The primary impetus behind this research was to provide a boost to the characterization of the Italian olive biodiversity by acquiring reliable and homogeneous data over the course of an eight-year trial on the floral and fruiting biology of 120 molecularly analyzed cultivars, most of which have either low or very low diffusion. The obtained data highlighted a considerable variability to almost all of the analyzed parameters, which given the uniformity of environment and crop management was indicative of a large genetic variability in the accessions under observation, as confirmed through the molecular analysis. Several cases of synonymy were reported for the first time, even among plants cultivated in different regions, whilst all of the varieties examined, with only one exception, showed very low percentages of self-fruit-set, indicating a need for the employment of suitable pollinator plants. Eventually, a fitted model allowed us to evaluate the clear effects of the thermal values on blossoming, particularly in the months of March and April, whereas the climatic conditions during the flowering time had only a modest effect on its duration. Olea europaea L.) usually blossoms rather abundantly, with up to half a million flowers per plant [The olive tree . Lack of precipitation, especially in summertime, was offset by a subsurface drip irrigation system. The field is maintained in accordance with sustainable management, with a vegetation cover (mowed in summertime), occasional fertilization using ternary fertilizers, and light annual pruning. The meteorological data  for the phenological model were collected in situ by means of a meteorological station. The meteorological trend over the eight years of study is reported in Measurements for each parameter were performed on five plants (four plants for rare cases of plants that are either heavily diseased or explanted) for each of the 120 cultivars selected, and were repeated for eight consecutive years, from 2000 to 2007, in order to account for any anomalous values in production and the influence of climatic conditions. The parameters considered are reported below.Visual observations were carefully conducted during the flowering phase (March and April) throughout the eight consecutive years through randomized comparisons of the differentiation percentages of the flower buds of a similar number of the plants’ tagged twigs. Specifically, every year at the time of inflorescence formation, an adequate number of twigs were tagged on the plants for a total of over 3000 inflorescences per variety. Meanwhile, an adequate number of twigs in blossom bearing an equivalent number of inflorescences were bagged in such a way as to enable the assessment of the fruit set from self-pollination.The plants were divided into four classes: those without inflorescences (or at most with an insignificant number of inflorescences), plants with a low blossom profile (1 to 30% flower differentiation), plants with intermediate flowering (30–60% differentiation), and plants with an elevated blossom percentage (60–90% or higher).The observations were carried out three times a week over the relevant period (April–June). The flower development stages were recorded according to the BBCH -scale . In partIn the stage prior to flower opening, a variable number of flowering buds  were cut so as to have at least 200 blossoms per variety every year (which correspond to an average of over 3400 flowers for every variety) before being transported to the laboratory to tally the length of each inflorescence, the number of floral sprouts per inflorescence, as well as the pistil abortion percentage.Fifteen to 20 days after the blossom process had concluded (in June), the number of small fruits present on the tagged twigs as calculated along with the percentages of fruit set per inflorescence and per number of fertile flowers. At the same time, the bags were removed, and as mentioned above, the small fruits on the selected twigs were counted and compared with the total number of flowers and flowering buds. Eventually, the self-pollination index (SI) was calculated by taking the percentage of small fruits obtained through self-pollination and dividing it by the percentage of those obtained through open pollination. Values nearing 1 indicate a heightened degree of self-fertilization; values equaling or approaching 0 indicate a self-sterile variety.The summer fruit drop was evaluated between July and September depending on the variety, observing the number of small fruits that remained on the tagged small branches during the first growth phase.The harvest of the drupes took place between October and December (depending on the variety) in order to have the olives at the same degree of veraison. Between 30 and 40 drupes per plant were weighed so as to have at least 150 values per variety each year.Cultivar-dependent variation of flowering time was further characterized by means of a phenological model fitted to observational full flowering dates. In this way, the cultivar earliness classification was enriched with physiologically based information by providing quantitative estimates of the specific thermal requirements.The model was chosen among a set of 11 candidate models of varying complexity, which are listed in Sampled leaves were dried using silica gel ground to a fine powder and then stored at −80 °C until the time of analysis. Total genomic DNA was extracted from leaves using a commercial kit . The DNA quality was checked with a NanoDrop 2000 spectrophotometer . A set of twelve labelled microsatellites (SSRs), most of them widely used in literature, were chosen based on their amplification consistency via polymerase chain reaction (PCR), polymorphism, and ease of allele scoring in both conventional and multiplex amplification strategies ,33,34,35Two reference varieties (Leccino and Frantoio) were included in PCR amplification to check experimental conditions (data not shown). The GeneScan 500 LIZ  was used as internal standard, and amplification products were separated on an ABI PRISM Genetic Analyzer 3130xl . The allelic assignment was performed using GeneMapper 3.7v software. Standardization of raw data was conducted in comparison to the authenticated molecular profiles of Leccino and Frantoio reference varieties in accordance with Ben Mohamed et al. .Genetic diversity was evaluated using a cluster analysis of the 120 SSR profiles scored. A similarity matrix using Dice’s coefficient ,42 was fThe Wright’s inbreeding coefficients, Fis, Fit, and Fst, and gene flow (Nm) estimates were calculated using PopGene 1.32 .A population structure analysis was also conducted using STRUCTURE v. 2.3.4 software  to estabEventually, a parentage analysis was computed using Cervus v.3.0.7 software. An approach based on the LOD (logarithm of the odds) score significance was adopted and the following parameters were run: (i) number of offspring: 100,000; (ii) number of candidate parents: 120; (iii) proportion of candidate parents sampled: 0.4; (iv) proportion of loci typed: 0.7208. Default values were adopted for the parameters “proportion of loci mistyped” and “error rate in likelihood calculations”. The relaxed and strict confidence levels were set to 95% and 99%, respectively.The amount of flowering was high in more than half of accessions, with variable percentage of flower differentiation ranging from 60% to over 90% of buds. The highest and most constant values were in the cultivars of Aitana, Cavalieri, I77, Nasitana a frutto grosso, Ogliastro grande, Olivone di Viterbo, Ritonnella, San Benedetto, and Tombarello.Fifty-five varieties showed a medium amount of bloom, whilst the flowering was low in only two varieties (Cornia and Zarbo), an indication of late entry into production.The duration of the interphase, i.e., the time needed to pass from the beginning to full flowering, is almost constant amongst the varieties and over the years, equaling 6–7 days .The stable relationship amongst flowering phenophases indicates the presence of a mechanism regulating the development pacing, which was further analyzed through modeling analysis (see below).A negative correlation (Pearson’s r = −0.84) was observed between the timing of BBCH60 (as well as BBCH65) and the average temperature in March, namely during the phase of development of the flower buds. In confirmation of this, an even higher correlation was found between the accumulated growing degrees (growing degree-days -GDD-) for the month of March and BBCH60 (r = −0.87). Growing degree-days for the month of March have been calculated with the formula:and 9 °C ,50,51.2 = 0.81, where y is the beginning of flowering (number of the day in the year) and x is the GGD for the month of March. A rather similar correlation (Pearson’s r = −0.75) was observed between BBCH60 and rainfall in March. This may suggest that in the flowering-inducing hot period, given the same temperature, a greater amount of rainfall favors an earlier opening of the flowers as a likely effect of factors such as an increased absorption of nutrients from the roots.A polynomial equation can be proposed here as:m is the mean and s is standard deviation. The Bernoulli distribution was further employed as a reasonable criterion for unambiguous classification of M + L or M + E varieties, where it provides the probability that an event having an a priori probability of occurring in a test may occur x times in N repeated tests by pure effect of the case. Based on these considerations, we could reasonably classify the following varieties.Once it was established that the annual data were normally distributed, we established the lower and upper limits in order to divide the varieties into “early” (E), “medium” (M), and “late” (L) for each year. With this criterion, each variety is identified by a string—a series of eight combinations (one for each year) of characters E, M, and L. The threshold value was identified according to two parameters: (i) the probability (α) associated with the event was sufficiently low; (ii) each variety presented a maximum of two characters simultaneously (M + L or M + E) during the eight years. In our case, the optimal probability value corresponded to α = 9% and the fixed limits were: Early varieties: Abunara, Caprina di Casalanguida, Dolce di Andria, Giusta, Provenzale, Rosciola Coltodino, Riminino, Sammartinenga, San Benedetto, Spezzanese, Tonda dolce, and Paesana Bianca;Late varieties: Ascolana dura, Aurina, Caprina Vastese, Carpellese, Erbano, Fosco, Gnagnaro, Grossa di Venafro, Morellona di Grecia, Palmarola, Resciola di Venafro, Saligna, and Santa Maria.The flowering phenogram  highlighThe same methodology used to set the optimal threshold for the identification of early and late flowering varieties was implemented to investigate the overall duration of flowering (BBCH60-BBCH69 interphase) and whether some olive varieties have intrinsic characteristics of “short flowering” or “long flowering”. Again, a 9% probability threshold allowed us to distinguish 15 varieties with a short duration of flowering, including Aurina, Gnagnaro, Morellona di Grecia, Procanica and Rizzitella. Eighteen cultivars had a longer flowering period, amongst them Abunara, Dolce di Andria, Gentile dell’Aquila, Rotondella campana, and Tunnulidda, while 87 showed medium flowering. The varieties of Resciola di Venafro Aurina, Caprina Vastese, Fosco, Gnagnaro, Morellona di Grecia, Resciola di Venafro, and Santa Maria presented both late and the short flowering features, whilst