There is an increasing appreciation for the role of the human Y chromosome in phenotypic differences between the sexes in health and disease. Previous studies have shown that genetic variation within the Y chromosome is associated with cholesterol levels, which is an established risk factor for atherosclerosis, the underlying cause of coronary artery disease (CAD), a major cause of morbidity and mortality worldwide. However, the exact mechanism and potential genes implicated are still unidentified. To date, Y chromosome-linked long non-coding RNAs (lncRNAs) are poorly characterized and the potential link between these new regulatory RNA molecules and hepatic function in men has not been investigated. Advanced technologies of lncRNA subcellular localization and silencing were used to identify a novel intergenic Y-linked lncRNA, named lnc-KDM5D-4, and investigate its role in fatty liver-associated atherosclerosis. We found that lnc-KDM5D-4 is retained within the nucleus in hepatocytes. Its knockdown leads to changes in genes leading to increased lipid droplets formation in hepatocytes resulting in a downstream effect contributing to the chronic inflammatory process that underpin CAD. Our findings provide the first evidence for the implication of lnc-KDM5D-4 in key processes related to fatty liver and cellular inflammation associated with atherosclerosis and CAD in men. It is becoming increasingly clear that the biological functions of the Y chromosome transcend the male fertility and reproduction2, with associations between genetic variation within the male-specific region of the Y chromosome (MSY) and complex polygenic phenotypes including blood pressure3, circulating cholesterol levels4, and coronary artery disease (CAD)5 being reported. The mechanisms of these associations are not entirely clear although it appears that they are (at least in part) mediated by the expression of MSY genes in the target cells and tissues6. To date, over 200 Y-linked genes have been identified and those that encode Y proteins have been well-characterised7. However, MSY harbours not only 27 protein-coding genes but also a number of long non-coding RNAs (lncRNAs) – important new regulatory molecules implicated in cardiovascular health8 and disease9. So far, there is no data on Y chromosome-linked lncRNAs in relation to any human phenotypes. To this end, we sought to investigate Y-linked long intergenic non-coding RNAs (lincRNAs) - the largest class within the lncRNA group10 - in relation to phenotypes of relevance to CAD such as fatty liver-associated atherosclerosis.The human Y chromosome is best known for its key role in spermatogenesis and testicular developmentin vitro model system used in previous studies on liver steatosis and CAD14. We also investigated the expression of a novel intergenic Y-linked lincRNA, named lnc-KDM5D-4, across 21 normal tissue types. Utilising advanced technologies of lncRNA subcellular localization and silencing, we further characterized lnc-KDM5D-4 within HepG2 cells, and investigated the effects of its knockdown on atherosclerosis-related genes in these cells. Throughout the text, ‘lnc-KDM5D-4′ refers to the long non-coding RNA gene, and ‘lnc-KDM5D-4:1′ refers to the long non-coding RNA transcript.Here, we studied for the first time eleven novel Y-linked lincRNAs by real-time PCR gene expression analysis in the human hepatoma HepG2 male cell line, an 16 were used to select Y-specific lincRNA genes based on their expression in tissues relevant to the liver and CAD, such as heart, liver and white blood cells (WBCs) . Primers for SYBR® Green-based real-time polymerase chain reaction (PCR) gene expression analysis were designed using the free online software Primer-BLAST  following the recommendations for probe melting temperature (Tm) values, GC content, and amplicon length provided by Thornton & Basu17. All the primers for lincRNAs were designed from the DNA template sequence annotated on LNCipedia, the database for human lncRNA genes and transcripts18. Primer sequences are available in Supplementary Table The human lincRNA catalogues®, Life Technologies™) supplemented with 10% (v/v) heat inactivated foetal bovine serum (HI-FBS) , 100 U/mL penicillin, 100 µg/mL streptomycin  and were incubated at 37 °C in a humidified atmosphere containing 5% (v/v) CO2 in the air. The HepG2 cell model of palmitate-induced steatosis and insulin-resistance were developed as previously described19 .The human hepatoma cell line, HepG2, obtained by the Charchar Laboratory, was grown as a monolayer culture in Dulbecco’s modified Eagle medium   from 20 different normal, human tissues including adipose, bladder, brain, cervix, colon, esophagus, heart, kidney, liver, lung, ovary, placenta, prostate, skeletal muscle, small intestine, spleen, testes, thymus, thyroid, and trachea. Each pool is comprised of RNA from 3 tissue donors  and underwent a stringent DNase treatment. After storage at −80 °C on arrival, RNA samples were thawed at 37 °C then placed on ice prior use as recommended by the manufacturer’s instructions.The Human Tissue RNA Panel was obtained from AmbionTo study the expression of lincRNAs expressed in leucocytes in men, five healthy male volunteers (positive control group) and two healthy females (negative control group), between 18–50 years old were recruited at the Federation University Australia (Mount Helen campus). Participants were required to meet the following inclusion criteria: current non-smokers, free of recent surgery, ambulatory aids, and acute joint injury, free of underlying medical conditions such as heart disease, un-medicated hypertension and respiratory disorders in order to avoid skewing the results. Participants’ blood was withdrawn in Ethylenediaminetetraacetic acid (EDTA) blood collection tubes (tubes commonly used in routine haematology and furnished by the phlebotomist) by a qualified phlebotomist at Mount Helen campus. Then, blood samples were immediately stored on ice and total RNA was purified within an hour of collection to preserve the RNA integrity. This study was approved by the Federation University Ethics Committee at Mount Helen campus and carried out in accordance with Federation University relevant guideline and regulations. All the subjects gave informed consent [Ethics Project Number: B13-020].®) .To confirm steatosis induced by palmitate in HepG2 cells, cellular lipid droplets were stained using Oil Red O (ORO) from Hepatic Lipid Accumulation/Steatosis Assay  was resolved on a 4–15% SDS-PAGE gradient gel (BIORAD®) and transferred to a nitrocellulose membrane .®). Total RNA was resuspended in 50 µL RNase-free water followed by a column DNase treatment (1 µL per RNA sample) using Recombinant DNase I  according to the manufacturer’s instructions. RNA yield and purity were measured by absorbance using a NanoDrop™ 2000 Spectrophotometer . The ratio (A260 nm/A280 nm) of ~2.0 was accepted as “pure” for RNA. Finally, RNA samples were stored at −80 °C until use.Total RNA was extracted from cells using TRIzol reagent (Ambion®). Real-time PCR was assessed in triplicate and carried out using SYBR® Green (Bioline®) according to the manufacturer’s instructions. Data were analysed using the 2-delta delta CT method20. CT results were normalised with two housekeeping genes, ACTB (or GAPDH) and UBC .RNA (2 µg) was reverse transcribed into cDNA with the High Capacity Reverse Transcription Kit (Life Technologies® ViewRNA ISH Cell Assay (Affymetrix®). The permeabilisation procedure was amended and 1% glacial acetic acid was added to the fixation solution to detect nuclear RNAs21, then the RNA FISH assay from Four-Chambered Dish Format protocol was performed according to the manufacturer’s instructions .RNA FISH was performed using the advanced QuantiGene®, Canada) according to the manufacturer’s instructions. Both RNA fractions, nuclear and cytoplasmic, were resuspended in 50 µL RNase-free water followed by a column DNase treatment (1 µL per 1000 ng of RNA) using the Recombinant DNase I  according to manufacturer’s instructions. RNA yield and purity were measured by absorbance using a NanoDrop™ 2000 Spectrophotometer . The ratio (A260 nm/A280 nm) of ~2.0 was accepted as “pure” for RNA. Finally, the RNA samples were stored at −80 °C until use.Nuclear and cytoplasmic RNA fractions were isolated separately from confluent cells using the Cytoplasmic & Nuclear RNA Purification Assay (Norgen Biotek Corp.™) longRNA GapmeRs - a class of high-affinity RNA analogues that exhibit high thermal stability when hybridized to a complementary DNA or RNA strand - were used to silence lnc-KDM5D-4. Antisense LNA™ longRNA GapmeRs were custom designed (except for the control GapmeR ‘Negative control A’) and synthesized by Exiqon®. Three different GapmeRs targeting three different sites of lnc-KDM5D-4:1 RNA were used following the Exiqon’s recommendations. The transfection of the following GapmeRs, lnc-KDM5D-4_GapmeR_1 (Product sequence 5′-3′: GAGATGAAGCGGAATT), lnc-KDM5D-4_GapmeR_2 (Product sequence 5′-3′: CTTACTTTAGACTTC), lnc-KDM5D-4_GapmeR_3 (Product sequence 5′-3′: ACGTAAAATAGGATTA), and Negative control A (Product sequence 5′-3′: AACACGTCTATACGC) were performed using Lipofectamine® 2000 (Thermo Fisher Scientific®) following Exiqon manufacturer’s recommendations as well as those for knockdown of lncRNA and AOs provided by Integrated DNA Technologies®’s webinars on www.youtube.com/user/idtdnabio. HepG2 cells were seeded into 6-well plates at a density of ~2 × 105/mL and cultured in DMEM supplemented with 10% HI-FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) until the cells reached ~70% confluence. Then, the cells were transfected with 10 nmol/L of AO in Lipofectamine® 2000 reagent. After 48 h, total RNA was extracted using TRIzol reagent followed by a RNA clean-up step using miRNeasy kit (Qiagen®). Then, the knockdown was confirmed by real-time PCR.Advanced antisense oligonucleotides (AOs) Locked Nucleic Acid (LNA2 Profiler PCR Array (PAHS-038ZE) from SA Biosciences  was performed following the manufacturer’s recommendations . Then, the human tissue-specific network webserver GIANT22 was used to generate the potential tissue-specific functional interactions between the atherosclerosis-relevant genes.The Human Atherosclerosis RTt-test to test significance of data comparisons between cell treatments, with p-values < 0.05 considered as statistically significant.Statistical analysis of data was performed using Prism (GraphPad Software). Data were analysed using a Student’s unpaired 16 which include the Y chromosome, we identified 11 candidate MSY lincRNA transcripts and measured their expression in the human hepatoma HepG2 cell line. Seven MSY lincRNA transcripts such as lnc-KDM5D-4:1, lnc-ZFY-1:1, lnc-ZFY-2:1, lnc-RBMY1B-1:1, lnc-RBMY1B-1:4, lnc-USP9Y-1:4, and lnc-HSFY2-3:6 were found to be expressed in the HepG2 cells  in HepG2 cells. No significant increase in the expression