TSH beta subunit gene (TSHB) result in severe, isolated, central congenital hypothyroidism (CCH). This entity evades diagnosis in TSH‐based congenital hypothyroidism (CH) screening programmes in the UK and Ireland. Accordingly, genetic diagnosis, enabling ascertainment of affected relatives in families, is critical for prompt diagnosis and treatment of the disorder.Homozygous mutations in the TSH deficiency from three unrelated families in the UK and Ireland were investigated for mutations or deletions in TSHB. Haplotype analysis, to investigate a founder effect, was undertaken in cases with identical mutations (c.373delT).Four cases of isolated TSHB mutation (c.373delT). In kindreds 2 and 3, the affected individuals were compound heterozygous for TSHB c.373delT and either a 5·4‐kB TSHB deletion  or a novel TSHB missense mutation . Neurodevelopmental retardation, following delayed diagnosis and treatment, was present in 3 cases. In contrast, the younger sibling in kindred 1 developed normally following genetic diagnosis and treatment from birth.Two siblings in kindred 1 were homozygous for a previously described TSHB deletion, expands the molecular spectrum of TSHB defects and suggests that allele loss may be a commoner basis for TSH deficiency than previously suspected. Delayed diagnosis and treatment of profound central hypothyroidism in such cases result in neurodevelopmental retardation. Inclusion of thyroxine (T4) plus thyroxine‐binding globulin (TBG), or free thyroxine (FT4) in CH screening, together with genetic case ascertainment enabling earlier therapeutic intervention, could prevent such adverse sequelae.This study, including the identification of a second, novel,  TSHB), the immunoglobulin superfamily member 1 (IGSF1) or, less frequently, the TRH receptor (TRHR) genes.TSHB mutation cases, thyroid hormone deficiency is profound, resulting in neurodevelopmental retardation.Isolated congenital central hypothyroidism is rare (incidence 1 in 65 000),Heterodimeric TSH, secreted by anterior pituitary thyrotroph cells, comprises a hormone‐specific beta subunit (TSHβ) and a common alpha subunit (αGSU) shared with other members of the glycoprotein hormone family . The integrity and bioactivity of the heterodimer are maintained by a ‘seat belt’ structure formed by the β‐subunit peptide that is stabilized by disulphide bridges including the so‐called buckle between amino acids 39 and 125.TSHB mutation is a single‐nucleotide deletion (c373delT), resulting in a cysteine 125 to valine conversion (C125V) and disruption of the Cys‐Cys 39–125 disulphide bridge, with a subsequent shift in reading frame and premature stop codon at position 134.TSHB deletion, have also been reported.The human TSHβ subunit gene is organized into 3 exons: exon 1 is untranslated, and exons 2 and 3 encode a 138‐amino acid peptide. The twenty N‐terminal amino acids encode a signal peptide that is cleaved to yield a 118‐amino acid mature protein detectable in serum.letion c33delT, reTSHB mutations. In kindred 1, two siblings (P1a and P1b) are homozygous for the previously described c.373delT mutation. In kindred 2, the affected child (P2) is compound heterozygous for c.373delT together with a maternally derived 5·4‐kb deletion involving TSHB alone (c.1‐4389_417*195delinsCTCA). In kindred 3, the proband (P3) is compound heterozygous for c.373delT and a novel missense mutation , disrupting the methionine residue from which translation is initiated. We describe these TSHB defects and associated clinical phenotypes in the context of the wider literature.We report three kindreds from the UK or Ireland in which four cases exhibit isolated central hypothyroidism secondary to Clinical phenotype, auxological parameters and biochemistry for each case are summarized in Table P1a is the second son of healthy, nonconsanguineous Caucasian (UK) parents. Although his TSH on neonatal screening was reported normal, he presented aged 3 weeks with a lower respiratory tract infection and was noted to have clinical features of hypothyroidism, including somnolence, constipation and dry skin. Thyroid function tests were consistent with severe central hypothyroidism , with no TSH response to TRH stimulation. Subsequent dynamic testing, showing preserved corticotroph, somatotroph and gonadotroph function, excluded combined pituitary hormone deficiency (data not shown), and his basal prolactin level was 554 mU/l (NR <700). Levothyroxine treatment was commenced at the age of 5 weeks and 2 days, despite good compliance development was delayed. Formal assessment of age 2 years revealed significantly impaired hearing and speech. At age 7 years, growth is normal, and he is in mainstream education, but requires additional support.P1b, the younger brother of P1a, underwent screening for central hypothyroidism at birth following this diagnosis in his older sibling. Thyroid function was consistent with TSH deficiency . Levothyroxine was commenced at age 1 week, and he subsequently grew and developed normally. Both parents are clinically and biochemically euthyroid.P2 is the daughter of healthy, nonconsanguineous Caucasian (Irish) parents. Her TSH in neonatal screening was normal. She presented aged 8 weeks with prolonged jaundice, poor weight gain and feeding since birth, somnolence and constipation. She was clinically hypothyroid, with cool peripheries, hypotonia, an umbilical hernia, cutis marmorata, coarse facial features and macroglossia. Severe biochemical central hypothyroidism was confirmed . In a TRH stimulation test, the TSH response was absent, but prolactin levels rose , suggesting a defect downstream of the TRH receptor. Dynamic evaluation of corticotroph and somatotroph function was normal. Following commencement of levothyroxine aged 8 weeks, her growth was normal. Despite good compliance with treatment, she exhibits mild motor delay at age 7 years. Both parents are clinically and biochemically euthyroid.P3 is the only daughter of healthy, nonconsanguineous Caucasian (UK) parents. Her TSH in neonatal screening was reported as normal. She presented aged 3·5 months with feeding difficulties, somnolence, constipation and severe growth retardation. She was clinically hypothyroid with a small umbilical hernia and depressed nasal bridge. Thyroid biochemistry confirmed profound central hypothyroidism . Subsequent evaluation of pituitary–adrenal, gonadotrophins and basal serum prolactin was normal. Although her growth response to levothyroxine treatment was excellent, she has multiple neurodevelopmental deficits including bilateral sensorineural deafness and nystagmus without optic nerve hypoplasia. Motor and language development was significantly delayed; at the age of 10 years, she is on the autistic/Asperger's spectrum. She has a statement of Special Educational Needs and is unable to attend mainstream school. Her mother is clinically and biochemically euthyroid; her father is not available for evaluation.All investigations were part of an ethically approved protocol and/or clinically indicated, being undertaken with the consent from patients and/or next of kin.TSHB subunit gene exons and exon/intron boundaries were amplified by PCR using specific primers: 5’‐TCCAGGTAAAGATATTGTGAGCT‐3’ and 5’‐TTACAGCCTGTTGAAGCAAATT‐3’ (exon 1), 5’‐CCTAGATTTCTGAGTTAGCCCCT‐3’ and 5’‐TGCGTATCCATTGTGCTGAG‐3’ (exon 2) and 5’‐TGTTTCCTAAAGTCCTGTCACA‐3’ and 5’‐ACCTTCAACTGAGCCCAAAAG‐3’ (exon 3). To characterize the chromosomal deletion around TSHB in P3, the homozygous region delineated by SNP markers rs7523360 and rs1998008 was amplified by long‐range PCR using the primers 5’‐AACACACATCCAACATTCTTCCA‐3’ and 5’‐AAATCCCGCAGTAATTCTTTGCT‐3’.Genomic DNA was extracted from peripheral blood leucocytes using the standard techniques. All three TSHB variants listed in this study are described using the systematic nomenclature approved by the Human Genome Variation Society . Nucleotide numbering starts from the A (+1) of the translation initiation codon (ATG) of the NCBI reference sequence NM_000549.4 and includes the 20‐amino acid signal peptide. Amino acid residues are numbered according to the NCBI reference sequence NP_000540.2.PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit  and 3730 DNA Analyzer (Applied Biosystems). The TSHB locus  and spanning 225 kb 5’ and 205 kb 3’ of the In silico analysis of the novel missense variant (c.2T>C) was performed using Alamut as an interface  and the SignalP V4.1 signal peptide prediction package  to determine whether translation from Met‐7 would alter the probability of the protein sequence acting as a signal peptide.TSHB copy number and genomic THRA in P3, her parents, and 10 unrelated, euthyroid controls. TSHB: primer/probe set Hs02759015_s1 (ABI Life Technologies); THRA: forward 5’‐TTCGAGCACTACGTCAACCA‐3’; reverse 5’‐CCCGATCATGCGGAGGTCAG‐3’; probe 5’‐FAM CACAACATTCCGCACTTCTG‐TAMRA‐3’ using qPCR as previously described.TSHB normalized to genomic THRA.The following intra‐exonic primer/probe sets were used to quantify genomic TSHB, c.373delT, p.C125Vfs*10 , described previously, with both euthyroid parents being heterozygous. Despite paternal heterozygosity for this mutation, but wild‐type maternal TSHB sequence , disrupting the start site for translation at the beginning of the signal peptide of TSHβ. As the maternal TSHB locus only showed heterozygosity for the c.2T>C, p.Met1? change, these mutations were assumed to be compound heterozygous, occurring on separate alleles.P1a and P1b were homozygous for a single‐nucleotide deletion in TSHB copy number in P2, her parents, and ten unrelated, euthyroid controls. The results, showing a fifty per cent decrease in amplified signal from the TSHB locus in P2 and her mother, were consistent with a maternal, heterozygous TSHB deletion , 4·4 kb proximal to TSHB, confirmed uniparental  inheritance at this site (A/A), as no maternal allele (G/G) was inherited  , April 8th, 2016). Alamut predictive software suggests that this substitution will adversely affect translation of the TSHβ polypeptide.P3 exhibits a novel missense mutation (c.2T>C), in which a methionine codon, from which translation is