the varieties Dolce di Andria, Provenzale, Sammartinenga, San Benedetto, and Spezzanese had early and long flowering. In general, data analysis relating to timing and duration of flowering highlighted a certain trend towards long flowering for the cultivars with early anthesis and typically short ones for late-blooming varieties.The length of the inflorescences was on average 29.6 mm, ranging from 20 (Olivo di Castiglione) to 46.65 (Riminino) mm. Regarding the number of flowers per inflorescence, this parameter varied from 10.34 to 28.78, with a general average being equal to 17.59 ± 3.86.Amongst the varieties with small inflorescences were Cornia, Olivo di Castiglione and Pennulara; those with a higher number of flowers were Grossa di Venafro, Riminino and Olivone di Viterbo, all with an average of over 28 flowers.The percentage of pistil abortion for these 120 varieties turned out to have extreme variability, from 1% to 90%, with a general average of 38.72%.The varieties with a lower incidence of morphological sterility included Procanica (1.07%), Crognolo (1.41%), Remugnana (2.26%), and Palmarola (2.60%). Those with a higher percentage of abortion were Oliva grossa (90.75%), Sivigliana da olio (89.24%), and Olivone di Viterbo (88.93%).The fruit set by open pollination, expressed in fruits formed per hundred inflorescences, was extremely diversified, varying from 2.27% to 115.48%, with a general average of 44.86%.The varieties with the highest productive potential —with percentages of fruit set over 100%— were Grappolo (115.48%), Racioppa della Basilicata (107.62%), and Arnasca (103.34%), followed by Nebbia, Procanica, and Caprina Vastese, with fruit sets over 90%.The varieties Oliva grossa, Pizzutella, Ritonnella, Oliva da mensa, Vigna della Corte, and Nasitana a frutto grosso showed the lowest fruit sets at below 15%.If fruit set is expressed in drupes formed per 100 fertile flowers, a parameter calculated to evaluate the combined incidence of the cytological and factorial forms of sterility, then the values obtained were obviously lower, and the variability reduced, ranging from 1.04 to 16.67%.The varieties with fruit set per fertile flower at over 10% were Pesciatino, Olivastro Frentano, Sivigliana da olio, Olivone di Viterbo, Tenacella, and Grappolo; those with fruit set below 2% were Pizzutella, Nasitana a frutto grosso, Oliva grossa, Lumiaru, Aitana, Vigna della Corte, Olivo di Castiglione, and Oliva di Casavecchia.The percentage of self-fertility for the accessions considered in this study, intended as drupes formed per 100 bagged flowering buds, varied from 0 to 33.76.Nine varieties displayed a total self-incompatibility, namely Ascolana dura, Caiazzana, Dolce di Cerchiara, Faresana, I77, Oliva grossa, Ruveia, Sammartinenga, and Sivigliana da olio; only 10 varieties had a fruit set scope exceeding 10%, and of these, only two surpassed 30%,namely Cellina di Rotello and Tenacella.These data clearly indicate that all 120 varieties are associated with an adequate number of suitable pollinator varieties.Fruit set from self-pollination, expressed as fruits formed per 100 fertile bagged flowers, varied from 0 to 4.64; only 11 varieties surpassed 1%, and the ones with higher values were Pennulara (2.38%), Olivone di Viterbo (2.49%), and Tenacella (4.64%).This index generally yielded very low results. Indeed, 22 varieties had a result equal or close to zero, 93 had a result below 0.4, and four were between 0.4 and 0.5; one exception is the Calabrian variety Pennulara, which registered a value of 1.01, indicative of a self-fertility identical to open fertilization, and therefore not in need of pollinizers.The summer fruit drop ranged between 0  and 83.69% (Biancolilla), with an average value of 32.43%, confirming this phase as a crucial step for overall production.This parameter varied from 0.79 (Gnagnaro) to over 6 g , with an average value of 3.13 g.In terms of prediction accuracy, i.e., the capacity to predict the flowering date, all the models behaved better than the null model . The traThe model was re-optimized using the whole 8-year dataset to obtain a final parameterization. The range of GDD sum, expressing the thermal requirements of the cultivars, was plotted in The thermal requirement varies between 1251 and 1381 °C d.Based on the optimized thermal time, the earliest cultivar (GDD sum < 1260) were Tonda dolce, Rosciola coltodino, Giusta, Spezzanese, Dolce di Andria, San Benedetto, Sammartinenga, and Caprina di Casalanguida, while the latest ones (GDD sum > 1350) were Grappolo, Piangente, Grossa di Venafro, Saligna, Cellacchia, Santa Maria, Ascolana dura, Erbano, Racioppa, Fosco, Aurina, Gnagnaro, and Carpellese.P < 0.001), ranging between 188 and 316. Cultivars with the highest thermal requirements (GDDs > 300 °C d) were Cacaredda, Rustica, and Tunnulidda, whereas those with the shortest requirements (GDDs < 195°C d) were Lumiaru, Santa Maria, Aurina, Caprina vastese, and Tenacella.The thermal requirements to complete flowering  were variable among the cultivars  was on average 1.64. indicating that a gene movement occurred among populations.Allele frequencies varied from a minimum of 0.0042 to a maximum of 0.52, with at least one rare allele for almost all the loci, aside from the locus UDO12. The most frequent allele was 206 bp at the locus DCA5. Unique alleles found and the corresponding varieties are shown in The dendrogram was divided in two main groups . The firFor completeness of information, considering a wider dataset of over 300 Italian varieties whose genetic profile has been previously characterized in our laboratories , the culThe population structure analysis detected two main groups (K = 2), termed “Red” and “Green”, and a few admixed accessions. Overall, this is consistent with the cluster analysis. The “Red” group included most of the cultivars from Abruzzo, Basilicata, Campania, and Sicilia, as well as all of the olive cultivars from Calabria . With thA correspondence of this genetic division into two major groups has been sought for the physiological characteristics through means of hierarchical clustering . PearsonThe non-exclusion probability between two unrelated individuals (NE-I) and two hypothetical full siblings  ranged from 0.021 (DCA9) to 0.34 (OLEST23) and from 0.31 (DCA9) to 0.6 (OLEST23), respectively. Parentage analysis revealed critical LOD scores of 18.99 and 16.43 for the parent pair analysis, with unknown sexes for strict (99%) and relaxed (95%) confidence levels. The main putative pairs of parents, with a maximum of one mismatch for each one, are shown in Of note is the finding that three Sicilian cultivars, Aitana, Abunara, and Zarbo, are very likely siblings; their putative parents were Giarfara and Scarpetta from Sicily and Basilicata, respectively.An overall examination of all the reported data highlights that almost all of the analyzed parameters showed considerable variability, which given environmental and crop-management uniformity, is indicative of a large genetic variability in the accessions under observation.All of the examined varieties, with one single exception (Pennulara), showed low percentages of self-fruit-set, indicating the need for the employment of suitable pollinator plants, whereas the flowering time amongst the different cultivars in the same environment was found to vary from three to four weeks .On average, the fruit set per flowering bud was of an order 10 times greater than that per fertile flower, be they open-pollinated or self-pollinated, whilst the fruit set by open pollination was approximately 13 times greater than that from self-pollination. This confirms, from an agronomic point of view, the need for the presence of pollinators, and particularly the need for the selection of suitable pollinators, whereas some cultivars are genetically or physiologically  inter-incompatible ,55. For The data on the fruit set calculated per fertile flower highlighted that regardless of the imponderable likelihood of pollen grains settling on the fertile flower during receptivity, the aspects linked to cytological and factorial sterility—already described for olive trees —quite siWith regards to the correlation between the number of flowers per inflorescence and pistil abortion, a positive and statistically significant correlation of 99% was found . This coFitting the GDD model on the whole dataset allowed us to rank cultivars according to the thermal requirement required to reach full flowering. Overall, the GDD requirement ranged between 1090 and 1380 °C per day, but it is to be noted that two-thirds of the cultivars under study (67.5%) exhibited a much narrower variation, between 1292 and 1330 °C per day . As a coAnother aspect to be evidenced is that fitting the GDD model converged to base temperatures between 1 °C and 4.5 °C (not shown), with 2.25 °C as the average. In general, higher temperatures are reported, varying from 6 °C to 12.5 °C ,59.Calculation of base temperature may give varying results depending on the calculation methodology, and currently there is no agreement as to the best values to use ,61,62.Duration of flowering, expressed in thermal time, was also found to be cultivar specific, with no apparent correlation with earliness, unlike what was found when the period was expressed in calendar days. A likely explanation of this latter effect is that when flowering starts early it takes longer to complete because air temperature is colder, while late flowerings occur in a warmer period, which makes them shorter.The analysis of the genetic variability and population structure confirmed a high genetic variability and low degree of differentiation (only two groups), with no clear geographic division, apart from the Calabrian varieties. Our results only partially overlap with those of Muzzalupo et al. . This woSome of the synonymies recorded in this work have previously been partially described ,53, althEventually, the normally distributed physiological variables of the varieties found to be synonyms were subjected to a principal component analysis  to evaluThis work provides an important contribution to the characterization of the Italian olive biodiversity through the study, at both molecular and physiological levels, of 120 varieties that have traditionally been marginalized. The bio-agronomic characterization provides a basic understanding of the varieties of olive, which is indispensable for not only proper classification, but also for the design and development of future studies that, given the incredibly rich germplasm, are definitely needed in order to increase knowledge about this valuable species. For instance, the identification of a self-fertile cultivar represents a notable datum within what is generally considered a self-sterile species. Furthermore, a greater knowledge of the floral and fruiting biology might allow for the rediscovery and revaluation of many of these varieties, for the purpose of eventually reintroducing them into their areas of origin (or taken beyond to other areas), or for utilization in future breeding programs. In the same regard, the proper varietal identification using molecular markers is an extremely important requirement for the use and marketing of propagation material of agricultural plants. Finally, the observations, based on a large number of varieties and repeated over an eight-year period, aside from providing interesting information on individual cultivars, have allowed us to draw up general rules on the floral biology of the olive tree.


