaves representing the maximum photochemical efficiency of photosystem II (PSII) (Fv/Fm) were measured with a Handy PEA . Leaves were incubated in the dark for 30 min before fluorescence measurements. Chlorophyll content was determined by spectrophotometry, as described by 2O2 reduction by following the methodology of 2O2, and 150 μl enzyme extract to a final volume of 1.5 ml. Each treatment of the above indexes was repeated three times.Plant height was measured from the base of the shoot to the tip of the tallest leaf. Main root length was measured from the base of the shoot to the end of the root, and leaf area was calculated using the formula: leaf area = leaf length (leaf base notch to leaf tip) × leaf width (the widest part of the leaf) × 0.87. Each treatment of the above indexes had 15 replicates. Leaf water content (LWC) was measured by taking 5–10 leaves from the top of the plant, immediately determining the fresh weight, and then placing them in an 80 ° C oven for 16 h to measure their dry weight. The LWC was calculated with the following equation: LWC = (fresh weight – dry weight)/fresh weight × 100 . RelativDNA from soil and rhizosphere samples was isolated using the PowerSoil DNA Isolation Kit  according to the manufacturer’s instructions. The concentration and quality of the DNA were evaluated with a NanoDrop 2000 spectrophotometer . The V3–V4 region of the 16S rRNA gene was amplified using the universal primers (341F: 5′ -CCTACGGGNBGCASCAG-3′ and 806R: 5′ -GACTACNVGGGTATCTAATCC-3′) . Every 5t-test were used for significance analysis between different groups . Bar graphs were plotted with Prism 8.4.3 . The alpha diversity of each sample was analyzed based on four alpha diversity indices: Chao1, Shannon, Simpson, and Ace. All sample indexes were calculated using QIIME (v.1.9.1) and t-tests were used to evaluate the differences in alpha diversity index between different treatments. Principal Coordinate Analysis (PCoA) based on weighted_unifrac distance was performed with the R Package vegan (v.2.5.7). A histogram of species distribution and a heat map were generated with the ggplot2 (v.3.3.5) package. Linear discriminant analysis effect size (Lefse), for which the logarithmic LDA score was set to 4.0 with statistical significance (p < 0.05), and the functional potential of a bacterial community predicted by PICRUSt2 were both performed on the BMK Cloud platform.Statistical analysis was performed with SPSS software (v.26.0.0.2). Student’s We found that the unsterilized treatment significantly increased the drought tolerance of alfalfa and improved the growth of alfalfa under drought stress compared to the sterilized treatment. Interestingly, soil microbiomes were more effective for the drought-tolerant variety AH . SpecifiWe found that drought stress significantly reduced the leaf area of alfalfa under sterilized conditions, but soil microbiomes could effectively improve this situation, with AHWDr having a significantly higher leaf area than AHMDr ,B. Drouga, Chlb, or Chlt content. Drought stress inhibited the synthesis of chlorophyll content, but the microbiomes were able to significantly increase the Chla content of AH and QS during drought compared to the sterilized treatment  is one of the most important indicators of the extent to which plants are affected by drought. We found that microbiomes significantly increased the LWC of AH under well-watered and drought conditions, while LWC of QS only increased under drought conditions and was even lower than the sterilized treatment under well-watered conditions . REC of reatment , and werficantly , with noantly  of weighted_unifrac distance was performed to reveal the overall dissimilarity of rhizosphere bacterial compositions . The samProteobacteria, Actinobacteria, and Gemmatimonadota. Drought had a small effect on bacterial community composition and abundance in BS, with only Cyanobacteria increasing in abundance. Compared to BS, alfalfa was specifically enriched for certain types of bacteria in soil, and drought, in turn, led to changes in the abundance of various types of bacteria. We found that when AH was well-watered, the rhizosphere significantly enriched Proteobacteria (74%) and Bacteroidetes (14%), while under drought, the abundance of Proteobacteria (48%) and Bacteroidetes (4%) decreased, and the abundance of Acidobacteria (19%) and Gemmatimonadota (10%) increased. However, QS had the opposite trend. Under well-watered conditions, the rhizosphere of QS significantly enriched Proteobacteria (64%) and Acidobacteria (10%), while under drought the abundance of Acidobacteria (2%) decreased, and the abundance of Proteobacteria (68%) and Bacteroides (10%) increased. Interestingly, the diversity of microbial composition of AH was low under well-watered conditions, but higher under drought conditions. By contrast, QS was high under well-watered and low under drought conditions. Perhaps AH can recruit more microbiomes to help with drought stress, while QS