initiated, is replaced by a threonine. This mutation was absent from the published normal genome data sets , Cambridge, MA , with a purine  at position ‐3 (5’) and guanine at position +4 (3’), relative to the adenine (A) of the initiator codon  lacks both a purine at position ‐3  and a guanine at position +4 , suggesting that efficient translation from this alternative site is unlikely. Additionally, as the first 20 amino acids of TSHβ form a signal peptide, even if limited synthesis from Met‐7 of TSHβ occurs, the resulting N‐terminally deleted TSHβ polypeptide would lack hydrophobic residues within its signal sequence that may be critical for correct protein translocation . Mutation of a normal mammalian ATG start codon to ACG usually results in markedly diminished protein translation in silico predictions, in vivo data from P3 strongly suggest that negligible bioactive TSH is synthesized from the c.2T>C allele, as profound hypothyroidism (undetectable fT4) is associated with barely measurable serum levels (0·45 mU/l) of intact TSH. Some of this TSH immunoreactivity may reflect translation of TSHβ from the residual c.373delT TSHB mutant allele, which is known to mediate the limited synthesis of biologically inactive TSH that retains immunoreactivity in some assays.Our cases exhibit three different genetic aetiologies including one novel missense TSHB alone, has been mapped precisely. Biallelic TSHB deletions were recently described in a Turkish patient.TSHB mutation exhibited apparent uniparental inheritance; compound heterozygosity of the c.373delT mutation with a TSHB deletion would also be a plausible alternative explanation in this patient.TSHB deletion together with a monoallelic c.373delT mutation, suggesting that TSHB deletions may be a more common cause of isolated TSH deficiency than previously thought.P2 is the first recorded patient in whom a heterozygous deletion , encompassing TSHB mutation reported, occurring in the unrelated cases from Western Europe, South America and the USA.TSHB either mono‐ or biallelically. Previous functional studies have shown that it is the cysteine to valine amino acid change at codon125, rather than subsequent deletion of 13 carboxyterminal residues of TSHβ, which impairs the function of mutant TSH.TSHB, the gene defect is present as a founder mutation in some populations.Worldwide, c.373delT is the commonest Screening for congenital central hypothyroidism (CCH) can be achieved by inclusion of thyroxine (T4) plus thyroxine‐binding globulin (TBG), in the CH screening programme, as in the Netherlands or fT4 as in Japan. Combined T4/TSH/TBG evaluation in Dutch neonates enables diagnosis of permanent central CH with an incidence of 1:21 000 newborns.TSHB mutations. Moreover, Zwaveling‐Soonawala et al. recently demonstrated that more than 50% of children diagnosed with CCH in the Netherlands exhibit moderate or severe hypothyroidism, rather than mild thyroid hormone deficits, thus emphasizing that the potential neurological sequelae of delayed diagnosis should not be underestimated .The benefits of including T4 measurement in CH screening continue to be debated with arguments against citing the relative rarity of congenital central hypothyroidism, with the presumption that it is usually mild and unlikely to be associated with adverse neurological sequelae. However, screening for CCH would enable prompt diagnosis and treatment of affected cases, improving neurodevelopmental outcomes particularly in severe cases due to TSHB mutations, whose genetic aetiologies include a novel missense mutation, a compound heterozygous TSHB deletion and a known frameshift mutation. As well as expanding the known repertoire of TSHB mutations, our observations suggest that TSHB deletions may be more common than previously thought and show that c.373delT TSHB mutation is the most common genetic defect in cases of central hypothyroidism in the UK and Ireland. Concordant with other recorded cases, our patients with isolated TSH deficiency exhibit neurodevelopmental delay when overt hypothyroidism is diagnosed and treated later postnatally. As measurement of T4 is not included in UK or Ireland CH screening programmes, genetic ascertainment in families with isolated TSH deficiency will be particularly important, enabling prompt diagnosis of affected children and early treatment to prevent the adverse neurodevelopmental outcome.We report the first cases from the UK and Ireland harbouring Nothing to declare.


N = 106) with different levels of self-reported autistic traits using an approach based on negative afterimages. The afterimage strength, as indexed by duration and vividness, was also quantified. In opposition to the Hellendoorn and colleagues’ model, we were unable to demonstrate any kind of relationship between abilities in size constancy and autistic traits. However, our results demonstrated that individuals with higher degrees of autistic traits experienced more persistent afterimages. We discuss possible retinal and post-retinal explanations for prolonged afterimages in people with higher levels of autistic traits.Deficits in perceptual constancies from early infancy have been proposed to contribute to autism and exacerbate its symptoms . Here, we examined size constancy in adults from the general population (The online version of this article (doi:10.1007/s10803-016-2971-6) contains supplementary material, which is available to authorized users. If one were to view the world only by the image on the retina, it would appear distorted and unstable. For example, as we watch a car drive away from us, the size of its image on the retina would shrink as it speeds away. Although the car is shrinking on the retina, we perceive it exactly the same size, but just moving further away from us. This perceptual ability is known as size constancy. Size constancy is a scaling mechanism that allows us to make accurate judgments about an object’s size regardless of variations in the retinal image size arising from changes in viewing distance ; American Psychiatric Association (APA) To our knowledge, no study has investigated the perception of afterimages induced by a bright source of light in autism before. The reasons for this gap in knowledge may relate to the risk of inducing discomfort in this population. Indeed, the presentation of a bright flash of light is reported to distress individuals with autism  took part in the experiment. All participants reported to have normal or corrected-to-normal visual acuity, and to have never been diagnosed with an autism spectrum disorder or any other neurological or psychiatric condition. Participants received either 4 study credits or £7 for their time. Written consent was obtained prior to testing. All procedures were approved by the local Institutional Review Board.One hundred and six volunteers  ranging in age from 18 to 37 years of age  on an Acer Travel Mate 240 laptop running Windows XP. Negative afterimages were induced by means of a ring of white light emitting diodes (LEDs) with a luminance of 2686 cd/mo et al. . The indo et al.  was usedo et al. . The ciro et al. .The procedures for inducing and measuring afterimages are almost identical to those used previously by Sperandio et al. , 2013. Tp values were based on two-tailed criteria and corrected for multiple comparisons using the Bonferroni method . Unless specified otherwise, all reported r) were calculated between each measure of the afterimage  and each of the quotient scores , as well as between each of the AQ subscales. Finally, a median-split approach was used to compare each perceptual report of the afterimage  between participants with low versus high scores on the AQ, EQ, and SQ questionnaires. Results from this additional analysis are reported in Supplementary Materials.Mean duration and mean vividness of the afterimages were also calculated. Pearson’s correlation coefficients (p < .001) in the expected direction: a positive correlation between AQ and SQ (r(92) = .55) and negative correlations between AQ and EQ (r(92) = −.64) as well as SQ and EQ (r(92) = −.39).The distributions for AQ, EQ, and SQ are reported in Fig. M = 0.96, SD = 0.07). Only 5 participants out of 106 (4.7%) did not show a significant relationship with Emmert’s law . Then, we calculated the slope of the regression line. A slope of 1 would indicate perfect size-distance scaling whereas a shallower or steeper slope would indicate less adherence to Emmert’s law. The slope values  were then correlated with quotient scores . The correlation revealed that there was no significant relationship between slope size and AQ (r(104) = −.001, puncorr = .99), SQ (r(92) = −.02, puncorr = .81), or EQ (r(92) = .14, puncorr = .17). In addition, the amount of deviation of the slope from perfect size constancy was computed for each participant as an absolute difference between the slope value and 1. The deviation values  were then correlated with quotient scores. Once again, the correlation revealed that there was no significant relationship between slope size and AQ (r(104) = −.01, puncorr = .94), SQ (r(92) = .01, puncorr = .89), or EQ (r(92) = −.17, puncorr = .11). Therefore, the results show that size constancy is unaffected by autistic traits  = .25, puncorr = .01) and SQ (r(92) = .31, puncorr = .003) scores, and negatively with EQ (r(92) = −.38, puncorr < .001) scores. That is, the higher the degree of autistic traits, the longer the duration of the afterimage  = .26, pcorr = .02; r(104) = .27, pcorr = .01, respectively) whereas social skill (r(104) = .16, pcorr = .3), attention to detail (r(104) = .13, pcorr = .46), and imagination (r(104) = .1, pcorr = .76) were not correlated with perceived afterimage duration. These findings indicate that those participants with higher autistic traits related to attention switching and communication experienced longer afterimages.To further investigate the relationship between afterimage duration and autistic traits, the perceived afterimage duration was correlated with scores from the different subcategories of the AQ. One-tailed criteria were applied given we had already demonstrated a