of the transcripts lnc-ZFY-1:1, lnc-ZFY-2:1, lnc-RBMY1B-1:1, lnc-RBMY1B-1:4, lnc-USP9Y-1:4, lnc-HSFY2-3:6, and HULC was observed   in steatosis hepatocytes have been previously demonstrated ent Fig.  and exament Fig. . To confent Fig. . The absent Fig.  suggesteent Fig. . Real-ti05) Fig. .Figure 2To investigate the possible correlation between steatosis and insulin-resistance, the potential effect of insulin resistance on the expression of lnc-KDM5D-4 was studied. To create an insulin-resistant cell line, HepG2 cells were stimulated with 10 nM of insulin solution over 0, 0.25, 0.5, 2, 4, 8, 24, and 48 h replacing the media and insulin every 8 h. Total cellular protein was extracted using RIPA buffer and 20 μg of total cellular protein was subjected to SDS-PAGE, and subsequently blotted onto Immobilon-PVDF membrane. The relative levels of phosphorylated protein kinase B (pAkt) or phosphorylated insulin-receptor (pIR) were detected relative to Total Akt and Total IR respectively Fig. . WesternTo gain a better understanding about the biological role of lnc-KDM5D-4, we examined its expression across 21 normal human tissue types including adipose, bladder, brain, cervix, colon, esophagus, heart, kidney, liver, lung, ovary, placenta, prostate, skeletal muscle, small intestine, spleen, testes, thymus, thyroid, trachea, male and female leucocytes from WBCs. The real-time PCR analysis revealed that lnc-KDM5D-4 shows ubiquitous expression with the highest level of expression in the spleen and the heart. As expected, no expression was found in female tissues such as cervix, ovary, and placenta as well as in female leucocytes Fig. .Figure 324, we examined its subcellular localization using RNA fluorescence in situ hybridization (RNA FISH) with single-molecule sensitivity. To achieve a controlled system as background image, the assay was performed without addition of glacial acetic acid (AA) to the fixation solution  to target three different sites of lnc-KDM5D-4:1. A random scrambled sequence that contains no known homology to the human, mouse, or rat genome was used as a negative control. Cells without transfection (Untreated) were also used to verify the Scramble (negative control probe) for non-specificity. Cells were harvested 48 h after transfection. AO-mediated silencing of lnc-KDM5D-4 was assessed in real-time PCR. The highest knockdown efficiency was obtained when the cells are treated with 10 nmol/L of the lnc-KDM5D-4_GapmeR_1 with a transcript reduction of 88% . The comparison of transfected cells with GapmeRs and Scramble (control GapmeR) to Untreated samples showed that there was no significant effect of transfection reagent or transfection reagent plus GapmeR respectively, on the cell ) that blocked lnc-KDM5D-4:1 activity was performed. This was followed by a gene expression analysis using the RTell Fig. .Figure 52 Profiler PCR Array was used to compare cells transfected with the lnc-KDM5D-4_GapmeR_1 and cells transfected with the control GapmeR (Scramble) , EGR1 (early growth response 1), MMP3 , IL1A , BCL2 , SERPINE1 (serpin family E member 1), VEGFA , PLIN2 (perilipin 2), ITGA2 , ITGA5 , and TNF (tumor necrosis factor)  Fig. . This aror) Fig. . Most ofor) Fig. . Finallycis by regulating their immediate neighbouring protein-coding genes leading to either an increase or reduction of their expression26. After confirmation of the lnc-KDM5D-4 knockdown in HepG2 cells, we examined the potential transcriptional activity of the nearest neighbouring protein-coding genes by lnc-KDM5D-4 such as KDM5D (lysine demethylase 5D), EIF1AY , and RPS4Y2  located within 1.1 MB window centred on lnc-KDMM5D-4. Real-time PCR analysis showed that there were no significant changes  in the expression of these neighbouring Y chromosome protein-coding genes in cells transfected with the lnc-KDM5D-4_GapmeR_1 compared to the control cells (Scramble) , that in turn, lead to increased lipid droplets formation in hepatocytes. We propose that this link may, in part, contribute to the association between certain Y chromosome haplogroups and atherosclerosis.Genetic variation within the Y chromosome has been linked to human lipid levels and cardiovascular disease30. Indeed, due to the haploid nature of the Y chromosome, the usual methods of analysis (such as genome-wide association studies (GWAS)) cannot be employed to investigate variations. This study provides the first evidence of the expression of Y-linked lincRNA transcripts such as lnc-KDM5D-4:1, lnc-ZFY-1:1, lnc-ZFY-2:1, lnc-RBMY1B-1:1, lnc-RBMY1B-1:4, lnc-USP9Y-1:4, and lnc-HSFY2-3:6 in human hepatocellular carcinoma (HCC). Further research focusing on these lincRNAs between primary human hepatocytes and HepG2 cells should be done as these lincRNAs may also play a role in HCC, and could be potential biomarkers for the diagnosis of this cancer in men. Our data also provides the first evidence for the up-regulation of the Y-linked lincRNA, known as lnc-KDM5D-4, in response to palmitate treatment in HepG2 cells. However, lnc-KDM5D-4 was not differentially expressed or affected in insulin-resistance HepG2 cells, demonstrating that the results found with the steatosis cell model were independent of those found in the insulin-resistance cells. These results confirmed that the significant changes in expression of lnc-KDM5D-4 after the FFA-palmitate treatment was triggered by the steatosis phenotype, and not by the insulin-resistance phenotype. It was suggested that lnc-KDM5D-4 was then implicated in hepatic steatosis which can occur independently of insulin resistance in the liver. Given the role of FFAs in the pathogenesis of CAD33, these results suggest that lnc-KDM5D-4 may play a role in the hepatic metabolism of lipids – a process of well-known relevance to atherosclerosis. Whether aberrant expression of this lincRNA plays an insignificant role in this context, or if it is a mere consequence of disease pathology remains an open question. Further research on lnc-KDM5D-4 should be done to study the role of this Y-linked lincRNA in steatosis-associated atherosclerosis.There is currently no information regarding Y chromosome-linked lncRNAs in human traits and disease. This is due to the fact that the Y chromosome is routinely excluded from genome analysis studies into the identification and the functions of lncRNAs in mammalsLnc-KDM5D-4:1 transcripts were found to be expressed in adipose, bladder, brain, colon, esophagus, heart, kidney, liver, lung, prostate, skeletal muscle, small intestine, spleen, testes, thymus, thyroid, trachea, and leucocytes suggesting that this lincRNA is widely expressed in male tissues. These findings provide a novel expression profile for lnc-KDM5D-4 across human healthy tissues. Based on these data, we believe that this lincRNA may have other molecular and physiological roles in men.34. Lnc-KDM5D-4:1 RNA FISH results suggested that lnc-KDM5D-4:1 is a lincRNA retained within the nucleus of hepatocytes. Concomitantly, these findings were in agreement with the previous observations showing that lncRNA transcripts were enriched in the cell nucleus in HepG2 cells16. This clearly showed that lnc-KDM5D-4 plays a potential role in biological functions taking place within the nucleus35 such as the establishment and maintenance of nuclear domains36, shaping of the 3D organization37, or acting as enhancer-like RNA by activating its neighbouring genes using a cis-mediated mechanism39. Indeed, an exclusively nuclear localization would argue against putative lncRNAs encoding short peptide sequences as translation occurs in the cytoplasm. However, given that its silencing does not affect the expression of its surrounding protein-coding genes, this may argue that it acts in trans rather than in cis.The main known function of lncRNAs to date is regulation of gene expressionPLIN2 is known to be implicated in lipid metabolism. This protein-coding gene is located on chromosome 9 and belongs to the perilipin gene family that regulate intracellular lipid storage droplets and very low-density lipoprotein (VLDL) secretion40. Overexpression of PLIN2 has been shown to trigger an increase in lipid droplets formation within hepatocytes via the up-regulation of the peroxisome proliferator-activated receptor gamma isoform (PPAR gamma)41. Furthermore, the up-regulation of PLIN2 was previously associated with liver steatosis42 and atherogenesis43. More recently, PLIN2 expression was also associated with atherosclerosis in patients with carotid stenosis44. On the other hand, the loss of PLIN2 has resulted in reduction of liver steatosis and inflammation45. An overview of the involvement of lnc-KDM5D-4 and its potential interaction with PLIN2 in the context of atherosclerosis and CAD is proposed in Fig. Among the genes that were affected by lnc-KDM5D-4 knockdown in HepG2 cells, 18 which could be due to the unique forces that drive the evolution of the Y chromosome. It is therefore not possible to study further this lncRNA in rodents.Lnc-KDM5D-4 seems to be not conserved between human and rodentsPLIN2 in hepatocytes. Overall, our data adds to the evidence that the human Y chromosome plays an important role in cardiovascular disease in a male specific manner and provides novel insight into potential new therapeutic targets for CAD.In conclusion, this is the first study on lincRNAs on the human Y chromosome and gene expression analysis. We provide evidence for the potential involvement of one of these lncRNAs, lnc-KDM5D-4, in atherosclerosis and CAD, possibly through the lipid metabolism-associated gene Supplementary Information


