Androstenedione (AD) is an important steroid medicine intermediate that is obtained via the degradation of phytosterols by mycobacteria. The production process of AD is mainly the degradation of the phytosterol aliphatic side chain, which is accompanied by the production of propionyl CoA. Excessive accumulation of intracellular propionyl-CoA produces a toxic effect in mycobacteria, which restricts the improvement of production efficiency. The 2-methylcitrate cycle pathway (MCC) plays a significant role in the detoxification of propionyl-CoA in bacterial. The effect of the MCC on phytosterol biotransformation in mycobacteria has not been elucidated in detail. Meanwhile, reducing fermentation cost has always been an important issue to be solved in the optimizing of the bioprocess.Mycobacterium neoaurum (MNR), prpC, prpD and prpB in the prp operon encode methylcitrate synthase, methylcitrate dehydratase and methylisocitrate lyase involved in MCC, and PrpR is a specific transcriptional activator of prp operon. After the overexpression of prpDCB and prpR in MNR, the significantly improved transcription levels of prpC, prpD and prpB were observed. The highest conversion ratios of AD obtained by MNR-prpDBC and MNR-prpR increased from 72.3 ± 2.5% to 82.2 ± 2.2% and 90.6 ± 2.6%, respectively. Through enhanced the PrpR of MNR, the in intracellular propionyl-CoA levels decreased by 43 ± 3%, and the cell viability improved by 22 ± 1% compared to MNR at 96 h. The nitrogen transcription regulator GlnR repressed prp operon transcription in a nitrogen-limited medium. The glnR deletion enhanced the transcription level of prpDBC and the biotransformation ability of MNR. MNR-prpR/ΔglnR was constructed by the overexpression of prpR in the glnR-deleted strain showed adaptability to low nitrogen. The highest AD conversion ratio by MNR-prpR/ΔglnR was 92.8 ± 2.7% at low nitrogen level, which was 1.4 times higher than that of MNR.There is a complete MCC in prp operon and glnR of mycobacteria was investigated for the first time. The overexpress of prpR in MNR can increase the transcription of essential genes  of MCC, reduce the intracellular propionyl-CoA level and improve bacterial viability. The knockout of glnR can enhance the adaptability of MNR to the nitrogen source. In the MNRΔglnR strain, overexpress of prpR can achieve efficient production of AD at low nitrogen levels, thus reducing the production cost. This strategy provides a reference for the economic and effective production of other valuable steroid metabolites from phytosterol in the pharmaceutical industry.Improvement in phytosterol biotransformation after the enhancement of propionyl-CoA metabolism through the combined modifications of the  Steroid drugs are widely used in anti-inflammation, endocrine regulation, and fertility management . The gloβ-oxidation, and several propionyl-Coenzyme A (CoA) and acetyl-CoA are produced [Mycobacterium smegmatis and M. tuberculosis produces a toxic effect, which seriously affects the growth of mycobacteria on propionate or mixtures of propionate and glucose [Mycobacterium neoaurum (MNR) promotes the conversion of propionyl-CoA to nontoxic succinyl-CoA and improves the transformation of phytosterols [The production of AD and 9α-OH-AD from phytosterol bioprocessing mainly involves the degradation of aliphatic side chains through produced –16. For produced . Propionproduced . Previou glucose , 20. Throsterols . TherefoprpC) catalyzes the condensation of propionyl-CoA with oxaloacetate to 2-methylcitrate, which is then dehydrated to produce methylaconitate. This step is accomplished by either methylcitrate dehydratase  or by the combined activity of methylcitrate dehydratase (AcnD) and methylaconitate cis–trans isomerase (PrpF). Following dehydration, methylaconitate is rehydrated by aconitase (AcnB) to yield methylisocitrate and cleaved by methylisocitrate lyase  or by isocitrate lyase to produce pyruvate and succinate [prp operon plays a key role in the assimilation of propionyl-CoA in Mycobacterium tuberculosis [, and Mycobacterium smegmatis [prpR) that regulates the MCC by induces self-transcription and activating the prp operon in M. tuberculosis. PrpR is essential for the utilization of odd-chain-length fatty acids, and the prpR knockout strain exhibited an inhibited growth on propionate as a sole carbon source. Therefore, speculation is that the effector of PrpR may be propionyl-CoA, which is an intermediate metabolite of both odd-chain-length fatty acids and propionate.Previous findings have shown that in the MCC of mycobacteria, propionyl-CoA is converted to pyruvate at a molar ratio of 1:1 , 23. Theuccinate . The prprculosis , and Mycmegmatis  to obtaimegmatis  identifiMycobacterium tuberculosis and Mycobacterium smegmatis, which are mainly related to the function of MCC at low cholesterol concentrations and other substrates. The effect of MCC on the transformation ability of steroid precursor production strains at a high concentration of phytosterol has not been reported, which is worthy of further study.Current research on MCC in mycobacteria is focused on Nitrogen is an essential element for the microbial synthesis of proteins, nucleic acids, and substances required for growth. The supply of nitrogen sources strongly influences the growth and metabolism of heterotrophic microorganisms and autotrophic algae, which is very important for the efficient generation of target products during fermentation –30. The prpDBC and inhibits its transcription in M. smegmatis. However, little is known about the effects of regulatory factors, PrpR and GlnR, on the transformation of phytosterols by mycobacteria. Improving the nitrogen source adaptation level of a strain by nitrogen regulation factors is significant, and to the best of our knowledge, this process has not been reported.Considerable research has shown that the utilization of nitrogen sources by microorganisms is controlled by regulatory proteins such as GlnR . StudiesMycobacterium. The transcriptional level of prpDBC and the changes in cell growth and AD production after the overexpression of prpR in MNR were studied. The effects of glnR deletion on the transcription level of prpDBC and the AD molar conversion rates were investigated at different nitrogen source levels. To further improve the productivity of AD, a prpR overexpressing strain MNR-prpR/ΔglnR was constructed under glnR deletion, and the AD productivity of MNR-prpR/ΔglnR at different nitrogen source levels was evaluated. In summary, strategies for effectively enhancing AD production by using prp operon and glnR were first reported.In the present work, we show for the first time that genetically manipulating transcription factors can improve the AD production in a Escherichia coli DH5α was used for plasmid replication. Genes overexpression and deletion methods have been reported in previous studies [R) was used to overexpress the target gene. Genes of PrpR and PrpDBC were amplified from the genome of MNR. The prpR and prpDBC genes were recombined with the linearized pMV261 using the In-Fusion HD Cloning method to generate recombinant plasmids, which were named pMV261-prpR and pMV261-prpDBC, respectively. The recombinant plasmid introduced to MNR through electroporation to gain the recombinant strains (MNR-prpR and MNR-prpDBC). The empty plasmid control strain (MNR-pMV261) was constructed by the introduction of pMV261 into the MNR. The in-frame deletion mutants of glnR were built by referring to the methods of Yao et al. [glnR were obtained as a recombinant fragment by PCR. Two fragments were ligated into the plasmid p2NIL and then digested with PacI and ligated with a selection marker cassette from pGoal19 to construct a homologous recombinant plasmid. The constructed plasmid was transferred into mycobacterial cells by electroporation, and the glnR knockout strain was screened according to the previously reported protocol [prpR/ΔglnR and MNR-prpDBC/ΔglnR by electroporation of plasmids pMV261-prpR and pMV261-prpDBC into MNRΔglnR.All modified strains and plasmids used in this work are listed in Table  studies . In MNR,o et al. . The 113E. coli DH5α was cultured at 37 °C in Luria–Bertani (LB) medium , where appropriate, kanamycin was added to the medium with a final concentration of 50 μg/mL. Mycobacteria seeds were prepared according to the previous description [4)2HPO4, 0.05 g/L ferric ammonium citrate, 0.25 g/L MgSO4, 25 mM hydroxypropyl-β-cyclodextrin, and 5 g/L phytosterol. The normal nitrogen source level was set to 1 (N1), and one-tenth (N0.1), one-half (N0.5), three-fifths (N0.6), seven-tenths (N0.7), four-fifths (N0.8), and nine-tenths (N0.9) of its weight were added to the fermentation medium to investigate the effects of different nitrogen source levels on the strain.All reagents and substrates were prepared as previously described , 21. E. g for 10 min at 4 °C. Isolation of RNA was carried out according to the method described by our previous description [−ΔΔCt method) [For qRT-PCR analysis, the cells were cultured for 48 h and collected by centrifugation at 8,000×cription . The qRTcription . The pricription . Relativ method) .prpR and MNR-prpDBC, samples were taken at 24-hour intervals, and the samples were divided equally. One part was used to detect the dry cell weight (DCW) according to the previous method [g for 10 min at 4 °C and transferred to an equilibrated solid-phase extraction column . After loading the sample, wash the extraction column with 0.1% trifluoroacetic acid (TFA). The extraction column was subsequently eluted with 40% acetonitrile containing 0.1% TFA. The collected eluent was freeze-drying performed in a freeze-dryer  and stored at − 80 °C until analysis.During the fermentation of MNR, MNR-s method , and thes method . Cells cFor the analysis of propionyl-CoA, the prepared samples were assayed by high-performance liquid chromatography (HPLC) using Agilent 1260  equipped with UV detection at 260 nm. HPLC analysis was realized on a reversed-phase C18 column (250 mm × 4.6 mm) at 25 °C, and the two mobile-phase solvents used were buffer A (acetonitrile) and buffer B . The linear gradient elution was carried out at 0.8 mL/min, and elution condition was as follows: 2–12% A (0–5 min), 12–38% A (5–15 min), 38% A (15–17 min), 38–2% A (17–19 min), data collection was stopped at 30 min. The content of propionyl-CoA was calculated by the external standard method.Propionyl-CoA  was used for the preparation of the standard curve. The standard stock solution of propionyl-CoA (100 μmol/L) was prepared and stored at − 80 °C. Propionyl-CoA standard working solutions were prepared by diluting the stock solutions to 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 2.5, 5.0, and 10 μmol/L. The calibration curve was prepared using the standard working solutions of propionyl-CoA. For each sample, the propionyl-CoA concentration was calculated by interpolating the sample measurement in the standard curve. Finally, the intracellular propionyl-CoA concentrations were calculated according to the DCW value of each sample.600 value to 1 using a Tris–HCl buffer (pH 7.2). Add 190 μL fermentation broth and 