positive correlation with total AQ. Attention switching and communication were positively correlated with perceived afterimage duration . Vividness did not correlate with AQ (r(104) = −.08, puncorr = .42), SQ (r(92) = .05, puncorr = .59), or EQ (r(92) = .03, puncorr = .79) score, suggesting that the level of autistic traits did not affect the visibility of the afterimage  = .11, p = .25). We argue that the lack of relationship between these two perceptual attributes of the afterimage may be due to a reduced sensitivity in measuring vividness. In fact, vividness ratings were exclusively based on memory processes. This was not the case for afterimage duration. As a consequence, ratings of vividness were more subjective than judgments about duration.To verify if afterimage visibility affected perceived duration, vividness scores were correlated with duration. As it turned out, vividness did not correlate with duration . It could be the case that hypersentivity to sensory stimuli may hinder a person’s volition to shift towards these same stimuli. It is also conceivable that this hindrance may have undesirable consequences during development. Spatial shifting of orientation emerges in early infancy and is thought to be important for the development of communication  correlated positively with scores on the Glasgow Sensory Questionnaire  require photochemical reactions of rhodopsin to properly transduce light energy into a neural signal. These photochemical changes occur every time the photoreceptor cells capture and convert light into a neural signal. When light stimulation reaches a certain point, the rhodopsin in the photoreceptor cells gets depleted and photoreceptors are no longer responsive to light until the rhodopsin has been restored. It is known that this photochemical bleaching plays a necessary role in the creation of afterimages  has been observed in children with autism Rubin  and theiRegarding the second possibility, previous research has shown that saccadic eye movements reduce retinotopic adaptation, generating shorter afterimages


Extreme precipitation events are predicted to occur more frequently and will have significant influences on terrestrial ecosystem carbon (C) cycling in the future. However, response patterns of soil respiration to precipitation changes remain uncertain in terrestrial ecosystems. A field experiment with seven precipitation treatments (i.e. from −60% to +60% of ambient precipitation to form a drought to wet precipitation gradient) was conducted over three growing seasons (2010–2012) in a semiarid temperate steppe of Northern China. Results showed a nonlinear response pattern of soil respiration along the experimental precipitation gradient, with soil respiration suppressed by decreased precipitation and enhanced by increased precipitation. Over the three growing seasons, soil respiration was reduced more under the three drought treatments  than stimulated under the three wet treatments . Our results indicate that soil respiration was more sensitive to decreased than increased precipitation treatments. The nonlinear and asymmetric responses of soil respiration to precipitation changes should be built into ecosystem models to project ecosystem C cycling associated with climate change. Soil respiration is the second largest carbon (C) flux between terrestrial biosphere and the atmosphere1346711In arid and semiarid regions, water is the predominant limiting factor for plant growth, net primary productivity, and other biological processes131415161819162012et al. (2008) simulated soil heterotrophic respiration and found that non-linear relationship between soil respiration and precipitation, largely due to non-proportional increases in surface runoff with increasing precipitationLarge spatial-scale investigations have demonstrated that soil respiration tends to linearly increase with precipitation amount among different sites2728In this study, a field manipulative experiment was conducted over three growing seasons (June-September) of 2010–2012 to examine soil respiration responses along an experimental precipitation gradient  in a semiarid temperate steppe of northern China. The study site is water limited and sensitive to climate change2432P = 0.06). In contrast, precipitation changes significantly influenced SWC  and 17% (P < 0.05) in the P − 6 and P − 4 treatments, respectively, and an increase of 19% (P < 0.01) in the P + 6 treatment, compared to that of control  relative to that of control  relative to that of control  appeared in 2011 and the lowest value (1.14 μmol m−2 s−1) in 2012 in the control plots (−2 s−1) appeared in July, and the lowest value (0.54 μmol m−2 s−1) in October in the control plots over the three growing seasons  and ANPPforb (P < 0.05). ANPP were 137.75 and 260.77 g m−2 yr−1 under the P − 6 and P + 4 treatments, respectively, which accounting for a change of −37.28 (P < 0.01) and 18.37% (P < 0.05) relative to that of control  and −30.74% (P < 0.05) relative to that of control, but no differences in ANPPforb were found among the other treatments (grass and belowground net primary productivity (BNPP) were found  in different years . The sen2 = 2.67, P = 0.26, df = 2, GFI = 0.98, RMSEAR = 0.09). Results of SEM explained 76% of the variations in SR  revealed that the interactive networks of abiotic and biotic factors regulated SR  in Inner Mongolia, China. The study site has been fenced to exclude grazing disturbance since 2001. Mean annual precipitation (1951–2010) was 379 mm, with 78.4% of the total precipitation fell from June to September. Mean annual temperature was 2.2 °C. The sandy soil in this area is classified as Haplic Calcisols with soil bulk density is 1.31 g cm2 with a 3-m distance between any two adjacent plots, a core area with 3 × 3 m2 within each plot was used for measurements. V-shaped rainout shelters made by transparent plexiglass were used in the decreased precipitation plots to intercept different amounts of incoming rainfall, corresponding to different numbers of bandsThis experiment used a randomized block design with a precipitation gradient from −60% (P − 6), −40% (P − 4), −20% (P − 2), to ambient precipitation (C), and to +20% (P + 2), +40% (P + 4), and +60% (P + 6) of ambient precipitation2 flux system  was used to measure SR three times per month from 09:30 AM to 12:30 PM  during each year (May-October) of 2010–2012. Green plants inside the collar were cut to eliminate the effects of aboveground autotrophic respiration on the soil respiration. Soil temperature (ST) was measured with a thermocouple probe of Li-Cor 8100 when measuring SR. Volumetric soil moisture content (SWC) (0–10 cm) was measured using a portable SWC device , recording every 3–5 days a time for a month in 2010–2012.One PVC collar (11 cm in diameter and 5 cm in height) was permanently inserted at the depth of 3 cm at the center of each plot for soil respiration (SR) measurement. A LI-8100 portable soil CO2 quadrats in each plot in August from 2010 to 2012, when plant biomass reached its peak levelforb) and grass (ANPPgrass)2 frame with 100 equally distributed grids (10 × 10 cm2) was put above the canopy in permanent quadrat (1 × 1 m2) in each plot for measuring plant coverage in August of 2010 to 2012. Species richness was recorded as the number of plant species in the quadrat.Aboveground net primary productivity (ANPP) was estimated by clipping living biomass in August each year. All living plant tissues were harvested from two 0.15 mgrass, ANPPforb, coverage, species richness, ANPP and BNPP. Multiple comparisons with Tukey HSD test were used for comparing the mean difference of the above variables. These statistical analyses were conducted using the R version 3.2.1 RCet al. (2015) and Wilcox et al. (2015) examined the drought sensitivity of ANPP that the response of a variable (ANPP) to a unit change in a driver (millimeters of precipitation)51The repeated measure linear mixed-effects models were used to test the effects of year, sampling, and precipitation for ST, SWC and SR. Linear mixed-effect model was also used to test the effects of year, precipitation, for ANPPT is the soil respiration of precipitation treatments, SRC is the soil respiration of the control; and PT is the precipitation amounts of in the treatment plots, PC is the precipitation amount in the ambient plots.where SR2-testFurthermore, structural equation modeling (SEM) was used to identify causal linkages between explanatory variables and SRHow to cite this article: Miao, Y. et al. Nonlinear responses of soil respiration to precipitation changes in a semiarid temperate steppe. Sci. Rep.7, 45782; doi: 10.1038/srep45782 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The syndrome of inappropriate antidiuresis is often undertreated with most patients discharged with persistent hyponatraemia. This study tested the hypothesis that an endocrine input is superior to routine care in correcting hyponatraemia and can improve patient outcomes.This single-centre prospective-controlled intervention study included inpatients admitted at a UK teaching hospital, with serum sodium ≤ 127 mmol/l, due to syndrome of inappropriate antidiuresis over a 6-month period. The prospective intervention group (18 subjects with mean serum sodium 120.7 mmol/l) received prompt endocrine input, while the historical control group (23 patients with mean serum sodium 124.1 mmol/l) received routine care. The time needed for serum sodium increase ≥ 5 mmol/l was the primary endpoint.P = 0.005). In the intervention group, the mean total serum sodium increase was 12 mmol/l with only 5.8 % of patients discharged with serum sodium < 130 vs. 6.3 mmol/l increase (P < 0.001) and 42.1 % of the subjects discharged with serum sodium < 130 mmol/l in the control group (P = 0.012). The mean length of hospital stay in the intervention group (10.9 days) was significantly shorter than in the control group .The inpatient mortality rate was 5.5 % in intervention arm vs. 17.4 % in control arm, but this difference was not statistically significant.The intervention group achieved serum sodium rise by ≥5 mmol/l in 3.5 vs. 7.1 