CCPG1 transcripts are inducible by ER stress, providing a direct link between ER stress and reticulophagy. In vivo, CCPG1 prevents the hyper-accumulation of insoluble protein within the ER lumen of pancreatic acinar cells and alleviates ER stress. Accordingly, CCPG1 loss sensitizes the exocrine pancreas to tissue injury.The importance of selective macroautophagy/autophagy in cellular health is increasingly evident. The selective degradation of portions of the endoplasmic reticulum (ER), or reticulophagy, is an emerging example but requires further mechanistic detail and broad evidence of physiological relevance. In a recent study, we identified CCPG1, an ER-resident transmembrane protein that can bind to Atg8-family proteins and, independently and discretely, to RB1CC1/FIP200. Both of these interactions are required to facilitate CCPG1's function as a reticulophagy cargo receptor.  The endoplasmic reticulum is a key intracellular organelle that oversees the synthesis and quality control of transmembrane and secretory proteins. If environmental conditions become unfavorable or the ER becomes overwhelmed, proteins can become misfolded and are retained in the ER lumen. This can be defined as a form of ER stress. The best understood mechanism for resolving ER stress is the unfolded protein response (UPR). The UPR employs sensors that detect unfolded protein levels in the ER lumen and activate downstream signalling cascades in the cytosol and nucleus. This leads to inhibition of global translation and transcriptional upregulation of a subset of genes that act to restore ER homeostasis.ATG genes, there is no evidence for this having a specific effect on the ER. In recent years, our understanding of the selective degradation of the mammalian ER, known as reticulophagy, has grown with the discovery of a number of reticulophagy receptors, namely RETREG1/FAM134B, SEC62 and RTN3. Despite this, no direct link has been proposed between ER stress, the UPR and the induction of reticulophagy.Whereas it is known that the UPR drives increased global autophagic flux through the transcriptional upregulation of both Atg11 and Atg8. In the case of CCPG1, the dominant FIR has sequence homology to the Atg11-binding consensus sequence of yeast SARs. Additionally, CCPG1 binds to a C-terminal portion of RB1CC1 that encompasses its Atg11-homology region. These observations raise the possibility that CCPG1 employs an analogous mechanism to that of yeast SARs. In yeast, the interaction between SARs and Atg11 allows recruitment of active Atg1 to the cargo. This should be tested for CCPG1 and ULK1 recruitment.We recently identified CCPG1 in an affinity purification-mass spectrometry screen for GABARAP-interacting proteins. CCPG1 is an ER-resident, vertebrate-specific, single-pass transmembrane protein that possesses both cytosolic and ER-lumenal regions . CCPG1 tRetreg1/Fam134b mutant mice display a loss of maintenance of cellular health of peripheral sensory neurones, which correlates with distended ER in these cells. However, a role for reticulophagy in most organs remains unaddressed. Thusly, Ccpg1 hypomorphic mice were generated. Prior to this, we had also shown that CCPG1 transcripts were inducible by ER stress, in human established cell lines in vitro. Thus, in vivo, we decided to look at the acinar cells of the exocrine pancreas, which, due to their high biosynthetic workload, are under constitutive low-level ER stress. Ccpg1-deficient mice exhibit hyper-accumulation of insoluble protein within the ER lumen of these cells. This is associated with elevated, sustained levels of ER stress, tissue injury and an inflammatory response in the pancreas (The physiological importance of reticulophagy requires further elucidation. Most evidence for ER disruption following autophagy loss comes from models where all autophagy has been removed. As such, interpretation of the effects on ER that are observed is often confounded by other, clearly primary, effects, such as loss of mitophagy and the subsequent bioenergetic compromise of the cell. A more direct study showed that pancreas . We therCCPG1 gene is inducible by the UPR, providing insight into how cells are able to respond to ER stress through reticulophagy. In vivo, CCPG1 is required to maintain the health of pancreatic acinar cells and, as such, future work in disease-state scenarios, such as pancreatic cancer formation, which is regulated by autophagy status, will further illuminate the role of CCPG1 and reticulophagy in health and disease.In summary, CCPG1 constitutes an example of a noncanonical reticulophagy cargo receptor, due to its functional requirement to bind Atg8-family proteins as well as RB1CC1, independently of one another. The


We aimed to conduct a meta-analysis concerning the frequency and risk factors of reduced bone mineral density (BMD) in systemic lupus erythematosus (SLE) with evidence from published studies. A comprehensive literature search was conducted based on the EMBASE, Web of Science, PubMed, and Cochrane Library databases up to March 5th, 2017. Eligible studies reported any prevalence of reduced BMD in SLE patients. All risk factors with odds ratios or risk ratios associated with reduced BMD were extracted. 71 reports with 33527 SLE patients were included. Low BMD, osteopenia, and osteoporosis at any site were presented, respectively, in 45%, 38%, and 13% of the SLE patients. The prevalence of osteoporosis increased with the advancing of age, while U-shaped associations between age and the prevalence of low BMD and osteopenia were found. Lumbar spine was indicated to have higher prevalence of osteoporosis. Age, disease duration, drugs use, and many other factors were identified as predictors of reduced BMD. Low BMD, osteoporosis, and osteopenia appeared to be prevalent in patients with SLE. Risk factors of reduced BMD were various. Nowadays the long-term complications of systemic lupus erythematosus (SLE) have become great concerns as the survival of SLE has improved dramatically . PublicaRisk factors associated with decreased BMD are still under debate in SLE patients. SLE occurs commonly in females , 9. WomeThe aim of this study was to conduct a meta-analysis concerning the frequency and risk factors of osteopenia, osteoporosis, or low BMD in SLE patients with evidence from published studies.This study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) Checklist  Supplem. S1 fileWe performed a comprehensive literature search based on the PubMed, Web of Science, Cochrane Library, and EMBASE up to March 5th, 2017. We used the keywords including “(Systemic Lupus Erythematosus or SLE)” and “” and “” . S2 fileWe included published original articles and data on prevalence of decreased bone density, osteoporosis, or osteopenia in SLE patients. There was no restriction of language. Letter, review, conference abstract, editorial material, comment, case report, meta-analysis, and book chapter were excluded. If two articles from the same population reported different data, we included both of them. Otherwise, if the articles reported the same prevalence, we included the article with the larger sample size. In a longitudinal study that reported prevalence of decreased bone density in different time points, we included the baseline data.We extracted the information of interest by two authors (Y.Y. and J.M.X) from each study including study characteristics  and any prevalence  of low BMD  in SLE patients at any part of body. We classified the prevalence according to different sites and different degree of decreased bone density. If a study measured three or more sites of any one patient and gave whole prevalence of osteoporosis, osteopenia, or low BMD for this population or reported the prevalence without stating the sites, we classified this prevalence as “at any site.”χ2 based on Q-statistical test and quantified by I2 index. Roughly, heterogeneity was considered significant at P<0.10 and Higgins I2 values of 25%, 50%, and 75% were considered low, moderate, and high inconsistencies. Results of studies were pooled by random-effects models in the presence of high heterogeneity among our studies. All analyses and graphs were made using Stata 10.0  and GraphPad Prism 6. P values < 0.05 by two-tailed test were considered significant.Heterogeneity of prevalence across included studies was examined using We identified 1233 articles by systematic electronic searches; 70 of those were eligible for this meta-analysis. And 1 study was included through hand-searching the reference lists of all identified eligible papers , no matter in premenopausal patients (40%) or in postmenopausal patients (43%). The prevalence of osteopenia in all patients and premenopausal patients and postmenopausal patients was 38%, 42%, and 25%, while prevalence of osteoporosis was 13%, 9%, and 21%, respectively.Higher prevalence of osteoporosis was shown at lumbar spine 13%) compared with the femur (6%) and hip (4%) in our study, which was consistent with some observational studies , 3, 17. % compareSeveral factors may explain the prevalence of low BMD in SLE patients.α [First, SLE was a chronic autoimmune disease with multiorgan inflammation. The mean disease duration of SLE was 8.5 years and the mean SLEDAI score was 4.6 in our analysis. There were many disease-related variables playing a role in bone health. Increasing of tumor necrosis factor-α , interleα , and intα  in the sα .β-hydroxysteroid dehy- drogenase type 1 [Second, corticosteroid therapy was frequent among SLE patients. From our literature search, the percentage of corticosteroid use was reported to be from 51.6%  to 100% e type 1 , and othThird, postmenopausal status could lead to bone loss. In our study, postmenopausal patients  showed higher prevalence of osteopenia, osteoporosis, and low BMD in almost all sites. Most of our SLE patients undergo early menopause, which may be due to SLE disease per se or its treatment with cytotoxic substances , 40. PosFourth, degeneration of bone increased with age. Older age was independently associated with bone loss in both general population  and SLE Other risk factors might have effects on bone health in SLE patients. (1) The use of antimalarials might influence cytokines, lysosomal membranes, DNA, antigen processing, and other mechanisms that might lead to loss of bone. (2) The effect of body mass index (BMI) was still under debate. It was reported to be associated with increased risk of vertebral fracture  , but LeeWe acknowledged some limitations of our analyses. Firstly, BMD measurements were performed in SLE patients with different disease duration. Secondly, the treatments for patients varied greatly. Thirdly, the differences regarding race, genetics, geographical locations, and lifestyle across the different population studied were not well considered. Fourthly, few studies reported the bone condition in male SLE patients, so we failed to extrapolate gender-related differences. Finally, the heterogeneity was high for most of the analyses.SLE patients were at a great risk of developing low BMD, especially in lumbar spine and the postmenopausal patients. The risk factors that associated with low BMD might include low body weight, menopause duration, age, and disease-related factors.