Antibacterial screening of the novel cephalosporins revealed that all targets (4a–j) retained the antibacterial activity of cephalexin against Staphylococcus aureus (ATCC 6538). N-Nicotinylcephalexin (4c) and N-cephalexin (4g) exhibited a broader spectrum of antibacterial activity towards standard strains of Staphylococcus aureus (ATCC 6538), Paenibacillus polymyxa (ATCC 842), and Escherichia coli (ATCC 10536) as well as a resistant strain of Pseudomonas aeruginosa (ATCC 27853).Cephalexin ( Effective antibiotics are needed to combat the growing bacterial resistance . CephaloAcylation reactions enabled the development of various generations of cephalosporins . In addiN-acylbenzotriazoles are advantageous acylating agents showing numerous merits over acid chlorides because: (i) they are usually isolated in high yields; (ii) they form crystals easily; (iii) they are stable in air; and (iv) chirality is preserved during the course of their preparation and reaction. These carboxylic acid surrogates are widely used when the corresponding acid chlorides are unstable or difficult to prepare oct-2-ene-2-carboxylic acid (4a): White microcrystals; yield: ; m.p. 210–212 °C; 1H-NMR  δ 13.21 , 9.35 -CH-), 8.53 -Ph), 7.49–7.43 , 7.35–7.24 , 5.70 -Ph), 5.62 -CH-), 5.03 , 4.96 , 3.73 , 3.46 , 3.28 , 1.98 . 13C-NMR  δ 170.6 (CO-NH-CH), 168.7 (CO-NH-CH-Ph), 163.9 (CO-N-), 163.5 (COOH), 156.5(CO-O-CH2), 138.1 (Ar-C), 137.1 (Ar-C), 128.3 (Ar-C), 128.2 (Ar-C), 127.8 (Ar-C), 127.6 (Ar-C), 127.0 (CH3-C=C), 122.8 (-C=C-COOH), 65.4 (O-2CH-Ph), 58.4 (-CH-S-), 57.1 (CH-CH-S-), 55.4 (CH(CO)Ph), 43.3 (2CH-NH-CO-O-), 28.9 (-S-2CH-), 19.4 (CH3). Anal. Calcd. for C26H26N4O7S: C, 57.98; H, 4.87; N, 10.4; S, 5.95; found: C, 58.13; H, 4.91; N, 10.62; S, 6.04.-7-((R)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)-2-phenylacetamido)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4b): White microcrystals; yield: ; m.p. 216–218 °C; 1H-NMR  δ 9.37 -CH-), 8.83 -Ph), 7.56 CH2-Ph), 7.40–7.19 , 5.74 -Ph), 5.63 -CH-), 4.96 , 4.94 , 4.50–4.45 -CH2-Ph), 3.46 , 3.25 , 2.96 -2CH-Ph), 2.71 -2CHPh ), 1.97 . 13C-NMR  δ 171.2 (CO-NH-CH(CO)-Ph), 170.6 (CO-NH-CH(CO)-Ph), 163.9 (CO-N-), 163.5 (COOH), 155.8 (CO-O-CH2), 138.3 (Ar-C), 137.9 (Ar-C), 137.0 (Ar-C), 129.8 (Ar-C), 129.3(Ar-C), 128.3 (Ar-C), 128.2 (Ar-C), 128.0 (Ar-C), 127.7 (Ar-C), 127.4 (Ar-C), 126.8 (Ar-C), 126.2 (Ar-C), 125.8 (CH3-C=C), 122.7 (-C=C-COOH), 65.2 (O-2CH-Ph), 58.4 (-CH-S-), 57.2 (CH(CO)-CH-S-), 56.0 (CH(CO)Ph), 55.3 (CH(CO)-NH-CO-O-), 28.9 (-S-2CH-), 19.4 (CH-2CH-Ph), 13.9 (CH3). Anal. Calcd. for C33H32N4O7S: C, 63.04; H, 5.13; N, 8.91; S, 5.1; found: C, 63.21; H, 5.19; N, 9.04; S, 5.18.-7-((R)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-(1H-indol-3-yl)propanamido)-2-phenylacetamido)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4c): White microcrystals; yield: ; m.p. 168–170 °C; 1H-NMR  δ 13.16 , 10.80 , 9.34 -CH-), 8.75 -Ph), 7.65 , 7.46 , 7.37–7.22 , 7.14 , 7.05 , 6.95 , 5.71 -Ph), 5.65 -CH-), 4.99 , 4.95 , 4.53–4.47 , 3.48 , 3.28 , 3.09 , 2.91 , 1.99 . 13C-NMR  δ 171.6 (CO-NH-CH(CO)-Ph), 170.6 (CO-NH-CH(CO)-Ph), 163.9 (CO-N-), 163.5 (COOH), 155.8 (CO-O-CH2), 138.2 (Ar-C), 137.0 (Ar-C), 136.1 (Ar-C), 129.8(Ar-C), 128.3 (Ar-C), 128.2 (Ar-C), 127.7 (Ar-C), 127.6 (Ar-C), 127.4 (Ar-C), 127.3 (Ar-C), 126.9(Ar-C), 124.0 (CH3-C=C), 122.8 (-C=C-COOH), 120.8 (Ar-C), 118.7 (Ar-C), 118.2 (Ar-C), 111.2 (Ar-C), 109.9 (Ar-C), 65.3 (O-2CH-Ph), 58.5 (-CH-S-), 57.2 (CH(CO)-CH-S-), 55.5 (CH(CO)Ph), 55.4 (CH(CO)-NH-CO-O-), 28.9 (-S-2CH-), 27.9 (2CH-CH-NH-(CO)-O-), 19.4 (CH3). Anal. Calcd. for C35H33N5O7S: C, 62.96; H, 4.98; N, 10.49; S, 4.8; found: C, 63.14; H, 5.06; N, 10.67; S, 4.91.-7-((R)-2-benzamido-2-phenylacetamido)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4d): Yellow microcrystals; yield: ; m.p. 132–134 °C; 1H-NMR  δ 13.11 , 9.27 -CH-), 8.86 -Ph), 7.92 , 7.59–7.44 , 7.37–7.28 , 5.89 -Ph), 5.66 -CH-), 5.00 , 3.48 , 3.28 , 1.99 . 13C-NMR  δ 170.7 (CO-NH-CH(CO)-Ph), 166.2 ((CO)-Ph), 163.9 (CO-N-), 163.4 (COOH), 137.7 (Ar-C), 133.8 (Ar-C), 132.8 (Ar-C), 131.4 (Ar-C), 129.7 (Ar-C), 129.2 (Ar-C), 128.5 (Ar-C), 128.2 (Ar-C), 128.1 (Ar-C), 127.7 (Ar-C), 127.5 (CH3-C=C), 122.7 (-C=C-COOH), 58.5 (-CH-S-), 57.1 (CH(CO)Ph), 56.5 (CH(CO)-CH-S-), 28.9 (-S-2CH-), 19.3 (CH3). Anal. Calcd. for C23H21N3O5S: C, 61.18; H, 4.69; N, 9.31; S, 7.1; found: C, 61.42; H, 4.76; N, 9.43; S, 7.19.-3-methyl-7-((R)-2-(nicotinamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4e): White microcrystals; yield: ; m.p. 200–202 °C; 1H-NMR  δ 9.29 -CH-), 9.21 -Ph), 9.04 , 8.70 , 8.27–8.24 , 7.54 , 7.51–7.48 , 7.38–7.29 , 5.90 -Ph), 5.59 -CH-), 4.94 , 3.42 , 3.18 , 1.95 . 13C-NMR  δ 171.1 (CO-NH-CH(CO)-Ph), 165.5 (CO-NH-CH(CO)-Ph), 164.9 (CO-N-), 163.8 (COOH), 152.5 (C=N-C=C-CO), 149.4 (C=N-C=C-CO), 138.0(Ar-C), 136.0 (Ar-C), 130.0 (Ar-C), 128.8 (Ar-C), 128.7(Ar-C), 128.3 (Ar-C), 128.1 (Ar-C), 127.9 (CH3-C=C), 123.8 (-C=C-COOH), 58.8 (-CH-S-), 57.6 (CH(CO)-CH-S-), 57.1 (NH-CH(CO)-Ph), 29.2 (-S-2CH-), 19.8 (CH3). Anal. Calcd. for C22H20N4O5S: C, 58.40; H, 4.46; N, 12.38; S, 7.09; found: C, 58.63; H, 4.53; N, 12.61; S, 7.21.-3-methyl-7-((R)-2-(3-nitrobenzamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4f): Yellow microcrystals; yield: ; m.p. 226–228 °C; 1H-NMR  δ 13.44 , 9.40 -Ph), 9.33 -CH- ), 8.76–8.62 , 8.48–8.46 , 8.40–8.34 , 7.83–7.75 , 7.54 , 7.39–7.30 , 5.91 -Ph), 5.67 -CH-), 5.00 , 3.48 , 3.28 , 1.99 . 13C-NMR  δ 170.6 (CO-NH-CH(CO)-Ph), 165.5 (CO-NH-CH(CO)-Ph), 164.5 (CO-N-), 163.6 (COOH), 147.60 (C-NO2), 137.4(Ar-C), 135.4 (Ar-C), 134.4 (Ar-C), 132.5 (Ar-C), 130.5 (Ar-C), 129.9 (Ar-C), 128.3 (Ar-C), 127.3 (CH3-C=C), 126.1 (Ar-C), 123.7(Ar-C), 122.7(-C=C-COOH), 58.6 (-CH-S-), 57.2 (NH-CH(CO)-Ph), 56.9 (CH(CO)-CH-S-), 28.9 (-S-2CH-), 19.4 (CH3). Anal. Calcd. for C23H20N4O7S: C, 55.64; H, 4.06; N, 11.28; S, 6.46; found: C, 55.78; H, 4.03; N, 11.52; S, 6.52.-3-methyl-8-oxo-7-((R)-2-phenyl-2-acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4g): Yellow microcrystals; yield: ; m.p. 197–199 °C; 1H-NMR  δ 9.26 -CH-), 8.92 -Ph), 7.52 , 7.37–7.29 , 7.26 , 5.92 -Ph), 5.57 -CH-), 4.92 , 3.83 , 3.70 , 3.39 , 3.14 , 1.93 . 13C-NMR  δ 170.8 (CO-NH-CH(CO)-Ph), 165.8 (CO-NH-CH(CO)-Ph), 164.0 (CO-N-), 163.5 (COOH), 152.5 (m-OCH3-C=), 140.2 (p-OCH3-C=), 137.8 (Ar-C), 129.7 (Ar-C), 129.0 (Ar-C), 128.2 (Ar-C), 127.7 (Ar-C), 127.6 (CH3-C=C), 122.7 (-C=C-COOH), 105.4 (Ar-C), 60.1 (-CH-S-), 58.6 (P-OCH3), 57.2 (NH-CH(CO)-Ph), 56.7 (CH(CO)-CH-S-), 56.1 (M-O3CH), 28.9 (-S-2CH-), 19.4 (CH3). Anal. Calcd. for C26H27N3O8S: C, 57.66; H, 5.03; N, 7.76; S, 5.92; found: C, 57.94; H, 5.11; N, 7.88; S, 6.11.-7-((R)-2-((S)-3-(1H-indol-3-yl)-2-(4-methylphenylsulfonamido)propanamido)-2-phenylacetamido)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4h): Brown microcrystals; yield: ; m.p. 125–127 °C; 1H-NMR  δ 12.56 , 10.76 , 9.32 -CH-), 8.70 -Ph), 8.09 , 7.91 , 7.46 , 7.40 , 7.30–7.24 , 7.17 , 7.08–7.02 , 6.92 , 5.68 -CH-), 5.58 -Ph), 4.98 , 3.88 , 3.40 , 3.37 , 3.04 , 2.84 , 2.31 , 2.00 . 13C-NMR  δ 172.6 (CO-NH-CH(CO)-Ph), 170.3 (CO-NH-CH(CO)-Ph), 163.9 (CO-N-), 163.4 (COOH), 142.1 (SO2-C=), 137.9 (Ar-C), 136.0 (Ar-C), 129.0 (Ar-C), 128.9 (Ar-C), 128.0 (Ar-C), 126.9 (Ar-C), 126.6 (Ar-C), 126.2 (Ar-C), 126.0 (CH3-C=C), 123.9 (-C=C-COOH), 120.7 (Ar-C), 120.6 (Ar-C), 118.3 (Ar-C), 117.7 (Ar-C), 111.3 (Ar-C), 108.8 (Ar-C), 64.83 (-CH-S-), 57.18 (CH(CO)-CH-S-), 56.76 (NH-CH(CO)-Ph), 56.48 (CH-NH-SO2), 28.61 (2CH-CH-NH-SO2), 28.22 (-S-2CH-), 20.88 (3CH), 15.07 (3CH). Anal. Calcd. for C34H33N5O7S2: C, 59.37; H, 4.84; N, 10.18; S, 9.32; found: C, 59.51; H, 4.89; N, 10.26; S, 9.46.-3-methyl-7-((R)-2-(2-(4-methylphenylsulfonamido)benzamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid(4i): White microcrystals; yield: ; m.p. 137–139 °C; 1H-NMR  δ 13.16 , 11.07 , 9.32 -CH-), 9.11 -Ph), 7.84 , 7.67 , 7.59–7.26 , 7.15–7.10 , 5.81 -Ph), 5.68 -CH-), 5.01 , 3.49 , 3.28 , 2.30 , 1.98 . 13C-NMR  δ 170.1 (CO-NH-CH(CO)-Ph), 167.7 (CO-NH-CH(CO)-Ph), 163.8 (CO-N-), 163.4 (COOH), 137.0 (SO2-C=), 129.7 (Ar-C), 129.7 (Ar-C), 129.5 (Ar-C), 129.4 (Ar-C), 128.2 (Ar-C), 127.8 (Ar-C), 127.7 (Ar-C), 126.7(Ar-C), 126.7 (CH3-C=C), 122.7 (-C=C-COOH), 121.2 (Ar-C), 120.0 (Ar-C), 58.5 (-CH-S-), 57.1 (NH-CH(CO)-Ph), 56.6 (CH(CO)-CH-S-), 28.8 (-S-2CH-), 20.9 (CH3), 19.3 (CH3). Anal. Calcd. for C30H28N4O7S2: C, 58.05; H, 4.55; N, 9.03; S, 10.33; found: C, 58.31; H, 4.62; N, 9.14; S, 10.52.-3-methyl-7-((R)-2-thiazole-5-carboxamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4j): Yellow microcrystals; yield: ; m.p. 179–181 °C; 1H-NMR  δ 9.28 -CH-), 8.88 -Ph), 7.56 , 7.52 , 7.38–7.30 , 7.26 , 5.91 -Ph), 5.67 -CH-), 5.01 , 3.83 , 3.71 , 3.48 , 3.28 , 1.99 , 1.91 . 13C-NMR  δ 170.8 (CO-NH-CH(CO)-Ph), 165.7 (CO), 164.0 (CO), 163.4 (COOH), 152.8 (Ar-C), 152.4 (Ar-C), 140.2 (Ar-C), 137.7 (Ar-C), 129.8 (Ar-C), 128.9 (Ar-C), 128.2 (Ar-C), 127.6 (Ar-C), 127.6 (CH3-C=C), 122.7 (-C=C-COOH), 122.7 (Ar-C), 105.4 (Ar-C), 60.0 (-CH-S-), 58.5 (p-OCH3), 57.2 (CH(CO)-CH-S-), 56.7 (NH-CH(CO)-Ph), 56.0 (m-OCH3), 28.9 (-S-2CH-), 21.0 (CH3), 19.3 (CH3). Anal. Calcd. for C31H31N5O9S2: C, 54.62; H, 4.58; N, 10.27; S, 9.41; found: C, 54.89; H, 4.61; N, 10.38; S, 9.5.-3-methyl-7-((R)-2-(2-((S)-4-methylphenylsulfonamido)propanamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4k): White microcrystals; yield: ; m.p. 234–236 °C; 1H-NMR  δ 9.33 -CH-), 8.57 -Ph), 7.99 , 7.65 , 7.39 , 7.35–7.23 , 5.65 -CH-), 5.57 -Ph), 4.95 , 4.07–3.95 , 3.44 , 3.23 , 2.39 , 1.97 , 1.03 .13C-NMR  δ 170.8 (CO-NH-CH(CO)-Ph), 170.4 (CO), 163.9 (CO), 163.4 (CO), 142.5 (Ar-C), 138.1(Ar-C), 137.8(Ar-C), 129.4 (Ar-C), 128.2 (Ar-C), 127.6 (Ar-C), 126.9 (Ar-C), 126.6 (Ar-C), 126.5 (CH3-C=C), 122.7 (-C=C-COOH), 58.3 (-CH-S-), 57.1 (CH(CO)-CH-S-), 55.3 (NH-CH(CO)-Ph), 51.7 (NH-CH-CH3), 28.9 (-S-2CH-), 21.0 (p-CH3), 19.4 (CH3), 18.9 (NH-CH-3CH). Anal. Calcd. for C26H28N4O7S2: C, 54.53; H, 4.93; N, 9.78; S, 11.20; found: C, 54.80; H, 4.96; N, 9.88; S, 11.30.-3-methyl-7-((R)-2-(2-((RS)-4-methylphenylsulfonamido)propanamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid : White microcrystals; yield: ; m.p. 224–227 °C; 1H-NMR  δ 9.35 -CH-), 8.57 -Ph), 8.01 , 7.72–7.58 , 7.45–7.22 , 5.73–5.62 -CH-), 5.57 -Ph), 5.46 , 4.07–3.92 , 3.45 , 3.27 , 2.36 , 1.98 , 1.07–1.03 . 13C-NMR  δ 170.8 (CO-NH-CH(CO)-Ph (k)), 170.7 (CO-NH-CH(CO)-Ph (k′)), 170.4 (CO), 163.9 (CO), 163.4 (CO), 142.6 (Ar-C (k′)), 142.5 (Ar-C (k)), 138.2(Ar-C (k′)), 138.1(Ar-C (k)), 137.8 (Ar-C), 129.9 (Ar-C (k)), 129.8 (Ar-C (k′)), 129.4 (Ar-C (k)), 129.3 (Ar-C (k′)), 128.2 (Ar-C), 126.9 (Ar-C), 126.6 (Ar-C), 126.5 (CH3-C=C), 122.7 (-C=C-COOH), 58.4 (-CH-S-), 57.2 (CH(CO)-CH-S-), 55.3 (NH-CH(CO)-Ph), 51.7 (NH-CH-CH3 (k)), 51.5 (NH-CH-CH3 (k′)), 28.9 (-S-2CH-), 21.0 (p-CH3 (k)), 20.9 (p-CH3 (k′)), 19.4 (CH3), 19.1 (NH-CH-3CH (k)), 18.9 (NH-CH-3CH(k)). Anal. Calcd. for C26H28N4O7S2: C, 54.53; H, 4.93; N, 9.78; S, 11.20; found: C, 54.77; H, 4.98; N, 9.86; S, 11.32.4a–j) and Cephalexin (Ceporex®) were dissolved in DMSO in the concentration 1 mg/mL. The microorganisms were cultivated overnight, and optical densities were adjusted 0.2–0.8 and diluted 1:100. Constructed compounds were tested against standard microbial strains in Mueller Hinton agar; Ceporex® and DMSO were tested in parallel as a reference and negative control, respectively. Diameters of inhibition zones were measured in mm.Compounds (4a-j) and Cephalexin (Ceporex®) were dissolved in DMSO in the concentration 2 mg/mL as a stock solution and then diluted two-fold serially to 1 µg/mL in a Mueller Hinton broth . The microorganisms were cultivated overnight, and optical densities were adjusted to 0.2–0.8 and diluted 1:100. The constructed compounds were tested against the standard microbial strains; Ceporex® and DMSO were tested in parallel as a reference and negative control, respectively.Compounds (N-tosyl-l-tryptophan (2h), N-tosylanthranilic acid (2i), N-tosyl-l-alanine (2k), and N-tosyl-dl-alanine  were prepared according to the reported procedure by the reaction of the corresponding amino acid with p-toluenesulfonyl chloride in the presence of triethylamine TEA [2j) (5) in 95% yield [5) was then hydrolyzed using sodium hydroxide to give the free carboxylic acid (6) [6) with N-benzotriazole (3g) afforded 4-methyl-2-thiazole-5-carboxylic acid (2j) in 90% yield.mine TEA . The preTEA [2j)  19]. Co. CoN-tos5% yield . Compounacid (6) ,22. CoupN-acylbenzotriazoles 3a–k′ , Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 10536), Paenibacillus polymyxa (ATCC 842), and Candida albicans (ATCC 10231). Cephalexin (Ceporex®) manufactured by: SmithKline Beecham, Harm-Giza, Egypt was used as a reference compound. Sensitivity testing (agar diffusion method) and MIC (broth microdilution method) were performed according to the reported method [Synthesized compounds d method . The ant4a–j showed antimicrobial activity against S.aureus comparable to Ceporex®. Furthermore, compounds 4c,e,g exhibited bactericidal activity against E. coli (G-ve bacteria) and P. polymyxa (Gm+ve anaerobic bacteria) close to that of Ceporex®. The most interesting result was the strong antimicrobial activity of 4b,c,e,g against the resistant strain of P.aeruginosa which was significantly higher than that of Ceporex®. N-nicotinylcephalexin (4c) and N-cephalexin (4g) enjoyed a broader spectrum of antibacterial activity than cephalexin.All the synthesized compounds N-acylbenzotriazole methodology. The protocol described herein enabled the modulation of the antibacterial activity of cephalosporins. The preliminary antimicrobial susceptibility testing of the novel synthesized targets led to two promising cephalosporin antimicrobials with a broader spectrum of activity.In conclusion, we have utilized a mild and efficient protocol for acylating cephalexin as an example of cephalosporins containing amino nucleophiles using