days in the control group (Since the endocrine input improved time for correction of hyponatraemia and shortened length of hospitalisation, widespread provision of endocrine input should be considered. Hyponatraemia is associated with considerable morbidity and mortality –4, and cto compare the total rise in sNa and the percentage of patients discharged with sNa < 130 mmol/l between intervention and control groupto examine the effect of endocrine input on inpatient mortality and length of hospital stayto study whether correction of hyponatraemia improves cognitive function.Despite suboptimal standards of care for SIAD, no prospective studies have examined the effect of widespread provision of endocrine input on its management. Also there is paucity of data about whether correcting hyponatraemia can improve patient-related outcomes, such as mortality, length of hospital stay, and readmission rate. The primary hypothesis of our study was that prompt and intensive endocrine input was superior to non-specialised ‘standard’ clinical care in correcting hyponatraemia with the primary endpoint being time to achieve an increase in serum sodium (sNa) by ≥5 mmol/l. Secondary objectives were:st October 2014–31st December 2014), all patients underwent ‘routine’ care (control group), while in the following 3-month period (1st January 2015–31st March 2015), patients received intensive endocrine input (intervention group). The rationale behind the use of a historical control was that undertaking a randomised controlled trial in a real-life clinical setting was considered impractical since clinicians might often seek expert help from the investigators about patients allocated to the control arm, resulting in possible ‘dilution’ of the control group with patients receiving the intervention. The time required to achieve sNa increase of ≥5 mmol/l was chosen as the primary endpoint since this magnitude of correction can be sufficient to improve symptoms of hyponatraemia   22] indi27 formula using the urine/plasma electrolyte ratio (U/P) = (UNa + UK)/(PNa + PK), for example 500 ml/day if U/P is 0.5–1.0 and 1000 ml/day if U/P is <0.5 [2O [Our proposed algorithm for optimal SIAD management includes various therapeutic options with individualised treatment decisions being based on several factors, including duration and degree of hyponatraemia, severity of symptoms, the capacity of the nephron to excrete free water, urine osmolality, the safety and efficacy of each treatment modality, cost implications of each therapy and patient compliance . The onl is <0.5 . If urin<0.5 [2O . Tolvapt<0.5 [2O . Serum N<0.5 [2O . FinallyOur findings highlight the need for multicentre prospective-controlled studies to examine the impact of specialist input on clinical endpoints such as mortality rate, length of stay, symptoms and readmission rate. Since SIAD represents a potential target for intervention to reduce healthcare expenditures for a large population of inpatients, it is also essential to test the cost-effectiveness of widespread provision of expert input, taking into account on the one hand potential reduction in length of hospitalisation and readmission rate, and on the other hand additional cost related to clinical and nursing time and cost of pharmacological therapies. Finally, studies are warranted to develop tools measuring hyponatraemia-specific symptoms which could be used longitudinally for assessment of symptoms and neurocognitive performance in response to any sodium-correcting therapy.In conclusion, these preliminary data demonstrated that intensive endocrine input not only was superior to ‘routine’ care in correcting hyponatraemia, but also improved patient-important outcomes such as length of hospital stay and symptoms. If these results could be generalised, provision of systematic endocrine care for patients with SIAD should be widely adopted to improve clinical outcomes and potentially reduce utilisation of hospital resources.


D. armandi larvae in each month from October 2014 to March 2015. The sorbitol, trehalose and glycerol values initially tended to increase as the ambient temperature decreased, before declining until the end of the winter. The activities of four enzymes  decreased, whereas POD, PK and MDH showed opposite trends in activity. Other enzyme activities  were low during the overwintering period and later increased and stabilized during spring. In this study, a polymerase chain reaction (PCR) genes of SDH, TPS and GLK was utilized to identify DarmSDH, DarmTPS and DarmGLK in D. armandi. They were found to be abundantly expressed during the overwintering stage by quantitative real-time PCR (qRT-PCR) analyses; by contrast, these three genes showed higher expression levels in December 2014 than in May 2015. The qRT-PCR results demonstrated that the reduction of mRNA expression levels was significant in DarmSDH-, DarmTPS- and DarmGLK-dsRNA-treated D. armandi compared with water-injected and non-injected controls. The mortality responses at low temperature were also increased in the dsRNA-treated D. armandi compared with the controls.The values of physiological indices and the enzymes activities involved in the overwintering stage were studied in  In temperate regions, insects face a great challenge in surviving at extremely low temperatures1Dendroctonus armandi (Tsai and Li) (Coleoptera: Curculionidae: Scolytinae) belongs to a group of insects whose biochemistry and physiology have not yet been fully recognized. Cold tolerance is controlled by more than one environmental condition131115The cold-tolerance of insects is an intricate adaptive response that relates to many defined biochemical, physiological and endocrinological adjustments in insectset al.8D. armandi are not well understood. To investigate the comprehensive mechanisms of D. armandi physiological adaptation to environment changes, its antioxidant enzymes  were determined. In addition, its glycometabolic enzymes , esterase  activities and the quantitative expression of its genes were measured. We hypothesize that the physiological and biochemical alterations and metabolic adjustments to these changes are connected with changes in the enzyme defence system. The antioxidant defence system, which prevents oxidative injuries from developing during normal metabolic activity, may be significantly altered by cold stress, due to changes characteristic of cold-hardiness mechanisms.Many studies have proved that low-molecular-weight sugars and polyols are significant intermediate metabolites and energy substances in many insect species during the overwintering stage3181115D. armandi, is the most damaging forest insect that invades the phloem of Pinus armandi Franch in the Qinling Mountains, Shaanxi, ChinaP. armandi in the Qinling Mountains have been susceptible to ruinous damage caused by approximately 20 species of bark beetles, with D. armandi, in particular, a leading serious forest pest, giving rise to large numbers of deaths among otherwise healthy P. armandi trees over the age of 30 yearsBark beetles (Coleoptera: Curculionidae: Scolytinae) are endophytic parasites of shrubs and trees and have been broadly recognized for their economic and ecological significance in forestsTrehalose  is an important disaccharide2527183031323032+ as a cofactorBombyx exposed to temperature of 0 °CBombyx eggs, SDH is normally expressed at a very low level. However, when the eggs are transferred from approximately 0 °C to 25 °C, SDH activity strongly increasesDrosophilaSDH is a member of the zinc-dependent alcohol dehydrogenase-like (ADH) family+. as seen in Epiblema scudderiana (Clemens)Spodoptera exigua47Hyalophora cecropia eggs, which accumulate glycerol, GLK plays a role in overwintering termination by converting glycerol to glycerol-3-phosphate for other intermediary metabolismBombyx mori, at least three GLK isozymes have been reported, among which only one isoform, GK3, appears to be associated with the utilization of glycerolGlycerol can be biosynthesized via two biosynthetic pathways via glycolysis and the pentose phosphate cycle43Caenorhabditis elegans53D. armandi, microinjection is considered an excellent method because of its convenience and low rate of injury. By silencing different genes, the roles of diverse proteins have been researched under environmental stress, especially at low temperature56RNAi is a mechanism for post-transcriptional gene silencing that has been developed as a powerful tool for studying gene function in a variety of organismsD. armandi larvae during overwintering. Determining how Chinese white pine beetles respond to the physiological problems posed by winter conditions is vital for predicting epidemic situations and their long-term impact. Therefore, the study of these problems in D. armandi has an important role in understanding the coordinated effects on the overwintering larval body.The main objectives of the current study were to assess cold tolerance and to determine the means by which the physiological index values and enzyme activities, as well as the DarmSDH , DarmTPS  and DarmGLK , affect F = 2.163 × 104, df = 5, P < 0.001), then declined to a level of 12.72 ± 0.16 mg/g in March , and then steadily declined to a value of 2.86 ± 0.18 mg/g in March  and then declined again to a level of 9.32 ± 0.28 mg/g in March  and CAT  activities consistently decreased at a very low rate in October, then decreased slightly during the overwintering period (between November and February), and weakly increased in March  activity changed inversely with the activities of the first two enzymes. Compared with the SOD and CAT activities, the POD activity changed significantly in each month during the winter. The POD activity increased from October to January, when the maximum POD activity was 6.503 ± 0.3 (g mass)−1, almost three times the value recorded in October (beginning of the overwintering stage). After reaching a maximum, the POD activity sharply decreased to the March level  activity reached its highest level of 5622.66 ± 60.34 (g mass)−1 from a starting point of 3390.43 ± 41.70 (g mass)−1 in October. In February, the PK activity