Objective: To investigate the relationship between serum leptin and reproductive hormones in females with unexplained infertility (UI).Methodology: It was a case-control study conducted in the Gynecology and Obstetrics Department and Infertility Clinic, Jinnah Postgraduate Medical Center (JPMC), Karachi, Pakistan. A total of 235 primary infertile females with an unidentified cause of infertility were selected from the Infertility Clinics. The patients were excluded if they were found to have polycystic ovary syndrome, endometriosis, tubal blockage, irregular menstrual cycles, hyperthyroidism, hypothyroidism, hyperprolactinemia, hyperandrogenemia, fasting blood sugar >110 mg/dl, and male factor infertility. A total of 205 healthy, fertile females were selected from the general population. The blood samples of both groups were collected on the 12th and 21st day of their menstrual cycle. Serum leptin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and E2 levels were measured. Statistical analysis was executed using the SPSS version 16 .Results: No significant difference was observed in leptin values of fertile and UI females, 37.110±1.19 vs. 35.321±0.901. In the preovulatory phase (12th day) of the cycle, infertile subjects with body mass index (BMI) <20 and 20-24.9 had significantly higher values for leptin (p<0.05), whereas, with an increase in BMI, leptin levels were reduced in these females. Leptin was reduced further in the luteal phase of infertile females with BMI 25-30, with a significantly lower value for FSH (p<0.005), LH (p<0.005), and estradiol , BMI , and progesterone .Conclusion: Low levels of leptin observed to have an increase in the BMI of UI females were associated with a reduced estradiol and progesterone production in the luteal phase of the cycle.  Infertility is the inability of a sexually active couple, not using any contraceptive techniques in a year or more, to get pregnant . InfertiIt seems that the central and peripheral signals of the hypothalamic-pituitary-gonadal axis play an important role in the action of leptin in the menstrual cycle. Although the physiological impact of the alteration in the plasma leptin during the menstrual cycle is uncertain, it emerges that the lowest threshold of the circulating leptin is essential for a normal ovulatory function . TherefoHigher leptin levels are considered to be the cause of infertility . On the The present study was carried out at the Department of Biochemistry, University of Karachi, Karachi, in collaboration with the Atomic Energy Medical Centre, Jinnah Postgraduate Medical Centre (JPMC), Karachi.A total of 235 primary infertile female subjects were selected from the Infertility Clinic, JPMC, Karachi, Civil Hospital Karachi, Sobhraj Maternity Center Karachi, and Infertility Consultants. A total of 205 healthy female subjects were included in the study and were selected from the general population. The sample size was calculated using Open Epi software. It estimated 220 infertile females based on a 5% level of significance and 80% power of the study with a mean difference of serum leptin levels of -1.5 among fertile and infertile females. The approval for this research was granted by the Board of Advanced Studies and Research (No. BASR/9728/Sc), University of Karachi, Karachi, Pakistan, and subjects' anonymity was maintained, and strictly followed the Declaration of Helsinki. For this research, spanning a period of two and a half years, more than 1,000 infertile couples were interviewed and after taking their history and thorough investigations, 235 subjects were selected. Those subjects that were not fulfilling the inclusion criteria, due to the involvement of the male factor, PCOS, endometriosis, tubal blockage, irregular menstrual cycles, hyperthyroidism, hypothyroidism, hyperprolactinemia, hyperandrogenaemia, and fasting blood sugar >100 mg/dl, were excluded from the study. The selected women had no endocrine syndromes, and their husbands had proper parameters of sperm. Only those primary infertile female subjects, whose husbands were neither azoospermic nor oligospermic, that is, with sperm motility >70%, were selected. The blood samples were collected on the 12th and 21st day of their menstrual cycle. The inclusion criteria for infertile subjects were being between 20 and 40 years of age, primary infertile female, no uterine or pelvic pathology, no known cause of infertility, females having a regular menstrual cycle, and her husband having normal sperm parameters (normospermic and sperm motility >70%). We excluded the infertile subjects if they were suffering from PCOS, hirsutism, blocked ovarian tube, thyroid disease, being a diabetic patient, suffering from hyperprolactinemia, oligospermic or azoospermic husband, and husband’s sperm motility <50%. Their blood pressures were measured to check if they had a history of the disease, or any other disease, such as sexually transmitted diseases, hypertension, tuberculosis, and so on.The inclusion criteria for fertile subjects were being female and having one or more children (control group), age ranging from 20 to 40 years, no uterine or pelvic pathology, and females having a regular menstrual cycle. The exclusion criteria for fertile subjects included diabetes, PCOS, hirsutism, thyroid disease, galactorrhea (hyperprolactinemia), pregnancy, lactation, and irregular and prolonged menstrual cycle.All selected infertile female subjects and control fertile female subjects were grouped according to their body mass index (BMI) and were further grouped according to their phases of the menstrual cycle, that is, follicular and luteal phases. Group A included infertile subjects having BMI <20, group B included infertile subjects having BMI <25 (20-24.9), group C included infertile subjects having BMI <30 (25-29.9), and group D included infertile subjects having BMI >30.The general and physical examination of subjects was done by measuring the height, weight, and hip and waist circumference of subjects, in order to determine the BMI and waist/hip (W/H) ratio. Subjects were weighed in light clothing and height was recorded, along with waist circumference measured at the level of the umbilicus and the hip circumference measured at the level of the greater trochanter. BMI (weight in kilogram/height in square meter) and W/H ratio was calculated using the formula. To characterize both groups of women, we gathered detailed information concerning their age, social background, jobs, and the length of time they have been trying to conceive. After obtaining written consent from the patients and normal subjects, 10 ml of blood was drawn by venipuncture from each subject in the follicular phase and 5 ml in the luteal phase of the menstrual cycle, the morning after an overnight fast (12 to 14 hours) using disposable plastic syringe under aseptic measures.oC for subsequent analysis. Samples were analyzed in the batches of a 100 to omit in between analytical variations. Before analysis, samples were allowed to attain room temperature.The blood was collected in a test tube and centrifuged for the separation of serum within an hour of blood collection, and then, the serum was stored in a deep freezer at a temperature of 70Serum estradiol, progesterone, FSH, LH, testosterone, T4, T3, TSH, and prolactin were measured by radioimmunoassay, using kits supplied by the Diagnostic System Laboratories . Immunoradiometric kit (DSL) was used for assaying serum leptin levels. The sensitivity of leptin IRMA kit (lowest detectable limit) was 0.1 ng/ml, and the intra- and interassay coefficients of variations were less than 8%. Glucose determination was done immediately by the enzyme oxidase method.A total of 235 primary infertile (UI) female subjects and 205 healthy female subjects were included in the study. All selected infertile female subjects and control fertile female subjects were categorized according to their BMI and were further grouped according to their phases of the menstrual cycle, that is, preovulatory and luteal phases. No significant difference in serum leptin level was observed in both fertile and UI females, 37.110 ± 1.19 vs. 35.321 ± 0.901 ng/ml.Table Table In the luteal phase (21st day) of their menstrual cycle, significantly lower values for FSH (p<0.005), LH (p<0.005), and estradiol (p<0.005) were found in the UI subjects when compared with those of the control subjects. Comparison by BMI showed a significantly low FSH, LH, and E2 in infertile females of all BMI groups Table .In the preovulatory phase (12th day) of the menstrual cycle, a significant correlation of serum leptin was observed with estradiol  and the BMI  in the fertile group, whereas in the infertile group, the serum leptin level was correlated with estradiol  and BMI . In the luteal phase (21st day) of the menstrual cycle, a significant correlation of serum leptin was found with estradiol  and BMI  in the fertile subjects, whereas in the UI subjects, it correlated with estradiol , BMI , and progesterone r=0.146, p<0.05) . Wunder et al. have reported an increase in circulating leptin levels from the follicular to the luteal phase . AnotherChange in leptin levels can be considered as one of the factors responsible for causing infertility, as a result of its impact on estradiol (E2) production by human granulosa cells, in response to LH . An assoA study by Farooq et al. reported that serum leptin has a strong negative correlation with LH, FSH, and testosterone in the fertile obese as well as in normal males and females, which were statistically significant . This stAs serum E2 has a significant correlation with leptin in infertile subjects, it is likely that an increased fat mass may play some role in establishing a relationship between serum estradiol and serum leptin. Serum leptin levels of females with UI were similar to females with a PCOS, although a high leptin content was observed in the peritoneal fluid of the UI females . The perLimitation of the study is the consideration of BMI as a measure of body fat storage that masks the underlying excess adiposity that can be depicted by an increased percentage of fat and a reduced muscle mass. The study should have compared serum and follicular leptin levels. At the same time, cases of UI may be due to the contributions from minimal factors from both partners, as the borderline semen analysis and poor ovarian responsiveness were not excluded from the study.The increase in leptin levels during the luteal phase of the menstrual cycle and the occurrence of leptin receptors in the ovaries need to be further explored.An association of leptin and E2 levels in non-obese to obese females with UI was observed in our population. Low leptin levels can explain UI in obese females with reduced E2 and progesterone production, required for implantation of the fertilized ovum.