can recruit more microbiomes to promote growth when well-watered.A phylum-based population structure analysis of the rhizosphere bacteria under different treatments revealed the dominant microflora in the rhizosphere based on their relative abundance . The topGemmatimonadaceae, Massilia, and unclassified_Vicinamibacteraceae , and the highest abundance in QSWCK was unclassified_ Comamonadaceae (7%). The top 30 genus from all samples were selected to further investigate the composition of alfalfa rhizosphere bacteria . In this study, microbes with LDA score > 4 were identified as biomarkers. Results showed that the rhizosphere microbes we identified as biomarkers to alfalfa differed significantly under well-watered and drought stress conditions. Moreover, different varieties had their own specific biomarkers under different water treatment conditions. Specifically, at the phylum level, ectively . At the ectively , pointinAlthough our results showed that alfalfa has an increased ability to recruit certain bacteria when facing drought stress and that the recruited bacteria are variety-specific, the functions of these bacteria remain largely unknown. Therefore, we performed a functional prediction analysis of the rhizosphere bacterial communities of the two varieties of alfalfa. The rhizosphere bacterial community differences of the two varieties of alfalfa were exhibited by contrasting the KEGG pathways (Level 3) of the functional abundance TOP10 . We founDrought is probably the abiotic stress that has the strongest effect on soil biota . DroughtOur first hypothesis was that soil microbiomes play a key role in enhancing drought stress in alfalfa. This was supported by our data. We found that a sustained decrease in water content significantly reduced the relative growth rate and biomass production of alfalfa seedlings, which is consistent with previously reported studies . HoweverProteobacteria, Acidobacteria, and Bacteroidetes  demonstrated that drought-induced rhizosphere bacteria rescue AH and QS from drought challenges. This implies that, despite the importance of genetic differentiation, plants appear to exhibit broadly similar phenotypic and physiological and biochemical response characteristics in the presence of drought. That is, the recruitment of specific root-associated rhizosphere bacteria capable of enhancing plant adaptation to drought stress mitigates the plant’s exposure to drought stress and enables it to sustain growth. However, although drought-induced rhizosphere bacteria could maintain sustained growth in alfalfa, drought-resistant and drought-sensitive varieties recruited different bacterial species and associated functions when challenged by drought, respectively. This means that plants employ variety-specific strategies to recruit beneficial soil bacteria, which can help them solve water challenges. In fact, plants are powerful drivers and selective forces in the evolutionary history of natural microbiomes. For example, the rhizosphere of alfalfa shows a strong preferential enrichment mainly for the three major phyla roidetes . Even diroidetes , as was roidetes , which sroidetes . In addit stress . Proteobbundance . Interesarieties , 8A. Actt stress . Our res. We found that the drought-tolerant varieties already had high abundance of its rhizosphere drought-resistant bacteria under well-watered conditions, whereas the abundance of rhizosphere drought-resistant bacteria of drought-sensitive varieties started to increase only after being subjected to drought stress. Thus, drought-tolerant varieties can respond to drought stress in a timely and more efficient manner, whereas drought-sensitive varieties need more time to respond to drought stress and are therefore more susceptible to damage. Overall, our study provides some new insights into the importance of soil microbiomes in improving drought tolerance in alfalfa and cultivar-driven rhizosphere bacterial variation under drought stress conditions. We consider that in the future, we will try to isolate drought-tolerant rhizosphere bacteria from drought-tolerant varieties for future development as PGPR inoculants. In addition, it is important to consider the influence of microbiomes on plant yield and stress tolerance when breeding new varieties with the rhizosphere microbiome as a selective trait. Future efforts to clarify the mechanisms of recruitment of drought-tolerant bacteria in drought-tolerant varieties in terms of gene regulation and rhizosphere secretions are recommended to lay the foundation for future breeding of new varieties that can autonomously recruit beneficial bacteria against environmental stresses using biological breeding.https://www.ncbi.nlm.nih.gov/, PRJNA900679 (SRR22266587- SRR22266604).The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: WF designed the experiments, analyzed the data, and wrote the manuscript. WF, JW, and JX conducted the experiments. JX and JD performed the data interpretation. JD revised the manuscript critically. FT provided the experimental materials and technical support. FS conceived the experiment and manuscript revision. All authors contributed to the article and approved the final manuscript for submission.This work was supported by the National Natural Science Foundation of China (grant no. 32160323), Key Projects in Science and Technology of Inner Mongolia (grant no. 2021ZD0031), and the major demonstration project of “the open competition” for Seed Industry Science and Technology Innovation in Inner Mongolia (grant no. 2022JBGS0016).