significantly decreased to 1808.79 ± 31.02 (g mass)−1 and reached its lowest level of 292.86 ± 17.28 (g mass)−1 in March  activity changed insignificantly in each month on six sampling times, only increasing gradually in November to a level of 2010.28 ± 87.76 (g mass)−1 and then decreasing slowly to 1777.11 ± 14.84 (g mass)−1 in March  in December compared with October and November. The MDH activity reached a level of 3331.02 ± 50.91 (g mass)−1 in December, declined to 2723.59 ± 44.91 (g mass)−1 in January and then recovered slightly. Finally, the level of MDH activity fell to 876.25 ± 20.83 (g mass)−1, its lowest value, in February  activity was 0.20 ± 0.02 (mg prot)−1 in October. With decreasing environmental temperature, the AchE activity declined quickly to 0.02 ± 0.01 (mg prot)−1 in November. After that, the AchE activity dropped further to reach a minimum value of 4.081 × 10–3 ± 0.01 (mg prot)−1 in March 2015.Information on the AchE activity is given in F = 4.267 × 102, df = 5, P < 0.001) activity to decrease significantly from 400.66 ± 1.63 (g mass)−1 in October to 306.79 ± 1.33 (g mass)−1 in Deember when it reached its minimum level. Then, the TPS activity increased sharply in January to 385.39 ± 1.24 (g mass)−1. After that, the TPS activity fluctuated within a narrow range  dropped sharply to 2999.59 ± 41.40 (g mass)−1 in January. After that, the activity kept rising into March to a value of 5376.54 ± 40.30 (g mass)−1, recovering the same level as that before overwintering  declined during the winter to reach a minimal level in January of 685.51 ± 21.40 (g mass)−1. The GLK activity levels remained high before December and stable between February (929.03 ± 18.26 (g mass)−1) and March (935.95 ± 20.30 (g mass)−1). The levels decreased from 1000.66 ± 11.40 (g mass)−1 in October to 707.82 ± 28.31 (g mass)−1 in December  were −5.7,−5.4, −4.8, −4.4, −4, −2, −0.9, 0, 4.4, 4.7, 4.9 and 5.4 °C, respectively. However, the LT50 values in December 2014 were −10.4, −9.0, −8.0, −8.0, −7.8, −6.5, −5.9, −3.7, −2, 4.1, 4.3 and 5 °C, respectively  by PCR using specific primers . BLAST s species . Identit species . The insD. armandi larvae during different months from October 2014 to March 2015 . Finally, we chose to induce their expression by a low-temperature exposure for 12 h . The expD. armandi larvae remained unchanged.The transcriptional expression level of DarmGLK reached its maximum value in November, while DarmSDH and DarmTPS reached their peak values in December . We knocD. armandi larvae to low temperature by mortality analysis. . The mortality changed significantly with different durations of low-temperature exposure , lipid peroxides, and hydroxyl radicals. If not eliminated, ROS propagate further oxidative processes, leading to the damage of cellular molecules and resulting in disturbed homeostasis and cellular death2O2 in insects can be achieved in two ways: (1) by CAT and glutathione recycling and (2) by an ascorbate recycling antioxidant mechanism2O2 elimination by CAT and the glutathione recycling mechanism is well balanced and sufficient to prevent oxidative damage, the second mechanism could be less active, and vice versa. This situation points to a precisely coordinated regulation in the levels of ROS and redox-active molecules by antioxidant enzymes. Furthermore, during the overwintering stage, glycogen was converted into other substancesOverall, the activities of many enzymes in D. armandi larvae from a new perspective. The LT50 values were low in November and decreased further in December. A possible explanation for this LT50 drop might be that low temperature was a limiting factor for larval survival during the overwintering stage. In addition, Boardman et al.Thaumatotibia leucotreta (Meyrick) are more tolerant than those that are fed, using another way of stating the case. Langford50 for D. armandi overwintering larvae declined during winter months to reduce mortality.Insects are poikilothermic animals, and their cold-tolerance capacity is a key aspect of their adaptation to their geographical environment, including factors such as altitude, latitude, and ambient temperature. Therefore, the determination of mortality at low temperature can help us understand the principles of overwintering in D. armandi larvae in the overwintering stage increased significantly in December compared with November. The effects of low temperature on mortality have also been demonstrated in other insects, such as Monochamus alternatusSarcophaga bullataDrosophila melanogaster71The transcript levels in the larvae of the Chinese white pine beetle reached their highest levels for DarmSDH and DarmTPS in December 2014 and for DarmGLK in November 2014; therefore, we chose November and December for the measurement of mortality. The low-temperature-mortality rate increased with longer exposure times and with decreasing temperatures. Additionally, under natural conditions, the cold-tolerance capacity of D. armandi larvae. In the insect, Bombyx mori, SDH has been shown to control the utilization of sorbitol in overwintering eggsIn addition, we quantified changes in the DarmSDH, DarmTPS and DarmGLK concentrations in each month from October 2014 to March 2015 and detected the quantitative expression of these three genes under different low-temperature conditions in May 2015 and December 2014. Sorbitol dehydrogenase is a widely distributed enzyme that, together with aldose reductase, constitutes the sorbitol pathwayD. melanogaster7576256D. melanogasterHelicoverpa armigeraSpodoptera exiguaCatantops pinguisD. armandi larvae. Therefore, the characterization of the DarmTPS gene and its enzyme activity in overwintering larvae was of interest. In our study, the expression pattern of the DarmTPS gene was observed in summer-sampled larvae (May 2015), in overwintering (December 2014) larvae and in larvae collected from October 2014 to March 2015. Nevertheless, significantly greater expression of DarmTPS was observed during winter in the overwintering larvae S. exigua)E. scudderianaS. exiguaD. armandi larvae. The real-time PCR of overwintering larvae from different months showed that DarmGLK was expressed at relatively high levels in November but at low levels in December and January. Even so, the expression of DarmGLK was higher in December 2014 than it was in May 2015. This indicates that this protein is crucial for the overwintering process.The biosynthesis of glycerol follows two independent pathways depending on the insect. One pathway uses polyol dehydrogenase catalysing glyceraldehyde with NADPH + HD. armandiD. armandi reduced the expression of the target gene by 80%D. armandi can now be considered a tenable hypothesis. We have demonstrated the feasibility of injecting RNAi and shown its injection can demonstrably inhibit the transcription level of a target gene in D. armandi larvae. This effect was also observed after abdominal injections of dsRNA: the midgut aminopeptidase N gene in Spodoptera lituraD. armandi larvae treated with DarmSDH, DarmTPS and DarmGLK dsRNA were significantly reduced compared with the levels in the two controls , Shaanxi, China.D. armandi larvae were collected from host trees Chinese white pines, P. armandi on the 10th of each month between October 2014 and March 2015 at six occasions from the above site. The larvae were collected from three sample plots in each month, each of which was 20 m × 20 m. We chose 5 trees in each sampling plot according to the five-point sampling method. We peeled off the phloem (20 cm × 20 cm) of each host tree from four directions to collect the larvae. The larvae were then transferred to the laboratory in the dark environment and used to examine the survival rate with exposure to low temperature, measure the physiological parameters, determine the activities of enzymes and extract the DNA.Overwintering Chinese white pine beetle, D. armandi were collected on the December 10th, 2014 and May 10th, 2015. The larvae were treated with different temperature  for different times . We used 2520 larvae in total for the treatments with dry electrothermostat . After exposure to low temperatures, the specimens were maintained at room temperature for 1 h, before detecting body movements when stimulated with a tweezers5The larvae of Three physiological indices were measured monthly from October 2014 to March 2015 to quantify the physiological variations in larvae, including the contents of sorbitol, trehalose and glycerol in the bodies of larvae.et al.Total body glycerol: A whole larva was treated as one sample, and twenty larvae were used for each treatment with 3 replicates. The fresh weight (FW) of an individual larva was measured with an electronic analytical balance  at nine sampling time points. Twenty larvae were placed into a 2.5 mL Eppendorf tubes and dried at 60 °C for 72 h. The dried larvae were homogenized with 2 mL of 70% ethanol and centrifuged at 3000 r/min for 10 min. The pooled supernatants from three replicates of the process were abandoned, and the rest of the pellet was used to isolate glycogen, according to the method reported by Ohtsu The supernatant was used to detect the glycerol levels. The glycerol content was determined using the phenol and sulfuric acid method892+ in alkaline solution. These two substances’ content levels were determined with a spectrophotometer  using the relevant content kit , Suzhou Comin Biotechnology Co., Ltd., Jiangsu, China) according to the kit protocol.The concentration of trehalose and sorbitol: 0.1 g larvae were treated as one sample, and used for each treatment with 3 replicates. The fresh weight (FW) of an individual larva was measured with an electronic analytical balance  at nine sampling time points. Anthrone colorimetry method was adopted to determine the concentration of trehalose. This compound have specific absorbing peak at 620 nm after grinding, extraction, quiescence, centrifugation, boiling and cooling. The principle of determining the sorbitol content is that sorbitol can form a blue complex with CuTen kinds of enzyme activities were determined monthly from October 2014 to March 2015 according to each protocol