Brain abscess, a severe intracranial infectious disease, refers to the parenchyma abscess caused by local infection or remote spread. Recently, advancements in modern medicine, especially the wide application of antimicrobial drugs, have contributed to the gradual decrease in the prevalence of this disease. However, cases of cryptogenic brain abscess that feature an unknown origin and atypical symptoms are rising. In this retrospective study, we report and analyze two cases of cryptogenic brain abscess. The first patient was a 30-year-old healthy man who was admitted to our hospital due to 1 week of headache and 3 days of headache aggravation, accompanied by nausea and vomiting. Head MRI shows a circular space-occupying as well as apparently enhanced DWI signals were observed in the right parietal lobe, and the ring wall manifested an apparent increase in signal intensity after enhancement. The patient was diagnosed as a brain abscess before operation and given craniotomy. The postoperative pathology confirmed brain abscess and recovered well after surgery. The second patient was a 45-year-old healthy woman who was hospitalized in a local hospital due to symptoms of headache and right limb weakness for 1 week. Head MRI shows a circular space-occupying lesion in the left basal ganglia, and the ring wall manifested an apparent increase in signal intensity after enhancement. The patient was suspected of glioma at the local hospital and was transferred to our hospital. Twelve hours after hospitalization, the patient was suspected of developing cerebral palsy and thus underwent emergency surgery including lesion resection in the left basal ganglia, resection of the polus temporalis, and a decompressive craniotomy. Postoperative pathology confirmed brain abscess. The patient was eventually conscious, but left the right limb hemiplegia. Hence, when a patient develops the classical triad of fever, headache, and focal neurologic deficits, the possibility of brain abscess should be investigated. Early diagnosis and treatment are crucial to minimize various complications and the number of deaths. Brain abscess is a severe intracranial infectious disease that has a prevalence of 0.4–0.9 per 100,000 population  as well 9/L , 70.4%  neutrophils, 20.1%  lymphocytes, and C-reactive protein (CRP) level of 9.0 mg/L . A cardiac echo reminded the heart that everything was normal. Head magnetic resonance imaging (MRI), including a plain scan and an enhancement scan, showed the following: (i) irregularly circular, slightly long, aberrant T1 and T2 signal shadows with sheet-like, long T1 and T2 signals as well as apparently enhanced diffusion-weighted imaging (DWI) signals were observed in the right parietal lobe; (ii) an edge ring wall with a relatively even thickness presented as slightly short T1 and T2 signals; (iii) the ring wall manifested an apparent increase in signal intensity after enhancement; (iv) the lesion was surrounded by large patches of T1 and T2 oedema shadow; (v) the adjacent ventricles and parenchyma displayed compression-resulted deformation; (vi) the midline structures were slightly shifted toward the left  scan showed that the space-occupying lesion in the right parietal lobe had disappeared, the surrounding area had large patches of low-density shadows with a blurred boundary, and the midline structures displayed local left-shift  score of 15, a soft neck, normal limb muscular strength, normal deep, and superficial sensations. After hospitalization, a complete blood count reported a white blood cell count of 8.4 × 10 Figures –G. The p Figures . After tt Figure . The pat9/L, 72.7% neutrophils, 19.6% lymphocytes, and CRP level of 18 mg/L. A cardiac echo reminded the heart that everything was normal. After hospitalization, the patient received an intravenous infusion of 20% mannitol 250 ml q8h and was prepared for relevant preoperative examinations. Twelve hours after hospitalization, the patient fell unconscious and had a GCS score of 6, enlargement of the left pupil, loss of light response, avoidance of the left limbs upon needling, and slight buckling of the right limbs upon needling. An emergency head CT showed a spheroid shadow of slightly low density present in the left basal ganglia, the surrounding area had large patches of low-density oedema shadows, the left ventricle displayed severe compression-resulted deformation, and the midline structures exhibited a prominent rightward shift  an aberrant, circular space-occupying lesion with long T1 and long T2 signal shadows was observed in the left basal ganglia; (ii) a ring wall with a relatively even thickness presented as slightly short T1 and T2 signals; (iii) the shadow displayed apparent intensification after enhancement; (iv) large patches of long T1 and long T2 oedema shadows were present in the surrounding area of the lesion; (v) the left ventricle exhibited slight compression-resulted deformation; and (vi) the midline structures were shifted lightly toward the left Figures –D. The pt Figure . The patt Figure . After te Figure . HoweverBrain abscess is a severe, life-threatening infection. It is often found in young and middle-aged individuals, most of whom are male  . Its mosThe two patients in the current study had histories physical health and normal immunological function; patient 1 had no record of chronic otitis media or sinusitis, no definitive foci of infection, and no history of head trauma. Patient 2 had only the symptom of headache, and a laboratory examination did not find elevated levels of white blood cells, neutrophils, or CRP.N-acetylaspartic acid, choline, and cholic acid) but has elevated levels of cytoplasmic amino acids and lactic acid. In addition, some patients display concurrent elevations in acetic acid and succinic acid. These are classical resonance signs of an abscess cavity. Although lactic acid and lipid signals can be detected in cases of brain abscess and brain tumor, valine, leucine, and isoleucine are the key markers for diagnosing brain abscess. However, the absence of the three amino acids does not preclude the presence of brain abscess , no apparent increase in intracranial pressure, or multiple brain abscesses without apparent space-occupation . It is rPatient No. 1 had an abscess with an intact capsule. The abscess was on the surface of the brain tissue and not in an important functional area, and the capsule wall was relatively thick. Based on these findings, this patient received stereotactic resection, during which surgery towels were used to protect the surgical field. Because of the high brain pressure, approximately 20 ml of yellow, viscous pus was aspirated, with care taken to avoid spillage. After the brain pressure was relieved, the abscess was completely excised along the capsule edge. Patient No. 2 had an acute onset. Twelve hours after hospitalization, her condition worsened, and she became unconscious and exhibited other symptoms . Emergency surgery was performed including the resection of lesions in the left basal ganglia, the resection of the polus temporalis, and a decompressive craniotomy. Because of the relatively high brain pressure, the left side of the polus temporalis was first removed, followed by an apparent pressure reduction. Lesion resection was then performed. The brain tissue surrounding the lesion was clearly yellow, suggesting the possibility of brain abscess. Diagnostic puncture allowed the aspiration of approximately 10 ml of yellow, viscous pus; subsequently, the patient was diagnosed with brain abscess. The lesion was completely excised along the capsule edge, during which surgery towels were used protect the exposed tissue.Streptococcus. Streptococcus spp. and anaerobic cocci. Staphylococcus (21.2%) and Streptococcus (20.2%). These bacteria are often found in post-neurosurgery patients or patients with sinusitis or otitis media. Postoperative patients with brain abscess should receive intravenous antibiotics for 4–6 weeks. In principle, the administration of antibiotics should be maintained until the postoperative body temperature has remained at a normal level for 10–14 days. At this point, follow-up examination using CT or MRI should be performed. For patients with cystic space-occupying lesions, DWI is recommended. If the abscess cavity remains or DWI indicates that the abscess cavity is not close to the cerebrospinal fluid, then the intravenous administration of antibiotics should be continued, even when the body temperature is normal (The postoperative bacterial culture of either patient indicated a single microbe of s normal . In thiss normal , 2F.In summary, cryptogenic brain abscess is rare in people with normal immune function. However, when a patient develops the classical triad of fever, headache, and focal neurologic deficits, close attention should be paid to his or her disease history and physical examination results, after which the possibility of brain abscess should be investigated. Although we are currently equipped with the most advanced imaging techniques, a large arsenal of antibiotics, and many surgical options, brain abscess remains a disease associated with a high mortality rate. As such, early diagnosis and treatment are crucial to minimize various complications and the number of deaths.WZ and XJ wrote the article. XS collected the materials.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.