In addition, ChIP assays demonstrated that Lnc-Myd88 might increase Myd88 expression through enhancing H3K27Ac in the promoter of Myd88 gene, thus resulting in the activation of both NF-κB and PI3K/AKT signal pathways. In conclusion, we proposed that Lnc-Myd88 might serve as a novel diagnosis and therapeutic target for HCC.Enhanced Myd88 expression has been found in various parenchymal tumors especially in hepatocellular carcinoma with little mechanism of its upregulation known. A lot of long non-coding RNAs are reported to regulate the protein-coding genes which have location association through various mechanisms. In our study we confirmed a new long non-coding RNA Myd88 aberrant upregulated in HCC located upstream of Myd88 and verified a positive regulation relationship between them indicating that Lnc-Myd88 might participate in the enhanced expression of Myd88 in HCC. The gain- and loss-of-function analysis revealed that Lnc-Myd88 could promote the proliferation and metastasis of HCC both  Finally, a recognized lncRNA named LOC105377033 was chosen as the candidate lncRNA located upstream of Myd88 with a distance of 20kp, and presented a reverse transcription direction with Myd88. We further named it as Lnc-Myd88. Myd88 is defined as a carcinogenic gene in HCC, promoting growth and metastasis of human HCC,25 which can serve as a prognostic and therapeutic target factor for HCC,26 while the function of Lnc-Myd88 in HCC still remains unclear.With the advancement of sequencing and annotation of whole genomes, a novel study reveals that almost >65% lncRNAs located within 10 kb of known protein-coding genes which is known as the 'Flank10kb' theory, suggesting that there may exist In this study, we tried to figure out the role of Lnc-Myd88 in HCC, including hepatocarcinogenesis and progression. In addition, we attempted to identify the mechanism by which Lnc-Myd88 regulate the expression of Myd88.de-novo lncRNA, we performed experiments to figure out its protein-coding ability and subcellular localization in liver tumor tissues and cell lines. To determine whether the transcript of Lnc-Myd88 might encode proteins, we consulted the Coding Protein Calculator (http://cpc.cbi.pku.edu.cn/)  . Compariedu.cn/) .P-value of <0.001 and r2=0.5665, suggesting there was a positive regulation relationship between Lnc-Myd88 and Myd88 . The qRT-PCR was conducted to evaluate the expression level of Lnc-Myd88 and mRNAs of all relevant genes. To extract RNAs from cytoplasma and cytonucleus, we used the SurePrep Nuclear or Cytoplasmic RNA Purification Kit of Thermo Fisher Scientific . GAPDH were used as the internal control.To overexpress Lnc-Myd88, full-length of Lnc-Myd88 was subcloned into the lentivirus vector GV367 . The shRNA sequence targeting Lnc-Myd88 was cloned into the lentivirus vector pLL3.7 (Gene) to knock down Lnc-Myd88 expression, and the negative control hairpin shRNA with no sequence homology to human genes was provided by the same manufacturer. Lentiviral plasmid vectors encoding short hairpin RNAs (shRNAs) targeting Myd88 or scramble shRNA were generated and designated as sh-Myd88 and sh-NC, respectively. All the vectors were labeled with enhanced green fluorescence protein (EGFP). Transfection of cells was conducted according to the manufacturer instructions, and the efficiency of transfection was validated by qRT-PCR, and then the cells were subjected to RNA extraction or functional assays.μm pore size; Millipore). In the migration assays, cells were cultured in the upper chamber with serum-free medium. However, in the invasion assays, the lower chamber was added in media supplemented with 10% FBS. After 48 h, cells that had migrated or invaded through the membrane were fixed with methanol, stained with crystal violet and counted.The proliferation ability of HCC cells was tested by Clone Formation assay, Cell Counting Kit-8 , EDU (5-ethynyl-2′-deoxyuridine) immunofluorescence staining assay  according to the manufacturer’s instructions. Cell migration and invasion assays were conducted using transwell chamber .7) in the right groin and Lv-NC SMMC-7721 cells (1 × 107) in the left groin. We calculated the volume of tumors every 5 days after transplantation and killed them 30 days after implantation. For the tail vein xenograft model, eight mice in each group were injected with cells (1 × 107 suspended in 200 μl PBS) through the tail vein and killed 5 weeks later. One group was sh-Lnc-Myd88-Huh7 cells and the other was sh-NC-Huh7 cells which were all labeled with EGFP. Tumors of lungs were visualized by fluorescence using a 470-nm light source .BAB/c nude mice, 6 weeks of age or older, were purchased from the animal center of Nanjing University , raised and permitted by the Nanjing medical University animal studies committee. In the subcutaneous transplantation model, five mice were implanted with Lv-Lnc-Myd88-SMMC-7721 cells  overnight at 4 °C and then washed in PBS for three times. After incubated with secondary antibodies, sections were subjected to DAB reaction. Photograph of the sections by using a digitalized microscope camera .The tissue samples were fixed in 4% paraformaldehyde at 4 °C and sectioned into slices. After deparaffinage and rehydration, the sections were put into pressure cooker for 5 min to restore the antigen by using the citrate method. H7–5x107 cells were collected. Formaldehyde is used to crosslink the proteins to the DNA for 20–30 min. Then sonicate lysate to shear DNA to a fragment size of 200–1000 bp. After determination of DNA concentration and fragment size, we add the primary antibody, anti-H3K27m3, anti-H3K27ac, anti-H3K4m3 and IgG, and protein A/G beads into the samples and incubated overnight at 4 °C. The crosslinking was reversed by incubation at 65 °C for 4 h. The DNA was recovered by phenol/chloroform extraction. The primers were used to detect the human Myd88 promoter region by PCR.ChIP was performed by using the ChIP assay kit according to the manufacturer’s protocol . 1x10κB and NF-κB , p-AKT/AKT , GAPDH (CST). The results were visualized by a chemiluminescent detection system  and exposure to autoradiography film.To get protein, tissue samples and cultured cells were dissolved by RIPA regent plus phenylmethanesulfonylfluoride . Consistently, 30 mg of the protein was loaded each lane, fractionated by SDS PAGE, transferred onto a PVDF membrane. And then the membrane was incubated at 4 °C overnight with human-specific antibody of Myd88 , p-NF-t-test was used to assess the statistical differences between groups. All statistical data were carried out using Statistical Program for Social Sciences 19.0 software  and presented with Graphpad prism 5.0 . P-value less than 0.05 was considered as significant.All experimental assays were repeated independently in triplicate. Data were expressed as mean±S.E.M. Two-tailed Student's Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


To evaluate functional outcomes, care needs and cost-efficiency of hyperacute (HA) rehabilitation for a cohort of in-patients with complex neurological disability and unstable medical/surgical conditions.A multicentre cohort analysis of prospectively collected clinical data from the UK Rehabilitation Outcomes Collaborative (UKROC) national clinical database, 2012–2015.Two HA specialist rehabilitation services in England, providing different service models for HA rehabilitation.All patients admitted to each of the units with an admission rehabilitation complexity M score of ≥3  years; males:females 63:37%). Diagnoses were acquired brain injury , spinal cord injury , peripheral neurological conditions  and other .Specialist in-patient multidisciplinary rehabilitation combined with management and stabilisation of intercurrent medical and surgical problems.Rehabilitation complexity and medical acuity: Rehabilitation Complexity Scale—version 13. Dependency and care costs: Northwick Park Dependency Scale/Care Needs Assessment (NPDS/NPCNA). Functional independence: UK Functional Assessment Measure (UK FIM+FAM). Primary outcomes: (1) reduction in dependency and (2) cost-efficiency, measured as the time taken to offset rehabilitation costs by savings in NPCNA-estimated costs of on-going care in the community.The mean length of stay was 103 (SD66) days. Some differences were observed between the two units, which were in keeping with the different service models. However, both units showed a significant reduction in dependency and acuity between admission and discharge on all measures (Wilcoxon: p<0.001). For the 180 (95%) patients with complete NPCNA data, the mean episode cost was £77 119 (bootstrapped 95% CI £70 614 to £83 894) and the mean reduction in ‘weekly care costs’ was £462/week (95% CI 349 to 582). The mean time to offset the cost of rehabilitation was 27.6 months (95% CI 13.2 to 43.8).Despite its relatively high initial cost, specialist HA rehabilitation can be highly cost-efficient, producing substantial savings in on-going care costs, and relieving pressure in the acute care services. Hyperacute (HA) rehabilitation is an emerging field about which there is currently very little in the published literature.This 3-year national consecutive cohort analysis compares two different service models of HA rehabilitation from opposite ends of England.Prospective routinely collected data are reflective of real clinical practice.Missing data are inevitable in routine clinical data