of the kit , Superoxide dismutase (SOD), peroxidase (POD), Lactate dehydrogenase (LDH), malate dehydrogenase (MDH), Pyruvate kinase (PK), trehalose-6-phosphate synthase (TPS), Sorbitol dehydrogenase (SDH), glycerol kinase (GLK) and Acetylcholin esterase (AchE), respectively. All of the enzyme activity kits were purchased from the Comin Biotechnology Company .50), the temperature that caused 50% mortality was estimated with logistic regression.Larvae were collected from November and December 2014 and then transferred to a dry electrothermostat  . Separate larvae were exposed to seven different constant temperatures  for different time . Each individual larva was placed in a 200 μL perforated Eppendorf PCR tube and kept dry. After exposure to low temperatures, the specimens were maintained at room temperature for 1 h before the detection of body movements when they were stimulated with tweezers5Total RNA was extracted following the protocol of the RNA extraction kit  for all of the above treatments. The quality of total RNA was detected by NanoDrop ND-1000 Spectrophotometer . Finally, 500 ng total RNA (OD260/OD280 = 1.80–2.10) was used for cDNA synthesis. First-strand cDNA was synthesized by using PrimeScriptTM RT reagent Kit with gDNA Eraser   according to the manufacturer’s instructions.Escherichia coli. The transformants (white colonies) were selected on Amp/X-gal/IPTG plates, and a total of 15 clones with inserts were sequenced directly by GenScript USA Inc . The obtained partial sequences were manually edited with DNAMAN and blasted against the NCBI database.The synthesized cDNA obtained from the sample was used as a template in PCR reactions. Each pair of specific primer was designed by Primer Premier 5.0  , includiD. armandi was used as endogenous control to normalize the target gene expression. The primers of the target and reference genes were designed by Primer Express 5.0  ; 60 °C (TPS and GLK) for 30 s and 72 °C for 25 s. Experiments for test samples, endogenous control, and negative control were performed in triplicate to ensure reproducibility. Relative quantification was performed by using the comparative 2−ΔΔCt methodThe transcripts of different low temperature, different time and RNAi treated larvae were measured by using a CFX-96 real-time PCR Detection System  and the Roche SYBR Green system . Actin gene (GenBank accession number: KJ507200) of bad, CA) . qRT-PCRAccording to the manufacturer’s instructions of the T7 Ribo-MAX™ Express RNAi System , primers  were desD. armandi larvae under 70% ethanol anesthesia were immobilized on the agarose plate with the abdomen directed airward using manual forceps. Afterwards, 0.05 μL DEPC treated water or dsRNA solution (0.1 μM) was injected in each D. armandi larvae using a PLI-100 Pico-Injector . Each treatment contains 90 larvae in triplicates. After injection, D. armandi larvae were kept in a refrigerator at 4 °C. 15 larvae in triplicate were selected per 24 h, 48 h and 72 h, frozen in liquid nitrogen, and then stored at −80 °C before qRT-PCR analysis (see above). Larvae injected for 24 h, 48 h and 72 h were tested for the mortality, and repeated three times each.Before injection, a 1% agarose plate was made and placed on an ice tray. D. armandi were performed using ANOVA followed by Tukey’s tests for multiple comparisons to detect significant differences. To determine the LT50 value of the different exposure time, binary logistic regressions were used to calculate the temperature at which 50% mortality occurred−ΔΔCt values were subjected to one-way ANOVA to determine if the gene expression was different among the treatments. The data and figure analyses were performed with SPSS 18.0  using Sigma Plot 12.5 software . All of the data were expressed as the mean ± SE, except for the survival at low temperatures.Statistical analyses of overwintering larvae data among trehalose, sorbitol and glycerol content, and enzyme activity assays in How to cite this article: Wang, J. et al. Cold tolerance and silencing of three cold-tolerance genes of overwintering Chinese white pine larvae. Sci. Rep.6, 34698; doi: 10.1038/srep34698 (2016).


Chondroblastoma is a rare benign cartilaginous tumor, which primarily occurs in children and adolescents. Chondroblastoma commonly originates in the epiphyseal plate of long bones. An aggressive curettage treatment is recommended to manage lesion, which may jeopardize an open epiphyseal plate and result in limb shortening and deformity as the limb grows and develops. The purpose is to observe surgical effects of chondroblastoma on open epiphyseal plate of long bones in children and adolescents and explore influences on limb growth and development.We retrospectively reviewed 18 cases of long bone chondroblastoma with open epiphyseal growth plate during March 2004 to October 2010 in our center. Seven females and 11 males with mean age of 11.6 ± 2.0 years old (8–15 years) were included. Patients, who suffered from trauma and pathological fracture of the epiphyseal plate or congenital diseases such as poliomyelitis, congenital dementia, and cartilage malnutrition, were excluded. All patients were treated with meticulous intralesional curettage and inactivity with alcohol followed by bone grafts. All cases were followed up 8.2 ± 1.7 years (5–11.5 years).P = 0.061). Meanwhile, there were obvious relativity with the greatest dimension of the lesion (TGD) (P = 0.003), the vertical dimension between edge of lesion and epiphyseal line (TVD) (P = 0.010), and area ratio of lesion to local epiphysis (lesion/growth plate) (P = 0.015). The MSTS93  and SF-36  had been significantly improved (P < 0.01).All had no local recurrence and distance metastasis. The length of the affected limb was short, 18.47 ± 7.22 mm (1.5–30 mm). There was no obvious relativity with tumor activity (Managing of chondroblastoma located in open epiphyseal plate of a long bone with meticulous curettage, inactivity, and bone grafts can control tumor progression and recurrence effectively. Meanwhile, early detection and prompt surgical treatment intervention, which reduced significantly the tumor to influence limb growth and development, get encouraging limb function.This is a retrospective study, which was not registered in any trial registry. Chondroblastoma is a rare benign bone tumor with an incidence of 9% in benign bone tumors . ChondroSurgery is recommended for treatment with chondroblastoma mainly , 9, 10. To our knowledge, there were rare study series exclusively focusing on limb length of chondroblastoma after surgery in young patients with open epiphyseal growth plate , 10. EpiAll patients with open epiphyseal growth plate of long bone chondroblastoma were accepted during March 2004 to October 2010 in our center. The chondroblastoma was confirmed by pathology. All patients received the first tumor resection. Operation agreement and rehabilitation protocol were signed.The patient with closed epiphyseal plate was excluded. Trauma and pathological fracture of epiphyseal plate was not involved. Patients who suffered from poliomyelitis, congenital dementia, cretinism, cerebral palsy, and cartilage malnutrition were not unaccepted.We performed a retrospective study of 18 children and adolescents with chondroblastoma in our hospital from March 2004 to October 2010. The diagnosis was based on recognized image data and histological criteria including intraoperative frozen pathological and postoperative paraffin tissue biopsy. The general information is described in Table All patients underwent chest radiography and radionuclide bone imaging on presentation as a screening procedure for metastatic disease, and no metastatic disease occurred. The status of adjacent epiphyseal plate was determined by radiographs. An epiphyseal plate was classified as open if a wide and clearly defined radiolucency was apparent. Meanwhile, an epiphyseal plate was closed if an epiphyseal scar was present . The les2/πab, Fig. We measured the length of each long bone according to X-ray on computer  Fig. . All lesFirstly, all patients have undergone meticulous intralesional curettage from the local cortical window and polished tumor cavity around the lesion edge, 2–5 mm by bone drill  software was used. The values are presented as mean ± standard deviation (SD). Rank correlation was used to determine the relationship between the shorten length with tumor activity, focus/epiphyseal plate, TGD, and TVD. P < 0.01) (Table All patients were received follow-up with the median of 8.2 ± 1.7 years (5–11.5 years). The outcomes were summarized in Table 1) Table . EighteeP = 0.061), but obvious relativity focus/epiphyseal plate (P = 0.015), TGD (P = 0.003), and TVD (P = 0.010)  compared with non-surgery limb . They all joined in social activities in adulthood as farmer, construction worker, college students, civil servants, and teachers, and so on.Limb length discrepancy and angular deformity may not necessarily lead to clinical problems during childhood and puberty, but psychosocial problems may occur. Social withdrawal, practical problems relating to clothing and shoes, fearing about future compatible partners, and career planning would suffer –23. LoweManaging of chondroblastoma located in an open epiphyseal plate of a long bone with meticulous curettage, inactivity, and bone grafts can control tumor progression and recurrence effectively. Meanwhile, early detection and prompt surgical treatment intervention, which reduced significantly the tumor to influence limb growth and development, get encouraging limb function.First, as a retrospective study, we had no control group to assess the superiority of our study compared with different tumor management. This was a retrospective review and is, therefore, limited by the heterogeneity of the available data and follow-up. Second, the sample size was few. We needed more cases and time to observe local recurrence, distant metastasis, limb discrepancy, and function. Furthermore, because of few cases in our study, we could not observe more factors, which might influence limb growth and development after surgical treatment, such as age and gender.