The critically ill polytrauma patient is characterized by a series of metabolic changes induced by inflammation, oxidative stress, sepsis, and primary trauma, as well as associated secondary injuries associated. Metabolic and nutritional dysfunction in the critically ill patient is a complex series of imbalances of biochemical and genetic pathways, as well as the interconnection between them. Therefore, the equation changes in comparison to other critical patients or to healthy individuals, in which cases, mathematical equations can be successfully used to predict the energy requirements. Recent studies have shown that indirect calorimetry is one of the most accurate methods for determining the energy requirements in intubated and mechanically ventilated patients. Current research is oriented towards an individualized therapy depending on the energy consumption  of each patient that also takes into account the clinical dynamics. By using indirect calorimetry, one can measure, in real time, both oxygen consumption and carbon dioxide production. Energy requirements  and the respiratory quotient (RQ) can be determined in real time by integrating these dynamic parameters into electronic algorithms. In this manner, nutritional therapy becomes personalized and caters to the patients’ individual needs, helping patients receive the energy substrates they need at each clinically specific time of treatment. The critically ill polytrauma patient represents a very complex, multifactorial case of associated pathologies that significantly increase mortality rates ,2,3. BotIn an international multicenter study, Heyland et al. reported that over 70% of the patients included in the study did not receive the minimum 80% of their energy requirements . They shFor decades, clinicians have been using mathematical equations to predict energy requirements. However, numerous studies have reported a disparity between the clinical status of the patient and the values rendered by these calculations, especially in the case of critically ill patients. A recent study published by Zusman et al. concluded that mathematical equations cannot replace the indirect calorimetry monitoring of energy requirements for critical patients. This study included a high number of patients, with over 3500 quantifications .Regarding the metabolic disorders of critically ill polytrauma patients, some of the most important clinical symptoms are hyperglycemia and increased insulin resistance, rapid loss of muscular mass, and disturbance of the nitrogen balance ,25. The In the literature, there are a series of controversies regarding the adjustment of nutrition in the case of critically ill polytrauma patients, as several studies present advantages and disadvantages for both enteral and parenteral nutrition ,29,30. OThe aim of this paper is to connect and present in a clinical setting the strong relationship between inadequate nutrition and a series of secondary complications, specific to the critically ill polytrauma patient. Furthermore, we wish to detail the modern methods used to determine energy expenditure, as well as new guidelines for adapting nutritional therapy in a specific manner to these patients.From a clinical point of view, the critically ill polytrauma patient is characterized by a series of primary traumatic injuries, as well as by a multitude of trauma-associated secondary injuries such as hemorrhagic shock, tissue hypoxia, generalized inflammation, oxidative stress, and infections. All of these subsequently lead to a multiple organ dysfunction syndrome (MODS) and to a significant increase in mortality rates ,32,33,34From a molecular point of view, the above-mentioned events lead to the activation of a series of molecular systems and mechanisms, such as coagulation ,36,37,38Redox balance is significantly affected, with significant quantities of free radicals being released. In this case, the metabolic imbalance is strongly affected at a molecular level, with the free radicals being involved in a series of protein and lipid denaturation reactions ,44,45,46An important aspect in the pathophysiology of critically ill polytrauma patients is represented by the negative nitrogen balance ,49,50. ORegarding the metabolic answer to trauma, a series of articles described the following three phases of the event: the ebb phase, the catabolic phase, and the anabolic phase ,55,56. OFrom a biochemical, molecular, and cellular point of view in trauma, we distinguish the ebb phase during the first 8 h to 24 h post-trauma, a phase characterized by important hemodynamic changes. From a clinical point of view, during this phase, volemic resuscitation through fluid and blood products is the basis of the therapy. Afterwards, during the next three days, patients are characterized by an aggressive production of cytokines and inflammatory molecules. During this phase, the metabolic disaster continues at a cellular level, with the redox imbalance being augmented and extensive. The last phase, described as an anabolic phase, is considered to be the one in which molecular and metabolic mechanisms are oriented towards recovery ,60,61,62The existence of an inflammatory response without the clear presence of bacterial sources leads to an alarming activation of the immune system a short time after the traumatic event. These underlying signaling events are also called alarmines and influence the metabolic status of these patients considerably. The most researched endogenous signaling pathways responsible for the excessive augmentation of the immune response are represented by defensins, heat shock proteins (HSPs), cathelicidin, high-mobility group box 1 (HMGB1), and eosinophil-derived neurotoxin (EDN). Moreover, post-traumatic coagulopathy is responsible for a series of other side effects that lead to a metabolic imbalance ,64,65,66In this manner, we can frame these molecular mechanisms in the so-called acute phase response, through which the liver’s protein synthesis is redistributed depending on the severity of trauma. In these situations, adapting the nutrition becomes an impossible task without adequate and correct monitoring of each individual patient.Although enteral nutrition is recommended by the majority of clinical studies, it was concluded that in certain patients, the desired enteral nutrition cannot be ensured because of digestive intolerance. In the case of critically ill polytrauma patients, this aspect is present in the majority of the situations ,67,68. Fwww.espen.org) recommends early initiation of enteral nutrition, in the first 24 h after admission to the ICU. On the other hand, The Canadian Society for Nutritional Science  recommends initiation of enteral nutrition in the 24–48 h interval after admission to the ICU.The European Society of Parenteral and Enteral Nutrition . Under conditions of stress and hypoxia, HIF-1α is used as a transcription substrate for a series of genes that modulate cellular metabolism, cellular motility, and angiogenesis. Recent studies have proven the existence of biochemical links between microRNAs and the HIF-1α activity. In this manner, they have identified changes in the microRNAs expression that were closely correlated with the hypermetabolic status, such as microRNA-140 (a genetic regulation reaction specific for chondrocyte), microRNA-140 and let-7 , microRNA-199a (differentiation reactions for chondrocytes through the SMAD1 transcription factor), and microRNA-365 (proliferation reactions of chondrocytes through histone deacetylase). Another study regarding the expression of regulatory T-lymphocytes and their interaction with microRNAs under metabolic stress showed that in under-nutrition conditions, the expression of transforming growth factor beta 1 (TGF-β1) is altered. Moreover, a series of interactions have been identified between the TGF-β1 and microRNA-29a, microRNA-146a, microRNA-21, microRNA-181a, microRNA-181c, and microRNA-155 ,111,112.In the past, there were several mathematical formulas capable of estimating energy needs. After numerous studies, however, it has clearly been proven that these formulas are, in fact, not capable of correct estimations of energy requirements for critically ill patients, especially for polytrauma patients. This is not surprising, as there is no correlation between the mathematical factors included in the formulas and clinical dynamics of these patients ,77,78,79In such circumstances, calculating the energy expenditure is important in order to accurately determine the number of calories they need. During the early phases of critical illness, the number of calories consumed is lower than the energy expenditure because the body utilizes the inhabitable glucose. For this reason, there is a risk of overfeeding. In a similar manner, more calories are needed when the critical illness is subsiding, and therefore there is a risk of underfeeding. Significantly, inappropriate energy intake can affect the outcome of the patient during critical illness. To avoid this, energy expenditure estimation methods, such as predictive equations, indirect calorimetry, use of double-labeled water, and reference methods have been applied . All of Nevertheless, studies have shown that these equations sometimes give inaccurate answers despite the age, gender, or weight  Table 1Table 1. 2) and carbon dioxide production (VCO2) [2 production and O2 consumption (RQ = VCO2/VO2) [Indirect calorimetry is one of the most widely discussed methods in the literature, because it is the ideal parameter for determining the energy requirements for critically ill patients who are intubated and mechanically ventilated ,90. Althn (VCO2) ,92,93. Cn (VCO2) ,95,96. ACO2/VO2) . BasicalNumerous studies have shown that a series of complications can arise after inadequate nutritional therapy. Among these are infections, acute kidney injury, acute respiratory distress syndrome, multiple organ dysfunction syndrome and, in the case that the patient survives, late and prolonged recovery ,97,98. A2) measured through indirect calorimetry and by the Fick method that uses central venous oxygen saturation (ScVO2) and cardiac output (CO) measured by pulse dye densitometry (PDD). This was a prospective study and included mechanically ventilated patients. Following certain tests, they identified major differences between the two studied methods. In the case of indirect calorimetry, VO2 was 148 ± 28 mL/min/m2 as compared with 110 ± 29 mL/min/m2 measured through the Fick method (p < 0.01). The authors concluded that, for the measurement of VO2, indirect calorimetry remains the gold standard due to its increased accuracy [Regarding the accuracy of the method, there are a series of studies that have shown an increased specificity for indirect calorimetry. Inadomi et al. conducted a study in which they compared oxygen consumption  that the patient can tolerate at that certain critical moment.It goes without saying that a critically ill polytrauma patient represents a real challenge for the intensive care physician in regard to selecting an approach to nutritional therapy. This is because of the complex immune response, the aggressive pro-oxidative status, and the generalized inflammatory response. In particular, the pathophysiological changes, molecular alterations, and dynamics of inflammatory status modify the clinical status differently from one patient to another, as well energy requirements  and protein turnover are modified continuously, and therefore the estimation of their values through mathematical methods becomes impossible.With respect to adapting nutritional therapy during the first week from admission to the ICU, in the case of critically ill polytrauma patients, it is recommended that over 50% of their caloric target is administered through enteral feeding. Obviously, if this is not feasible, then, parenteral nutrition is also an option. Another important recommendation is that proteins should not be included in the calculations for the caloric requirements, because, from a biological viewpoint, they are not used in the deposited of muscle mass or the metabolism reactions to cover the energy requirements ,98,102.A significant proportion of polytrauma patients is represented by patients with traumatic brain injury (TBI) ,103,104.p < 0.001) and protein balance (p < 0.001) for patients whose nutritional therapy was based on indirect calorimetry, however, following this study, there were no significant differences regarding mortality or time spent in the ICU and hospital days [Allingstrup et al. carried out a study on the topic of early nutrition versus standard nutritional therapy. Regarding the study design, the authors divided the patients into two groups. One group received