10 μL of WST-8 to the 96-well plate, detect absorbance at 450 nm after 1 h incubate at 30 °C. For product analysis, samples were taken every 24 h during the conversion of phytosterol, and the same volume of ethyl acetate was added to 1 mL fermentation broth for 30 min. After 12,000×g centrifugation for 10 min, 200 μL extract was dissolved in 80% methanol after vacuum drying and centrifuged at 12,000 × g for 20 min for HPLC analysis. HPLC analysis was prepared according to the method described by the previous description [The bacterial viability using the modified CCK-8 method was determined as described previously . The fercription .M. smegmatis contains prpD, prpD and prpC, but M. tuberculosis contains only prpD and prpC. The activity of PrpB is provided by isocitrate lyases of M. tuberculosis , which are two isoforms involved in the glyoxylate pathway. The amino acid sequence analysis showed that the PrpD, PrpB and PrpC of MNR were similar to M. smegmatis and M. tuberculosis with sequence identity between 76 and 88%  [prpDBC upstream region revealed a typical PrpR binding motif (TTCGCAAA), and GlnR binding motif (GGACCGGCACCGTAAC) were observed in the upstream region of prpDBC in MNR   had a coprpR and prpDBC overexpression strains (MNR-prpR and MNR-prpDBC) were constructed for studying the effect of PrpR on prpDBC in MNR. The relative transcription levels of prpD, prpB and prpC in MNR, MNR-prpR and MNR-prpDBC were compared in the presence of phytosterols. As shown in Fig. prpDBC increased significantly in the prpR-overexpressed strain compared with that in the wild type strain. The prpD, prpB and prpC transcripts increased in MNR-prpR 16.5 ± 2.4-, 10.5 ± 3.1- and 36.0 ± 4.9-fold, respectively. The overexpression of prpDBC increased the transcription levels of prpD, prpB and prpC 5.6 ± 2.0-, 6.5 ± 2.8- and 5.3 ± 2.0-fold, respectively. Therefore, prpR is a positive transcription regulator of prpDBC in MNR. In addition, the relative transcription level of prpD, prpB and prpC in strain MNR-prpR was higher than that in MNR-prpDBC. The reason may be that the PrpR protein produced by overexpression of prpR in MNR-prpR can promote the transcription of prpD, prpB and prpC on the genome, and also promote the transcription of prpR gene, thus producing a dual promoting effect on prpD, prpB and prpC transcription. Therefore, as far as the relative transcription level of prpD, prpB and prpC is concerned, overexpression of prpR alone has a better effect.The prp operon enhancement on cell growth and AD production were studied. It was found that the conversion rate of a single gene  overexpression was always lower than that of co-expression of three genes (data not shown). Therefore, the prpR and prpDBC overexpression strains (MNR-prpR and MNR-prpDBC) were selected for comparative study with the parental strain (MNR) and the control strain (MNR-pMV261). Although all strains have the same growth trend, the introduction of plasmids could reduce the biomass, which is more pronounced when overexpressing foreign genes. The biomass of the MNR-prpR and MNR-prpDBC were lower than the MNR and control strain, and MNR-prpR had the smallest biomass  and control strain (70.1 ± 2.7%). The AD conversion rate of MNR-prpDBC was always lower than that of MNR and MNR-pMV261 before 48 h, and the conversion rate of MNR-pMV261 was consistently lower than that of MNR. The main reason for this phenomenon may be due to the increased metabolic load caused by the generation of antibiotic resistance and genes overexpression. The above results showed that the overexpression of prp operon enhanced the AD production capacity of MNR, and the effect of prpR overexpression was most evident.The effects of n MNR-pMV61. Althon MNR-pMV61. AlthoprpR and MNR-prpDBC intracellular propionyl-CoA level assays showed that the recombinant strains had similar trends to the parental strains. The overexpression of the prpR and prpDBC can effectively reduce the accumulation of propionyl-CoA in the middle and late stages of biotransformation  were reduced by 43 ± 3% and 23 ± 4% compared to MNR (10.2 ± 0.9 μM). Moreover, the cell viability of recombinant strains was improved. The MNR-prpR always showed higher cell viability than MNR and MNR-prpDBC  was 22 ± 1% higher than that of MNR (1.3 ± 0.04). Although the cell survival rates of the three strains decreased after 96 h, MNR-prpR and MNR-prpDBC showed a slower downward trend than MNR. At 144 h, the cell viability of MNR was only 53 ± 1% of the highest value, whereas the ratio of MNR-prpR was 83 ± 1%. In M. tuberculosis, MCC is essential for growth on propionate or cholesterol. Masiewicz et al. [M. smegmatis [The MNR, MNR-ion Fig. a, and thz et al.  confirmemegmatis .Fig. 3Inprp operon can enhance the MCC, reduce the intracellular accumulation of propionyl-CoA and maintain cell viability. The reduction of propionyl-CoA also has a promoting effect on the side chain degradation of phytosterols, effectively improving the AD production capacity of MNR.The MCC plays an essential role in the detoxification of propionyl-CoA in mycobacteria. The overexpression of the 4)2HPO4 for use was 3.5 g/L, and the yield of AD decreased significantly with the decrease of (NH4)2HPO4 usage (data not shown). During the growth of M. smegmatis, nitrogen restriction increases the level of glnR transcription and inhibits the transcription of prpDBC involved in the MCC [glnR and prpDBC in MNR were studied at different nitrogen source levels. As shown in Fig. glnR transcription level increased 12.9 ± 1.4-fold at the nitrogen-limited level (N0.1), but a 96 ± 1% decrease for prpD, a 71 ± 2% decrease for prpB, and a 91 ± 1% decrease for prpC. At a low nitrogen source level (N0.5), the same trend was observed for prp operon. A prpR knockout (MNRΔglnR) and a replenishing strain (MNRΔglnR::glnR) were constructed and used in further confirming the effect of PrpR on the transcription of prpDBC. The transcription levels of prpDBC genes were studied under N0.1 conditions  [The transcription levels of nyl-CoA) .glnR is a global transcriptional regulator. In Saccharopolyspora erythraea, glnR can not only regulate nitrogen metabolism, but also regulate carbon metabolism [Streptomyces [prpDBC involved in the MCC at low nitrogen levels. The findings revealed a unique link between nitrogen metabolism and propionyl-CoA assimilation involved in fatty acid or cholesterol utilization [prpDBC. PrpD, PrpB and PrpC are the core enzymes of MCC and play an important role in the metabolism of propionyl-CoA. The overexpression of prp operon in MNRΔglnR affects the AD production of MNR. The prpR and prpDBC overexpression strains of MNRΔglnR (MNR-prpR/ΔglnR and MNR-prpDBC/ΔglnR) were constructed, and the cell growth and biotransformation of different strains were studied at different levels of nitrogen sources. As shown in Table 0.1 conditions. With the increase nitrogen source level, the two indices of each strain increased significantly. At the same nitrogen source level, glnR deletion was beneficial to the transformation of phytosterols, and the overexpression of prp operon gene cluster can further enhance the transformation ability of MNR. Consistent with the above results, the overexpression of prpR in glnR-deleted strains was more effective than prpDBC. Under the conditions of N0.1, N0.5 and N1, the biotransformation rates of MNR-prpR/ΔglnR were 21.6 ± 2.4%, 57.7 ± 2.8% and 94.3 ± 3.4%, which were 1.6, 1.4 and 1.2 times higher than MNR , respectively. These results indicated that the nitrogen source level is an important factor affecting the cell growth and AD production of MNR. At low nitrogen level, the deletion of glnR relieved the inhibition of GlnR on prpDBC and increased the metabolism of propionyl-CoA by MCC.The existing data indicate tabolism  and hometabolism , and affptomyces . Recent lization . The GlnprpR/ΔglnR were investigated in a shake flask. The result was used in studying the feasibility of reducing the cost of nitrogen sources. The biotransformation rates of MNR and MNR-prpR/ΔglnR increased with nitrogen source level from N0.5 to N1  under N0.7 condition was 1.2 times than that of MNR under N1 condition (75.7 ± 3.9%). The AD biotransformation rates of MNR-prpR/ΔglnR increased slightly with nitrogen content. The AD conversion rates of MNR-prpR/ΔglnR were 92.8 ± 2.7%, 93.7 ± 3.6% and 94.2 ± 3.0% under N0.8, N0.9 and N1 conditions, respectively. Therefore, in subsequent experiments, N0.7 conditions were selected for the investigation of the phytosterol biotransformation of MNR-prpR/ΔglnR. As shown in Fig. prpR/ΔglnR had higher phytosterol biotransformation ability during fermentation. At 144 h, the highest biotransformation rate of MNR-prpR/ΔglnR was 92.8 ± 2.7%, which was 28.4 ± 1.0% higher than that of MNR (64.4 ± 2.5%). A comparison of the AD formation rate (qp) of MNR and MNRΔglnR-prpR showed that the highest qp (0.045) of MNRΔglnR-prpR at 72 h was 2 times higher than the highest qp (0.023) of MNR at 96 h. Moreover, the production time for the highest qp for MNRΔglnR-prpR was shorter by 24 h. Therefore, the AD production performance of MNRΔglnR-prpR is better than that of MNR at the low nitrogen source level.The impact of varying nitrogen source levels on the AD production of MNR and MNR- N1 Fig. c. The ADR 90.6 ± .2% underprp operon and glnR in MNR, a novel strategy of improving phytosterol biotransformation in a low nitrogen level was established (Fig. prp operon enhanced MCC to reduce the accumulation of propionyl-CoA and its endotoxic, thus, enabling the strains to maintain high activity to achieve efficient production of AD. The combination strategy of knocking out glnR and overexpressing prpR can eliminate the inhibitory effect of glnR on prpDBC, increase the low nitrogen adaptation ability of MNR and enable the MNR-prpR/ΔglnR to produce AD efficiently under low nitrogen sources.Our previous studies showed that the enhancement of the propionyl-CoA carboxylase gene in the MMC of propionyl-CoA metabolism could improve the ability of MNR to transform phytosterols. In the present study, based on the identification of hed Fig. . In MNR,prp operon and the glnR in MNR and studied the regulatory effects of PrpR and GlnR on prpD, prpB and prpC. The enhancement of prpR and prpDBC can effectively reduce the accumulation of propionyl-CoA and enhance the phytosterol biotransformation ability of MNR. Besides, glnR-deficient strains can better adapt to a low nitrogen source environment and reduce nitrogen source consumption in AD production. These findings provide new insights into the enhancement of the production capacity of steroid precursors of mycobacteria and the reduction of the cost of using nitrogen sources.In this study, we identified the