This study investigated the combined effect of drought and heat wave on physiological responses of tomato seedlings. We found that the negative impacts of drought stress were exacerbated by heat wave through stomatal and biochemical limitations. Additionally, the recovery of leaf gas exchange in the combination of drought and heat wave was delayed more than in other treatments. Our results suggest that future climates characterized by drought and extreme heat may have substantial impacts on tomato productivity and survival. i.e. well watered and drought stress) at ambient temperature (26 °C) for 10 days. Afterwards, half of the seedlings were exposed to a 7-day 42 °C heat wave. All the drought-stressed seedlings were then rehydrated upon relief of the heat wave. Leaf gas exchange, the maximum carboxylation capacity (Vcmax), plant growth, relative chlorophyll content and leaf water potential were examined during the experimental period. The heat wave reduced leaf gas exchange rates, Vcmax and relative chlorophyll content, while it had no impacts on leaf water potential. In contrast, drought stress led to greater reductions in leaf gas exchange rates, growth and water potential than heat wave alone. Seedlings underwent a greater degree of stress in the combination of drought and heat wave than under the single drought treatment. The recovery of leaf gas exchange from drought stress lagged behind the water potential recovery and was delayed by heat wave. Our results show that drought stress had a predominant role in determining plant physiological responses and the negative impacts of drought stress were exacerbated by heat wave. The greater stress in the combination of drought and heat wave translated into the slower recovery of leaf gas exchange. Therefore, drought combined with heat wave may induce greater risks on crops under future climates.Heat waves in combination with drought are predicted to occur more frequently with climate warming, yet their interactive effects on crop carbon and water balance are still poorly understood. Hence, research on the capacity of crops to withstand and recover from the combined stress is urgently needed. This study investigated the effects of drought and heat wave on a crop species as well as the recovery from the combined stress. Seedlings were grown in growth chambers under two soil water conditions ( Leaf gas exchange, plant growth, relative chlorophyll content, leaf water relations and the maximum carboxylation capacity (Vcmax) were measured or estimated during the course of the experiment. The key research question is how plant carbon and water relations are coordinated during the recovery from the combined stress of drought and heat wave, which is crucial for tomato management strategies under climate change. We hypothesized that: (i) single heat wave treatment would lead to lower steady state leaf gas exchange rates, decreased water use efficiency, lower Vcmax, reduced plant growth and more negative leaf water potential compared with ambient temperature control; (ii) heat wave would exacerbate drought stress by reducing steady state leaf gas exchange rates, Vcmax, plant growth, leaf water potential, chlorophyll content to a greater extent in the combination of drought and heat wave treatment than in the single drought treatment; (iii) the coordination of leaf gas exchange and leaf water potential during recovery from drought would be shifted between ambient temperature and heat wave treatments, i.e. the recovery of leaf physiology from drought would be slower in the combination of drought and heat wave than in the single drought treatment.This study aimed to examine the leaf physiology and growth of cherry tomato  were sown and germinated in seed raising tube stocks in a poly-tunnel under ambient environmental conditions for one month. Healthy seedlings with similar heights (15–20 cm) were then transplanted into 3.7-L plastic pots filled with soil (one seedling per pot) and were grown for another month. Twenty four potted seedlings were then randomly assigned into two illuminated growth chambers  (twelve seedlings per growth chamber) with a 26 °C/19 °C day/night temperature cycle and 15 h/9 h day/night photoperiod, 60% relative humidity (RH), and 250 µmol m−2 s−1 photosynthetic photon flux density (PPFD). The seedlings were well irrigated daily and fertilized weekly with a commercial liquid fertilizer . Seedlings were rotated within and between growth chambers every week to minimise potential effects of growth chambers on plant performance.Seeds of cherry tomato (i.e. six seedlings) in each growth chamber were randomly assigned to two water treatments (i.e. well watered and drought treatments) on Day 0. The drought treatment consisted of one cycle of drought and recovery. Seedlings in the drought treatment in both growth chambers were treated under moderate drought stress until they were rehydrated on Day 20. During that time, the heat wave was imposed on one of the growth chambers for a period of 7 days. Specifically, from Day 10, one growth chamber was subjected to heat wave treatment (i.e. 42 °C/35 °C day/night temperature cycle), while the other one was maintained at ambient temperature treatment (i.e. 26 °C/19 °C day/night temperature cycle)  see . The temgs) is non-destructive, and that gs is a good indicator of plant and leaf water stress, we controlled this variable in order to establish the target drought stress. Water was then withheld in seedlings in the drought treatment until gs declined to the range of 0–0.05 mol m−2 s−1  each day to determine gravitational water loss and the corresponding soil volumetric water content (SWC) . About 50–100 ml water was then added to each pot to maintain the target SWC through the cycle of drought stress (25 % on average from Day 6 to Day 19)  see . Note thi.e. 30 °C, 34 °C, 38 °C, 42 °C and was maintained at each step for 4 h to allow plants to acclimate to the temperature changes. The temperature was then maintained at 42 °C/35 °C day/night before it declined back to 26 °C/19 °C day/night on Day 17. In this case, plants in HW and HD were subjected to a 7-day of + 16 °C heat stress treatment.On Days 10 and 11, the temperature in the heated growth chamber was raised from 26 °C to 42 °C in 4 steps, n = 4) using a portable open path gas exchange system  supplying photosynthetic photon flux density (PPFD) by red-blue light source (6400-02B). Leaf photosynthesis under saturating light , stomatal conductance , transpiration , leaf-to-air VPD , air temperature (Tair) and leaf temperature (Tleaf) were measured at mid-day (between 0930 h and 1400 h) throughout the experiment, at PPFD of 1200 µmol m−2 s−1, [CO2] of 400 µmol mol−1 and mid-day growth temperatures. Besides, the ratio of intercellular to atmospheric [CO2] (Ci/Ca) and the instantaneous water use efficiency  were also calculated. Leaf dark respiration  was measured during the day after Asat measurements at zero PPFD, [CO2] of 400 µmol mol−1 and mid-day growth temperatures.Leaf gas exchange measurements were taken on recently, fully expanded leaves from four seedlings per treatment . Four seedlings per water treatment (n = 4) were randomly chosen for the measurements.On Days 10 and 11, when the growth chamber was heated along the temperature steps, the responses of leaf gas exchange to leaf temperature were simultaneously measured at PPFD of 1200 μmol mVcmax (µmol m−2 s−1) was estimated using the ‘one-point method’ which was described in Asat is the leaf photosynthesis under saturating light (µmol m−2 s−1); Ci is the intercellular CO2 concentration (µmol mol−1); Γ* is the CO2 compensation point in the absence of mitochondrial respiration (µmol mol−1); Km is the Michaelis–Menten constant, which is determined byKc is the Michaelis constant for CO2 (µmol mol−1); Ko is the Michaelis constant for O2 (mmol mol−1); Oi is the intercellular concentrations of O2. The determination of Γ*, Kc, Ko and Oi can be estimated according to The H, cm) was measured from the stem base to the highest shoot tip and basal diameter  was measured at 1-cm height. 