nutritional therapy based on values obtained through indirect calorimetry and urinary urea nitrogen, whereas the other group received nutritional therapy based on a recommended administration of 25 kcal/kg/day. The authors randomly enrolled 203 patients in the study, and the study results showed a statistically significant difference regarding negative energy  values and RQ in the two groups .Sunderland et al. carried out a study including critical patients with TBI, in which they compared the measured energy expenditure through indirect calorimetry to the energy requirements calculated using different mathematical formulas. They included 102 patients in the study, with over 385 measurements. They showed an increased accuracy for indirect calorimetry, validating once again the clinical accuracy of the method . AnotherA revolutionary study was conducted by Strack et al. on the clinical outcomes and implications of indirect calorimetry-guided nutritional therapy. They proved, when guided by indirect calorimetry, that nutritional therapy and the administration of a minimum of 1.2 g proteins/kg/day brings significant improvements in survival rates after 28 days in the ICU. An important aspect of this study was that they did not identified any significant differences regarding mortality in the case of men .In addition, the critically ill polytrauma patient is characterized by a series of pathologies and imbalances, all of which are interconnected and have accelerated dynamics. These rapid pathophysiological and metabolic changes lead to an extremely dynamic energy expenditure profile that changes on a daily basis ,106,107.2 moles are needed for the oxidation of glucose, with the further production of six moles of CO2 for each mole (180 g) of glucose. In this particular situation, the RQ (VCO2/VO2) equals one. Through a similar mechanism, the oxidation of one mole of fat , there is a consumption of 78 moles of O2 and a production of 55 moles of CO2 for each mole of fat. Therefore, the RQ for fat (VCO2/VO2) will be 0.7. Other studies have also presented similar RQ values following fat metabolism, such as RQ for tripalmitin (fat) 0.71, oleic acid (fat) 0.71, and triolein (fat) 0.7 [RQ is another important aspect when using indirect calorimetry. It can be obtained directly by monitoring respiratory gases and can guide nutritional therapy in a patient-centered, individualized manner. The complexity of nutritional substrate biochemical and metabolic oxidation processes makes adapting nutrition for each particular segment impossible. Six Ofat) 0.7 ,109,110.2  in order to produce six moles of fat and 52 moles of CO2 (1165 L CO2). The biosynthesis and oxidation of lactate is another important process, which is also very specific to the critically ill patient. The mechanisms involved in lactate metabolism are represented by gluconeogenesis and oxidation. For the oxidation of one mole of lactate, the body uses three moles of O2 with the production of three moles of CO2, however, one mole of CO2 will be converted to bicarbonate due to the intervention of a proton (H+) in the oxidation process of lactate [In the case of proteins, the calculations are based on empirical formulas using the urinary nitrogen excretion. Studies have reported that from the metabolism of 6.25 g of protein, 1 g of urinary nitrogen will be produced. In the context of the critically ill patient, the metabolic processes are more complex and interfere with one another, leading to important dynamic changes in RW, such as oxidation of glucose to fat, metabolism of lactate, and reactions involved in ATP production. An example is the transformation of glucose into fat that needs the intervention of pyruvate and acetyl-CoA. Basically, through this complex mechanism, 27 moles of glucose  will consume six moles of O lactate ,114.Jeon et al. conducted a study on 215 adult patients with severe burns focusing on EE using IC in comparison with mathematical equations and proved a high accuracy for measurements performed using IC as compared with the results obtained when solely using equations. They also showed rapid changes in EE that were only detected by IC, proving that the adjustment of nutritional therapy based on mathematical equations is impossible .2 in patients with sepsis was 180 ± 19 mL/min/m2, whereas the value for patients in septic shock was 120 ± 27 mL/min/m2 (p < 0.001). Statistically significant changes (p < 0.01) were reported regarding the mean resting metabolic rate in sepsis (+55% ± 14%) and in septic shock (+2% ± 24%). The O2 extraction capacity was also studied, with maximum values being reported for sepsis as compared with septic shock . An increase in the resting metabolic rate with +61% ± 21% during recovery from sepsis and septic shock was also proven. Following their study, the research group underlined the importance of IC monitoring for detecting dynamic metabolic changes, as well as their association with the clinical context and with guiding nutritional therapy based on each patient’s hypermetabolic status [p = 0.01). The group also highlighted a statistically significant difference in the amount of protein administered per day  [Zusman et al. developed a similar study regarding the differences between measured EE and calculated EE including 1440 patients. They concluded that equations have low accuracy as compared with indirect calorimetry and cannot replace this method in guiding nutritional therapy . The stuc status . Singer  = 0.05) . Heidegg = 0.05) .p < 0.05) between measured EE and the Harris–Benedict method , WHO , as well as Schofield . While investigating the accuracy, they reported a lower accuracy for equations as compared with IC [The study by Tamura et al. investigated REE in cardiac surgery patients as compared with the REE values obtained by IC and the Harris–Benedict equations. The study included 47 patients and demonstrated significant differences between calculated and measured EE values. They also showed that EE calculated by using the Harris–Benedict equation was 1.14 higher than the EE monitored through IC . Dias Ro with IC . In a si with IC .p < 0.05). This study also showed that 59% of the patients were hypermetabolic with a measured EE of >110% than the predicted EE [When looking at the pediatric critically ill population, the energy requirements are of utmost importance. In the first two years of life, the nutritional status has a significant impact on the development of all biological and morphological structures. In preterm newborns, it has been observed that the nutritional status has important implications in organ development, especially due to organ immaturity and low nutritional reserve . A high icted EE . Howell icted EE . The stuicted EE .p = 0.001); IC vs. Schofield ; IC vs. Maffies ; IC vs. Kleiber ; IC vs. Dreyer ; and IC vs. Hunter  [Vazquez Martinez et al. also carried out a study on EE in the critically ill pediatric patient. They compared EE from IC and from mathematical equations for 43 mechanically ventilated pediatric patients. They showed a statistically significant difference regarding measurement accuracy, as follows: IC vs. Harris–Benedict  . Suman e= 0.001) . Bott et= 0.001) ,128.In conclusion, we can state that each critically ill polytrauma patient is special and unique because of their characteristic hypermetabolic and hyperinflammatory status. The time spent on mechanical ventilation, the multiple tissue injuries, the organ lesions, multiple site infections, and increased oxygen consumption all lead to dynamic changes in energy consumption for each individual patient. The literature shows that mathematical equations, for the most part, are far removed from the clinical reality of the patient, with significant discrepancies between the results. Therefore, indirect calorimetry becomes the gold standard for monitoring the personalized energy requirements of the patients, respecting the individual clinical dynamics.


Generally, crop straw degrades slowly in soil, which is unfavorable for tillage and next crop growth. Thus, it is important to develop a promising technology to boost degradation of straw. Herein, a nanobiosystem has been developed by loading bacterial mixture in nanostructured attapulgite (ATP) and using it as a straw returning agent (SRA). Therein, ATP could effectively anchor bacteria to the surface of straw and greatly facilitate the adhesion and growth of bacteria. Consequently, this technology could effectively accelerate the degradation and transformation of straw into nutrients, including nitrogen (N), phosphorus (P), potassium (K), and organic matters (OM). Pot and field tests indicated that SRA displayed significant positive effects on the growth of the next crop. Importantly, SRA could effectively decrease greenhouse gas emissions from farmland, which is beneficial for the environment. Therefore, this work provides a facile and promising method to facilitate the degradation of straw, which might have a potential application value. In many regions, such as the USA, UK, and China, the usual strategy of crop straw treatment is to return them to the soil after grinding ,2. ReturSeveral approaches of speeding up straw degradation have been explored in recent years, including physical grinding ,10,11, d4(Si)826·nH2O) (ATP) is a kind of natural crystalline hydrated magnesium aluminum silicate, consisting of nanorods approximately 800–2000 nm in length and 30–40 nm in width [Attapulgite in width ,17,18. Nin width ,20, carrin width ,22, and in width . Citrobacter freundii, Arthrobacter woluwensis, and Bacillus licheniformis), obtaining a nanobiosystem that was used as a straw returning agent (SRA). The promotion effects of ATP on the adhesion and growth of bacteria on the surface of wheat straw were investigated. Additionally, the influences of SRA on the degradation of wheat straw, growth of the maize, and the emission of greenhouse gas were also studied. Moreover, the morphology of SRA on the straw was observed to elucidate the mechanism. Therefore, this work provides a low-cost, facile, and environmentally friendly method to promote the degradation of straws, which has a huge application value in the development of sustainable agriculture all over the world.ATP was used as a carrier to load bacterial mixture (BM)  was located on the experimental farm of Hefei Institutes of Physical Science, Chinese Academy of Sciences, China. The primary soil in the area is classified as a yellow clunamon soil, with 15.30 g·kgC. freundii, A. woluwensis, and B. licheniformis) and then transformed them to powders (100–200 mesh) by freeze drying and grinding. Maize seeds were purchased from Fengle Seed Co., Ltd. . Deionized water was used in all the experiments except the pot experiment. Soil used in the pot experiment was taken from Dongpu Island . Natural ATP powder  was purchased from Mingmei Co., Ltd. . We isolated bacteria  at 30 °C for 20–24 h in a rotary shaker incubator (180 rpm), respectively. Then, the cells were harvested by centrifugation for 20 min at 8000× g and dissolved in sterile MilliQ water to obtain a final cell concentration of 107 CFU/mL, respectively. The bacterial strains were added and suspended in the freeze-dried protective agent containing dried skim milk and trehalose. After freezing for 12 h at an ultra-low temperature, the three bacterial strains were freeze-dried for 24 h at −50 °C with a vacuum freeze drier . Finally, the three bacteria were evenly mixed with ATP with a weight ratio of 1:2:2:5 to form the SRA.  studies ,25. For SRA aqueous solution  was evenly spayed onto 20 g of wheat straw  after harvest, and the resulting straw was placed in a cylindrical nylon net bag . After that, the bag was buried in soil (humidity of 30%) with a depth of 5–10 cm. The bag was dug out after 20, 40, 60, 80, and 100 d. Finally, length and weight of the remaining straw were determined after air drying. Nitrogen (N) content by alkali hydrolyzable method , phosphoSRA  or BM  aqueous solution was evenly spayed onto wheat straw  after harvest, and the resulting straw was evenly mixed with 3.5 kg of soil (humidity of 30%). The resulting mixture was then placed in a pot  and 10 cm (bottom)). Subsequently, three maize seeds were put in the soil with a depth of 5 cm. The resulting system was placed in a greenhouse under a temperature of 25 °C, and 50 mL of water was sprayed evenly to the system every three days. At given intervals after seeding, plant height, stem diameter, chlorophyll content, and photosynthetic rate in leaves were measured. All experiments were performed in triplicate.4 region using the 515F primer  and barcoded versions of 806R . Equimolar concentrations of the purified amplicons were pooled together and subjected to paired-end multiplex sequencing using an Illumina MiSeq instrument At Majorbio Bio-pharm Technology Co., Ltd. . For raw data analysis, only illumine reads over 175 nt were analyzed . Operational taxonomic units (OTUs) were defined at 97% nucleotide similarity. Taxonomic classification was done using the Bayesian classifier provided by the mothur software package  [To characterize the bacterial composition and community diversity under different treatments , soil samples of pot experiment of each treatment were randomly selected for pyrosequencing analysis. The soil total DNA was extracted from 0.25 g of cryopreserved samples by DNeasy PowerSoil Pro Kit , according to instructions from the manufacturer. Then the DNA were detected by 1% agarose-gel electrophoresis, and the concentration and purity of the DNA were detected by NanoDrop 2000 Microvolume Spectrophotometers and Fluorometer . The sample was frozen at −80 °C until pyrosequencing analysis. Bacterial 16S rRNA gene amplification  and barcoded pyrosequencing were performed on an Illumina MiSeq instrument . Briefly, triplicate polymerase chain reaction (PCR) products of each sample were pooled, purified, and quantified using a NanoDrop 2000 Microvolume Spectrophotometers and Fluorometer  after PCR amplification of the 16S VMI, USA) . The rel2, 2 g/L) or BM  aqueous solution was evenly sprayed onto wheat straw (average length of approximately 5 cm) in a field (100 m2) after harvest, and the resulting straw was returned to soil  using a rotary tiller . A cubic glass box (1 cubic meter) was then placed in the field with the open side downward  and photosynthetic rate apparatus , respectively. All the above indexes were determined from the top leaf of the plant, nine strains were selected for each treatment and the average value was taken.