Cervical component of thymus is noted more in children and young adults than in older age group. CT texture (lobules of soft tissue interspersed with fat), similarity with CT density of mediastinal thymus and continuity with mediastinal thymus on sagittal/coronal images, are given as the criteria for diagnosis of the cervical thymus. But CT densities of cervical and mediastinal components of the thymus may vary. The purpose of our study was to compare CT densities of cervical and mediastinal parts of the thymus, in cases where ultrasonography correlation was available.We retrospectively identified 22 patients who had undergone CT between May 2015 and May 2017 and in whom ultrasonography (USG) correlation was available. CT densities of cervical and mediastinal components of thymus were measured.CT density of cervical thymus is lower than the CT density of mediastinal thymus by ~ 25 HU.There is a moderate positive correlation between CT densities of cervical and mediastinal parts of the thymus.CT densities of both cervical and mediastinal thymus were found to reduce with age, but the reduction was statistically significant only in the cervical thymus in this study.CT densities of cervical and mediastinal components of the thymus may vary, with CT density of cervical thymus being lower. There is a positive correlation between CT densities of cervical and mediastinal parts of the thymus.CT density of cervical thymus reduces with age.Understanding these may help avoid confusion on CT and avoid the need for correlative USG, saving time and effort.The online version of this article (10.1186/s13244-019-0781-z) contains supplementary material, which is available to authorized users. CT densities of cervical and mediastinal components of the thymus may vary, with CT density of cervical thymus being lower.CT density of cervical thymus reduces with age.Understanding these may help avoid confusion on CT and avoid the need for correlative USG, saving time and effort.Although the thymus is predominantly a mediastinal organ, part of it can often be seen in lower neck normally , 2. WhenIt is a common practice at our center to do USG correlation when there is any ambiguity in the neck  on CT scan. We encountered some cases where the cervical component of thymus had a density different from the mediastinal thymus, but ultrasound  confirmed that the cervical lesion was indeed a normal variant and not some small lymph node/other pathology. We did a retrospective study of cases of the cervical thymus in which USG correlation was available, to compare CT densities of cervical and mediastinal parts of thymus.The institutional review board approved this retrospective study of existing imaging data, and written consent was waived. We retrospectively searched our USG and CT (PACS) records to identify patients in whom USG correlation Fig.  had beenCT was performed on a 16-channel scanner  with variable milliampere and kilovolt (peak) of 120. Display FOV varied with patient size but was approximately 25 mm; coronal and sagittal reformats were used in this evaluation.Cervical thymic tissue was defined as an oval focus of soft tissue above the level of sternal notch that had continuity with mediastinal thymus on axial/sagittal/coronal images. Each CT examination was evaluated by two radiologists  to determine densities of mediastinal and cervical components of thymus Fig.  and the t test. The comparison of density with age (groups) was tested using ANOVA test and the comparison of density with sex was tested using Student’s t test. A p value of .05 was chosen as the threshold for statistical significance. SPSS, Version 11  was used for all statistical calculations.The difference in density of cervical and mediastinal parts of thymus was tested using paired p 0.001). Mean value of CT density of cervical thymus is lower than CT density of mediastinal thymus by ~ 25 units . That is, if the CT density of mediastinal part of thymus is high, CT density of cervical thymus is also likely to be high; and vice versa.CT densities of both cervical and mediastinal thymus were found to reduce with age, but the reduction was statistically significant only in the cervical thymus in this study Table .After adjusting for age, there is no difference demonstrated in densities  between males and females.The 22 studied patients included 10 males (45.5%) and 12 female patients (54.5%); age varied from 1 to 34 years (mean age 14); 19 patients had contrast-enhanced CTs and 3 patients had non-contrast CTs Additional file Embryological pathway of thymic descent is from the angle of mandible to the superior mediastinum ; hence, thymus can be found anywhere along this path. Continuity with mediastinal thymus distinguishes cervical component of thymus from ectopic thymic tissue. Cervical component of thymus may be present at all ages , though On ultrasound, thymus of infants may be homogeneous, with echogenicity slightly less than liver and spleen  or have Thymic size and density are affected by a number of factors. Thymic size decreases with age; acute size reduction may occur during stress ; true hyperplasia occurs in recovery from recent stress ; when reOur study demonstrates that CT density of cervical part of thymus may vary from that of mediastinal part of the thymus, with density of cervical thymus being lower than the density of mediastinal component. But densities of cervical and mediastinal parts of thymus show positive correlation. We did not study the inter-observer variation in CT density, based on the excellent intra- and inter-observer agreements noted previously , 13, 14.The difference in CT density between cervical and mediastinal parts of the thymus may be partially due to the non-involvement of the cervical thymus in thymic hyperplasia. Most of our oncology patients would have had stress  and reactive thymic hyperplasia after recovery from stress.Limitations of our study include the retrospective nature of the study and absence of histological correlation. All patients in the study were imaged for non-uniform clinical reasons. Sick patients may have a higher rate of thymic hyperplasia and hence increased the difference in densities between cervical and mediastinal components of the thymus, but this is acceptable because it mirrors the actual incidence that would be seen on medical imaging.CT densities of cervical and mediastinal components of thymus may vary, with density of cervical thymus being lower than the density of mediastinal component. When continuity with mediastinal thymus is seen on CT, diagnosis of the cervical thymus can be made even in the presence of different CT densities. Densities of cervical and mediastinal parts of thymus show positive correlation. Decrease in CT density of thymus with age, noted previously in the mediastinal thymus, has been demonstrated in cervical thymus also. Understanding these may help avoid confusion on CT and avoid the need for correlative USG, saving time and effort.Additional file 1:Table S1. Patient data. (DOCX 20 kb)


Food environments have rapidly changed over the past years in China and children have more access to unhealthy food in convenience stores near schools. Since the studies on the association between convenience stores near schools and obesity had inconsistent results and no similar study in China, we conducted a study on the association in Beijing of China, which will provide scientific evidence for the intervention of childhood obesity.The study included 2201 students at grade 4 of 37 primary schools in Dongcheng or Miyun district of Beijing. The food environment data was acquired from AMAP, the free web-based geospatial service provider. The numbers of convenience stores were captured within the 800-m network buffer near schools using Geographic Information System. The weight and height of each student were measured by trained health professionals. Students’ dietary and physical behaviors and other information associated with obesity were collected with questionnaires for students and their parents. The generalized linear mixed model (GLMM) was used to analyze the data.CI): 1.03,1.24, P = 0.011). Compared with less than 24 convenience stores near schools, the students with more than or equal to 24 convenience stores near schools had an increased risk of obesity .The average age of the students was 10.2 years (Standard Deviation (SD) = 0.33). The prevalence of obesity in students was 14.9%. The median number of convenience stores within the 800-m network buffer near schools was 24 in two districts. The number of convenience stores near each school varied from 5 to 67 (median: 25) in Dongcheng district and from 1 to 57 (median: 22) in Miyun district. After adjusting for the confounding factors at the family and individual levels, the association between convenience stores and childhood obesity was statistically significant. Additional ten convenience stores near schools were associated with an increased risk of obesity (Odds Ratio (OR) = 1.13, 95% confidence interval (The students with more convenience stores near their schools had an increased risk of obesity. The findings provided evidence for developing public health policy to restrict the number of convenience stores near schools to prevent and control childhood obesity. Childhood obesity has emerged to be a major public health problem. Based on the Chinese National Survey on Students Constitution and Health, the prevalence of overweight and obesity in Chinese children and adolescents has increased continuously since 1985, reaching 19.4% in 2014 , evaluatChildhood obesity is a complex condition associated with the interaction of genetics and environmental factors which include physical activity, diet, and other relevant factors . RecentlBecause of the potential role of convenience stores near schools on students’ diet behaviors of the intake of fruits, vegetables, snacks, and fizzy drinks , some stThe mainland of China has been facing rapid economic growth and dramatic change of food environments, and children have more access to unhealthy food in convenience stores near schools. Thus, we conducted the study aiming to elucidate the association of convenience stores near schools and obesity of school-aged children in Beijing of China, providing scientific evidence for the intervention of childhood obesity.We recruited participants in grade 4 of 37 primary schools in Dongcheng or Miyun district of Beijing, China, from May to June in 2016. The inclusion criteria for schools were as follows: Firstly, non-boarding, non-specialty and non-minority schools. Secondly, schools with no less than 50 students in grade 4. Thirdly, schools’ leaders agreed to participate in the study. All the eligible primary schools (43 schools) which met the inclusion criteria were invited to complete the school-based obesity prevention survey. Finally, 37 of the 43 schools (86%) provided approval for the study, including 23 schools in Dongcheng district and 14 schools in Miyun district . We used the standard of Working Group on Obesity in China (WGOC) to define the nutritional status [Height (cm) and weight (kg) were measured according to the standard procedure. Participants were required to wear light clothes and stand straight, barefoot and at ease when being measured. Weight was measured to the nearest 0.1 kg with a standardized lever scale and height to the nearest 0.1 cm with a portable stadiometer. Both the scales and stadiometers were calibrated before measurements. Body mass index (BMI) was calculated as body weight (kg) divided by height (m) squared . The