2HD (cm3) was calculated as 2D times H, to estimate the aboveground growth  measurements were taken on one leaf from each of the four seedlings per treatment (n  = 4) during the daytime throughout the experiment using a Scholander-type pressure chamber . The leaves were cut with a blade, wrapped in moist paper towel and measured for Ψl immediately after collection.Leaf water potential . The SPAD values can reflect the relative chlorophyll content. Three leaves from each of six seedlings per treatments (n  = 6) were measured.Asat, gs, E, WUEi, Vcmax, Tair, Tleaf, Ψl, SPAD value and growth during the course of the experiment were analysed using a linear mixed-effects model (package ‘nlme’ in R), with water treatment (well-watered vs. drought), temperature treatment (ambient temperature vs. heat wave), and days as categorical fixed effects. Seedling number was treated as a random effect in all analyses. As we showed the data of SWC only in drought treatment, we analysed SWC with temperature and days as fixed effects and seedling number as a random effect. At each time step, the main and interactive effects of water and temperature on parameters were analysed using two-way ANOVA and followed by one-way ANOVA when interactive effects were significant. During the period of recovery from drought , Asat, gs, E, andΨl were analysed using a linear mixed-effects model, with temperature and days as fixed effects and seedling number as a random effect. Subsequently, Student’s t-tests were used to compare means between AD and HD treatments. The homoscedasticity and normality were checked prior to all the statistic analyses. In all cases, the results were considered significant if P ≤ 0.05.Statistical analyses were performed with R 3.2.2 . The pari.e. from Day 10 to Day 17) and returned to ambient temperature level afterwards. The leaf temperature varied in concert with air temperature throughout the experiment , growth parameters were not changed by the heat wave treatments. Hence, drought had a primary impact on seedling growth. At the end of the experiment (i.e. Day 22), heat wave had remarkably reduced seedling 2HD at both water conditions (Two-way ANOVA: P  = 0.017)  .Figure Asat of S. lycopersicum seedlings in AW control did not change too much over time, while Asat in HW treatment declined substantially as heat wave progressed, recovering to only 39 % of AW control in the end , Asat in droughted seedlings did not differ between temperature treatments, indicating that photosynthesis in response to drought was not altered by heat wave. By contrast, the recovery of Asat from drought was slower in HD treatment than AD treatment (t-test: P < 0.05). For instance, after two days rehydration, Asat in AD treatment almost returned to the value of the control, while that in HD treatment was only 71 % of Asat in AD treatment. the end , Table 1gs appeared to be one of the dominant factors in determining Asat responses . The time course responses of E were similar to those of gs in all treatments , while it had continuous declines as the heat wave was prolonged. Although Vcmax in HW treatment was recovered to some extent after the release of heat wave, it could not return to the control value even at the end of the experiment.achieved , Table 1riations . It was i.e. relative chlorophyll content) did not change too much in all treatments prior to the heat wave, while it had a pronounced reduction in the heat wave treatments (i.e. HW and HD) compared with the ambient temperature treatments (i.e. AW and AD), maintaining only 80% of ambient temperature values in the end  than the well watered treatments  .average) , Table 1Asat, gs and Rd of S. lycopersicum seedlings had weak but significant linear relationships with leaf temperature, only in HW treatment , gas exchange exhibited similar reductions between AD and HD treatment. After rehydration, however, seedlings in AD treatment exhibited sharper recovery of leaf gas exchange, particularly when leaf water potential was higher than -1 MPa. Generally, these results suggest that despite the fast recovery of leaf water potential, the recovery of leaf gas exchange lagged, to an even greater degree in HD treatment than AD treatment, suggesting the coordination of leaf gas exchange and leaf water potential was shifted between AD and HD treatments.The leaf water potential had a fast recovery from drought, returning to well watered control values in about 8 h after rehydration, similarly in AD and HD treatments , Table 2S. lycopersicum seedlings, and how leaf physiology recovered from the combined stress. Our results showed that the 7-day heat wave treatment (i.e. HW), as predicted in the first hypothesis, reduced growth, leaf gas exchange rates, Vcmax and water use efficiency compared with ambient temperature control (i.e. AW), while it did not have significant impacts on leaf water potentials. Compared with well watered conditions, drought stress led to stomatal closure, lower Vcmax, reduced growth and more negative leaf water potentials, similarly in AD and HD treatments. However, the observed lower relative chlorophyll content in HD treatment can partially support our second hypothesis that heat wave would exacerbate drought stress. Our results also demonstrated that recovery of leaf gas exchange lagged behind water potential recovery and leaf gas exchange exhibited much slower post-drought recovery in HD treatment than AD treatment, which agrees with our third hypothesis.This study investigated the individual and interactive effects of drought and heat wave on leaf physiology and growth of i.e. 42 °C) had negative impacts on the growth of S. lycopersicum seedlings, which is consistent with previous studies (e.g. R2= 0.76) illustrated that the high VPD associated with high temperature could have inhibited stomatal conductance. It has been demonstrated that the gradient in water potential between guard cells and epidermal cells rather than bulk leaf water potential, can affect stomatal responses to high VPD (The 7-day + 16 °C single factor heat wave (high VPD . Abscisihigh VPD .i.e. about 40 °C) could have caused leaf damage, thereby limiting photosynthesis. There is also evidence that Vcmax was substantially reduced in HW treatment compared with AW control towards the end of the heat stress, indicating that effects of non-stomatal limitation on photosynthesis were progressively enhanced. Together, it is suggested that the non-stomatal limitation can be a co-limiting factor affecting photosynthesis responses under heat stress and its contribution may vary depending on the duration of exposure to stress. The partial recovery of photosynthesis, stomatal conductance and Vcmax in the end indicated that the 42 °C heat stress had remarkably negative impacts on S. lycopersicum seedlings even after the heat stress was relieved. It is worth for further studies to quantify the relative contribution of stomatal and non-stomatal limitation on photosynthesis under stress and the following recovery, for better modeling plant responses to future climate change . Further studies are required to determine the detailed physiological processes and contributors during the recovery of plant from weather extremes. Altogether, our study demonstrated that heat wave largely affected the recovery of leaf gas exchange from drought in S. lycopersicum seedlings and highlighted the importance of studies on the interactive effects of drought and heat wave on plant recovery.More interestingly, recovery of leaf gas exchange was much slower in HD treatment than AD treatment, which suggests that previous imposed heat wave delayed the post-drought recovery of leaf gas exchange. This finding is in line with the recent study on black locust tree seedlings  and Jiangxi Provincial Department of Education  and Gan-Po 555 Talent Project.H.D., J.W. and H.F. conceived the experiment, H.D., X.Y. and Z.X. conducted the experiment, H.D., J.W., G.H, S.Z., W.L. and Y.L. analysed the results, H.D. wrote the manuscript with input from all of the other authors.None declared.The following additional information is available in the online version of this article —Figure S1. Leaf photosynthesis under saturating light (Asat) as a function of stomatal conductance (gs) of Solanum lycopersicum seedlings throughout the experiment. Data points are raw data of the measured variables. Data are fitted with exponential saturation functions: y  = a × (1−e(-bx)). The correlation coefficient (R2) in each treatment is 0.402 (AW), 0.744 (HW), 0.936 (AD) and 0.926 (HD), respectively. The fitted functions are not significant differences among treatments. Treatments: AW–Ambient temperature plus well watered; AD–Ambient temperature plus drought; HW–Heat wave plus well watered; HD–Heat wave plus drought.Figure S2. (A) Leaf-to-air vapour deficit (VpdL) and (B) ratio of intercellular to atmospheric [CO2] (Ci/Ca) of Solanum lycopersicum seedlings throughout the experiment. Values are means ± SE (n = 4–6). The grey area represents the period during which a 7-day 42 °C heat wave was applied. The vertical line represents the day when droughted seedlings were rehydrated. Treatments: AW–Ambient temperature plus well watered; AD– Ambient temperature plus drought; HW–Heat wave plus well watered; HD–Heat wave plus drought.Table S1. The raw data of leaf gas exchange and estimated Vcmax in this paper.Supplementary DataClick here for additional data file.