sets, but the 95% capture is high compared with many such analyses.The NPCNA estimations of cost savings should be interpreted with some caution, as they differ from techniques applied in traditional health economic studies. However, they offer the advantage of assessing care needs and costs independently of who provides the care, and are thus not biased by individual circumstances, such as the availability of informal carers or local policies in statutory care provision.There is a growing body of evidence for the effectiveness of early rehabilitation following acquired brain injury (ABI) and other complex disabilities. There is evidence from the trial-based literature and cohort studies that early rehabilitation can lead to reduced stay in hospital, earlier functional gains and improved rates of home discharge once patients are fit to engage in a rehabilitation programme.Recent UK policy documents from NHS England (NHSE)In the UK, the National Health Service (NHS) provides one of the most comprehensive health and social service systems in the world.11The national UK Rehabilitation Outcomes Collaborative (UKROC) database collates episode data for all inpatients admitted to specialist rehabilitation services in England, providing national benchmarking on quality, outcomes and cost-efficiency of rehabilitation. Within the UKROC data set, cost-efficiency is computed as the length of time taken to offset the initial costs of the rehabilitation episode through savings in the on-going costs of community care as estimated by the Northwick Park Dependency Scale and Care Needs Assessment.The objective of this article was to present the first national cohort analysis of the UKROC database to describe functional outcome, change in care needs and cost-efficiency following specialist HA rehabilitation for adults who have complex rehabilitation needs, but are still medically unstable. We also compare the costs, outcomes and cost-efficiency of the two different models of HA rehabilitation service that currently report their full data to the UKROC database.Since 2012, tertiary specialised rehabilitation services in England have been commissioned centrally by NHSE. Currently, a total of 36 NHSE-designated services are categorised as HA (n=3): Level 1a, b or c (n=17) and Level 2a (n=16). A further 33 district specialist rehabilitation services are commissioned locally, and a large number of local general (Level 3) rehabilitation services. Detailed information about the different service levels is available on the BSRM website.15In Liverpool (in the North West of England), the Walton Centre is the Regional Neurosciences Centre, within a network that also includes Broadgreen Hospital  and St Helens Hospital. The network's tertiary specialised rehabilitation services include the Lipton Hyperacute (HA) Rehabilitation Unit (10 Beds) and the ‘Complex Rehabilitation Unit’ (CRU) (20 beds) providing rehabilitation for patients with neurological or complex trauma conditions on the same site within the Walton Centre. The majority of patients from the HA unit will step down to the CRU once they are medically stable. The CRU is designated as a Level 1b (mixed disability) specialised rehabilitation service.The Regional/Hyperacute Rehabilitation Unit at Northwick Park Hospital, London, is a 24-bed unit in which HA and Level 1a  specialised rehabilitation beds are colocated. Patients in the immediate postacute stages of recovery from severe illness or injury often have periodic medical instability interspersed with periods when they are relatively well. The beds are flexibly allocated on a weekly basis to either ‘Level 1a’ or ‘HA’ according to the individual medical resource requirements of each patient as determined by serial recording of the Rehabilitation Complexity Score Medical subscale see . TypicalThe three designated HA rehabilitation services are sited in Manchester, Liverpool and London, but only the latter two currently report the full data set to UKROC. Between them, they provide about 18–19 beds, but these two units operate on rather different models:The study is a two-centre cohort analysis of prospectively collected clinical data from the UK Rehabilitation Outcomes Collaborative (UKROC) national clinical database 2012–2015. Participants were all adults with complex disability who were medically unstable when admitted to the specialist HA in-patient rehabilitation units in London or Liverpool.http://www.csi.kcl.ac.uk/ukroc.html). The staff from units were fully trained in the administration of the tools in the UKROC data set.The UKROC database was established in 2009 through funding from a programme grant from the UK National Institute for Health Research (NIHR),An RCSE-M score of 3 indicates a potentially unstable medical condition requiring the patient to be managed in a setting with on-site 24-hour emergency medical support immediately available. A score of 3 usually denotes that the unit's day-time medical team formally ‘hand over’ information to the out-of-hours medical service about the patient's current condition and likely needs for treatment, in case they are called to attend in the coming out-of-hours period. Typical medical conditions would be an unstable tracheostomy, ventilation, unstable dysautonomia, active/high risk of sepsis requiring intravenous antibiotics, unstable epilepsy likely to require intervention etc.An RCSE-M score of 4 indicates that the emergency medical/surgical services actually attended the patient out-of-hours within the previous week.The Rehabilitation Complexity Scale (RCSE) is a simple measure of rehabilitation requirements (resource use) in rehabilitation services.The UK FIM+FAM is a global measure of disability.21The Northwick Park Dependency Score (NPDS) is an ordinal scale of dependency on nursing staff time (number of helpers and time taken to assist with each activity) designed to assess needs for care and nursing in clinical rehabilitation settings.22The NPDS also translates via a computerised algorithm to Northwick Park Care Needs Assessment (NPCNA),Within the UKROC data set, the cost-efficiency is calculated as the time taken to offset the cost of rehabilitation by the resulting savings in the cost of on-going care in the community. This is calculated from ‘episode cost of rehabilitation’ divided by ‘reduction in weekly cost of care’ from admission to discharge, as estimated by the NPCNA. The cost of episode was calculated per patient as ‘bed-day cost×length of stay’ in the HA unit. The cost per bed-day was calculated using a previously published costing methodology.De-identified data were extracted for all recorded in-patient completed episodes for adults admitted to the HA rehabilitation services in Liverpool and London between March 2012 and July 2015, if they had an RCS-E v.13 M score of ≥3 on admission . To minimise bias, all episodes were included that met this criterion—there were no exclusions for age, diagnosis or length of stay. Data were collated in MS Excel and transferred to SPSS V.22 for analysis.As the proportion of missing data were small (≤5%), no data were imputed.Parametric statistical techniques were used to describe and compare interval quality data (such as care hours and costs). To minimise the effect of any skewed data, 95% CIs were calculated using bootstrapping based on 1000 bootstrap samples.Non-parametric techniques were used to compare ordinal data, including the RCSE, NPDS and FIM+FAM scores.2 tests for dichotomous data and unpaired t-tests for interval data—except where very highly skewed, in which case non-parametric Mann-Whitney tests were used.Demographic differences between the two services were examined using χWithin-group changes were examined using paired t-tests for interval data and Wilcoxon signed-rank tests for ordinal data.Between-group changes were examined using unpaired t-tests  and Mann-Whitney tests .In this non-interventional observational study, size was not predetermined but dictated by the accruals to the national data set over the 3-year period that met the inclusion criteria.From a total of 414 registered episodes admitted to the two units during this period, 190 had an admission RCSE-M score of ≥3 and were included in this analysis. These included 88 of 125 (70%) admissions to the Liverpool unit and 102 of 289 (35%) admissions to the London Unit.  The demographics of this main data set are given in The study sample comprised ∼3:2 males:females, with a mean age at admission of 46.0 (SD=15.7) years. The mean length of stay in the rehabilitation programme was 103 (SD=65.6) days. The large majority of patients (87.4%) had ABI, of which 37% had vascular pathology , 33% traumatic, and 18.7% anoxic aetiology. Nine had spinal cord injury ; 78% were cervical and 22% thoracic level injuries. Peripheral neurological conditions (5%) included inflammatory neuropathies  and critical illness neuropathy and complex polytrauma involving visceral and/or chest injuries were the commonest ‘other’ conditions (3%). Significant differences were seen between the two services. The London unit had a higher proportion of acquired brain injuries—especially due to anoxia and a significantly younger patient group . The time since onset of injury was quite long for both units, as these are complex and sick patients who often require a prolonged period in the intensive therapy unit before they are ready for transfer even to an HA unit. However, the time since onset was significantly longer in the London unit , which is likely to reflect the relative lack of HA rehabilitation capacity in London (see Discussion). Although not significantly different, the mean length of stay was slightly longer in the Liverpool unit (106 vs 100 days). This made up for the unit's slightly lower OBD costs so that the total episode costs (including MFF) were similar for the two units at £76 279 (Liverpool) and £74 409 (London)  on costs excluding MFF).At discharge, over three-quarters of the Liverpool patients were transferred to the associated Level 1b Unit (CRU) for continued rehabilitation. From the London unit, over half of the patients were discharged to a specialist nursing home/residential unit once they were medically stable enough and nearly a quarter progressed sufficiently to allow discharge home. Approximately 8% of both groups were transferred back to an intensive care or acute hospital setting.2 27.2, p<0.001) on admission. By discharge, the distribution was similar . Item level scores are shown in The RCSE summed Nursing and Medical subscale (RCSE−N+M) scores were recorded as a measure of medical and nursing acuity. Both units showed a significant reduction in acuity between admission and discharge (Wilcoxon p<0.001). The London unit had significantly higher acuity scores on admission (Mann-Whitney p<0.001) and on discharge.All case episodes had complete RCSE data. Of 190 (95.3%) paired FIM+FAM scores, 181 were available. Of the nine