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In addition, analytical performance of porous copper electrodes was ascertained and compared to that of copper film screen-printed carbon electrodes, prepared ex-situ by the galvanostatic deposition of metal in the plating solution. After calculating the current densities with respect to the geometric area of working electrodes, the porous electrodes exhibited much higher sensitivity to changes in concentration of analytes, presumably due to the larger surface of the porous copper deposit. In the future, they could be incorporated in detectors of flow injection systems due to their long-term mechanical stability.A porous layer of copper was formed on the surface of screen-printed carbon electrodes via the colloidal crystal templating technique. An aqueous suspension of monodisperse polystyrene spheres of 500 nm particle diameter was drop-casted on the carbon tracks printed on the substrate made of alumina ceramic. After evaporation, the electrode was carefully dipped in copper plating solution for a certain time to achieve a sufficient penetration of solution within the polystyrene spheres. The metal was then electrodeposited galvanostatically over the self-assembled colloidal crystal. Finally, the polystyrene template was dissolved in toluene to expose the porous structure of copper deposit. The morphology of porous structures was investigated using scanning electron microscopy. Electroanalytical properties of porous copper film electrodes were evaluated in amperometric detection of selected saccharides, namely glucose, fructose, sucrose, and galactose. Using hydrodynamic amperometry in stirred alkaline solution, a current response at +0.6 V vs. Ag/AgCl was recorded after addition of the selected saccharide. These saccharides could be quantified in two linear ranges (0.2–1.0 μmol L Nowadays, non-enzymatic glucose sensors (NEGSs) are of increasing interest to the scientific community, unlike traditional sensors employing an enzyme layer containing a glucose oxidase due to the possible decrease in catalytic activity of the enzyme arising from the immobilization and low guarantee of long-term stability . HoweverNon-enzymatic sensors designed to determine simple carbohydrates operate predominantly in the amperometric mode, where glucose represents the most common target analyte. Because glucose, galactose, and fructose are the end products of carbohydrate digestion and only glucose enters the bloodstream, NEGSs have to be selective for common interferences, such as ascorbic acid, dopamine, acetaminophen, and uric acid . Unlike Generally, the electrocatalytic process for glucose oxidation to gluconolactone, which is subsequently hydrolyzed to gluconic acid, is usually mediated by the Me(II)/Me(III) redox couple in the alkaline solution, where Me(III) species acted as an electron transfer mediator . NanostrTo increase the sensitivity of amperometric non-enzymatic sensors, different approaches have been used for the three-dimensional extent of active surface area. Several approaches were utilized for this purpose, namely nanomaterials decorated by island structures of electrochemically deposited metal films ,18, fabrIn this paper, a colloidal crystal templating technique  has beenThe results showed that the developed non-enzymatic porous copper sensors (NEPCSs) are selective to glucose in the presence of common electroactive interferences (ascorbic acid and uric acid) and show enhanced current response compared to non-porous copper film electrodes, allowing their feasible application in clinical and food analysis. However, it was also found that NEPCSs provide an overall current response for all saccharides tested. Therefore, a pre-separation step is necessary if quantification of individual saccharides is required in terms of their application in food and environmental analysis, where they could serve as sensitive electrochemical detection systems ,29,30,31® system from Merck .D-glucose (p.a), D-(−)-fructose (≥99%), sucrose (p.a), D-(+)-galactose (98%), copper(II) nitrate trihydrate (p.a.), ascorbic acid (p.a.), uric acid (≥99%), and suspensions of monodisperse polystyrene beads with particle sizes of 200 or 500 nm were purchased from Sigma Aldrich . Other chemicals, such as toluene (p.a.), nitric acid (65%), sodium hydroxide (≥97.0%), and acetone (≥99.0%) were obtained from Lach-Ner s.r.o . All aqueous solutions were prepared