validity study showed that the correlation between BKFS and three 24-h dietary recalls was 0.526, 0.600, 0.681, 0.478, 0.869 in the intake of vegetables, fruits, meat, sugar-sweetened beverages, and milk, respectively . We askeWe used AMAP, the free web-based geospatial service provider, to geocode and locate the schools’ addresses. By using the Application Programming Interface (API) on the AMAP open platform, we accessed to Point of Interest data (POI). Convenience stories were defined as the POI from 060200 to 060202 in AMAP, which included all small convenience stores and grocery stores such as 7-Eleven, Good Neighbor, Lin Jia, and other chain and non-chain convenience stores. We calculated the density of convenience stores located within an 800-m street network buffer of school centroid. We used the 800-m buffer for the reason that it approximately corresponded to 10- min walk, which was commonly used in previous studies .We also undertook an on-foot ground audit in 8 schools. We recorded the actual information of the convenience stores and compared the differences in the information between the AMAP and the ground audit. We also calculated the accuracy of the convenience stores from the map, by the number from both map and ground audit divided by the number from the ground audit. Finally, the accuracy for the convenience stores was 80.9%, which was similar to the previous study .Means and standard deviations were calculated for continuous variables with normal distribution, while medians and interquartile ranges for non-normally distributed variables. Frequencies were calculated for categorical variables. We defined more than or equal to the median number of convenience stores as more convenience stores and less than the median number as fewer convenience stores. We tested differences between Dongcheng district and Miyun district in categorical characteristics with the chi-square test, normally distributed continuous characteristics with t test, non-normally distributed continuous characteristics with Wilcoxon signed-rank test.We used Generalized Linear Mixed Model to explore the association of childhood obesity and convenience stores near schools. We used childhood obesity as the outcome variable and the number of convenience stores as the continuous predictor or more convenience stores as the categorical predictor. PROC GLIMMIX procedure was run with schools modeled as a random effect because of the cluster sampling of schools, using logit as the link function. Based on the previous studies on the association between food environment and childhood obesity , we chosβ0: the fixed intercept.μj0: the random intercept.i: individual level.j: school level.βj1 × schoolj: the random effect of the school.convenience stores: either as a continuous variable or a categorical variable.ε: the residual effect.All statistical analyses were performed using SAS 9.4.The 2201 students in grade 4 of 37 schools participated in the study. The characteristics of students were shown in Table Table The number of convenience stores near schools was shown in Table CI: 1.03,1.24). After dividing the number of convenience stores according to the median, the association was more significant. Compared with fewer convenience stores near schools (less than 24), students with more convenience stores near schools  had an increased risk of obesity .In the analysis of the generalized linear mixed model Table , we adjuCI: 1.03,1.24). Compared with less than 24 convenience stores near schools, the students with more than or equal to 24 convenience stores near schools had an increased risk of obesity .We found a significant association between convenience stores near school and childhood obesity in our study. Additional ten convenience stores near schools were associated with an increased risk of obesity (OR = 1.13, 95% et.al [et.al [et.al [Our findings of a positive association between convenience stores near schools and the prevalence of obesity corresponded to some studies carried out in North America. Sanchez et.al  found thl [et.al  and Langl [et.al  showed tSeveral studies in western countries were not in accordance with our study, which couldn’t identify a significant association between convenience stores near school and childhood obesity. The inconsistency may be due to the differences in types of the buffer zone, buffer distance, source of information about food environments, definitions of food environments, measurements of participants’ weight and height, adjustment of the potential covariates of dietary and physical behaviors, and sample size. Some of the studies investigated the food environments in a circular buffer instead of a street network buffer –27, 29. In China, there was no study on the association between convenience stores near school and childhood obesity in mainland, and only two studies were conducted in Chinese Taiwan , 33. TheStudies have also shown that school-aged children would like to buy sugar-sweetened beverages and energy-dense snacks when they went to school or went back home . Intake Our study had the significance not only for parents but also for policymakers. Although China has announced the policy that schools are banned to have retail shops and vending machines within the campus, the significant role of the convenience stores near the campus in childhood obesity was ignored. The findings provided evidence for developing the public health policy to restrict the number of convenience stores near schools to prevent and control childhood obesity.There are some strengths in our study. First, to our knowledge, it was the first study using the geographical information system to investigate the association between convenience stores near schools and childhood obesity in mainland of China. Besides, we included information at the district, family, and individual levels, which allowed us to adjust for the potential covariates at different levels. Especially, we adjusted for the dietary and physical behaviors, which were the important influencing factors of obesity, to assess the true association between convenience stores near schools and childhood obesity.There are some limitations in the present study. Firstly, given the cross-sectional design of our study, it has the limited capability to infer causality between convenience stores near school and childhood obesity. Secondly, although we adjusted for several confounding factors at the district, family, and individual levels, we can’t rule out the possibility of residual confounding factors. We didn’t adjust for other food environments like the density of fast food outlets, supermarkets or other markets in the region and vending machines in the school. However, according to the situation in China, the fast food shop serves more expensive foods than convenience stores, and students are unlikely to go to supermarkets or other markets alone. The Education Committee in Beijing has banned the retail shops and vending machines on the campus since 2013, so there’re no retail shops within our study schools. So our study results indicated convenience stores near schools had a more important role in primary students in China with little potential for the residual confounding of other types of food stores, which need further studies for validation. What’s more, our study was conducted in the two districts of Beijing. Further studies are needed to investigate the association between convenience stores near schools and childhood obesity in a large number of Chinese schools in different regions of China.The students with more convenience stores near their schools had an increased risk of obesity. The findings provided evidence for developing the public health policy to restrict the number of convenience stores near schools to prevent and control childhood obesity.Additional file 1: Figure S1. Flowchart of primary schools in the study


Identifying possible new biological activities of psychoactive substances belonging to various chemical classes may lead to a better understanding of their mode of action and side effects. We report here that amines structurally related to amphetamine, a widely used psychoactive substance, such as amphetamine, methamphetamine, phentermine, mephentermine, and chlorphenteramine, potently activate several carbonic anhydrase  isoforms involved in important physiological functions. Of the 11 investigated human (h) isoforms, the widespread hCA I and II, the secreted hCA VI, as well as the cytosolic hCA XIII, and membrane-bound hCA IX and XIV were poorly activated by these amines, whereas the extracellular hCA IV, the mitochondrial enzymes hCA VA/VB, the cytosolic hCA VII, and the transmembrane isoform hCA XII were potently activated. Some of these enzymes are abundant in the brain, raising the possibility that some of the cognitive effects of such psychoactive substances might be related to their activation of these enzymes. Here, we report the first such study, showing that amphetamine, methamphetamine, phentermine, mephentermine, and chlorphenteramine, potently activate several CA isoforms, some of which are highly abundant in the brain, where they play important functions connected to cognition and memory, among others,As mentioned above, many psychoactive compounds possess the phenethylamine scaffold and the general formula Ar–CH2.2.1.1–4 were commercially, highest purity available derivatives from Sigma-Aldrich  and were used without further purification. Amines 5–9 were certified standards from the standards collection of University of Florence, Struttura di Tossicologia Forense, furnished under authorisation from the Italian Ministry of Health by LGC standards SRL .Compounds 2.2.2 hydration activity with Phenol red as indicator, working at the absorbance maximum of 557 nm, following the initial rates of the CA-catalysed CO2 hydration reaction for 10–100 s. For each activator at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of activator (0.1 mM) were prepared in distilled–deionised water and dilutions up to 0.1 nM were done thereafter with the assay buffer. The activation constant (KA), defined similarly with the inhibition constant KI, was obtained by considering the classical Michaelis–Menten equation (A]f is the free concentration of activator.A stopped-flow methodKM ([S] ≪ KM), and considering that [A]f can be represented in the form of the total concentration of the enzyme ([E]t) and activator ([A]t), the obtained competitive steady-state equation for determining the activation constant is given by Equation (2)v0 represents the initial velocity of the enzyme-catalysed reaction in the absence of activator. All CA isozymes used in the experiments were purified recombinant proteins obtained as reported earlier by our groupWorking at substrate concentrations considerably lower than 3.3.1.5–9 are widely used psychoactive substances  stimulants and were originally used as drugs, for the treatment of attention deficit hyperactivity disorder (ADHD), narcolepsy, obesity, nasal congestion, and depressionAmines 5–9 against 11 catalytically active and physiologically relevant hCA isoforms, that is, the cytosolic hCA I, II, VII, and