missing ratings, eight were at discharge, and all were in the London service. The commonest reasons for missing scores were: very short stays, eg, for assessment only (5 of 8), and/or unexpected transfer to ITU or acute care ward (3 of 8). .Of 190 (94.7%) NPDS scores, 180 were available. The three cases missing in London were the three unexpectedly transferred back to ITU/acute care. The seven missing in Liverpool were due to the lack of a rating within the required time period at discharge . On admission, the London patients had significantly higher complexity scores in terms of medical and nursing acuity (p<0.001).A trend towards greater dependency on admission did not reach the threshold for statistical significance, after correction for the use of multiple tests. By discharge, the dependency and acuity were similar between the two groups.The London patients had a tendency to lower FIM+FAM scores, on admission (18-point mean difference) and on discharge (47-point mean difference) that were likely to be clinically significant.2 test, p<0.01). Functional outcomes were therefore examined, including and excluding patients who remained in PDOC at discharge from either unit  of the London patients were in prolonged disorders of consciousness (PDOC) with the lowest possible FIM+FAM score, and by discharge 31 (30%) remained in this state. In Liverpool, 20 (23%) were in PDOC on admission, of which only 11 (12.5%) remained in this state at discharge. These differences reached statistical significance . The mean time taken to offset the costs of rehabilitation was slightly longer in London (29.9 vs 24.9 months), but the CIs were wide and the between-centre difference did not reach significance .While the emerging evidence for better outcomes from early and continuous chain rehabilitation creates a strong ethical incentive to provide HA rehabilitation services, planners and commissioners still require evidence that this provision also provides value for money. Cohort analyses of routinely collected outcome data may not provide definitive evidence that changes are attributable to rehabilitation, but they, nevertheless, make an important contribution to our understanding of the gains that can be made from rehabilitation in the course of real-life clinical practice and provide the opportunity for comparing different populations and practices. This first multicentre analysis of the UK national clinical data set for hyperacute (HA) specialist rehabilitation demonstrates that patients with complex neurological disability who are still medically unstable have the potential to gain from specialist rehabilitation across a wide range of conditions. Our findings confirm benefits for the patients and their families in terms of gains in functional independence and reduction in on-going care needs. But, in addition, there are also gains for the payers. Although the costs of HA rehabilitation were quite high . This unit is one of the two specialist centres in London for assessment and management of PDOC, and the only one based in an acute hospital setting that is able to take patients who are medically unstable. Moreover, within London, the specialist rehabilitation service network includes four other Level 1 and nine other Level 2 services. As the only HA/Level 1a rehabilitation service within the London network, this unit is expected to take an undiluted caseload of complex and profoundly dependent patients. In contrast, the Liverpool unit would be expected to have a population of more mixed disability, as indeed the FAM-Splat confirms.In both services, over 60% of patients had become medically stable by the time of discharge, in that their RCSE-M scores had fallen to below 3 see . In the Despite the differences in service model and in daily costs, both units proved to be cost-efficient with no significant difference between them. Around the world, many health services planners are currently making plans for the provision of services to meet sudden unexpected demands, such as in the event of major atrocity or serial terrorist attacks. In that context, just as important as the provision of frontline and emergency services is the provision of HA rehabilitation units to transfer patients immediately after initial stabilisation and acute management, in order to free up the acute services for a further wave of casualties. Hence it is important to explore and share the relative merits of different service models.At present, there are no reliable data to inform demand and capacity planning. It is worthy of note that the Liverpool unit has 10 beds for a catchment population of ∼3.5 million, whereas the 8–9 beds in the London unit represent only the HA rehabilitation provision for London and the Home Counties, serving a catchment population of ∼18–20 million. At present, provision is based on a commissioning decision rather than a needs assessment, but the significantly longer time between the onset of injury and admission to HA rehabilitation in London compared with Liverpool suggests a substantial shortfall in capacity and this information may help to inform future service planning.The data were collected in the course of routine clinical practice, and inevitably there are some missing data points. In this series, 10 patients had missing NPDS/NPCNA data. However, these constituted <5% of the population, so no data were imputed.The NPCNA estimates of continuing care costs are not true assessments as applied in traditional health economic studies. On the other hand, the instrument has been in use for over 15 years. It is now quite widely taken up in clinical practice and in research,We also acknowledge that a small number of patients with multiple comorbidities may have a significantly shortened life expectancy and might not survive the 28 months to offset the cost of the HA rehabilitation episode. However, for the majority of cases, the period of medical instability is a transitional stage on the pathway towards recovery, and most had stabilised by the time they move on to the next phase of treatment.Although this paper provides an overall view of the acuity and dependency of patients presenting with medical instability, it does not give any data about the nature of the acute needs or the types of interventions provided by the emergency medical/surgical teams, which would provide further insights into the codependencies of an HA rehabilitation unit. This information will be detailed in an article that is being presented separately for publication.The authors recognise the following limitations to this study:It should also be noted that ‘Specialist rehabilitation’ denotes something rather different in the UK from other countries. In the USA and Australia, a ‘specialist rehabilitation centre’ would be one in which the central focus of treatment is rehabilitation, often in diagnosis-specific programmes . In the UK, a stroke unit that provides rehabilitation as part of a specialist stroke programme would be classed as a Level 3  rehabilitation service. The term ‘specialist rehabilitation’ is reserved for tertiary (Level 1 and 2) centres, serving a large catchment population  and admitting a selected population of patients with highly complex rehabilitation needs, regardless of diagnosis. Patients who would progress satisfactorily with more standard rehabilitation programmes were not included in this analysis, which, therefore, represents a small subgroup of more complex patients, in comparison with other international rehabilitation cohorts.The above limitations accepted, findings from this study provide evidence for the cost-efficiency of specialist hyperacute rehabilitation for patients with complex disability who are still medically/surgically unstable.


Hydra, one of the classical model systems for evolutionary developmental biology and regeneration, transcription factor FoxO modulates both stem cell proliferation and innate immunity. This provides strong support for the role of FoxO as a critical rate-of-aging regulator. However, how environmental factors interact with FoxO remains unknown. Here, we find that deficiency in FoxO signaling in Hydra leads to dysregulation of antimicrobial peptide expression and that FoxO loss-of-function polyps are impaired in selection for bacteria resembling the native microbiome and more susceptible to colonization of foreign bacteria. These findings reveal a key role of FoxO signaling in the communication between host and microbiota and embed the evolutionary conserved longevity factor FoxO into the holobiont concept.The aging process is considered to be the result of accumulating cellular deterioration in an individual organism over time. It can be affected by the combined influence of genetic, epigenetic, and environmental factors including life-style-associated events. In the non-senescent freshwater polyp  The aging process is considered to be the result of accumulating cellular deterioration in an individual organism over time. It can be affected by the combined influence of genetic, epigenetic, and environmental factors . In ordeHydra is considered non-senescent   according to the standard procedure  . All linrocedure . The aniH. vulgaris (AEP) embryos as previously described (The stable knockdown lines for FoxO  were achescribed . By seleescribed .Thermo Fisher Scientific) at -80°C until RNA extraction with the PureLink RNA Mini Kit (Ambion) according to the manufacturer’s protocol. Additionally, the optional on-column DNA digestion was performed. The RNA was eluted in 30 μl and checked for sufficient quality. If necessary, the RNA was purified using 1-butanol and diethyl ether (RRID:SCR_011841) (H. vulgaris (AEP) transcriptome (RRID:SCR_005476) (RRID:SCR_001905) (RRID:SCR_000154)  . Subsequcriptome  was perf_005476) . All dow_001905) . Differe_000154) . The RNA-) polyps were generated using the previously established protocol , Hydra oligactis, or Hydra viridissima were pooled and washed three times with sterile HM. After homogenization, the three suspensions were filled up to 1.3 ml and used for recolonization. Individual germ-free polyps of control and FoxO- animals were recolonized with 100 μl in 4 ml HM each of one of the suspensions in six biological replicates. The same source community suspension was used to recolonize six replicates of control and FoxO- polyps in a 1:1 ratio, meaning one homogenized polyp to one polyp to be recolonized. One adult polyp equated to the following colony forming units (CFU) on R2A media: H. vulgaris (AEP): 2.9 ∗ 104; H. oligactis: 6.7 ∗ 104; and H. viridissima: 2.3 ∗ 103. The remaining 100 μl was frozen at -20°C for source community analysis. After 24 h of incubation, all polyps were washed with sterile HM. On day 4 of recolonization, the polyps were washed three times with sterile HM and frozen at -20°C for 16S rRNA profiling until DNA extraction.Germ-free control and FoxO-deficient (FoxOprotocol  and incuprotocol . For