using deionized water with resistivity higher than 18.3 MΩ cm, which was obtained from the Millipore Milli-Q−1 KCl salt bridge (reference) from Metrohm Česká republika s.r.o. , and Pt sheet auxiliary electrode from Elektrochemické detektory, s.r.o. . The abovementioned electrodes were connected to a potentiostat/galvanostat AUTOLAB PGSTAT101 from Metrohm Česká republika s.r.o.  which was operated by the NOVA 1.11 software. Images of electrode surface structures were obtained by scanning electron microscope (SEM) JEOL JSM7500F  using a secondary electron detector and accelerating voltage of 15 kV or 20 kV.Electrochemical measurements were carried out in a voltammetric glass cell with a conventional three-electrode configuration with the use of a screen-printed carbon electrode (SPCE) plated with a porous copper layer (pCuFE) (working), a silver chloride electrode (length 12.5 cm) with 3.0 mol L2). The freshly printed electrodes were dried at 60 °C for 30 min. Furthermore, they were covered with a thin layer of PVC insulator to define a rectangular shape of the working electrode area (5 × 3 mm) while the other side was fitted with adhesive copper tape for better electrical contact.Bare screen-printed carbon electrodes were prepared by screen-printing of commercial carbon ink (type C10903D14) purchased from Gwent Electronic Materials Ltd.  onto the ceramic supports (10 × 40 mm). The printing itself was done using a semi-automatic screen-printer UL 1505 A from Tesla  through an etched stencil  in 0.01 mol L−1 HNO3 (pH 2) at −1.5 mA for 90 s. After rinsing with a stream of deionized water, the resulting CuFEs were ready for subsequent electrochemical measurements.To confirm an improvement of non-enzymatic amperometric sensing of saccharides using pCuFEs, it was important to make a comparison with conventional copper film electrodes (CuFEs). A rectangular shape with the size of working surface area (5 × 3 mm) was defined using a PVC insulator before the copper electrodeposition. Conventional CuFEs were prepared by galvanostatic electroplating from solution 0.1 mol L2 were applied onto the working surfaces of bare SPCEs. A volume of 30 μL of monodisperse polystyrene beads suspension  with a 200 or 500 nm diameter was pipetted into such prepared molds. After evaporating deionized water for two days in a steady environment, the plastic molds were removed. The self-assembled colloidal crystals were used as templates for preparing the porous copper layers. Prior to the electrodeposition of copper film, it was necessary to insert an SPCE with a colloidal crystal template into a copper deposition solution containing the 0.1 mol L−1 Cu(II) in 0.01 mol L−1 HNO3 (pH 2) for at least 15 min (infiltration time). The copper plating was then carried out galvanostatically at a constant current of −1.5 mA for different deposition times, namely 30, 60, and 90 s. After copper electroplating, the electrode was rinsed with a stream of deionized water and inserted into pure toluene to dissolve the polystyrene template for 15 min. Finally, the resulting pCuFE was rinsed again with water and, after drying, used for subsequent SEM characterization and electrochemical measurements.Circular plastic molds (rings) with an internal surface area of 10 mm−1 of each saccharide was measured using cyclic voltammetry (CV) within a potential range from 0.0 to +0.8 V vs. Ag/AgCl at the bare SPCE, CuFE, and pCuFE and in non-deaerated 0.1 mol L−1 NaOH of pH 13 at a potential step of 5.0 mV and scan rate of 50 mV s−1. Hydrodynamic amperometric measurements in the batch configuration were performed in 0.1 mol L−1 NaOH of pH 13 at a detection potential of +0.6 V vs. Ag/AgCl and a stirring speed of 600 rpm.To study the electrochemical behavior of the saccharides of interest , a model solution with 1.0 mmol LPorous copper films were formed using galvanostatic electrodeposition over the colloidal crystal template, made of monodisperse polystyrene spheres. The self-assembled colloidal crystal template was successfully formed on the surface of the screen-printed carbon working electrode. To properly develop the copper three-dimensional porous structure on the surface of the working electrode, several experimental parameters related to the formation of the template and electrodeposition of copper had to be optimized. After the electrodeposition, the electrode was immersed in toluene for 15 min, which readily dissolved the template and exposed the porous structure, as shown in w/w in the water suspension, drop-casted on the surface of the working electrode, were tested. Both types of spheres formed colloidal crystals, but in the case of lower amounts and smaller particles, the resulting porous copper film did not cover fully the surface of the electrode and consisted of isolated porous islands, presumably because it did not completely fill the rough screen-printed carbon layer. The porous copper coating was often cracked overall on the surface, and an overgrown deposit