XIII, the membrane-associated hCA IV, the mitochondrial hCA VA and VB, the secreted hCA VI, as well as the transmembrane isoforms hCA IX, XII, and XIV,,,,,,We investigated the CA activating properties of amines 5–9 and standard CAAs 1–4 are shown in CA activation data against these 11 isoforms with psychotropic amines 5–9 can be drawn from data of 5–9, with activation constants in the range of 24.1–79.5 µM. The best activators of hCA XIII were amphetamine 5 and methamphetamine 6, whereas phentermine 7, mephentermine 8, and chlorphenteramine 9 showed a decreased potency. It is interesting to note the differences between the activating effects of amines 5–9 and the standard CAAs 1–4: hCA I for example is effectively activated by compounds 1–4, whereas hCA II is poorly activated by histamine 1 and L-adrenaline 4, but highly effectively activated by amino acids such as L-His and D-Phe .The cytosolic, widespread isoforms hCA I and II were not significantly activated by these amines up to concentrations as high as 150 µM, whereas hCA XIII, another cytosolic isoform with a more particular expression pattern (as it is widespread in many organs but with quite low expression level)nd D-Phe . hCA XII,KAs ranging between 98 nM and 0.93 µM. The most effective hCA VII activator was chlorphenteramine 9 whereas the remaining derivatives, possessing the phenethylamine scaffold showed rather similar, submicromolar activation constants (KAs of 0.64–0.93 µM). No major differences in the activating capacity were observed between the primary and secondary amines, or between the derivatives possessing the relatively not sterically hindered α-methyl group near the amino functionality, such as 5 and 6, compared to the sterically hindered, structurally related amines 7 and 8 , followed by phentermine 7 and amphetamine 5 (KAs of 74–94 nM). The sterically hindered (at the amino moiety) mephentermine 8 was the least effective CAA with a KA of 1.03 µM. There is a huge difference of activity between the psychotropic amines investigated here 5–9, which act as efficient or highly efficient hCA IV activators, and compounds 1–4, which were quite ineffective such agents with KAs in the range of 7.3–45 µM .3–45 µM .5–9, with KAs ranging between 0.24 and 2.56 µM (8 against hCA VB (KA of 240 nM) and chlorphenteramine 9 against hCA VA (KA of 310 nM), with the remaining compounds (except amphetamine 5 against hCA VB which showed a KA of 2.56 µM) showing a rather effective, submicromolar activation profile against both isoforms and with a flat SAR , the standard CAAs 2–4 were generally less effective mitochondrial CA activators compared to the psychotropic amines 5–9  2.56 µM . The besines 5–9 .5–9 up until 150 µM concentration of activator, whereas compounds 1–3 showed a much better activating efficacy efficacy .5 and methamphetamine 6 (KAs >150 µM), whereas the remaining psychotropic amines 7–9 showed weak activating effects, with KAs of 25.8–34.6 µM. However, the second tumour-associated, transmembrane isoform hCA XII showed a very different activation profile with these compounds compared to hCA IX. Indeed, amphetamine 5, methamphetamine 6, and chlorphenteramine 9 were submicromolar activators (KAs of 0.64–0.97 µM) whereas the sterically hindered amines 7 and 8 were one order of magnitude less efficient as hCA XII activators (KAs of 3.24–6.12 µM). The primary amines 5 and 7 were in this case more effective activators compared to the corresponding secondary amines 6 and 8. Among the standard CAAs, only L-adrenaline 4 showed the same level of activity as amines 5 and 6, the remaining derivatives 1–3 being generally less effective as hCA XII activators  for the activation of these enzymes with psychotropic amines 4.5, methamphetamine 6, phentermine 7, mephentermine 8, and the structurally diverse chlorphenteramine 9, for their activating effects on 11 CA isoforms of human origin, hCA I, II, VII, and XIII (cytosolic isoforms), the membrane-associated hCA IV, the mitochondrial hCA VA, and VB, the secreted hCA VI, as well as the transmembrane isoforms hCA IX, XII, and XIV. The widespread hCA I and II, the secreted hCA VI, as well as the cytosolic hCA XIII and membrane-bound hCA IX and XIV were poorly activated by these amines, whereas the extracellular hCA IV, the mitochondrial enzymes hCA VA/VB, the cytosolic hCA VII, and the transmembrane isoform hCA XII were potently activated. Some of these enzymes  are abundant in the brain, raising the possibility that some of the cognitive effects of such psychoactive substances might be related to the activation of these enzymes. Furthermore, unlike the CAIs which are in clinical use for decades for a multitude of applications,We investigated psychotropic amines based on the phenethylamine scaffold, such as amphetamine


Automated continuous noninvasive ward monitoring may enable subtle changes in vital signs to be recognized. There is already some evidence that automated ward monitoring can improve patient outcome. Before automated continuous noninvasive ward monitoring can be implemented in clinical routine, several challenges and problems need to be considered and resolved; these include the meticulous validation of the monitoring systems with regard to their measurement performance, minimization of artifacts and false alarms, integration and combined analysis of massive amounts of data including various vital signs, and technical problems regarding the connectivity of the systems. Patient monitoring by definition is the repeated or continuous observation of vital signs or physiologic functions to ensure patient safety and guide therapeutic interventions. Today, most advanced cardiorespiratory monitoring systems depend on invasive sensors, cables, and bulky monitors to recognize, transfer, process, and display the bio-signals to be monitored. Therefore, continuous advanced cardiorespiratory monitoring is mainly restricted to the intensive care unit, the operating room, and the post anesthesia care unit. Most other monitoring in the hospital continues to be basic and intermittent—including monitoring on medical and surgical general care wards. When at home, before-and-after hospital admission, patients are usually not monitored at all .While most advanced monitoring is in place in intensive care units, nearly half of all adverse events in hospitalized patients occur on the general care ward –4. IroniCurrent ward monitoring protocols typically consist of intermittent spot checks by a nurse about every 4–8 h. This leaves patients unmonitored for most of the time during their hospital stay , 10. SimA closed claims analysis of opioid induced respiratory compromise on the general care ward identified nearly half of all these events occurred within 2 h of the last nursing check . In addiAutomated continuous noninvasive ward monitoring is a promising approach to closely follow changes in vital signs over time and thus identify patients who are deteriorating in a timely fashion Fig. . The ratThere is already some evidence that intensified and automated ward monitoring of vital signs may improve patient outcome by a reduction of rescue events . Before-Although continuous ward monitoring is not standard of care today, innovative monitoring systems—in theory—would already allow us to noninvasively and continuously monitor heart rate, blood pressure, respiratory rate, oxygen saturation, skin temperature, body posture, activity, and location within the hospital –28. BattFurthermore, before automated continuous noninvasive ward monitoring becomes a reality in routine clinical care outside of studies, several problems and limitations need to be considered. Most importantly, monitoring systems need to be reliable, accurate, and be able to provide readings of vital signs with a low rate of artifacts and false alarms. However, some of the currently available monitoring systems lack clinically acceptable accuracy and precision . TherefoAutomated continuous noninvasive ward monitoring of a variety of vital signs with one or more sensors will create a massive amount of data that need to be processed in real-time, stored, and secured. These data reflecting different bio-signals will need to be integrated and analyzed together to allow the identification of certain patterns of vital sign alterations instead of merely recognizing that single values of single variables are outside of their normal range. Several predictive statistical models have already been developed, validated, and embedded in electronic medical records as automated aggregated “early warning scores” that assign weights to altered vital signs proportionate to their deviation from normal ranges –37. IdenOther challenges concern the technical connectivity between sensors and monitoring systems. While the “internet of things”  became part of our daily life, wireless data transmission and processing are not yet well established in hospitals and other health care facilities. Problems for the implementation of wireless monitoring systems include—but are not limited to—range, power consumption, integration in electronic health records, and cybersecurity . Legal iFinally, before automated continuous noninvasive ward monitoring can be recommended for routine clinical use, we need to await the results of adequately powered randomized controlled trials demonstrating its effectiveness in improving the quality of care and carefully chosen patient-centered outcomes. As a next step, research may then focus on investigating which patients benefit from expanding ward monitoring to home monitoring in the period after hospital discharge , 40.Automated continuous noninvasive ward monitoring seems to be an intriguing opportunity to timely detect clinical problems by recognizing subtle changes in vital signs and improve patient outcomes on the general care ward. There is already some evidence—mainly from before-and-after studies—that automated ward monitoring can improve patient outcome. From a technical point of view, monitoring systems for automated continuous noninvasive ward monitoring are already available and will be further refined during the next years, probably resulting in small, wireless, and wearable sensors. However, before automated continuous noninvasive ward monitoring can be implemented in clinical routine, several challenges and problems need to be considered and resolved; these include the meticulous validation of the monitoring systems with regard to their measurement performance, minimization of artifacts and false alarms, integration and combined analysis of massive amounts of data including various vital signs, and technical problems regarding the connectivity of the systems. The primary scientific aim, though fairly simple, needs some thought and well-planned trial design, and would look to evaluate in robust and adequately powered trials whether automated continuous noninvasive ward monitoring can improve patient outcome compared with current standard spot-check monitoring. Till such time, it seems rather inappropriate to leave our patients under-monitored and unprotected for large periods of time as they recover from illness on our general care hospital wards.