−1 spectral range at a resolution of 4 cm−1 for each sample. The thermal gravimetric analysis (TG) and differential thermal analysis (DT) were performed by a thermogravimetric analyzer  at a scan rate of 10 °C /min from 40 to 800 °C in oxygen or nitrogen. The morphology of the straw samples was measured by a scanning electron microscope (SEM) . The structure and interaction were analyzed by an X-ray diffractometer  with Cu radiation . The samples were washed with sterile water several times to remove the bacteria from the surface of the straw and dried in a constant temperature drying oven  until the quality was constant. The dried straw samples were mixed with KBr and ground to power, where the sample content in KBr was 0.5%, then characterized by a Fourier transform infrared (FTIR) spectroscopy  at ambient temperature. 32 scans were recorded in a 400–4000 cmP < 0.05 using Fisher’s least significant different (LSD). Statistical analysis was performed using SPSS 16.0 software for Windows.A two-way analysis of variance (ANOVA) was performed to determine the effects of the treatments. Significant differences in the means of three replicates among all the treatments were considered at Wheat straw mainly consisted of cellulose, hemicellulose, lignin, and inorganic substances and thus degraded slowly in soil, which greatly limited tillage and growth of next crop. In order to evaluate the promotion effect of SRA on the degradation of straw, the dynamic influence of SRA on the length, weight, nitrogen, phosphorus, and potassium contents of wheat straw was investigated compared with wheat straw alone and BM. As shown in In order to understand the effect of SRA on maize growth, a pot experiment was carried out, wherein three maize seeds were placed in soil with wheat straw alone and those treated by BM and SRA, respectively A. As illA field test was also performed to further obtain the effect of SRA on the germination, growth, and yield of maize. As shown in 4 emission [4 emission is primarily regulated by the availability of C substrates in the soil, and wheat straw could provide available C sources as organic materials to support the growth of methanogenic populations under anaerobic conditions [2O emission is different. Xia et al. found that long-term straw incorporation can reduce N2O emissions compared to no straw return [2O emission by 15–39% [2, CH4, and N2O from a field after treatment for 60 days. As demonstrated in 2, CH4, and N2O in a certain degree compared with straw alone, and SRA could dramatically decrease the emission of those gases compared with BM. This result was in accordance with the preceding results, indicating that the decreased gas emission after SRA treatment probably resulted from the promotion effect of SRA on the degradation of straw. That is to say, SRA could effectively transform the degraded straw to nutrients rather than gas, which was favorable for improving soil quality and reducing its contribution to global warming. Wheat straw returning could significantly stimulate CHemission ,31. Soilnditions . Previouy 15–39% . We obtaSoil microorganisms are important drivers involved in the process of degradation, transformation, and utilization of crop straw . Some st2 on the surface, thus hydrogen bonds (H-bonds) tended to form between ATP and straw or BM, promoting the adhesion and growth of BM on the straw surface (In order to elucidate the mechanism of SRA on the degradation of straw, the morphologies of SRA on the longitudinal and cross sections of wheat straw were observed. As shown in  surface m–q, whic−1 corresponding to cellulose, galactose, mannose, Arabia sugar, and polysaccharide in the spectrum of straw alone left-shifted to 898 cm−1 and became weak, indicating that BM promoted the degradation of those substances. Additionally, after SRA treatment for 100 d, the relative strength of the peak at 898 cm−1 further weakened compared with BM, suggesting that SRA displayed a higher promotion performance on the degradation of those substances than BM. The result demonstrated that SRA mainly promoted the degradation of cellulose, galactose, mannose, Arabia sugar, and polysaccharide in wheat straw. In addition, XRD analysis was also performed to investigate the influence of SRA on the crystal structure of straw. As illustrated in To further investigate the mechanism of SRA, FTIR measurements were performed. As shown in TG analysis was conducted to evaluate the thermal stability of straw with different treatments. As shown in In large-scale practice , crop stIn this work, SRA was fabricated through incorporation of ATP and BM, wherein ATP nanonetworks acted as an ideal carrier for BM, anchored BM on the surface of straw, enhanced the adhesion, promoted the growth of BM, and thus accelerated the degradation of straw and release of nutrients. Thus, this technology could effectively promote the growth of the next crop. Importantly, this technology could also significantly reduce greenhouse gas emission and increase the number of bacteria, which were beneficial for the degradation of straw. This work provides a low-cost and simple approach to accelerate straw degradation and protect the environment, which has promising application prospects.


Low-temperature-processed ITO thin films offer the potential of overcoming the doping limit by suppressing the equilibrium of compensating oxygen interstitial defects. To elucidate this potential, electrical properties of Sn-doped In Transparent conductive oxides (TCOs) are key materials for electrodes in display and solar cell technologies ,3,4,5,6.40cm2/Vs . Even hiIf thermodynamic equilibrium of defect concentrations can be established, the concentration of free electrons in TCOs is limited by the formation of self-compensating intrinsic defects ,8,9,10. l oxygen ,13,14,15at 300∘C ,17,18,19If samples are processed at temperatures low enough to suppress oxygen diffusion and dopant segregation, defect equilibrium cannot be established. In such a case, the concentration of compensating defects can, in principle, be lower than in equilibrium. Therefore, low processing temperatures of TCOs offer the potential advantage of achieving higher carrier concentrations. In contrast, in the case of donor-doped Inerature ≤0∘C are ttrations ,22,23,24trations ,26. Enhad layers . TherebyRecently, Koida and coworkers have demonstrated that high conductivities of differently doped Ino ∼200∘C ,29,30. IAs the conductivity of ITO thin films is determined by a number of factors, including crystallinity, grain size and oxygen content, which impact carrier concentration and mobility differently, it is difficult to discriminate between the different contributions. This becomes particularly important for the identification of the conditions needed to achieve higher carrier concentrations in low-temperature-processed samples.In the present work, the effect of low processing temperature  measurements were performed without breaking vacuum using a Physical Electronics PHI 5700  spectrometer. Monochromatic Al Kposition .As a first step, the influence of substrate temperature on the electrical properties of ITO thin films with different thicknesses is presented in this section. Films deposited at elevated substrate temperature are expected to be crystalline but films grown at room temperature might be amorphous ,25,34,35The thinnest film does not show any diffraction peaks. This is not due to the low film thickness, as clear peaks are observed after annealing the same film in vacuum  and (ii) the diffusion of oxygen in the film, characterized by the diffusion coefficient D  iffusion  and withiffusion , indicatThe increase in carrier concentration and the decrease of mobility during heat treatment of the 200 nm-thick films in either atmosphere, can both be explained by crystallization and grain growth. Crystallization of amorphous regions, which might still be present in the 200 nm-thick films deposited at room temperature, would activate the Sn donors ,26 and tVacuum annealing: The substantial increase of carrier concentration during vacuum annealing of the 10 nm-thick film indicates a substantial increase in effective dopant concentration. According to the discussion above, two different effects can contribute to this observation: (i) donor activation by crystallization and grain growth; (ii) extraction of oxygen. Crystallization during vacuum annealing is clearly demonstrated by the GIXRD results in the insert of L and is given by D is the diffusion coefficient [The annealing of the 200 nm-thick films indicates that a change of the overall oxygen interstitial content in the film is not important for the 200 nm-thick films. However, it can be important in thin films as the time fficient . Given t studies .Air annealing: The decrease of carrier concentration and increase of mobility during air annealing of the 10 nm-thick film can also be explained by a change of oxygen content. Medvedeva and coworkers have demonstrated that the carrier concentration in amorphous Inhiometry . Incorpohiometry .To further check whether the removal of oxygen contributes to the strong increase of carrier concentration of the 10 nm film during vacuum annealing, an extended annealing experiment has been carried out. The measurement is shown in At the beginning of the annealing, the behaviour observed in 00∘C see . After cThe very high carrier concentrations reached after long vacuum annealing of the 10 nm film, can only be reached if oxygen is extracted from the films during annealing. This can be concluded by comparison with ITO films grown under identical process conditions in our laboratory at room temperature but on Fed layers . The Fe2The effect of annealing in dependence on film thickness and atmosphere is summarized in X-ray photoelectron spectra of 20 and 200 nm-thick ITO films deposited at room temperature before and after 5 ALD cycles of AlDeposition of Alsurfaces ,47,48,49surfaces .The Fermi energy in the Alubstrate ,32,50,51ate [2O3 , it is eVB≈3.3eV . This toVB≈3.3eV ,32,53,54In contrast to the results reported for SnO2 , the XPSConductivity and Hall effect measurements, performed on ITO films deposited at room temperature, are shown in The AlCarrier mobility: As already discussed in As the reduction of mobility of Al XPS see . The red is fast enough for 10 nm-thick films. Therefore, extraction of oxygen during annealing in vacuum results in a substantial increase of carrier concentration by almost an order of magnitude up to During annealing at Finally, realization of highest carrier concentrations in ITO thin films should be possible by using a low-substrate temperature-deposition process with a very low oxygen activity. Nevertheless, the inherently small grain size of low-temperature grown films has to be overcome, as this clearly limits the carrier concentration. The use of seed layers, such as the recently demonstrated Fe2O3 , might b