was visible even after short times of electrodeposition. On the contrary, colloidal crystal templating with larger particles and higher amounts showed a regular porous structure, which also followed the relief of the printed carbon layer and covered compactly and with only small irregularities on the surface of the working electrode. However, the use of 1% suspension often resulted in crystallite and overgrown copper deposits. Therefore, colloidal crystal templating with 0.5% suspension of 500 nm polystyrene spheres in deionized water was used as an optimum for further experiments.Two diameters of polystyrene spheres, namely 200 nm and 500 nm, and different amounts of the particles from 0.3% to 1% Electrodeposition of copper using constant current provided a regular and firm metal deposit, contrary to the electrodeposition using constant potential, and was selected for further experiments. Constant currents were varied within the range from −0.5 mA to −2.0 mA. Lower currents yield weak and incomplete coverage of the electrode surface with the copper deposit, whereas higher currents led to the formation of a high amount of copper crystallites. The value of −1.5 mA provided dense coverage of carbon substrate with the copper deposit, and hence it was chosen as an optimum. Subsequently, various periods for infiltration of the plating solution into the colloidal crystal and electrodeposition of copper were tested to obtain a porous metal film on the electrode surface as homogeneously as possible. Shorter infiltration times caused the collapse of the porous structure after the copper electrodeposition and dissolution of the template in toluene. Therefore, the infiltration time of 15 min prior to the electrodeposition was found necessary for the proper development of the porous copper film. Finally, different times from 30 s to 90 s were used for ex-situ copper electrodeposition to prepare a porous copper layer. A low deposition time of 30 s resulted in partial coverage of the electrode surface with copper and consisted mainly of isolated islands of porous metal deposits a, simila−1 saccharides in 0.1 mol L−1 NaOH using a scan rate of 50 mV s−1 (−1 NaOH (pH 13), which was then thoroughly used in the following experiments.The electrochemical properties of pCuFEs in the detection of saccharides were investigated using cyclic voltammetry and compared to a non-porous CuFE and bare SPCE. The general mechanism of direct electrooxidation of saccharides at copper-based electrodes includes adsorption and deprotonation of the organic molecule on the electrode surface and subsequent oxidation with Cu(II)/(III) redox couple in copper oxides formed at positive potentials in alkaline media  28,31].,31.28,310 mV s−1  showed aIt is generally known that porous electrodes exhibit several times higher sensitivity compared to non-porous electrodes in the electrochemical detection of compounds, which adsorb at the electrode surface . The pCu−1 NaOH  for saccharide detection were calculated as three times the standard deviation of currents recorded for the lowest values of the linear range and measured using five individual sensors, divided by the slope averaged from five individual calibrations of those sensors in the concentration range 2 × 10−1, whereas for glucose it is around 5 mmol L−1 [As can be expected, most sugars will provide a current response, and therefore for samples containing a mixture of different saccharides, the obtained current response will correspond to their total amount. Herein, it is important to realize that already developed non-enzymatic sensors have faced the same problem. It is evident that the applicability of pCuFEs only depends on the appropriate selection of samples. For example, in clinical analysis, the literature reports up to 100-fold higher glucose concentration than galactose in plasma ,37. The mmol L−1 ,41.−1 DA [−1 APAP in urine [−1, whereas juices or sweet drinks contain up to tens of grams per liter of saccharides [Ascorbic (AA) and uric acid (UA) can be considered as predominant interfering species for the non-enzymatic detection of saccharides in clinical and food analysis. As evident from −1 DA  in plasmin urine ,44. Regacharides . Hence, It is generally known that the electroanalysis of AA and UA is based on irreversible oxidation, which was proven to be a diffusion-controlled process. This fact was confirmed during the interference study when both AA and UA were analyzed together with saccharides using hydrodynamic amperometry. Both interferants exhibited almost the same current densities during amperometric detection regardless of the working electrode. The enlarged porous surface of the pCuFE in comparison with the CuFE did not cause increased current densities for AA and UA oxidation, which is in concordance with .