Hypomagnesaemia (blood Mg2+ concentration <0.7 mmol/l) has serious consequences as it increases the risk of developing type 2 diabetes and accelerates progression of the disease. This study aimed to determine the mechanism by which triacylglycerol levels affect blood Mg2+ concentrations.The blood triacylglycerol level is one of the main determinants of blood Mg2) who participated in the 300-Obesity study , we investigated the association between serum Mg2+ with laboratory variables, including an extensive lipid profile. In a separate set of studies, hyperlipidaemia was induced in mice and in healthy humans via an oral lipid load, and blood Mg2+, triacylglycerol and NEFA concentrations were measured using colourimetric assays. In vitro, NEFAs harvested from albumin were added in increasing concentrations to several Mg2+-containing solutions to study the direct interaction between Mg2+ and NEFAs.Using samples from 285 overweight individuals . After lipid loading, we observed a postprandial increase in plasma triacylglycerol and NEFA levels and a reciprocal reduction in blood Mg2+ concentration both in mice  and in healthy humans (Δ plasma Mg2+ −0.07 mmol/l at 6 h post lipid intake). Further, in vitro experiments revealed that the decrease in plasma Mg2+ may be explained by direct binding of Mg2+ to NEFAs. Moreover, Mg2+ was found to bind to albumin in a NEFA-dependent manner, evidenced by the fact that Mg2+ did not bind to fatty-acid-free albumin. The NEFA-dependent reduction in the free Mg2+ concentration was not affected by the presence of physiological concentrations of other cations.In the cohort of overweight individuals, serum Mg2+ levels, in part explaining the high prevalence of hypomagnesaemia in metabolic disorders. We show that blood NEFA level affects the free Mg2+ concentration, and therefore, our data challenge how the fractional excretion of Mg2+ is calculated and interpreted in the clinic.This study shows that elevated NEFA and triacylglycerol levels directly reduce blood MgThe online version of this article (10.1007/s00125-018-4771-3) contains peer-reviewed but unedited supplementary material, which is available to authorised users. The stuws et al . High-thws et al  was usedws et al . Detailsws et al . In the g and plasma was procured.This animal study was approved by the animal ethics board of the Radboud University Nijmegen (RU DEC 2015-0073) and by the Dutch Central Commission for Animal Experiments . Twelve male C57BL6/J mice  were obtained at an age of 9–10 weeks. Mice were acclimatised for 2 weeks in a temperature- and light-controlled room, with six mice per cage , and were allowed free access to acidified tap water and standard pellet chow . After the acclimatisation period, mice received experimental chow containing 18.3% protein (wt/wt), 4.1% crude fat (wt/wt), 25.1% starch (wt/wt) and 33.6% sugar (wt/wt) . After 2 weeks on the synthetic diet, mice were fasted overnight, from 21:00 to 09:00 hours, before receiving 200 μl intragastric olive oil  via oral gavage. Blood was drawn via tail-bleed, using chilled sodium-heparin capillary tubes  coated with paraoxon , before the gavage (0 h) and at 1, 2, 4, 6 and 8 h post gavage. The blood was centrifuged at 3000 All procedures for this experiment were conducted in compliance with the UK Animals Scientific Procedures Act (1986) and University of Oxford ethical guidelines. Kir6.2-p.Val59Met mice were generated in our laboratories, as previously published . All micThree days after tamoxifen injection, mice were fasted overnight from 17:00 to 09:00 hours. Successful induction of the Kir6.2-p.Val59Met transgene was validated by measuring fasted blood glucose levels using a StatStrip Xpress glucose meter . One female mouse did not have elevated fasting glucose levels and was excluded from subsequent analyses. The remaining mice then underwent oral gavage and blood sampling using an identical experimental set up to that described above for C57BL6/J wild-type mice.2 solution  or a physiological buffer, both set at pH 7.5 by adding NaOH. The physiological buffer contained the following : 27 mmol/l NaHCO3, 112 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l MgCl2, 1 mmol/l Na2HPO4  and 2.5 mmol/l CaCl2 dissolved in Milli-Q .BSA and fatty-acid-free BSA (FF-BSA) (both Sigma Aldrich) were separately dissolved at several concentrations in a 1 mmol/l MgClg to clog the protein in the filter. Extracted NEFAs were added to 250 μl FBS , 1 mmol/l MgCl2, or 0.5 mmol/l BSA in the amounts of 0, 25, 50, 100, 200, 350, 500 and 700 μl. Dilution factors were accounted for when measuring the concentrations of Mg2+ and NEFA.To extract endogenous NEFAs, BSA (0.2 g/ml) dissolved in Milli-Q was mixed (1:2 vol./vol.) with ice-cold ethanol–diethyl ether (3:1 vol./vol.). The lipid phase was evaporated overnight at room temperature. To remove trace amounts of BSA, the solution was centrifuged three times with an Amicon 50 kDa filter (Merck Millipore) for 15 min at 2500 2+  concentrations were measured using a spectrophotometric assay according to the manufacturer’s protocols. The Mg2+ colourimetric assay was based on a Xylidyl Blue-I method and the absorbance was measured at 600 nm. NEFAs were measured at 546 nm, triacylglycerols at 500 nm and protein at 562 nm on a Bio-Rad Benchmark plus microplate spectrophotometer . For inductively coupled plasma mass spectrometry (ICP-MS) analyses, serum samples were dissolved in HNO3  and diluted prior to being subjected to ICP-MS .Protein , NEFA , triacylglycerols  and MgClinicalTrials.gov registration no. NCT01967459), which aimed to examine the effect of vitamin D supplementation on postprandial leucocyte activation, 24 female volunteers underwent an oral fat-loading test. The study was approved by the Institutional Review Board of the Franciscus Gasthuis & Vlietland Rotterdam and the regional independent medical ethics committee of the Maasstad Hospital Rotterdam  × 100), where sMg and uMg are the serum and urinary Mg2+ levels, respectively, and sCr and uCr are the serum and urinary creatinine levels, respectively [2+ to albumin [Approximately 30% of blood Mg albumin . Howeverectively . As the  albumin . This pa2+, while the cream used in the human study contains 3.3 mmol/l Mg2+, leading to a potential underestimation of the reduction in serum Mg2+ in the healthy volunteers. Moreover, in the in vitro experiments, NEFAs extracted from BSA were used to increase NEFA levels in several solutions. However, the yield of this extraction procedure was not equal in each experiment performed, making it difficult to combine data from all experiments. Despite these differences in NEFA yield, the results were similar in all four replicate experiments. Finally, in overweight individuals and in individuals with type 2 diabetes, Mg2+ inversely correlates with triacylglycerols [2+ to NEFA molecules, which, in contrast to triacylglycerol molecules, possess a negative charge. It is unlikely that Mg2+ binds to uncharged triacylglycerol molecules. However, in humans, blood triacylglycerols and NEFA levels strongly correlate, meaning that most individuals with hypertriacylglycerolaemia also have elevated NEFA levels, which would underlie the inverse correlation between Mg2+ and triacylglycerols [Several limitations need to be considered with regard to this study. Hypertriacylglycerolaemia in mice was achieved using olive oil, while in human volunteers this was done by an oral load of cream. Olive oil contains no Mglycerols , 23, 24.lycerols –51.2+, which could explain why hypomagnesaemia is so prominent in diseases such as type 2 diabetes. Our data do not rule out the possibility that changes in Mg2+ concentrations could also influence lipid levels.This study has several strengths. Our data extend from molecule to population and have clinical implications. Moreover, we demonstrated the directionality of the inverse association between triacylglycerols and Mg2+ concentrations by the binding of Mg2+ ions to NEFA molecules. Our data explain the high prevalence of hypomagnesaemia in several metabolic diseases, characterised by elevated triacylglycerol levels [2+ levels should be routinely measured and monitored.In conclusion, we show that elevated blood NEFA and triacylglycerol levels directly reduce blood Mgl levels –3. In inESM(PDF 161 kb)


Despite sustained economic growth and reduction in money metric poverty in last two decades, prevalence of malnutrition remained high in India. During 1992–2016, the prevalence of underweight among children had declined from 53% to 36%, stunting had declined from 52% to 38% while that of wasting had increased from 17% to 21% in India. The national average in the level of malnutrition conceals large variation across districts of India. Using data from the recent round of National Family Health Survey (NFHS), 2015–16 this paper examined the spatial heterogeneity and meso-scale correlates of child malnutrition across 640 districts of India.I statistics and bivariate LISA maps were used to understand spatial dependence and clustering of child malnutrition. Multiple regression, spatial lag and error models were used to examine the correlates of malnutrition. Poverty, body mass index (BMI) of mother, breastfeeding practices, full immunization, institutional births, improved sanitation and electrification in the household were used as meso scale correlates of malnutrition.Moran’s I statistics was 0.65, 0.51 and 0.74 for stunting, wasting and underweight respectively suggesting spatial heterogeneity of malnutrition in India. Bivariate Moran’s I statistics of stunting with BMI of mother was 0.52, 0.46 with poverty and − 0.52 with sanitation. The pattern was similar with respect to wasting and underweight suggesting spatial clustering of malnutrition against the meso scale correlates in the geographical hotspots of India. Results of spatial error model suggested that the coefficient of BMI of mother and poverty of household were strong and significant predictors of stunting, wasting and underweight. The coefficient of BMI in spatial error model was largest found for underweight  followed by stunting  and wasting . Women’s educational attainment and breastfeeding practices were also found significant for stunting and underweight.The univariate Moran’s Malnutrition across the districts of India is spatially clustered. Reduction of poverty, improving women’s education and health, sanitation and child feeding knowledge can reduce the prevalence of malnutrition across India. Multisectoral and targeted intervention in