recRRID:SCR_008249) (RRID:SCR_013004). Chimeric sequences were identified with ChimeraSlayer (RRID:SCR_013283)  as described in the manufacturer’s protocol. Elution was performed in 50 μl. Extracted DNA was stored at -20°C until sequencing. Prior to sequencing, the variable regions 1 and 2 (V1V2) of the bacterial 16S rRNA genes were amplified according to the previously established protocol using the primers 27F and 338R . For bac_008249) . Paired-_013283)  and veriRRID:SCR_011921) (RRID:SCR_002830) (H. vulgaris (AEP) and H. oligactis. For analyses including data from the H. viridissima source community the number of reads was normalized to 3,200 since reads assigned to chloroplasts of the algal endosymbiont were removed in silico. Alpha diversity was calculated using the Chao1 metric implemented in QIIME using ten replicates of rarefication per sample. Beta diversity was depicted in a PCoA by 100 jackknifed replicates using binary Pearson distances (BPDs). Bacterial groups specifically associated with control or FoxO- polyps were identified by LEfSe (RRID:SCR_014609) . A q-value of 0.25, an effect size threshold of 3.0 , and a mean abundance of at least 0.1% in one of the treatments were used for all bacterial groups discussed in this study.Operational taxonomic unit (OTU) picking was performed using the pick_open_reference_otus.py protocol with at least 97% identity per OTU and annotation was conducted with the UCLUST algorithm (_011921)  against _002830)  implemen_002830) . The num_014609) . LEfSe ct-test or Mann–Whitney U-test if applicable. If multiple testing was performed, p-values were adjusted using the Benjamini–Hochberg correction  to disrupt FoxO-dependent signaling  animals for the epithelial tissue.In order to investigate the effect of FoxO on innate immunity and metaorganism homeostasis in a functional approach, we generated transgenic animals carrying a shRNA construct against the single - animals, resulting in 30–40 million reads per sample after quality control. In order to distinguish even closely related contigs within one AMP family, we utilized the uniquely mapped hits to calculate DE contigs. Principal component analysis of the transcriptomes revealed significant changes in gene expression by disruption of FoxO signaling. Clustering of control and FoxO- samples explained >50% of variance in the dataset , while the Kazal protease inhibitors . The largest family of AMPs, the Arminins . Most contigs respond with significant downregulation up to 52% in FoxO-. However, for four individual contigs of the Arminin AMP family , we could not find significant changes in expression. Remarkably, evaluating normalized read counts .For comparative expression analysis, we sequenced ten total RNA libraries with five biological replicates of control and FoxO- polyps . FoxO de lineage  and are  lineage , S4D,E.  lineage  are downhibitors  show dysArminins , is alsofoxO transcript in the epithelial tissue leads to an overall downregulation of epithelially expressed AMPs. The extent of regulation for all AMP contigs shows strong similarities within the families and seems to be independent of the expression level . Assuming that AMPs affect microbial colonization . We generated germ-free control and FoxO- polyps and separated them into single wells under sterile conditions. Independently, we provided the bacteria-free animals with source communities for 24 h from either their own species H. vulgaris (AEP) or from other Hydra species, H. oligactis or H. viridissima. After four days of recolonization, single polyps of the six biological replicates were subjected to 16S rRNA profiling of the bacterial colonizers via sequencing on the Illumina MiSeq platform .Our observations demonstrate that nization , we nextFigure 2B: pink, blue, green; Supplementary Table - polyps with the native bacterial community (H. AEP) did not result in significantly different outcomes   or H. viridissima , the recolonization resulted in distinct clusters for FoxO- and control polyps (Supplementary Table H. vulgaris (AEP) source community confirms that FoxO- polyps are significantly impaired in selecting bacteria resembling the native microbiome, when presented with foreign bacterial backgrounds . Their BPDs increase significantly when recolonized with bacteria from H. oligactis  or H. viridissima (0.81 ± 0.03 to 0.87 ± 0.04). Remarkably, even during recolonization with naturally occurring bacteria the resilience was drastically decreased, as the within treatment variability for the FoxO- animals was significantly increased compared to the control (0.57 ± 0.05 to 0.74 ± 0.21) . Evaluating the alpha diversity, FoxO-deficient polyps were less efficient in controlling the number of bacterial OTUs . While FoxO- polyps recolonized with bacteria from H. viridissima clearly showed a significant increase in OTUs (189 ± 21 to 245 ± 27) , animals provided sources from H. vulgaris (AEP)  or H. oligactis  showed significantly higher variation in alpha diversity compared to the control, suggesting a higher prevalence of stochastic events in FoxO- animals.Principle coordinate analysis (PCoA) on a binary Pearson distance (BPD) matrix of the composition data displayed distinct clustering for the different bacterial backgrounds, verifying the successful recolonization with the provided source communities , native bacteria could settle on the polyp and the bacterial communities almost resembled the diversity of the source community for control and FoxO- polyps already four days after recolonization. Remarkably, when recolonized with bacteria from other Hydra species, control and FoxO-deficient animals select for bacterial taxa resembling their native microbiota (H. AEP) from the source community. Even some highly abundant members of the foreign source communities, such as Sphingobacteriia (H. oli), Sapropirae, or Cytophagia (H. vir), were poorly transmitted. Interestingly, bacterial orders that are barely present in the source or the native H. vulgaris (AEP), like Bacteroida, Flavobacteriia, Alteromonadales, or Firmicutes, were able to expand at the drawback of bacteria resembling the native microbial composition  community are able to reach higher relative abundances and thereby reduce the portion of Beta- and Gammaproteobacteria , Flavobacteriia (2708478), Cyanobacteria (NewRefOTU159), and Actinobacteria (1098473) can establish in higher abundances on FoxO- animals , Cyanobacteria (YS2), and Actinobacteria could be observed consistently on the FoxO- polyps . Due to the presence of host species-specific bacterial taxa in the different source communities, there is only little overlap of commonly affected OTUs over the whole data set. However, all the rising bacterial groups are usually absent or only found in very low abundances in the naturally occurring H. vulgaris (AEP) microbiota  and Gammaproteobacteria (Pseudomonas sp.) .When evaluating the differences between control and FoxO-deficient animals for the foreign bacterial backgrounds using linear discriminant analysis effect sizes (LEfSe) , it becocrobiota  (Supplemcrobiota . Vice veH. vulgaris (AEP) source community and drastically reduces resilience when provided bacteria from other Hydra species. Especially bacterial taxa that are rarely found in the native community can expand at the drawback of bacteria resembling the naturally occurring microbiota.Taken together, our observations demonstrate that FoxO-dependent signaling not only affects the expression of AMPs, but also has the potential to mediate abundances and diversity of microbes by controlling selectivity for bacterial colonizers. FoxO deficiency significantly increases variability of recolonization outcome with a native bacterial Hydra holobiont is a valuable tool to study conserved mechanisms of host–microbe interactions in functional analyses . Most genes encoding epithelially expressed AMP families including Hydramacin, Kazal, and Arminin respond with downregulation to FoxO deficiency. Only one gene (contig 45266) was found to be upregulated in FoxO-deficient animals, suggesting a mainly activating function of FoxO signaling on AMP expression and innate immunity. Second, FoxO loss-of-function polyps were more susceptible to colonization of foreign bacteria and impaired in selection for bacteria resembling the native microbiome . Therefore, FoxO-induced decrease in AMP expression is correlating with differences in microbial colonization and highlights the inhibitory action of AMPs against non-commensal bacteria. In a state of intact FoxO signaling, secretion of numerous AMP families provides a highly selective milieu and shapes the microbial composition in a species-specific manner . FoxO deficiency reduces the expression of AMPs, which results in a decreased selection pressure on colonizing taxa and in establishment of higher abundances of foreign bacteria in the community. Consequently, especially during the process of colonization, reduced expression of FoxO compromises the resilience of the microbiome .The non-senescent analyses . By expl- animals. We would explain this by the early state of recolonization that we present in this study. The naturally occurring diversity was nearly completely restored for control polyps after 4 days of recolonization , while FoxO- animals were less efficient in controlling the bacterial taxa provided with the source communities . Since the host factor controlling the bacterial colonization is reduced in FoxO- animals, long-term effects may be shaped by a higher degree of bacteria–bacteria interactions resulting in overgrowth of few dominant taxa. This would reduce the overall diversity as observed in other studies. Consequently, the observed reduction in diversity during the aging process in other models may result from the decreasing control of dominant bacterial colonizers expanding in the community, while rare members are repressed. Since the structure of the bacterial community can be protective against pathogens, can shape the nutrient landscape, and may affect the inflammation status, evidence is accumulating that it has a direct influence on senescence in humans  there is a need to consider the holobiotic nature of an organism when thinking about longevity, that (ii) the microbial environment matters in the context of senescence and contributes to complex processes such as aging; and that (iii) the hub regulator FoxO presents a direct link between age-related processes and microbial colonization.Taken together, observations in different organisms indicate that, in contrast to the essentially static genome, the microbiome is rather dynamic throughout life history. Our observations in non-senescent Hydra.Ethical restrictions do not apply to cnidarian model organisms such as BM, SF, AK, and TB designed the experiments. BM performed the experiments. BM, JT, SF, AK, and TB analyzed the data. BM and TB wrote the paper.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.


In vitro, we found that UCH-L5 could inhibit migration and invasion of U87MG and U251 cells. It has been reported that the expression of SNRPN, SNRPF, and CKLF was abnormal in gliomas or other tumors. We also found that SNRPF-siRNA, SNRPN-siRNA and CKLF-siRNA could inhibit migration and invasion of U87MG cells. And knockdown of UCH-L5 expression improved both mRNA expression and protein level of SNRPF. The relationship between UCH-L5 and SNRPF was further confirmed in 293T cells. Our study showed that UCH-L5 could inhibit migration and invasion of glioma cells via down regulating expression of SNRPF. And the above findings suggest that UCH-L5 may inhibit occurrence and metastasis of gliomas.Ubiquitin C-terminal Hydrolase-L5 (UCH-L5/UCH37), a member of the deubiquitinases (DUBs), suppresses protein degeneration via removing ubiquitin from the distal subunit of the polyubiquitin chain. The activity of UCH-L5 is enhanced when UCH-L5 combines with proteasome 19S regulatory subunit by Rpn13/Admr1 receptor and inhibited when UCH-L5 interacts with NFRKB. But the role of UCH-L5 in gliomas remains unknown. In this study, analysis of 19 frozen and 51 paraffin-embedded clinic pathological cases showed that UCH-L5 expression in glioma tissues was lower than normal brain tissues.  Gliomas are the most common primary brain tumors and are thought to arise from a neural stem cell . About 3Proteins are normally degraded by ubiquitin proteasome system (UPS) and autophagy . The majAccording to the homology and the functional mechanism of DUBs, we mainly divided them into 5 categories, namely Ub C-terminal hydrolase (UCH), Ub specific protease (USP), ovarian tumor protease (OTU), Josephin/Machado–Joseph disease protease (MJD) and JAB1/MPN/MOV34 metalloenzyme (JAMM) , 12. UCHA spliceosome is a kind of enzyme in eukaryotic cells. It removes introns from a transcribed pre-mRNA. The enzyme comprises of more than 100 associated proteins and 5 small nuclear ribonucleoproteins including U1, U2, U4, U5 and U6 . Smith pIn the current study, we found that UCH-L5 was down expression in glioma tissue. Furthermore, we discovered that UCH-L5 could inhibit cell migration and invasion of glioma cell lines through downregulating SNRPF, a factor of Sm protein ring in the spliceosome. According to the abnormal protein level in high and low degree glioma , and mas19 frozen samples including 3 normal brain tissues and 16 glioma tissues were analyzed for UCH-L5 expression by real-time quantity PCR (RT qPCR), and 3 normal brain tissues and 5 glioma tissues picked up randomly were carried out for UCH-L5 expression by Western blot. The results showed that mRNA level Figure  and protTo further investigate the functions of UCH-L5 in gliomas, we firstly designed UCH-L5-siRNA (5′-GGAGACUGUAUGAAUUAGATT-3′), and it knocked down UCH-L5 efficiently in U87MG cells and U251 cells which were examined by RT qPCR and Western blot. Knockdown efficiency was about 70% in U87MG Figure  and 60% Since metastasis and recurrence represent the main malignant characteristics of high-grade glioma. We found that knockdown of UCH-L5 promoted the cell capability to migrate and invade in both U87MG and U251 cells.***P < 0.001, **P < 0.01. In an invasion assay, the number of invading U87MG cells increased from 223 ± 19 cells per field for control to 316 ± 79 cells per field for cells treated with UCH-L5-siRNA, **P < 0.01  . GliomasThe common roles of DUBs in various cellular processes are now well understood and new functions are continued to emerge gradually. DUBs are involved in degradative and non-degradative signaling such as signal pathway activation , gene tret al. and Vander Linden et al. uncover the detailed mechanism of deubiquitination domains in RPN13 (ADRM1) and INO80G (NFRKB), can either activate or inhibit UCH-L5 [Ubiquitination is regulated by DUBs. At present, there are lots of researches who proved that ubiquitination is involved in the regulation of glioma , 34. Andt UCH-L5 , 37.et al. found UCH-L5 promotes cell migration and invasion via interacting and deubiquitinating splicing factor PRP19 in hepatocellular carcinoma [UCH-L5 is also linked to TGF-β signaling , and ovearcinoma . TherefoHowever, in our study, we found that the expression of UCH-L5 both in low-grade gliomas and high-grade gliomas were lower than normal brain tissues. Furthermore, we found that knockdown of UCH-L5 expression promotes the migration and invasion of U87MG and U251 cells. And overexpression of UCH-L5 inhibits the migration and invasion of U87MG and U251 cells. These findings are different from the previous studies in other cancers. Therefore, we conclude that UCH-L5 plays a negative regulatory role in gliomas. However, further studies showed that the downregulation of UCH-L5 has no effect on the apoptosis and cell cycle of U87MG and U251 cells. These results suggest that UCH-L5 may inhibit the migration and invasion of glioma cells to inhibit the occurrence of glioma.A spliceosome is a large and complex molecular machine found primarily within the splicing speckles of the cell nucleus of eukaryotic cells. It consists of about 100 proteins and 5 Small nuclear ribonucleic acid (snRNAs), including U1, U2, U4, U5, and U6 . The splWe screened some candidate genes including SNRPF, SNRPN, and CKLF from previous study , 21. SNRIn previous study, the INO80 chromatin-remodeling complex has been implicated in DNA replication. And it has been reported that INO80 regulates gene transcription through binding to replication forks and promoting fork progression in human cells . And BRCA total of 19 frozen samples including 3 normal brain tissues and 16 glioma tissues, and 51 paraffin-embedded samples including 4 normal brain tissues, 16 low-grade gliomas and 31 high-grade gliomas were from the department of Neurosurgery of the Second Affiliated Hospital, Zhejiang University, School of Medicine . The patients were diagnosed as gliomas by preoperative CT and MRI examinations, and tumor tissues were obtained by surgical removal. 3 normal brain tissues were obtained from patients with cerebral trauma and cerebral hemorrhage. Therefore, 19 frozen samples were subjected to the analysis of the different expressions of UCH-L5 between gliomas and normal brain tissues by RT qPCR and Western blot. And 51 paraffin-embedded tissues samples were subjected to the analysis of the different expressions of UCH-L5 by Immunohistochemistry. We classified gliomas as different types according to the morphology or origin of tumor cells, and subtypes were categorized according to the malignancy of cells. And we defined the Low-grade glioma and High-grade glioma according to the WHO classification of tumors of the central nervous system 2007). More information about frozen and paraffin-embedded samples was showed in Table 007. More2O. Endogenous catalase activity was inactivated by H2O2. Sections were set in citrate buffer (PH6.0) and heated to fixed antigen. Then Sections were put in a wet box and blocked by 5%BSA, and incubated with 1:100 dilution of rabbit anti-UCH-L5 antibody  at 4°C overnight, and with the biotinylated goat anti-rabbit antibody with the streptavidin-peroxidase conjugate. Finally, 3′, 3′-diaminobenzene (DAB) was used for color reaction. All staining results were observed and evaluated under a light microscope. The percentage of positively stained cells and the staining intensity were calculated and analyzed by using imageJ software.Paraffin-embedded tumor tissues were cut into 5μm paraffin sections, Sections for Immunohistochemical SABC staining were dewaxed in xylene, soaked with 100%, 95%, 90%, 80%, 70% ethanol and HU87MG, U251, and 293T cells  were cultured in DMEM with 10% fetal bovine serum (FBS) and 1% myocilin.5 U87MG or U251 cells were inoculated on the 12-well or 6-well cell culture plate and placed in 37°C, 5% CO2 incubator overnight. After the screening concentration of siRNA, 30 nM UCH-L5-siRNA for U87MG and U251 cells, and 30 nM SNRPF-siRNA, SNRPN-siRNA, or CKLF-siRNA for U87MG cells were added to 200 μl DMEM medium, adding 4 μl (12 well plate)or 6 μl (6 well plate) interfering reagent and reacting at room temperature (RT) for 15 minutes, then added to transfection complexes into plates and following experiments were continued after 48 hours. The siRNA sequences of candidate target genes including: SNRPN-1:GCCAAAGAAUGCGAAGCAATT, SNRPN-2:UCUUCAUUGGCACCUUUAATT;SNRPF-1:CCUUUCCUCAAUGGACUAATT, SNRPF-2:UCCCAAACCUUUCCUCAAUTT; CKLF-1: GCACU AACUGUGACAUCUATT, CKLF-2: GGCCUUUGCUUGAUAUUAUTT The RT qPCR primers include: for SNRPN: CCC AGC TTG CAT TGT TTC TAG (Forward), CAT CTT GCT ACT CTT GCC AAC (Reverse). for CKLF: TGC TCA TCG TAT CTG TGT TGG (Forward), AGC TTC CGG TAA ATA AGG GC (Reverse); for SNRPF: AGA GTA GCC TGC AAC ATT CG (Forward), GAT AGC CCT TGT ACT CCA TTC C (Reverse). And for ACTB: ACCTTCTACAATGAGCTGCG (Forward), CCT GGA TAG CAA CGT ACA TGG (Reverse).108/ml. 3 days after infection, U87MG and U251 cells with stable knockdown UCH-L5 and stable overexpressing UCH-L5 cell lines were screened by GFP and puromycin. The knockdown and overexpression efficiency of UCH-L5 were determined by RT qPCR and Western blot. pCMV-Tag 2B and pCMV-HA plasmid were purchased from Invitrogen (USA). UCH-L5 ORF was inserted into pCMV-Tag 2B, SNRPF ORF (NM_003095.2) was reversed from total RNA from U87MG cells, subcloned and inserted into pCMV-HA. ORF PCR primers sequences for UCH-L5 and SNRPF were:4shRNA(shRNA1:GTCCCGACTTGACACGATATTTTCAAGAGAAATATCGTGTCAAGTCGGGATTTTTT.shRNA2:GAGCCAGTTCATGGGTTAATTTTCAAGAGAAATTAACCCATGAACTGGCTTTTTTT.shRNA3:GGAGACTGTATCAATTAGATTTCAAGAGAATCTAATTCATACAGTCTCCTTTTTT.shRNA4:GTGAAGGTGAAATTCGATTTAATTCAAGAGATTAAATCGAATTTCACCTTCATTTTT) targeting UCH-L5 (NM001199261) were designed, and shRNA3 was indicated effective in U251 and U87MG cells. shRNA were inserted into lentivirus vector pLent-4 in 1 shRNA-GFP-Puro . Lentivirus mediated overexpression vector of UCH-L5 was subcloned from pDEST-LTR-N-FLAG-HA-UCH-L5-puro . The UCH-L5 ORF was inserted into pLent-EF1a-FH-CMV-GFP . These constructs were confirmed by DNA sequencing. Lentivirus was packaged in 293T cells and lentivirus titer was 1 × 10for UCH-L5, 5′-CGCGGATCCATGACGGGCAATGCCGGG-3′ (forward) and 5′-CCGGAATTCTTTGGTTTCCTGAGCTTTCTTTGC-3′ (reverse); for SNRPF, 5′-CGGAATTCGGATGAGTTTACCCCTCAATCCCAAAC-3′ (forward) and 5′-GGGGTACCTTCTCTCATTTCCCCATCTTCTTC-3′ (reverse).Cells were lysed in cell lysis buffer  with a protease inhibitor cocktail . Proteins were separated by 10% or 12% SDS-PAGE and transferred onto PVDF membranes. After blocking in 5% non-fat milk, the membranes were incubated with primary antibodies overnight at 4°C and detected by incubating with specific secondary antibodies  for 2 h at room temperature. Then protein bands were exposed with ECL chromogenic substrate. β-Actin  served as an internal loading control. Other primary anti-human antibodies include UCH-L5 , SNRPF , Flag-tag  and HA-tag .5 U87MG or U251 cells were incubated into 6-well plates, adherent cells were treated with serum free medium for 24 hours to keep cells in the same period, continuing to cultivate 48 hours after interference treatment. Cells were digested with trypsin and centrifuged in 300 g for 5 minutes. After suspending in PBS buffer and centrifuging in 300 g for 5 min, cells were suspended in 500 μL PBS buffer and fixed with 4.5 ml 70% cold ethanol at 4°C for 12 h. Then centrifuging in 800 g for 5 minutes at 4°C and suspending with 1 ml PBS buffer containing 10% serum. After centrifuging in 800 g for 5 minutes at 4°C again and adding 500 μl staining buffer , cells were incubated at 37°C for 1 h in the dark. Flow analysis was carried out after adding 500 μl PBS buffer. Each sample had 3 repetitions accordingly.2 × 105 cells were trypsinized, centrifuged, washed with ice-cold PBS buffer. Absorbing the supernatant and resuspending cells with 100 μl Annexin V-FITC binding buffer. Then cells were incubated in 5 μl propidium iodide (PI) staining (PI dye and RNase A) solution and 5 μl Annexin V-FITC for 15 min at RT in the dark. Cell apoptosis was finally analyzed by flow cytometry within 1 h. Each sample had 3 repetitions.U87MG and U251 cells were treated with scramble-siRNA or UCH-L5-siRNA, SNRPF-siRNA, SNRPN-siRNA, and CKLF-siRNA for 48 h, 1 × 105 U87MG or U251cells were seeded on the 12-well plate, U87MG and U251 cells were treated with scramble-siRNA, UCH-L5-siRNA, SNRPF-siRNA, SNRPN-siRNA, and CKLF-siRNA for 48 h. The cell monolayers were scraped with a sterile yellow micropipette tip to create a denuded area of constant width. Cells were washed with PBS buffer to remove cell debris, and cultured in DMEM with 2% FBS. The wound widths were photographed every 12 hours in the same area.105/ml with serum free medium, and 100 μl cell suspensions were added to the top chamber with a matrigel-coat membrane. And DMEM containing 15% FBS was used as a chemoattractant in the lower chambers. After incubation for 48 h, cells were fixed with methanol and stained with 4’, 6-diamidino-2-phenylindole (DAPI). Then cells were counted in 5 independent fields with a light microscope.Cells were seeded on the 6-well plate, U87MG and U251 cells were treated UCH-L5-siRNA, SNRPF-siRNA, SNRPN-siRNA, and CKLF-siRNA for 48 h. Cells were digested, centrifuged and diluted to 5 × 10t-test using Prism GraphPad software. P < 0.05 was considered statistically significant.The identity of UCH-L5 in Immunohistochemical samples was tested using ImageJ software. Statistical significance was determined by