−5 mol L−1 AA and UA and 1 × 10−4 mol L−1 saccharides (ratio 1:10), the measured currents for AA constituted only 10–14% of the response of saccharides at pCuFE, whereas it was 14–24% when CuFE was used. The values within 6–11% and 12–25% for pCuFE and CuFE, respectively, were obtained in the case of UA injections. The porous surface of pCuFE improves the sensitivity of saccharides detection resulting in a smaller overall contribution from AA and UA.The advantage of the use of pCuFE over CuFE for the detection of saccharides in the presence of AA or UA was demonstrated in the following experiments . For seq−6 mol L−1 AA and UA and 6 × 10−5 mol L−1 saccharides (ratio 1:10). The AA, UA, and saccharides were sequentially analyzed eight times at one pCuFE electrode, and average currents from eight measurements together with the corresponding relative standard deviations (RSD) were plotted in The repeatability of amperometric detection of saccharides in the presence of AA and UA at the pCuFE was determined for repetitive analyses of 6 × 10−1. The procedure for the porous layer preparation is very simple, low cost, and demands only controlled conditions for the proper formation of the colloidal crystal template. With such an approach, porous structures of metals with defined pore dimensions can be easily prepared, and their properties can be tuned for particular electroanalytical applications. Due to their relatively high mechanical stability , it is planned to utilize these porous copper film screen-printed electrodes as amperometric detectors in flow injection analysis, allowing quick analysis of saccharides in many samples.The macroporous copper film electrode was successfully prepared with full coverage of the porous layer on the surface of the screen-printed carbon electrode using colloidal crystal templating and an optimized procedure for copper electrodeposition. The enlarged electrode surface enables more sensitive electrochemical detection of species, which adsorb firstly at the electrode surface prior to the electrochemical redox transformation, which is typical for electroanalysis of various saccharides. The contribution from interfering species is significantly reduced in the case of compounds with diffusion-controlled redox activity. We have demonstrated that it is feasible to detect saccharides at submicromolar concentrations with the aid of the porous copper film screen-printed carbon sensors. These sensors provide two linear dependencies for all examined saccharides, 0.2–1 and 4–100 µmol L


Therefore, in this study, the leaf structure and CFI curves of sunflower and sorghum seedlings were analyzed. Results revealed that there was a significant difference between the structures of palisade and spongy tissues in sunflower leaves. Their CFI curves, measured on both the adaxial and abaxial sides, also differed significantly. However, the differences in the leaf structures and CFI curves between both sides of sorghum leaves were not significant. Further analysis revealed that the differences in the CFI curves between the adaxial and abaxial sides of sunflower leaves almost disappeared due to reduced incident light scattering and refraction in the leaf tissues; more importantly, changes in the CFI curves of the abaxial side were greater than the adaxial side. Compared to leaves grown under full sunlight, weak light led to decreased differences in the CFI curves between the adaxial and abaxial sides of sunflower leaves; of these, changes in the CFI curves and palisade tissue structure on the adaxial side were more obvious than on the abaxial side. Therefore, it appears that large differences in sunflower leaf structures may affect the shape of CFI curves. These findings lay a foundation for enhancing our understanding of CFI from a new perspective.Chlorophyll  The time-course of chlorophyll a fluorescence is called fast chlorophyll a fluorescence induction kinetics (CFI), which reflects the closure of photosystem II (PSII) reaction centers and is related to reductions in QA to QA−. It can thus provide detailed information on the photochemical activity of PSII [The leaf is the main photosynthetic organ in higher plants. Chlorophyll ynthesis ,2. When  of PSII ,3,4,5. V of PSII ,7,8,9. A of PSII ,11,12,13If leaf structure affects CFI, then great differences in leaf structures may result in greater CFI differences. Therefore, the comparison and analysis of leaves or leaf tissues with great structural differences can elucidate this issue. Leaves can be classified into isolateral and bifacial leaves based on their main morphological and structural features. The adaxial and abaxial sides of isolateral leaves have no obvious palisade or spongy tissue differentiation and have similar mesophyll cells; thus, both sides of the leaf can photosynthesize efficiently. Unlike isolateral leaves, the adaxial side of bifacial leaves is composed of tightly arranged columnar palisade cells, while the abaxial side is mainly composed of loosely arranged irregular or globular spongy cells ,15. CompIn general, light enters leaf tissues from the adaxial side of the leaf. When the incident light on the adaxial side propagates to half the thickness of the palisade tissue inside the leaf, the light intensity decreases by ≥60% . When liSignificant differences in the CFI curves between the adaxial and abaxial sides of sunflower leaves were detected . As the Although there were significant differences detected in the CFI curves between the adaxial and abaxial sides of sunflower leaves in vivo, these differences almost disappeared after enhancing the light transmission within the leaves A–C. Enha−2·s−1 pulsed light; the increase was greater in the adaxial side  of sunflower leaves.To further confirm the differences between the two light treatment groups, the gas exchange was measured . The ligo plants . Comparea fluorescence mainly reflects the photosynthetic function of shallow cells in leaves. However, as pulsed light intensity increases, the pulsed light may penetrate through shallow leaf mesophyll tissue and release fluorescence from deeper chloroplasts [In this study, the difference in leaf mesophyll tissue within sorghum leaves was slight, which was consistent with the slight differences detected in the CFI curves of the leaf adaxial and abaxial sides D–F. In croplasts ,25,26. IWe noticed that the palisade tissues of sunflower leaves under full sunlight consisted of columnar cells, while the spongy cells were round or irregular and loosely arranged B. This sThe cells of sunflower leaves became smaller, and the difference between palisade and spongy tissues decreased when grown under low light C; the diIn this study, significant differences in the CFI curves of the adaxial side of sunflower and sorghum leaves were detected . The JIPMany environmental stressors decrease the electron transport activity of PSII in vivo, including high light, salt stress, and drought ,29,30. HThus, we suggest that large differences or variations in leaf structure may affect the shape of the CFI curves in sunflower leaves, which may further affect calculations of PSII electron transfer activity.v/v). Seedlings were placed outdoors and divided into strong light (full sunlight) and low light (10% full sunlight obstructed by shading) groups. On sunny days, the daily maximum photosynthetic photon flux density (PPFD) was 1400–1600 μmol m−2 s−1 at midday. Normal water was provided, and fertilizer management was performed throughout the experiment to avoid nutrient and drought stress. After plants had 7–8 leaves, all measurements were conducted using just fully expanded leaves.The experiment was conducted at the Institute of Botany, Chinese Academy of Sciences, from May to September in 2020 and 2021. Sunflower and sorghum were planted in pots . The substrate in the pots consisted of grass charcoal and soil  from 08:00 to 12:00 on a sunny day. During this process, CO2 concentration and humidity were maintained at 380 ± 20 μmol mol−1 and 75% ± 5%, respectively. An ambient temperature was maintained in the leaf chamber. Five replicates from each group were used for the measurements.Gas exchange was measured at an irradiance of 1200 µmol·mO). The following data were obtained: fluorescence intensity at 2 ms (J phase (FJ)), fluorescence intensity at 30 ms (I phase (FI)), and maximum fluorescence intensity (FP). The relative variable fluorescence was calculated as, Vt = (Ft − FO)/(FP − FO) [t is the measured fluorescence intensity at time t between FO and FP. The pulsed light intensities were 200, 1000, and 3000 μmol m−2 s−1. Twenty replicates were used for each pulsed light intensity measurement. All JIP-test parameters  . Fluorescence curves were recorded during 1 s pulses of red radiation. All samples were detached and fully dark-adapted (1 h) before measurements were taken. The first reliably measured point of the CFI was at 20 µs, which was used as the minimum fluorescence (FFP − FO) , where Frameters  were calLeaf discs were cut out of sunflower leaves and placed in a beaker with distilled water, and then pumped with a vacuum pump until the leaves were submerged in water . CFI wasLeaf segments (2 × 2 mm) without major veins were cut from the basal part of the leaf lamina using a razor blade. The segments were fixed in 0.1 µL PBS solution , rinsed three times with PSB solution, fixed overnight with <1% starvation acid, rinsed three times with PBS solution, dehydrated in an ethanol series, embedded in Spur resin, and cut into l µm thick sections. Then, the sections were stained with toluidine blue, observed under a light microscope (Nikon-E800), and photographed with a digital camera.Data were analyzed using one-way ANOVA and compared with the significant difference (LSD) multiple comparison test using SPSS (version 25). The least significant differences between the means were estimated at a 95% confidence level. Plots and curves were generated using the graphics software Sigmaplot v12.5.Large differences or variations in leaf structure may affect the shape of the CFI curves in sunflower leaves, which may further affect calculations of JIP-test parameters.