the geographical hotspots of malnutrition can reduce malnutrition in India.The online version of this article (10.1186/s12889-018-5873-z) contains supplementary material, which is available to authorized users. Reduction in malnutrition and poverty is the primary agenda among public health professionals, planners and policy makers at global, national and regional level. Though nutritional deficiency affects all age groups, children under five-year age group are at higher risk. Globally, 156 million children under five years of age are stunted, 93 million are underweight and 50 million are wasted [Malnutrition is the primary cause of immuno-deficiency among the infants and the five infectious diseases  contribute to the half of all deaths in children aged less than five years and are directly associated with one or other measures of malnutrition –4. About2) of mother is consistently found a high risk factor of poor intrauterine growth and low birth weight may lead to malnourished children [Malnutrition is primarily caused due to the immediate causes , underlying causes  and the basic causes  –17. The children , 29. Parchildren .During last two decade, India has experienced sustained economic growth (over 5% growth in GDP) and reduced the poverty level by half (from 50% in 1993–94 to 22% by 2011–12) but reduction in stunting, underweight and wasting has not been observed in the same scale . EvidencThe aim of this study is to understand the spatial heterogeneity and meso-scale correlates of malnutrition across the districts of India. The study has been conceptualised with the following rationale.First, most of the studies for India examined the levels of malnutrition and explored the social and demographic correlates of malnutrition at individual and household level. However, the districts of India are large and exhibit enormous variation in socio-demographic and economic indicators. And very little is known about the spatial heterogeneity of malnutrition, particularly poverty and the associated nexus with malnutrition at a meso scale, i.e., in the districts of India. Second, geographical location has a large impact on the distal determinants of nutrition which directly or indirectly controls the availability and accessibility of foods mainly due to agricultural patterns and yields. Third, a district level analysis of malnutrition is very much needed considering the spatial occurrence of poverty. Fourth, India ranked 97th globally (118 countries) in the hunger index and the hunger index shows huge variation across the states of India . Studieshttp://rchiips.org/NFHS/districtfactsheet_NFHS-4.shtml). The unit level data is available from Demographic Health Survey (DHS) data repository and could be accessed upon a data request through www.dhsprogram.com/data/ [Data from the recently conducted National Family Health Survey round four (NFHS-4), 2015–16 is used in the analysis. NFHS 4 provides comprehensive information on fertility, mortality, maternal health care and child health including child nutrition for the states and districts of India. NFHS-4 covered a nationally representative sample of 601,509 households, 699,686 women aged 15–49 years and 103,525 men aged 15–54 years in India . The aveom/data/ –37. The om/data/ . By malnThree anthropometric indicators of nutritional status of children namely stunting (height-for-age), underweight (weight-for- age) and wasted (weight-for-height) are used as the dependent variables in this study. These three measures are the standardized measures of nutritional status of children and commonly used in literature , 40. WhiA set of proximate and distal determinants of malnutrition within a meso scale framework were used based on prior literature and data availability . Child fI statistics and a set of regression models in the analyses. Differentials in malnutrition by its correlates were presented using bivariate analyses. Univariate LISA and bivariate LISA maps were utilized in this study to identify the spatial clusters. Univariate LISA map provided the geographical clustering of different variables used in this study while Bivariate LISA measured the correlation between the independent and the weighted average of the dependent variable in a particular location. To understand the significant correlates of malnutrition, a set of regression models were used to give the best fit of the data and to understand the association of malnutrition and its correlates. At first, we used the Ordinary Least Square (OLS) regression with each of the outcome variables and estimated the extent of spatial autocorrelation in the error term and the corresponding Moran’s I statistic. Since the OLS confirmed spatial autocorrelation in its error term for all three outcome variables, we further estimated spatial lag model (SLM) and Spatial Error Model (SEM). The underlying assumption of a spatial lag model is that the observations of the dependent variable are affected in the neighbourhood areas whereas the spatial error model is used to consider the effect of those variables which are not present in the regression model but have an effect on the outcome variable. The basic difference between the two models is that the spatial lag model unlike spatial error model does not consider the spatial dependence in the error term. Diagnostics tests for spatial dependence were carried out, and the value of Lagrange Multiplier was found significant in both the models(p < 0.0001) and next we compared the Akaike Information criterion (AIC) value for both the models to know the best spatial fit. This spatial models examined the non linear relationship between the correlates and malnutrition at a meso scale controlling other demographic and distal covariates subject to the spatial structure of the data [We have used descriptive statistics, estimated univariate and biravriate Moran’s the data . Each ofThe multiple linear regression model is given as;i denotes the proportion of children malnourished in the i-th district;Where Yα is the intercept.j denotes the regression coefficient for the jth variable where j = 1 (1) 8.β i  denotes the no of districts.2, CPOV is the poverty head count ratio for the i-th district, WEDU is the district level proportion of women with 10 or more years of schooling, ID gives the district level percentage of institutional delivery, FI is the percentage of full immunization in the district; CFP is the percentage of children in the i-th district who are breastfed and received adequate diet; IS is the percentage of households with improved sanitation and ELECT denotes the percentage of households in the i-th district with electricity connection.BMI denotes the percentage of women in the i-th district whose body mass index (BMI) is below 18.5 kg/mThe spatial lag model assumes that the dependent variable in one area is affected by the dependent variable in the nearby. A typical spatial lag model can be written as follows:i denotes the prevalence of malnutrition for the i-th district, δ is the spatial autoregressive coefficient, Wij denotes the spatial weight of proximity between district i and j, Yj is the prevalence of malnutrition in the j-th district, βj denotes the coefficient, Xj is the predictor variable and εj is the residual.Here YA Spatial Error Model (SEM) is expressed as follows:i denotes the prevalence of malnutrition for the i-th district, λ is the spatial autoregressive coefficient, Wij denotes the spatial weight of proximity between district i and j, Yj is the prevalence of malnutrition in the j-th district, βj denotes the coefficient, Xj is the predictor variable and εi is the residual.Here YArcGIS version 10.1, GeoDa version 1.6.7 and STATA 12.0 are used for analyzing the data.We begin the discussion by presenting the spatial pattern of stunting, underweight and wasting in districts of India Map . The disTable 2), the average prevalence of stunting was 27.5% and it was found 44.5% in those districts where 30% of the mothers were undernourished (less than 18.5 kg/m2BMI). Figure Table I statistics depicting the extent of spatial autocorrelation of malnutrition and the meso scale predictors. The Moran’s I values were 0.65 for stunting, 0.74 for underweight and 0.51 for wasting. All three coefficients were statistically significant. The district level estimates on poverty head count ratio (PHCR) showed Moran’s I value of 0.59. Among all the variables including the malnutrition variables, the Moran’s I value was highest for underweight followed by improved sanitation and lowest for full immunisation. Based on Moran’s I statistics values, we inferred that the pattern of spatial autocorrelation was highest for underweight followed by stunting and wasting in districts of India. This confirmed the clustering of districts in terms of malnutrition among the children under age five.Additional file I statistics for stunting, underweight and wasting against the correlates. It was found that the spatial autocorrelation of stunting and poverty was 0.46 and that with BMI was 0.53. Similarly, the spatial autocorrelation of underweight and stunting with BMI was 0.66 and 0.52 respectively. In general, poverty and women’s BMI showed high and positive spatial autocorrelation with each of the three measures of malnutrition while women’s education, institutional delivery, children’s immunization status, feeding practices and household level sanitation showed a negative spatial autocorrelation with stunting and underweight. Map Table 2. The bivariate LISA cluster map suggested that around 135 districts constitute the hot spots  whereas 112 districts constitute the cold spots  (Map Map ing) Map . SimilarI = 0.31, p value = 0.000001; Underweight: Moran’s I = 0.50, p value = 0.000001; Wasted: Moran’s I = 0.37, p value = 0.000001). This suggested that the prevalence of malnutrition among the children was not distributed uniformly across the districts of India and occurred in particular clusters. Hence, we rejected the null hypothesis and accepted that there is a positive spatial autocorrelation in the prevalence of malnutrition and we further estimated the spatial autoregressive models to consider the autocorrelation into account. Spatial lag and error models were fitted in the data. Based upon the model diagnostics we found the spatial error model to be better performing and hence the error model gave the final estimates of the association. Additional file Table p-value< 0.001). It was observed that, compared the other models, the stunting model showed a lower AIC value with a pseudo R square value of 0.72 indicating the explained variability of the model.The SEM model gave the final and the spatial endogeneity corrected estimates of the correlates for stunting, underweight and wasted prevalence across India Table . Among tp-value = 0.036; 95% CI: 0.01, 0.22) whereas this was 0.08  for poverty.The underweight model suggested that, BMI and poverty were two most closely associated predictors of underweight prevalence across the districts. The corresponding coefficients of BMI and poverty were observed to be 0.38  and 0.10  respectively. Contrary to the stunting model, maternal education did not seem to be statistically significant predictor for underweight. Among the other correlates, full immunization, institutional births and sanitation appeared to be the significant predictors of underweight. The underweight model suggested a lag of 0.75 and the corresponding pseudo R square value of the model was 0.81 with a slightly higher AIC value (3845) than the stunting model. From the wasted model we found that the district level prevalence of wasted children under age five was mostly determined by maternal nutrition (BMI status) and poverty situation in the districts. The corresponding association between BMI and wasted was 0.11  indicated that any shock in the omitted variables which were not present but may affect the prevalence of malnutrition was strongly likely to cause a change in the prevalence of malnutrition in the neighbouring districts.I statistics suggested the measure of spatial dependence and it was found to be highest for stunting followed by underweight and wasting which confirmed the geographical gradient of malnutrition in India. Though a high level of malnutrition spread was found across some selected states and geographical regions, but the clustering was higher in the districts belonging to the states of Uttar Pradesh, Bihar, Jharkhand, Madhya Pradesh and Rajasthan. Second, the spatial analyses suggested statistically significant association of malnutrition  with the factors such as BMI status of mothers, poverty, maternal education and improved sanitation. Study results confirmed that districts with higher incidence of poverty are at higher risk of increased prevalence of stunting, underweight and wasting. Though poverty and malnutrition nexus is complex in different country settings but our results confirmed that poverty significantly affects nutritional status among the children in districts of India. Previous studies for the developing countries proved that poverty is a major contributor to the burden of child malnutrition [2 were significantly more likely to have higher prevalence of malnutrition among the children and findings were consistent with previous studies. It is well established that maternal nutrition is an important risk factor of poor intrauterine growth and low birth weight during pregnancy and undernourished mothers cannot breast-feed their children adequately causing poor nutrition to their children [The following are the salient findings from the study. First, the study findings suggest a clear spatial pattern of stunting, underweight and wasting across the districts of India. The Moran’s utrition . Similarutrition , 45. Parutrition . Third, children , 29, 47.children , 49.Similarly, malnutrition was found to be negatively and significantly associated with women’s educational attainment. Our findings also support the positive linkage between women’s educational attainment and child’s nutrition. This finding is also consistent with the previous studies which establish the impact of mother’s education on child’s nutritional status , 51. FouThis study illustrates the spatial heterogeneity of malnutrition among the children in districts of India. The findings could be useful for public health planning and targeting the underlying meso scale factors associated with child nutrition in India. It also suggests allocation of health resources and the implementation of child health specific interventions in the geographical hotspots of higher malnutrition prevalence. The spatial clustering of malnutrition is found in those geographical pockets where poverty is high, women’s education is low, BMI level among women is below normal. The malnutriton indicators also  significantly falls in terms of other distal and proximate factors, which reinforces the need for intersectoral co-ordination in fighting malnutrition in India. An integrated approach; a multisectoral co-ordination of reduction of poverty, increasing sanitation coverage and maternal nutrition can help to reduce child malnutrition in India.Additional file 1:Appendix 1. Moran’s I Statistics showing the spatial dependence for the district level prevalence of stunting and the meso scale indicators in India, 2015–16. Appendix 2. Estimated results from Spatial lag model for stunting, underweight and wasted, India, 2015–16. Appendix 3. Bivariate LISA cluster maps, India, 2015–16. Appendix 4. Districts with higher prevalence of stunting (46–65)% among children under age five, India, 2015–16. Appendix 5. Districts with higher prevalence of underweight (44–67)% among children under age five, India, 2015–16. Appendix 6. Districts with higher prevalence of wasting (28–47)% among children under age five, India, 2015–16 (PDF 192 kb).


The cementation factor is necessary to determine porosity via the Archie equation, and its range of values has been suggested in many previous studies. However, the cementation factors in the literature are limited to fully saturated conditions, and it may thus be inaccurate to use the same value in other saturation conditions. The objective of this study is to characterize how the cementation factor varies depending on the saturation percentage. In this study, glass beads and soil are selected as the specimens, and two relative density values, 40% and 80%, are selected. Time domain reflectometry (TDR) is used to obtain both the saturation and electrical resistivity of the specimens. TDR is installed in the cell, and fluid is continuously circulated from the bottom to the top of the porous material for 30 min. The estimated saturation increases with time and the electrical resistivity is reduced during the circulation. Finally, the cementation factor at every saturated stage is determined, and the error ratio based on the porosity is calculated to show the importance of the cementation factor. The results show that there is a high error ratio when an unsuitable cementation factor that does not consider the saturation condition is used. This study demonstrates that the method for determining the actual cementation factor using TDR and the Archie equation can be applied in various saturation conditions.  Pore structure plays an important role when considering the characteristics of porous material because it is affected by various physical properties, including density, strength, friction, and water flow ,2. Many Electrical resistivity measurement has been a widely used technique because it is an efficient tool with a simple experimental method and high resolution . ElectriTime Domain Reflectometry (TDR) was initially used to measure the volumetric water content in porous material with a specifically calibrated equation . To imprThe theoretical background for obtaining porosity from electrical resistivity is introduced, and the method for predicting the cementation factor with the consideration of saturation using time domain reflectometry (TDR) is explained. The laboratory test for measuring electrical resistivity and saturation is also described. Finally, the behavior of the cementation factor under various saturation conditions is discussed, and the error ratio based on the porosity is also demonstrated.PM (Ω·m) denotes the electrical resistivity of porous material, and ρP (Ω·m) and ρEL (Ω·m) denote the electrical resistivities of the particle and electrolyte, respectively. n (-), γP (N/m3), g (m/s2), λddl (S) and Sa (m2/kg) indicate the porosity, unit weight, coefficient of gravity, surface conduction and specific surface area, respectively. In general, Equation (1) can be abbreviated as Equation (2), with the neglecting electrical resistivity of the particle in the first term because the soil particle is an electrically non-conductive material; Equation (2) is generally known as the Archie equation  al al15] altrue denotes the porosity calculated from the cementation factor that is estimated using the actual saturation. In addition, ninference indicates the inferenced porosity from the cementation factor based on the 100% saturated condition. The result shows that there is a high error ratio (over 400%) at low saturation conditions, but the error ratio becomes small when the saturation is close to 100%. In particular, in the case of an approximately 100% saturation, the error ratio approaches zero. The high and low error ratios are natural results because the appropriated cementation factor is not applicable. Note that if the exact cementation factor is not calculated using the actual saturation, there will be a large error in the estimation of the porosity of the medium. Overall, the TDR technique, which is applied in this study, is useful to determine a reliable porosity considering the saturation conditions.The Archie equation is mainly used to evaluate the condition of the porous medium, and it is also used to calculate the porosity as a design parameter ,10,11. HTime domain reflectometry is selected to measure the dielectric constant, and the value is converted into the saturation and electrical resistivity of each specimen. Additionally, the saturation factor at every saturation stage is determined from the relationship between the saturation and electrical resistivity ratio.The cementation factor, suitable for each degree of saturation, is derived, and the value ranges from negative to positive.The error ratio based on the porosity is computed, and a high error ratio indicates an inaccurate saturation range. The results show that the determination of the true cementation factor is necessary for enhancing the reliability.Finally, the result shows that the Archie equation can be applied under dried, unsaturated and saturated conditions when the suitable cementation factor is applied.In this study, the distribution of cementation factor values at every saturation stage was determined for unconsolidated granular materials, and the importance of selecting the appropriate cementation factor was also shown using the error ratio based on the porosity. A detailed summary of this study follows: