Astragali radix (AR) is one of the most widely used traditional Chinese herbal medicines. Modern pharmacological studies and clinical practices indicate that AR possesses various biological functions, including potent immunomodulation, antioxidant, anti-inflammation and antitumor activities. To date, more than 200 chemical constituents have been isolated and identified from AR. Among them, isoflavonoids, saponins and polysaccharides are the three main types of beneficial compounds responsible for its pharmacological activities and therapeutic efficacy. After ingestion of AR, the metabolism and biotransformation of the bioactive compounds were extensive in vivo. The isoflavonoids and saponins and their metabolites are the major type of constituents absorbed in plasma. The bioavailability barrier (BB), which is mainly composed of efflux transporters and conjugating enzymes, is expected to have a significant impact on the bioavailability of AR. This review summarizes studies on the phytochemistry, pharmacology and pharmacokinetics on AR. Additionally, the use of AR as a personalized medicine based on the BB is also discussed, which may provide beneficial information to achieve a better and more accurate therapeutic response of AR in clinical practice.  Astragali radix (AR), also well-known as Huangqi in China, is one of the most popular herbal medicines that has been widely applied for over 2000 years in Chinese clinics. It is derived from the roots of Astragalus membranaceus (Fisch.) Bge. or A. membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao. As a highly potent herb widely used to treat various diseases, AR has played an indispensable role in healthcare throughout Chinese history [ history ,2. Tradi history ,4,5. For history . Further history . Modern  history . Now, AR history ,11,12,13Astragalus polysaccharides (APS) have been identified [AR has been shown to contain isoflavonoids, triterpene saponins, polysaccharides and some trace elements . To dateentified . What isentified ,18,19. Because of the extensive use of AR in clinical practice and its effective pharmacological functions, a large number of studies on the pharmacokinetic characteristics of AR and its bioactive compounds have been performed ,21,22. TTo date, more than 200 compounds have been isolated and identified from AR, including saponins, flavonoids, polysaccharides, amino acids, trace elements and other compounds . Some ofAstragalus total triterpene saponins in AR ranges from 0.5 mg/g to 3.5 mg/g [Astragalus saponins have been isolated and identified. Four new saponins were isolated and two of them were shown to stimulate the proliferation of mouse splenocytes without significant cytotoxic [Astragalus triterpene saponins, astragaloside I–VIII, acetyl astragaloside, isoastragaloside I–IV and soyasaponins are the major four types, which accounts for about 80% of all the saponins [Astragalus saponins, which has been reported to exert extensive pharmacological actions in various diseases [AR abounds with saponins and the content of 3.5 mg/g . With th3.5 mg/g . As earl3.5 mg/g . Then, e3.5 mg/g . Subsequ3.5 mg/g . With thytotoxic . Then, fytotoxic . Among tsaponins . The stusaponins . In addidiseases . Modern diseases ,49,50. MAstragalus flavonoids in AR ranges from 0.5 mg/g to 3.0 mg/g [O-β-d-glucoside (CG), calycosin-7-O-β-d-glucoside-6″-O-malonate, ononin, -3-hydroxy-9,10-dimethoxypterocarpan-3-O-β-d-glucoside, calycosin, (3R)-7,2′-dihydroxy-3′,4′-dimethoxyisoflavan-7-O-β-d-glucoside, formononetin-7-O-β-d-glucoside-6″-O-malonate and formononetin [Astragalus flavonoids, including pendulone, isoliquiritigenin and sulfuretin, have also been isolated from AR [It is well known that flavonoids are also one of the main types of beneficial compounds responsible for the biological activities of AR. It was determined that the content of 3.0 mg/g . So far,3.0 mg/g . In 2000ononetin . Among tononetin ,54. Moreononetin ,56. What from AR . 6 as determined by the gel filtration method. Sugar compositional analysis by TLC and gas chromatography showed that it consisted of Rha, Xyl, Glc, Gal, Man and Fru in the molar ratios of Rha: Xyl: Glc: Gal: Man: Fru = 4.9: 4.7: 8.3: 122.2: 2.2: 3.1 [3 Da) was a neutral polysaccharide composed of arabinose, galactose, glucose and mannose (molar ratio: 1.00:0.98:3.01:1.52) with pyranose ring and α-type glycosidic linkages [6 Da determined by high-performance gel-permeation chromatography (HPGPC) analysis. APS4 is consists of rhamnose, arabinose, xylose, mannose and galactose, in a molar ratio of 12.1:0.3:0.6:1.0:1.0:1.7 through Chromatography (GC) analysis. More experiments indicated that APS4 had the potential application for cancer treatment [6 Da and AERP2 was a glucan with 2.11 × 103 Da [It is relatively difficult to isolate and characterize the individual APS because of their macromolecules with complicated chemical structures. To date, over 30 APS have been isolated and are mainly classified into dextran and heteropolysaccharides . From a 2.2: 3.1 . In anotreatment . The autreatment . In addi× 103 Da . In addition to the isoflavonoids, saponins and polysaccharides, AR also contains over 20 kinds of trace elements such as rubidium, manganese, copper, chromium, cobalt, scandium, selenium, cesium, iron, molybdenum and zinc . In addiAs a highly potent herb widely used to treat various diseases, AR has played an indispensable role in healthcare throughout Chinese history ,2. TradiAn increasing number of experiments have confirmed that AR can effectively inhibit cardiovascular diseases such as myocardial ischemia-reperfusion injury , myocard2+-mediated calcineurin/NFATc3 and CaMKII signaling cascades in isoproterenol-induced cardiac hypertrophy rats [Myocardial hypertrophy is usually considered as a compensatory response to pathological conditions . Long-tephy rats   in SR is responsible for the re-uptake of sarcoplasmic calcium. Numerous studies have shown that the expression of SERCA2a was markedly decreased in injured myocardium. It was reported that AR can promote the expression of SERCA2a, promote the re-uptake of calcium in SR, inhibit overload calcium, thereby inhibiting oxidative stress-induced cell apoptosis and cytoskeleton damage in adriamycin-injured rat hearts [Sarcoplasmic reticulum (SR) plays an important role in the regulation of intracellular calcium. Sarcoplasmic reticulum Cat hearts   proteins in the myocardium . In a stCoronary heart disease (CHD) is another major cardiovascular disease, which remains a serious public health burden . The vasType 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) are widely prevalent metabolic diseases with differing pathologies. T1DM manifests due to autoimmune destruction of the pancreatic beta cells, resulting in a diminished secretion of insulin. T2DM originates from a state of insulin resistance, resulting in hyperglycemia and reduction in beta cell mass. Both diseases can cause severe health consequences . Compell4 + T cells and CD8 + T cells. Under normal physiological conditions, CD4 + T cells and CD8 + T cells maintain a dynamic balance of CD4 +/CD8 + and keep a normal immune response in the body. Under the stimulation by antigens, the CD4 + T cells differentiate into Th1 and Th2 cells. Those cytokines secreted by Th1 cells include IL-1, IL-2, IL-6, IL-12, TNF-α and INF-γ, which can mediate the cellular immunity and cause damage to islet beta cells. Those cytokines secreted by Th2 cells include IL-4, IL-5, IL-6, IL-10, IL-13 and TGF-β, which can mediate fluid immunity and protect the islet beta cells. Th1 and Th2 cytokines together play important roles in immune regulation [8 + T cells, which may be an important mechanism by which APS protects β cells of the pancreatic islets from apoptosis induced by CD8 + T cells in T1DM in vivo [T1DM is an autoimmune disease characterized by the destruction of insulin-producing beta cells in the pancreas and the absolute insufficiency of insulin secretion . T lymphgulation . In clingulation ,86,87. Igulation  , which is responsible for sugar transport, is related to insulin sensitivity in different tissues and organs. On the one hand, the expression of GLUT4 can be regulated by the AMP-activated protein kinase (AMPK). On the other hand, the binding of insulin and insulin receptor can induce the phosphorylation of insulin receptor substrate 1 (IRS1), then activate PI3K/Akt signaling pathway and finally induce the GLUT4 expression. It was demonstrated that APS could not only activate the AMPK signaling pathway to promote GLUT4 expression and glucose metabolism ① but couThe cumulative findings show that microRNAs also play a vital role in regulating the expression of insulin secretion-related proteins. MicroRNAs microarray combined with bioinformatics has been used to screen differentially expressed micro (mi)RNAs after treatment with APS in T2DM Goto Kakizaki (GK) rats. By bioinformatics analysis, the authors found 12 differentially expressed microRNAs. The findings further provided evidence that miRNA-203a-3p, the most prominent differentially expressed gene, may have a functional role in ER stress signaling in the liver of T2DM GK rats. In addition, APS attenuated insulin resistance in T2DM, likely through upregulating or maintaining the miR-203a-3p expression levels, decreasing its target GRP78 mRNA and protein expression levels and regulating the protein expression of the ER stress signaling pathway  Figure ⑥.A growing number of studies suggested that AR and its active ingredients also possess effective curative effects on those diabetic complications, including diabetic cardiomyopathy, diabetic nephropathy, diabetic retinopathy and cognitive dysfunction. For example, diabetic cardiomyopathy is characterized by an imbalance between myocyte death and regeneration mediated by the progressive loss of cardiac stem and progenitor cells (CSPCs) by apoptosis and necrosis due to the activation of oxidative stress with diabetes. It was confirmed that APS could increase the CSPCs abundance by the inhibition of oxidative stress-mediated apoptosis in diabetic hearts of streptozotocin (STZ)-induced diabetic mice . SubstanHigh glucose-induced inflammation, renal microangiopathy, renal injury and fibrosis are the main characteristics of diabetic nephropathy, which is the leading cause of the end-stage failure of the kidney. NF-κB, an activation of the transcription factor, has been suggested to be a key step in the pathogenesis of diabetic nephropathy. Under physiological conditions, NF-κB is bound with an inhibitory protein of nuclear factor-κB (IκB) to form as an inactive transcription factor in the cytoplasm. After exposure to various stimuli, such as oxidative stress, angiotensins and various cytokines, NF-κB is released rapidly from IκB and then activate the gene expression of several cytokines, chemotactic and matrix proteins involved in inflammation, immunological responses and/or proliferation, which may contribute to the accumulation of renal extracellular matrix and tubulointerstitial fibrosis, resulting in kidney damage . APS wasOther diabetic complications, including diabetic retinopathy and cognitive dysfunction, also could be effectively alleviated by AR and its bioactive compounds. It was observed that AS-IV could prevent the activation of ERK1/2 phosphorylation and NF-κB and further relieve the retinal ganglion cells dysfunction in db/db mice with diabetic retinopathy, which provided a basis for investigating the clinical efficacy of AS-IV in preventing diabetic retinopathy . In addiAR and its main components are widely used in the treatment of cancer and rehabilitation after operations in patients because of their characteristics of increasing curative effect and reducing the toxicity of chemotherapeutic drugs ,118. SubThe anti-tumor activity of formononetin has been extensively studied and reported. Formononetin can effectively inhibit various tumors by inhibition of the proliferation, migration and invasion of tumor cells and induction of the tumor cell apoptosis. Genomic studies reveal that PI3K/AKT signaling is one of the most frequently deregulated pathways in several human cancers. This pathway plays a crucial role in cancer cell proliferation, survival, motility and metabolism and therefore could be an attractive therapeutic target. It was found that formononetin could significantly inhibit the PI3K/AKT signaling pathway to induce the apoptosis of cervical cancer HeLa cells and suppress xenograft tumor growth in nude mice, which indicated that formononetin may be used as an anti-cancer drug for cervical cancer in the future  Figure ①. AnotheCompelling studies have demonstrated that AS-IV also exhibits inhibitory activity against cancer. For example, AS-IV could inhibit the migration and proliferation of non-small cell lung cancer (NSCLC) cells and caused a noticeable increase in cell death via inhibition of the Akt/GSK-3β/β-catenin signaling axis. The authors, therefore, propose that AS-IV represents a promising novel agent for the treatment of NSCLC  Figure ⑤. Vav3, Accumulating evidence suggests that APS also possesses effective anti-tumor activity. For example, APS can delay the growth of human lung cancer A549 cell line xenograft in BALB/C nude mice in vivo and inhibit the proliferation of human lung cancer cell line A549 and NCI-H358 via the inhibition activity of NF-κB transcription activity . APS alsIn addition to the single bioactive compounds, the extracts of AR also exhibit inhibitory activity against cancer . For exaRespiratory diseases, which mainly include asthma, bronchitis, chronic obstructive pulmonary disease and pulmonary fibrosis, seriously reduce the quality of human life. The main characteristics of respiratory diseases include inflammation, excessive secretion of respiratory mucus and pulmonary fibrosis ,150. Sub4+ CD25+ regulatory T cells (Tregs) play a vital role in the regulation of immune function and their roles in asthma pathogenesis are increasingly recognized [4+CD25+ T cells, has been regarded as not only a faithful marker of Tregs but also a critical component for Treg development and function [4+CD25+Foxp3+ Treg cells, promote Foxp3+ mRNA expression, enhance Th2-mediated response and inhibit Th1-mediated response, thereby inhibiting the inflammation in a rat model of asthma, suggesting that the antiasthmatic effects of AR are at least partially associated with CD4+CD25+Foxp3+ Tregs [4+CD25+Foxp3+ T cells in ovalbumin-induced asthma [Asthma, a chronic airway inflammatory disease, is associated with a wide range of symptoms, including prolonged inflammation, airway hyperresponsiveness, mucus hypersecretion and airway remodeling . Airway cognized . The forfunction . It was 3+ Tregs   functions served as an important mediator of fibrogenesis. Plenty of works have identified that TGF-β1 is an important pro-fibrotic factor that has been shown to induce EMT in pulmonary fibrosis . Thus, tBronchopulmonary dysplasia (BPD) is defined as the most common form of chronic lung damage in premature infants, which includes barotrauma, volutrauma and oxygen toxicity . It is kThere is increasing evidence indicating that AR and its main components also exhibit inhibitory activity against a variety of nervous system diseases, mainly including cerebral ischemia injury , chemothIt has been proven that mitochondrial dysfunction emerges as a key event linking altered metabolism with neuronal death. The stability of mitochondrial structure and function is essential for neuronal survival during ischemic injury. The binding of hexokinase-II (HK-II) to mitochondria is demonstrated to protect mitochondrial function from ischemic injury . Thus, pThe isoflavonoids isolated from AR are also receiving much attention due to their various health benefits. Plenty of works have suggested that some of these beneficial effects of isoflavonoids mediated by their antioxidant activity . The neuParkinson’s disease (PD), an incurable progressive disease, is characterized by shaking, rigidity, slowness of movement and difficulty with walking . Some anIn addition to cardiovascular diseases, diabetes mellitus, cancer, respiratory diseases and nervous system diseases, AR and its bioactive compounds are also reported to have other various therapeutic uses. For example, AS-IV could protect against the progression of renal fibrosis by inhibiting inflammation via the TLR4/NF-κB signaling pathway . APS posConclusively, as an indispensable herbal drug widely used in clinical practice, AR has played a crucial role in healthcare. An increasing number of extracts and active compounds have been isolated and immunomodulation, antioxidant, anti-inflammation and antitumor activities, among others, were found. The effective treatment of cardiovascular diseases, diabetes mellitus, cancer, respiratory diseases and nervous system diseases by AR were also reported and verified by in vivo and in vitro experiments. With the increasing popular studies on AR and the development of models in the biological evaluation, more and more pharmacological activities of AR and its bioactive compounds will be clarified in the future.The pharmacokinetics of the AR water extract in rats have been reported in our previous study . Eight cmax of metabolite C-3′-G was highest and followed by metabolite F-7-G. While their parent compounds CG and ononin had lowest Cmax, which suggested that CG and ononin could rapidly transform to their glucuronides through hydrolyzation and glucuronidation. Moreover, the eight detected compounds had relative slow elimination [max at 2 h and t1/2 at 3.99 h , which mainly consists of drug-metabolizing enzymes (DMEs) and efflux transporters (ETs), abundantly distributed in the liver and intestine of the human body ,208. TheFormononetin, a methoxylated isoflavone, is one of the vital bioactive compounds of AR and possesses various pharmacological activities . The phaCG, a marker used routinely to monitor the quality of AR, exhibits several pharmacological activities . The phaOnonin, the glycoside form of formononetin, is also one of the major isoflavonoids in AR with biological activities . The phaAs the most abundant saponin and a characteristic compound of AR, AS-IV is documented as the chemical marker for the quality control of AR . The phaAs concluded above, AR possesses various biological functions, which has played an indispensable role in healthcare throughout Chinese history for thousands of years. However, so far, the relationship between the dose and efficacy of AR has still not been clearly clarified, which is reflected in the curative effects of AR vary greatly between different individuals. According to the historical literature and modern clinical reports, the dose of AR administration range from 0.1 g to 300 g , which fThe BB, which mainly consists of DMEs and ETs, abundantly distributed in the liver and intestine of the human body ,208. On From the results of the pharmacokinetic studies on AR concluded above, it has been well-recognized that the coupling of UGTs and ETs plays a crucial role in regulating the bioavailability of the bioactive compounds from AR. Thus, it can be speculated that the diverse polymorphisms of UGTs and ETs in patients with the similar syndrome could notably produce differences in the reactions to the AR efficacies, which would lead to blindness in treatment and could easily result in ineffective treatment. Transcriptome studies showed that the genetic polymorphisms of UGTs extensively exist in different individuals, which directly regulates the efficacy and toxicity of drugs in different individuals ,243,244.Collectively, the clinical use of AR as a personalized medicine is necessary to achieve a better and more accurate therapeutic efficacy. The polymorphisms in UGTs and ETs are important determinants of inter-individual differences in the toxicities and response to many drugs and also plays a crucial role in regulating the pharmacokinetic behavior of AR in vivo. Thus, the genetic polymorphisms of the UGTs and ETs in different individuals can be applied to characterize and optimize personalized therapies of AR in clinics in the future.As an effective and safe herbal drug widely used to treat various diseases, AR has played an indispensable role in healthcare in China and other countries. It is worth mentioning that AR is a popular herb frequently coadministered with many other herbal or chemical drugs for efficacy improvement in clinics. According to the historical literature and modern clinical reports, AR has been used as an essential ingredient in over 200 Chinese herb prescriptions for treating various complicated diseases. However, the potential risks of herb-herb or herb-drug interactions intensively exist during the combination therapy in clinics, which could easily cause an undesirable variation in the plasma concentrations of coadministered drugs, thereby resulting in treatment failure or toxicologically unsafe consequences ,255,256.Modern pharmacological studies and clinical practices provide more and more evidence for the effective functions of AR. According to the results from various validated in vivo and in vitro studies, AR has been proved to possesses multiple biological functions, such as immunomodulation, antioxidant, anti-inflammation and antitumor properties and thus widely ingested for the treatment of cardiovascular diseases, diabetes mellitus, cancer, respiratory diseases, nervous system diseases and other diseases. In the last decade, the underlying molecular mechanisms involved in the therapeutic effects of AR have been widely studied and reported. But further investigation is needed because of the various and complicated compounds contained in AR, which leads to the diversity and complexity of the molecular targets intervened by AR.The phytochemical studies indicate that AR mainly contains isoflavonoids, triterpene saponins, polysaccharides and some other trace elements. To date, more than 200 compounds have been isolated and identified from AR and various biological activities of the compounds have been confirmed. Among them, isoflavonoids, saponins and polysaccharides are the three main types of beneficial compounds responsible for the pharmacological activities and therapeutic efficacy of AR. Further studies need to be conducted to isolate and identify the new compounds from AR to get a more comprehensive understanding of AR.The pharmacokinetic characteristics of AR and its bioactive compounds have also been widely performed and clearly elucidated. It is well-recognized that the metabolism and biotransformation of the bioactive compounds were extensive after ingestion of AR. The isoflavonoids and saponins and their metabolites are the major type of constituents absorbed in plasma. The coupling of UGTs and ETs in vivo plays a crucial role in regulating the pharmacokinetic characteristics and determining the bioavailability of the bioactive compounds from AR. Thus, the varied polymorphisms of UGTs and ETs in different individuals are likely to be the critical factors contributing to the vast dosage differences in AR ingestion in individuals. In future clinical practice, the genetic polymorphisms of the UGTs and ETs in different individuals can be applied to characterize and optimize personalized therapies of AR to achieve better and more accurate therapeutic efficacy.


We investigated whether increased signaling by beta2-adrenergic receptors (β2-ARs), which mediate the action of catecholamines, enhances the progression of hepatocellular carcinoma (HCC). Mean age of patients with HCC, the most prolific form of liver cancer, has progressively increased over the last decade. Beta2-AR-mediated signaling in liver increases with age. We also observed increased β2-AR levels in liver tissues of patients with HCC compared to control subjects. We, therefore, hypothesized that increased β2-AR signaling enhances HCC progression while inhibition of β2-AR signaling by treatment with beta blockers suppresses its progression. To test this hypothesis, we used N-nitrosodiethylamine (DEN) to induce HCC in liver-specific β2-AR knockout (LKO) and control mice in the absence or presence of beta blocker propranolol. At the end of 25 weeks, we observed increased numbers of visible tumors, disarray of liver architecture, and mortality in DEN-induced control mice which was reduced by propranolol treatment. We also observed that DEN-treated LKO mice demonstrated reduced mortality, disarray of architecture, and phosphorylation of oncogene Src compared to DEN-treated control mice. Taken together, these results indicate that decreased β2-AR signaling because of a lack of receptors in the liver or inhibition of receptor action with propranolol reduces HCC progression. Studies are in progress to determine the β2-AR-mediated mechanisms involved in HCC progression. Our studies suggest that beta blocker propranolol, used to treat cardiovascular diseases, may be repurposed as a potential therapeutic option for treatment of HCC.


Mycobacterium tuberculosis (Mtb) infection are desperately needed to tackle the continuing global burden of this disease and the efficacy and cost limitations associated with current medicines. Herein, we report the synthesis of a series of novel benzoxa--diazole substituted amino acid hydrazides in a two-step synthesis and evaluate their inhibitory activity against Mtb and selected bacterial strains of clinical importance utilising an end point-determined REMA assay. Alongside this, their potential for undesired cytotoxicity against mammalian cells was assessed employing standard MTT assay methodologies. It has been demonstrated using modification at three sites  it is possible to change both the antibacterial activity and cytotoxicity of these molecules whilst not affecting their microbial selectivity, making them attractive architectures for further exploitation as novel antibacterial agents.Discovery and development of new therapeutic options for the treatment of  Mycobacterium tuberculosis (Mtb), the causative agent of TB -diazoles as potential inhibitors of Mtb ATP phosphoribosyl transferase (HisG) and APS reductase (APSR) -diazole, benzothia--diazole and benzotriazoles are a class of heterocycle which exhibit widespread therapeutic opportunities . In addie (APSR) ,13, we wAA) and the substituted aryl hydrazine (1R), towards Mtb selectivity, potency, efficacy, and avoidance of toxicity against mammalian cells  ,15,16, tbacteria  which re and Mtb .To gain an improved understanding of the antibacterial potency and scope of these compounds, a dose-range REMA assay was performed  .1–4 providing broad spectrum activity. Whilst the nitrobenzoxa--diazole 3 showed the highest levels of activity against Mtb, the lack of selectivity was a cause for concern. Replacing the nitro group with a sulphonamido amino acid moiety greatly improved specificity for mycolata bacteria, with substituted benzoxa--diazole 6 showing much higher activity than 5, suggesting poor cell wall penetration of 5 is due to the carboxylic acid moiety. Notwithstanding this, replacement of the benzoxa--diazole with benzothia--diazoles 7 and 8 led to a complete loss of activity suggesting that the benzoxa--diazole plays a crucial role in these compounds antibacterial activity.The results of the endpoint REMA assay revealed a mix of activity against the organisms tested, with simple substituted benzodiazole compounds 9–12 provided compounds more consistent activity across a range of structures. Consequently, substituted benzoxa--diazoles were chosen as the partner to amino acid hydrazides for further investigation, via a SAR study to further understand the importance of the amino acid (AA) and the hydrazine (1R) on anti-mycobacterial activity.Further analysis of the results revealed that conversion of the ester to an aryl hydrazide N-Boc amino acids . Introduction of an unsubstituted aromatic hydrazine 18a alongside halogenated hydrazines 19a–22a did not provide any significant enhancement in activity although a marked increase in cytotoxicity was observed. For both series, enhanced antibacterial activity was restored on coupling to the benzoxa--diazole 9, 10, 14b–22b albeit at the cost of increased cytotoxicity, as noted for this subunit -diazole substituted amino acid hydrazides have considerable potential as selective and potent agents against 19a or 21a provides a reduction in cytotoxicity , HOBt (2 equiv.) and DCC (1.2 equiv.) was dissolved in THF (7.5 mL), cooled to 0 °C and stirred for 15 min. The solution was treated with N-Aryl/Alkyl hydrazine * (1.2 equiv.) before warming to room temperature and stirring for a further 1.5 h. The mixture was then poured into sat. aq. NH4Cl (20 mL) before separating and extracting the aqueous layer with EtOAc (40 mL). The organic layer was further washed with sat. aq. NaHCO3 (20 mL) and then brine (20 mL). The combined organic layers were dried over MgSO4, filtered, concentrated and dried in vacuo. Flash chromatography  afforded the desired N-Boc amino acid hydrazides.A solution of 3N (1.2 equiv.) is added to neutralise the salt* When the hydrazine hydrochlorides were used, Ettert-Butyl 2-oxo-2-(2-(4-(trifluoromethyl)phenyl)hydrazinyl)ethylcarbamate, 13aN-Boc-l-Glycine  and 4-trifluorophenyl hydrazine  was transformed following flash chromatography into the title compound which was isolated as a white solid ; m.p. 185–187 °C; υmax (ATR) 3370 (NH), 3278 (NH), 3234 (NH), 3108, 3058, 2996, 1649 (C=O), 1615, 1518 (C=O), 1330, 1245, 1156, 1107, 1052, 828, 559 cm−1; δH  9.78 , 8.32 , 7.40 , 7.07 , 6.78 , 3.60 , 1.37 3COC(O)NH); δC  170.2 (CONH), 156.6 ((CH3)3COC(O)NH), 153.1 (Ar-C), 126.7 (Ar-C), 126.4 (Ar-C), 118.82 (Ar-C), 118.63 (Ar-C), 112.1 (Ar-C), 78.8 (NHCH2), 42.7 ((CH3)3CO), 28.8 ((CH3)3CO); δF  -59.30; m/z (ES+) 356 (MNa+), 689 (2M + Na+); HRMS (ES+) Found MH+, 334.13722 (C14H19F3N3O3 requires 334.13730).Following the general procedure outlined, tert-Butyl (S)-(2-oxo-1-phenyl-2-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)ethyl)carbamate, 14aN-Boc-l-phenylglycine  and 4-trifluorophenyl hydrazine  was transformed following flash chromatography into the title compound as a brown solid ; Rf 0.44 (DCM/EtOH/NH3 [200:8:1]); m.p. 138–143 °C; υmax (ATR) 3328 (N-H), 2982, 2930, 2851, 1678 (C=O), 1655 (C=O), 1524, 1322, 1158, 1113, 1067, 834, 696, 558, 488 cm−1; δH  8.00 , 7.46–7.37 , 6.68 , 6.24 , 5.69 , 5.35 , 1.42 3); δC  157.0 (NHCHCO), 150.3 (NHCOO), 129.3 (Ar-C), 128.9 (Ar-C), 128.4 (Ar-C), 127.2 (Ar-C), 127.1 (Ar-C), 126.5 (Ar-C), 125.1 (Ar-C), 123.6 (CF3), 112.7 (Ar-C), 80.3 (C(CH3)3), 59.4 (NHCHCO), 28.3 (C(CH3)3); δF  -61.60 ; m/z (ES+) 410 (MH+), 432 (MNa+), 841 (2M + Na+); HRMS (ES+) Found MH+, 410.1689 (C20H23F3N3O3 requires 410.1686).Following the general procedure outlined, tert-Butyl (S)-2-(2-(4-(trifluoromethyl)phenyl)hydrazine-1-carbonyl)pyrrolidine-1-carboxylate, 15aN-Boc-l-proline  and 4-trifluorophenyl hydrazine  was transformed following flash chromatography into the title compound as a golden-brown solid ; Rf 0.38 (DCM/EtOH/NH3 [200:8:1]); m.p. 116–120 °C; υmax (ATR) 3265 (N-H), 2979, 2932, 2851, 1672 (C=O), 1618, 1399, 1324, 1107, 1065, 833, 591 cm−1; δH  8.91 , 7.45 , 6.87 , 6.34 , 4.45–4.34 , 3.52–3.35 3), 2.06–1.87 3), 1.53 3); δC  172.2 (NCHCO), 156.1 (NCOO), 150.8 (Ar-C), 126.4 (Ar-C), 125.2 (Ar-C), 123.7 (CF3), 112.7 (Ar-C), 81.0 (C(CH3)3), 58.4 (NCHCO), 47.2 (NCH2(CH2)2), 28.4 (C(CH3)3), 25.5, (NCH2(CH2)2), 24.8 (NCH2(CH2)2); m/z (ES+) 374 (MH+), 396 (MNa+), 769 (2M + Na+); HRMS (ES+) Found MNa+, 396.1497 (C17H22F3N3O3Na requires 396.1505).Following the general procedure outlined, tert-Butyl (S)-(1-oxo-3-phenyl-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)carbamate, 16aN-Boc-l-phenylalanine  and 4-trifluorophenyl hydrazine  was transformed following flash chromatography into the title compound as a golden-brown solid ; Rf 0.39 (DCM/EtOH/NH3 [200:8:1]); m.p. 157–159 °C; υmax (ATR) 3324 (N-H), 3274 (N-H), 2931, 2851, 1686 (C=O), 1660 (C=O), 1520, 1328, 1156, 1103, 1067, 835, 716, 515 cm−1; δH  7.93 , 7.39 , 7.36–7.32 , 7.26–7.22 , 6.61 , 6.18 , 5.09 , 4.46 , 3.12 , 1.47 3); δC  171.6 (NHCHCO), 150.3 (NHCOO), 135.96 (Ar-C), 129.3 (Ar-C), 128.9 (Ar-C), 127.2 (Ar-C), 126.4 (Ar-C), 126.4 (Ar-C), 125.2 (Ar-C), 123.6 (CF3), 112.7 (Ar-C), 80.9 (C(CH3)3), 54.5 (NHCHCO), 33.5 (CH2CHCO), 28.3 (C(CH3)3); δF  -61.56 ; m/z (ES+) 424 (MH+), 446 (MNa+), 869 (2M + Na+); HRMS (ES+) Found MH+, 424.1847 (C21H25F3N3O3 requires 424.1843).Following the general procedure outlined, tert-Butyl (S)-(1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)carbamate, 17aN-Boc-l-alanine  and 4-trifluorophenyl hydrazine  was transformed following flash chromatography into the title compound as a dark brown solid ; Rf 0.37 (DCM/EtOH/NH3 [200:8:1]); m.p. 126–131 °C; υmax (ATR) 3321 (N-H), 2929, 2851, 1683 (C=O), 1664 (C=O), 1617, 1524, 1322, 1157, 1101, 1066, 830, 641 cm−1; δH  8.50 , 7.46 , 6.87 , 6.34 , 5.07 , 4.40 , 1.50 3), 1.43 ; δC  171.7 (NHCHCO), 158.7 (NHCOO), 129.3 (Ar-C), 128.9 (Ar-C), 127.2 (Ar-C), 126.4 (CF3), 112.8 (Ar-C), 80.9 (C(CH3)3), 54.6 (NHCHCO), 33.7 (CHCH3), 28.3 (C(CH3)3); δF  -61.55 ; m/z (ES+) 348 (MH+), 370 (MNa+), 717 (2M + Na+); HRMS (ES+) Found MNa+, 370.1352 (C15H20F3N3O3Na requires 370.1349).Following the general procedure outlined, tert-Butyl 2-oxo-2-(2-phenylhydrazinyl)ethylcarbamate, 18aN-Boc-l-Glycine  and phenyl hydrazine  was transformed following flash chromatography into the title compound as a white solid ; Rf 0.1 (n-hexane/EtOAc 7:3) as a mixture of rotamers in the ratio [5:1] by NMR @ 25 °C; m.p. 120–122 °C; υmax (ATR) 3348 (NH), 3274 (NH), 2922, 2852, 1746, 1652 (C=O), 1640 (C=O), 1519, 1488, 1418, 1375, 1260, 1192, 1106, 1036, 874, 760 cm−1; δH  9.59 , 9.08 , 7.85 , 7.65 , 7.17 , 7.08 , 7.02 , 6.74 , 6.71 –6.63 , 6.61 , 3.68 , 3.58 , 1.37 3COC(O)NH), 1.34 3COC(O)NH); δC  174.2 (CONH), 169.9 (CONH), 156.5 ((CH3)3COC(O)NH), 149.9 (Ar-C), 149.1 (Ar-C), 129.3 (Ar-C), 119.0 (Ar-C), 112.8 (Ar-C), 112.6 (Ar-C), 78.7 (NHCH2), 78.5 (NHCH2), 42.7 ((CH3)3CO), 31.9 ((CH3)3CO), 28.8 ((CH3)3CO), 22.8 ((CH3)3CO); m/z (ES+) 266 (MH+), 531 (2M + H+); HRMS (ES+) Found MH+, 266.24985 (C13H20O3N3 requires 266.14992).Following the general procedure outlined, tert-Butyl (2-(2-(4-fluorophenyl)hydrazineyl)-2-oxoethyl)carbamate, 19aN-Boc-l-Glycine  and 4-fluorophenyl hydrazine  was transformed following flash chromatography  into the title compound as a light brown solid  as a mixture of rotamers [4:1] by NMR @ 25 °C; Rf 0.32 (DCM/EtOH/NH3 [200:8:1]); m.p. 127–129 °C; υmax (ATR) 3364 (NH), 3269 (NH), 3132, 2984, 1652 (C=O), 1505, 1393, 1370, 1269, 1225, 1224, 1162, 1029, 1007 cm−1; NMR data given for major rotamer δH  8.35 3COC=ONH), 6.89 , 6.75 , 5.30 δ1H, bs, NHC=O), 4.07 , 3.85 , 1.45 3COC=ONH); δC  170.4 (C=O), 157.4 (C=O), 158.6 (Ar-C) 143.7 (Ar-CNH), 115.7 (Ar-C), 114.9 (Ar-C), 81.0 ((CH3)3COC=ONH) 43.5 (CH2), 28.7 ((CH3)3COC=ONH); δF  -123.19 (Ar-F); m/z (ES+) 284 (MH+), 306 (MNa+), 589 (2M + Na+); HRMS (ES+) Found MH+, 284.1412 (C13H19N3O3F requires 284.1410).Following the general procedure outlined, tert-Butyl (2-(2-(4-chlorophenyl)hydrazineyl)-2-oxoethyl)carbamate, 20aN-Boc-l-Glycine  and 4-chlorophenyl hydrazine  was transformed following flash chromatography into the title compound as a brown solid  as a mixture of rotamers [4:1] by NMR @ 25 °C; Rf 0.2 (n-hexane/EtOAc [1:1]); m.p. 118–120 °C; υmax (ATR) 3365, 3275 (NH), 3122, 3044, 2929, 2851, 1697, 1650 (C=O), 1525, 1491, 1441, 1391, 1369, 1311, 1268, 1241, 1158, 1091, 1053 cm−1; NMR data given for major rotamer δH  8.07 , 7.17 , 6.76 , 5.16 , 3.87 , 3.45 , 1.47 3COC=ONH); δC  169.0 (C=O), 156.5 (C=O), 146.2 (Ar-CCl), 129.1 (Ar-C), 126.2 (Ar-CNH), 114.8 (Ar-C), 80.4 ((CH3)3COC=ONH), 43.8 (CH2NH), 28.3 ((CH3)3COC=ONH); m/z (ES+) 300 ([35Cl]MH+), 302 ([37Cl]MH+), 322 ([35Cl]MNa+), 324 ([37Cl]MNa+), 623 , 625 , 627 ; HRMS (ES+) Found [35Cl]MH+, 300.1134 (C13H19N3O335Cl requires 300.1115).Following the general procedure outlined, tert-Butyl (2-(2-(3-chlorophenyl)hydrazineyl)-2-oxoethyl)carbamate, 21aN-Boc-l-Glycine  and 3-chlorophenyl hydrazine hydrochloride  was transformed following flash chromatography into the title compound as a pale brown solid  as a mixture of rotamers [4:1] by NMR @ 25 °C; Rf 0.38 (DCM/EtOH/NH3 [200:8:1]); m.p. 119.5–121.7 °C; υmax (ATR) 3343 (NH), 3269 (NH), 3077, 2980, 2932, 1659 (C=O), 1597, 1512, 1490, 1368, 1242, 1230, 1156, 1046, 1030 cm−1; NMR data given for major rotamers δH  8.09 , 7.12 , 6.85 , 6.81 , 6.70 , 5.17 , 3.88 , 1.47 3COC=ONH); δC  170.0 (C=O), 157.5 (C=O), 148.9(Ar-C), 135.1 (Ar-CNH), 130.2 (Ar-C), 121.3 (Ar-C), 113.4 (Ar-C) 111.8 (Ar-C), 81.2 ((CH3)3COC=ONH), 28.3 ((CH3)3COC=ONH); m/z (ES+) 300 ([35Cl]MH+), 302 ([37Cl]MH+), 322 ([35Cl]MNa+), 324 ([37Cl]MNa+), 623 , 625 , 627 ; HRMS (ES+) Found [35Cl]MH+, 300.1119 (C13H19N3O335Cl requires 300.1115).Following the general procedure outlined, tert-Butyl (2-(2-(2-chlorophenyl)hydrazineyl)-2-oxoethyl)carbamate, 22aN-Boc-l-Glycine  and 2-chlorophenyl hydrazine hydrochloride  was transformed following flash chromatography into the title compound as a light brown gum  as a mixture of rotamers [3:1] by NMR @ 25 °C; Rf 0.50 (DCM/EtOH/NH3 [200:8:1]); υmax (ATR) 3262 (NH), 3074, 2960, 2928, 2851, 2118, 1668 (C=O), 1593, 1536, 1498, 1456, 1393, 1365, 1281, 1254, 1226, 1171, 1053, 1035 cm−1; NMR data given for major rotamer δH  8.23 , 7.31 , 7.14 , 6.88 , 6.84 , 6.40 , 3.90 , 3.47 , 1.47 3COC=ONH); δC  169.7 (C=O), 156.9 (C=O), 143.5 (Ar-CCl), 129.5 (Ar-C), 127.6 (Ar-C), 121.4 (Ar-C), 119.5 (Ar-CNH), 113.5 (Ar-C), 80.9 ((CH3)3COC=ONH), 43.5 (CH2NH), 28.2 ((CH3)3COC=ONH); m/z (ES+) 300 ([35Cl]MH+), 302 ([37Cl]MH+), 322 ([35Cl]MNa+), 324 ([37Cl]MNa+), 623 , 625 , 627 ; HRMS (ES+) Found [35Cl]MH+, 300.1108 (C13H19N3O335Cl requires 300.1115).Following the general procedure outlined, N-Boc amino acid hydrazides (1.20 equiv.) were dissolved in 4 M HCl solution in dioxane (3 mL) and stirred for 30 min at room temperature. The solvent was then removed in vacuo, the resulting solid was suspended in THF (3 mL) and Et3N (3 equiv.) was added. The solution was cooled to 0 °C, before treating with 7-chlorobenzoxa--diazole-4-sulphonyl chloride  before warming to room temperature and stirring for 2 h. The mixture was then poured into sat. aq. NH4Cl (20 mL). The aqueous layer was separated and extracted with EtOAc (20 mL). The combined organic layers were dried over MgSO4, filtered, concentrated and dried in vacuo. Flash chromatography  afforded the desired sulphonamides.97-Chloro-N-(2-oxo-2-(2-(4-(trifluoromethyl)phenyl)hydrazinyl)ethyl)benzo[c]oxadiazole-4-sulfonamide, tert-Butyl 2-oxo-2-(2-(4-(trifluoromethyl)phenyl)hydrazinyl)ethylcarbamate, 13a  was transformed following flash chromatography into the title compound as a pale brown solid ; Rf 0.6 (DCM/MeOH 9:1) as a mixture of rotamers in the ratio [7:1] by NMR @ 25 °C; m.p. 211–213 °C; υmax (ATR) 332, 3158, 1685 (C=O), 1614, 1524, 1473, 1418, 1326, 1155, 1107, 1062, 952, 834, 632 cm−1; δH  9.88 , 9.31 , 8.73 , 8.46 , 8.41 , 8.26 , 8.02 , 7.95 , 7.91 , 7.85 , 7.57 , 7.43 , 6.79 , 6.68 , 3.94 , 3.88 ; δC  168.4 (C=O), 152.6 (Ar-C), 149.4 (Ar-C), 146.2 (Ar-C), 134.5 (Ar-C), 131.5 (Ar-C), 129.1 (Ar-C), 126.7 (Ar-C), 125.6 (Ar-C), 112.1 (Ar-C), 44.3 (NHCH2); δF  -59.71, -59.89; m/z (ES−) 448 ([35Cl]M-), 450 ([37Cl]M−), 897 , 899 , 900 ; HRMS (ES−) Found [35Cl]M−, 448.00931 (C15H1035ClF3N5O4S requires 448.00996).Following the general procedure outlined, S)-7-Chloro-N-(2-oxo-1-phenyl-2-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)ethyl)benzo[c]oxadiazole-4-sulfonamide, 14b(tert-butyl (S)-(2-oxo-1-phenyl-2-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)ethyl)carbamate, 14a  was transformed following flash chromatography into the title compound as a pale yellow solid ; Rf 0.46 (DCM/EtOH/NH3 [200:8:1]); m.p. 183–187 °C; υmax (ATR) 3275 (N-H), 2930, 1693 (C=O), 1618, 1521, 1325, 1161, 1105, 1067, 840, 800, 636, 619, 590, 570, 530, 504, 491 cm−1; δH  10.20 , 9.35 , 8.36 , 7.97 , 7.82 , 7.32–7.27 , 7.18–7.15 , 6.44 , 5.30 ; δC  168.9 (NHCHCO), 152.3 (Ar-C), 148.9 (Ar-CNO), 145.3 (Ar-CNO), 137.1 (Ar-C), 134.9 (Ar-C), 131.1 (Ar-C), 128.9 (Ar-C), 128.5 (Ar-C), 128.3 (Ar-C), 127.9 (Ar-C),127.7 (Ar-C), 126.4 (Ar-C), 125.6 (Ar-C), 124.5 (CF3), 111.6 (Ar-C), 59.0 (NHCHCO); m/z (ES+) 526 ([35Cl] MH+), 528 ([37Cl] MH+), 548 ([35Cl] MNa+), 550 ([37Cl] MNa+), 1073 , 1075 , 1077 ; HRMS (ES+) Found [35Cl]MNa+, 526.0565 (C21H16F3N5O4S35Cl requires 526.0558).Following the general procedure outlined, S)-1-sulfonyl)-N′-(4-(trifluoromethyl)phenyl)pyrrolidine-2-carbohydrazide, 15b(tert-butyl (S)-2-(2-(4-(trifluoromethyl)phenyl)hydrazine-1-carbonyl)pyrrolidine-1-carboxylate, 15a  was transformed following flash chromatography into the title compound as a yellow solid ; Rf 0.46 (DCM/EtOH/NH3 [200:8:1]); m.p. 166–170 °C; υmax (ATR) 3324 (N-H), 2929, 2851, 1682 (C=O), 1621, 1574, 1325, 1146, 1102, 1066, 946, 836, 616 cm−1; δH  10.09 , 8.39 , 8.08 , 7.86 , 7.44 , 6.82 , 4.43 , 3.61–3.57 2), 3.47–3.41 2), 1.26–0.96 2); δC  171.4 (NHCHCO), 152.8 (Ar-C), 149.5 (Ar-CNO), 146.3 (Ar-CNO), 136.8 (Ar-C), 131.2 (Ar-C), 127.7 (Ar-C), 126.5 (Ar-C), 126.3 (Ar-C), 125.5 (Ar-C), 124.7 (CF3), 111.9 (Ar-C), 60.7 (NCHCO), 49.3 (NCH2(CH2)2), 33.9 (NCH2(CH2)2), 24.9 (NCH2(CH2)2); m/z (ES+) 490 ([35Cl] MH+), 492 ([37Cl] MH+); HRMS (ES+) Found [35Cl]MH+, 490.0555 (C18H16F3N5O4S35Cl requires 490.0558).Following the general procedure outlined, S)-7-Chloro-N-(1-oxo-3-phenyl-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)benzo[c]oxadiazole-4-sulfonamide, 16b(tert-butyl (S)-(1-oxo-3-phenyl-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)carbamate, 16a  was transformed following flash chromatography into the title compound which was isolated as a brown solid ; Rf 0.41 (DCM/EtOH/NH3 [200:8:1]); m.p. 80–84 °C; υmax (ATR) 3321 (N-H), 2932, 2854, 1677 (C=O), 1617, 1323, 1156, 1109, 1065, 834, 700, 632, 618, 577, 488 cm−1; δH  10.13 , 9.06 , 8.43 , 7.76 , 7.71 , 7.41 , 7.00–6.96 , 6.84–6.79 , 6.70 , 4.16–4.12 , 2.87 , 2.68 ; δC  170.9 (NHCHCO), 152.6 (Ar-C), 148.9 (Ar-CNO), 144.7 (Ar-CNO), 136.9 (Ar-C), 133.9 (Ar-C), 130.7 (Ar-C), 129.5 (Ar-C), 128.2 (Ar-C), 127.6 (Ar-C), 126.5 (Ar-C), 126.2 (Ar-C), 126.2 (Ar-C), 125.5 (Ar-C), 124.6 (CF3), 111.8 (Ar-C), 57.4 (NHCHCO), 38.0 (CH2CHCO); m/z (ES+) 540 ([35Cl] MH+), 542 ([37Cl] MH+), 562 ([35Cl] MNa+), 564 ([37Cl] MNa+), 1101 , 1103 , 1105 . HRMS (ES+) Found [35Cl]MH+, 540.0642 (C22H1835ClF3N5O4S requires 540.0715).Following the general procedure outlined S)-7-Chloro-N-(1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)benzo[c]oxadiazole-4-sulfonamide, 17b(tert-butyl (S)-(1-oxo-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)propan-2-yl)carbamate, 17a  was transformed following flash chromatography into the title compound as a pale yellow solid ; Rf 0.34 (DCM/EtOH/NH3 [200:8:1]); m.p. 127–131 °C; υmax (ATR) 3388 (N-H), 3315 (N-H), 3274, 2929, 2851, 1667 (C=O), 1619, 1323, 1159, 1110, 1067, 949, 836, 633, 597, 579, 511 cm−1; δH  9.90 , 8.80 , 8.24 , 8.00 , 7.82 , 7.39 , 6.61 , 4.20 – 4.15 , 1.26 ; δC  171.4 (NHCHCO), 152.5 (Ar-C), 149.2 (Ar-CNO), 145.6 (Ar-CNO), 134.4 (Ar-C), 131.2 (Ar-C), 128.9 (Ar-C), 128.3 (Ar-C), 126.5 (Ar-C), 125.4 (Ar-C), 124.6 (CF3), 111.7 (Ar-C), 51.2 (NHCHCO), 19.8 (CHCH3); m/z (ES+) 464 ([35Cl] MH+), 466 ([37Cl] MH+); HRMS (ES+) Found [35Cl]MH+, 464.0400 (C16H14F3N5O4S35Cl requires 464.0402).Following the general procedure outlined, 107-Chloro-N-(2-oxo-2-(2-phenylhydrazinyl)ethyl)benzo[c]oxadiazole-4-sulfonamide, tert-butyl 2-oxo-2-(2-phenylhydrazinyl)ethylcarbamate, 18a  was transformed following flash chromatography into the title compound as a yellow solid ; Rf 0.4 (DCM/MeOH 9:1) as a mixture of rotomers in the ratio [4:1] by NMR @ 25 °C; m.p. 209–211 °C; υmax (ATR) 3262 (NH), 1738, 1647 (C=O), 1606, 1530, 1494, 1410, 1346, 1190, 1157, 947, 834, 747 cm−1; δH  9.70 , 9.13 , 8.68 , 8.35 , 8.00 , 7.94 , 7.90 , 7.84 , 7.60 , 7.22 , 7.10 , 6.80 , 6.71–6.66 , 6.58 , 3.96 , 3.85 ; δC  168.2 (C=O), 149.47 (Ar-C), 149.43 (Ar-C), 146.1 (Ar-C), 134.4 (Ar-C), 131.5 (Ar-C), 129.8 (Ar-C), 129.3 (Ar-C), 125.5 (Ar-C), 119.2 (Ar-C), 112.7 (Ar-C), 44.4 (NHCH2); m/z (ES+) 382 ([35Cl]MH+), 384 ([37Cl]MH+); HRMS (ES+) Found [35Cl]MH+, 382.03702 (C14H1335ClN5O4S requires 382.03713).Following the general procedure outlined, N-(2-(2-(4-fluorophenyl)hydrazineyl)-2-oxoethyl)benzo[c]oxadiazole-4-sulfonamide, 19b7-Chloro-tert-butyl (2-(2-(4-fluorophenyl)hydrazineyl)-2-oxoethyl)carbamate, 19a  was transformed following flash chromatography into the title compound as a brown solid  as a mixture of rotamers [4:1] by NMR @ 25 °C; Rf 0.48 (DCM/EtOH/NH3 [200:8:1]); m.p. 180–185 °C; υmax (ATR) 3364, 3245 (NH), 2918, 2850, 1673 (C=O), 1507, 1453, 1414, 1367, 1346 (S=O), 1216, 1157 (S=O), 1108, 1042 cm−1; NMR data given for major rotamer δH  9.69 , 8.63 , 7.98 , 7.81 , 6.91 , 6.56 , 3.81 ; δF  -126.45 (Ar-F); δC  167.9 (C=O), 155.6 (Ar-C), 149.1 (Ar-C), 145.8 (Ar-C), 145.7 (Ar-C), 134.1 (Ar-C), 131.2 (Ar-C), 129.0 (Ar-C), 125.3 (Ar-C), 115.5 (Ar-C), 113.8 (Ar-C), 44.1 (CH2NH); m/z (ES+) 400 ([35Cl]MH+), 402 ([37Cl]MH+), 422 ([35Cl]MNa+), 424 ([37Cl]MNa+), 799 , 803 , 804 , 821 , 823 , 825 ; HRMS (ES+) Found [35Cl]MH+, 400.0271 (C14H12N5O4FS35Cl requires 400.0283).Following the general procedure outlined, N-(2-(2-(4-chlorophenyl)hydrazineyl)-2-oxoethyl)benzo[c]oxadiazole-4-sulfonamide, 20b7-Chloro-tert-butyl (2-(2-(4-chlorophenyl)hydrazineyl)-2-oxoethyl)carbamate, 20a  was transformed following flash chromatography into the title compound as a brown solid  as a mixture of rotamers [6:1] by NMR @ 25 °C; Rf 0.37 (DCM/EtOH/NH3 [200:8:1]); m.p. 174–177 °C; υmax (ATR) 3326, 3277 (NH), 3151, 3932, 2856, 1668 (C=O), 1523, 1491, 1325 (S=O), 1158 (S=O), 1107, 1067, 1043 cm−1; NMR data given for major rotamer δH  9.72 , 8.62 , 7.98 , 7.81 , 7.72 , 7.10 , 6.55 , 3.81 ; δC  168.0 (C=O), 149.1 (Ar-C), 148.3 (Ar-C), 145.8 (Ar-C), 134.2 (Ar-C), 131.3 (Ar-C), 128.9 (Ar-C), 128.7 (Ar-C), 125.3 (Ar-C), 122.3 (Ar-C), 114.0 (Ar-C), 44.1 (CH2NH); m/z (ES+) 416 , 418 , 420 , 438 , 440 , 442 , 853 , 855 , 857 , 859 , 861 ; HRMS (ES+) Found MH+, 415.998 (C14H12N5O4S35Cl2 requires 415.9987).Following the general procedure outlined, N-(2-(2-(3-chlorophenyl)hydrazineyl)-2-oxoethyl)benzo[c]oxadiazole-4-sulfonamide, 21b7-Chloro-tert-butyl (2-(2-(3-chlorophenyl)hydrazineyl)-2-oxoethyl)carbamate, 21a  was transformed following flash chromatography into the title compound as a pale yellow solid  as a mixture of rotamers [5:1] by NMR @ 25 °C; Rf 0.29 (DCM/EtOH/NH3 [200:8:1]); m.p. 211–213 °C; υmax (ATR) 3332, 3266, 3163 (NH), 2927, 2852, 1688 (C=O), 1597, 1524, 1469, 1425, 1371 (S=O), 1339, 1159 (S=O), 1073, 1043 cm−1; NMR data given for major rotamer δH  9.75 , 8.66 , 8.10 , 7.98 , 7.82 , 7.08 , 6.68 , 6.61 , 6.52 , 3.82 ; δC  168.13 (C=O), 150.9 (Ar-C), 149.2 (Ar-C), 145.8 (Ar-C), 134.1 (Ar-C), 131.2 (Ar-C), 130.7 (Ar-C), 128.9 (Ar-C), 125.3 (Ar-C), 118.4 (Ar-C), 114.0 (Ar-C), 111.8 (Ar-C), 111.1 (Ar-C), 44.2 (CH2NH); m/z (ES+) 416 , 418 , 420 , 471 , 473 , 475 ; HRMS (ES+) Found MH+, 415.9981 (C14H12N5O4S35Cl2 requires 415.9987).Following the general procedure outlined, N-(2-(2-(2-chlorophenyl)hydrazineyl)-2-oxoethyl)benzo[c]oxadiazole-4-sulfonamide, 22b7-Chloro-tert-butyl (2-(2-(2-chlorophenyl)hydrazineyl)-2-oxoethyl)carbamate, 22a  was transformed following flash chromatography into the title compound as a yellow solid  as a mixture of rotamers [4:1] by NMR @ 25 °C; Rf 0.16 (DCM/EtOH/NH3 [600:8:1]); m.p. 173–175 °C; υmax (ATR) 3324 (NH), 2928, 2851, 1669 (C=O), 1626, 1574, 1526, 1343, 1311, 1243, 1159 (S=O), 1088, 1042 cm−1; NMR data given for major rotamer δH  9.85 , 8.69 , 8.00 , 7.84 , 7.27 , 7.21 , 7.10 , 6.71 , 6.58 , 3.85 ; δC  168.1 (C=O), 149.2 (Ar-C), 145.8 (Ar-C), 144.5 (Ar-C), 134.2 (Ar-C), 131.2 (Ar-C), 129.5 (Ar-C), 128.9 (Ar-C), 125.3 (Ar-C), 120.6 (Ar-C), 117.5 (Ar-C), 113.2 (Ar-C), 44.1 (CH2NH); m/z (ES+) 416 , 418 , 420 , 471 , 473 , 475 ; HRMS (ES+) Found MH+, 415.9972 (C14H12N5O4S35Cl2 requires 415.9987).Following the general procedure outlined, Bacterial strains and growth media used in this study .TM and incubated at 37 °C, unless 30 °C was stated as the optimum for the organism. Freshly prepared filter sterilised resazurin  was filter sterilised and 10 μL added to all wells and re-incubated at 37 °C or 30 °C for 24 h or until the positive and negative controls showed a clear result.The minimum inhibitory concentration of the compounds against all strains using stand REMA assay protocols . BrieflyIn vitro chemosensitivity of Human NCI-H460 lung carcinoma cells to the agents were determined using the MTT assay, described elsewhere . Cells wAs shown by the present study, this interplay between cytotoxicity and antibacterial activity can be readily manipulated through the substitution patterns on each component, aromatic hydrazides, the size of the amino acid side chain and the benzoxa--diazole . The eas


Acetyl-CoA carboxylase (ACC) plays an important role in the metabolism of various cancer cells, but its role in head and neck squamous cell carcinoma (HNSCC) is uncertain. Therefore, in the present study, we explored the role of ACC2 in HNSCC.Western blot and immunohistochemistry assays were used to determine ACC2 protein expression levels in laryngocarcinoma and adjacent normal tissues derived from patients with laryngocarcinoma. ACC2 expression was knocked down in the hypopharyngeal cancer cell line FaDu to determine its effect on apoptosis. Lipid oil red staining was used to test the change of intracellular lipid.The results showed that the ACC2 protein was highly expressed in laryngocarcinoma and that the ACC2 expression level was positively associated with the clinical cancer stage and negatively associated with the degree of laryngocarcinoma cell differentiation. Kaplan–Meier analyses indicated that compared with patients having low levels of ACC2, those with high ACC2 levels had a decreased 5-year survival rate. The results of western blot and terminal deoxynucleotidyl transferase dUTP nick-end labeling assays showed that knockdown of ACC2 accelerated apoptosis in FaDu cells. Furthermore, knockdown of ACC2 significantly reduced the intracellular lipid levels in FaDu cells.These findings suggest that ACC2 may be an important prognostic marker for patients with HNSCC and that ACC2 may be a potential target in the treatment of HNSCC. Approximately 62,000 new head and neck cancer diagnoses and more than 13,000 deaths associated with this cancer occur in the United States every year . AlthougThe liver kinase B1–AMP-activated protein kinase–acetyl-CoA carboxylase (LKB1-AMPK-ACC) pathway is a major signaling pathway engaged in cell energy and lipid metabolism . AMPK isIn the literature, the study in ACC2 is very little and its function is not well demonstrated. In the present study, the expression levels of ACC2 in HNSCC and normal tissues were compared using immunohistochemistry and western blot assays. Associations of ACC2 expression level with clinical stage, degree of cancer cell differentiation, and survival of patients with HNSCC were also investigated to explore the role of ACC2 in the development of HNSCC. In addition, FaDu cells, a hypopharyngeal cancer cell line, were transfected with ACC2 siRNA to determine whether knockdown of ACC2 enhanced cell apoptosis. We further used oil red staining to test whether ACC2 knockdown affected intracellular lipids in FaDu cells. The ultimate goal of these experiments was to determine whether development of compounds targeting ACC2 as therapeutic targets for HNSCC would be warranted.All tissue samples (carcinoma and para-carcinoma tissues) from patients diagnosed as having laryngocarcinoma were collected at the First Affiliated Hospital of Anhui Medical University from 2012 to 2013. No distant metastases were found in any patient before surgery, and these patients were followed-up for 5 years after surgery to determine the survival rate . Specime2. The FaDu and NP69 cells were transiently transfected with ACC2 siRNA   using Lipofectamine 3000  following the manufacturer’s instructions and were cultured for another 48 h before being used in experiments.The FaDu cells and NP69 cells were purchased from American Type Culture Collection. The FaDu cells or NP69 cells were cultured in minimum essential medium Eagle’s (Mod.) or 1,640 medium  supplemented with 10% fetal bovine serum  and antibiotics (100 KU/L penicillin and 100 mg/L streptomycin) in an incubator at 37 °C with 5% COg for 20 min. The protein was extracted from the supernatant, and the remaining supernatant was mixed with loading buffer using a ratio of 4:1 at 100 °C for 10 min. An equal amount of protein (30 μg) was loaded onto a gel for sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane . The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated in 5% nonfat milk containing primary antibodies at 4 °C overnight. The membranes were incubated with the secondary antibody for 1 h at room temperature. Visualization of the secondary antibody was accomplished using an ECL detection system . The optical density (OD) of each protein band was measured using the free software Image J  and was normalized to β-tubulin , which had been located in the same lane. The following primary antibodies were used in this study: rabbit anti-ACC2, rabbit anti-Bax, rabbit anti-Bcl-2, and rabbit anti-Caspase 3 .The protein levels in FaDu cells or NP69 cells or in specimens were assessed by western blot assay. Briefly, the cells from different treatment groups or specimens were treated with RIPA lysis buffer  on ice. The samples were then centrifuged at 4 °C and 12,000×Human laryngeal carcinoma tissues from patients were obtained during clinical surgery. Specimens were fixed with 4% paraformaldehyde and embedded in paraffin. The specimens were cut into five μm-thick sections, deparaffinized, and dehydrated. Antigen retrieval was accomplished by heating the sections in citrate buffer in a microwave oven for 15 min. The sections were then incubated with hydrogen peroxide (3%) for 30 min to destroy endogenous peroxidase activity. Subsequently, the sections were incubated with primary antibody to ACC2  overnight at 4 °C prior to incubation with an anti-rabbit secondary antibody . After being treated first with horseradish peroxidase and then with 3,3′-diaminobenzidine, the sections were counterstained with hematoxylin, dehydrated, cleared, and mounted. For the negative control group, the primary antibody was omitted. Images of stained sections were captured using a light microscope and analyzed with Image Pro Plus 5.1  software.The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was conducted using a TUNEL kit  following the manufacturer’s instructions. Images were captured using fluorescence microscopy and analyzed with Image J software.2 incubator. Then, 10 μL of CCK8 solution was added to each well. After following incubation for 2 h, the absorbance of each well was measured at 450 nm. The data were expressed as OD.The proliferation of FaDu and NP69 cells was tested by CCK8 Cell Counting Kit . The cells were seeded on 96-well plates and transfected with ACC2 siRNA and scrambled siRNA, respectively. Incubated for 48 h in a 37 °C 5% COThe intracellular lipids stain is following the manufacturer’s instructions of Oil Red O staining kit . All specimens were washed with 40% isopropyl alcohol and distilled water. Images were captured using an light microscope and analyzed with Image J software.t-tests were used to compare the results between groups. Values of P < 0.05 were considered statistically significant.SigmaPlot software was used to analyze all data. Data are expressed as means ± SEM. Two-tailed, unpaired Student’s We demonstrated that the p-ACC expression level was significantly higher in laryngocarcinoma tissue than that in the adjacent tissue , which wThe laryngocarcinoma specimens used in immunohistochemical analysis were then analyzed to determine whether an association existed between the level of ACC2 expression and the clinical cancer stage or the degree of cell differentiation. Each patient’s clinical stage was classified as one of three stages  based on the tumor-lymph node-metastasis (TNM) classification system. As shown in Immunohistochemistry was performed to determine the ACC2 expression level in laryngocarcinoma tissue. The patients were divided into two groups, low ACC2 and high ACC2, based on the level of ACC2 expression. A total of 5-year patient survival data were obtained and the Kaplan–Meier method was used to investigate the association of survival time with ACC2 expression. As shown in Because we demonstrated in the present study that ACC2 is highly expressed in laryngocarcinoma and the prognosis for patients with laryngocarcinoma is poor owing to its rapid metastasis and growth, we next explored whether ACC2 affects apoptosis and proliferation in HNSCC. The FaDu cell line was derived from a hypopharyngeal carcinoma, a type of HNSCC. The NP69 cell line was derived from human nasopharyngeal epithelial cells, which was used as control cell line. Western blot analyses results showed that the expression level of ACC2 in FaDu cells was significantly higher than that in NP69 cells  and 3B. In the present study, we explored the role of ACC2 in laryngocarcinoma based on patient cancer stage, degree of cancer cell differentiation, patient survival time, and cell apoptosis. Our major findings were as follows: (1) ACC2 was highly expressed in laryngocarcinoma vs. adjacent normal tissues; (2) tissue from patients with a more advance clinical laryngocarcinoma stage expressed more ACC2 than that from patients in earlier clinical stages. The ACC2 expression level was inversely associated with the degree of differentiation of laryngocarcinoma cells; (3) high ACC expression was associated with decreased 5-year survival after surgery among patients with laryngocarcinoma; (4) ACC2 was highly expressed in FaDu cells compared to NP69 cells. (5) Knockdown of ACC2 in FaDu cells enhanced cell apoptosis but not significantly affected cell proliferation. (6) Knockdown of ACC2 in FaDu cells significantly decreased intracellular lipids levels. Taken together, our results suggest that ACC2 may play an important role in HNSCC apoptosis and indicate that the prognosis of patients with laryngocarcinoma is associated with ACC2 expression level. Therefore, ACC2 may be a potential therapeutic target in the treatment of HNSCC.Laryngocarcinoma is one of the most common malignant neoplasms of the head and neck . In receSeveral studies have shown that the LKB1-AMPK-ACC signaling pathway plays a non-negligible role in the development of tumors, including breast, prostate, ovarian, and hepatocellular carcinomas . AcetyltWe demonstrated that ACC2 expression levels were higher in laryngocarcinoma tissues than in normal tissues, higher in later than in earlier clinical stages of laryngocarcinoma, and inversely associated with the degree of laryngocarcinoma cell differentiation. Higher ACC2 expression was associated with reduced five-survival rates in patients with laryngocarcinoma. Knockdown of ACC2 in FaDu cells enhanced apoptosis and decreased intracellular lipids levels, which suggested that knockdown of ACC2 enhanced FaDu cell apoptosis, possibly by interfering with intracellular lipid synthesis. Thus, we propose that the development of ACC2-based targeted therapy in the treatment of HNSCC is warranted.10.7717/peerj.7037/supp-1Supplemental Information 1The data is showing that ACC expression in laryngocarcinoma by immunohistochemistry and westernblots. The 12-L-C-1 is represented by 12- laryngocarcinoma-cancer-1 while 12-L-N-1 is represented by 12-laryngocarcinoma-normal tissue-1.Click here for additional data file.10.7717/peerj.7037/supp-2Supplemental Information 2The data is showing full-length uncropped blots  and apopClick here for additional data file.10.7717/peerj.7037/supp-3Supplemental Information 3Images showing apoptotic NP69 cells transfected with scrambled siRNA or ACC2 siRNA.Click here for additional data file.10.7717/peerj.7037/supp-4Supplemental Information 4Immunohistochemical images showing p-ACC immunostaining in the normal tissue adjacent to laryngocarcinoma tissue.Click here for additional data file.10.7717/peerj.7037/supp-5Supplemental Information 5Images showing the level of lipids in FaDu cells transfected with scrambled siRNA and ACC2 siRNA.Click here for additional data file.10.7717/peerj.7037/supp-6Supplemental Information 6Western blot images showing p-ACC expression in laryngocarcinoma tissue and the adjacent normal tissue.Click here for additional data file.10.7717/peerj.7037/supp-7Supplemental Information 7Numerical data including immunohistochemistry, westernblot, CCK8, apoptosis rate, and survival data.Click here for additional data file.10.7717/peerj.7037/supp-8Supplemental Information 8Numerical data containing NP69 cell apoptosis results, Oil red lipid stain results, p-ACC expression in HNSCC , and p-ACC expression in HNSCC .Click here for additional data file.10.7717/peerj.7037/supp-9Supplemental Information 9P < 0.05.(A-C) Representative immunohistochemical images (A and B) and summary data (C) showing p-ACC immunostaining in the normal tissue adjacent (A) to laryngocarcinoma tissue (B) . D–E. Representative images (D) and western blot summary data (E) showing p-ACC expression in laryngocarcinoma tissue (LC) and the adjacent normal tissue (AT). Values represent the mean ± SEM (n = 4-18). *Click here for additional data file.10.7717/peerj.7037/supp-10Supplemental Information 10P < 0.05..(A-C) Representative images (A and B) and summary data (C) showing apoptotic NP69 cells transfected with scrambled siRNA or ACC2 siRNA. The change in apoptosis was evaluated using TUNEL staining. Images of TUNEL-positive cells were captured by a fluorescence microscope. Values represent the mean ± SEM (n = 4). *Click here for additional data file.10.7717/peerj.7037/supp-11Supplemental Information 11The data is showing ACC expression levels, effect of ACC siRNA on expression of ACC in FaDu cells and role of ACC in apoptosis of FaDu cells.Click here for additional data file.


We aim to identify the key biomarker of acute rejection (AR) after kidney transplantation via bioinformatics methods. GSE75693 of 30 samples with stable kidney transplantation recipients and 15 AR samples were downloaded and analyzed by the limma package to identify differentially expressed genes (DEGs). Then, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were done to explore the biological functions and potential important pathways of DEGs. Finally, protein-protein interactions (PPIs) and literature mining were applied to construct the cocitation network and to select the hub protein.  The gene expression data P < 0.01 and |log2(fold change)| > 1.0. DEGs of AR are mainly located on membranes and impact the activation of receptors in immune responses. In the PPI network, Src kinase, lymphocyte kinase (LCK), CD3G, B2M, interferon-γ, CD3D, tumor necrosis factor, VAV1, and CD3E in the T cell receptor signaling pathway were selected as important factors, and LCK was identified as the hub protein.  A total of 437 upregulated genes and 353 downregulated genes were selected according to  LCK, via acting on T-cell receptor, might be a potential therapeutic target for AR after kidney transplantation. Kidney transplantation is widely accepted as a standard life-saving therapy for end-stage chronic kidney disease . FrequenAccording to the distinct key players in the pathogenesis, AR is classified into two types, i.e., acute T cell-mediated rejection (TCMR) and acute antibody-mediated rejection (AMR). Although the precise mechanism of TCMR is still unclear, cell-mediated cytotoxicity of parenchymal cells and local cytokine release are two possible causes . By contOnce AR occurs, a short course of intensive immunomodulation is needed. Pulse steroid therapy, alteration of immunosuppressants, monoclonal antibodies and combinations thereof are common therapies for AR . Among oGene expression analysis by bioinformatics methods has been widely used in genomics and biomedical studies, which helps clarify the molecular events underlying human biology and diseases . Data miGSE75693 from the public Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). The dataset GSE75693 consists of the gene expression information of kidney biopsies from 30 stable renal allograft recipients, 15 patients with AR, 15 with BK virus nephropathy, and 12 with chronic allograft nephropathy. All subjects were pediatric and young adult recipients who received transplantation between 2000 and 2011 at the Lucile Packard Children's Hospital of Stanford University [First, we downloaded the microarray dataset http://www.bioconductor.org/), including background correction, normalization, and calculation of gene expressions.The array data were based on the platform of GPL570 Affymetrix Human Gene U133 Plus 2.0 Array . The raw data were preprocessed by Robust Multiarray Average  algorithP values were calculated by the Bayesian t-test and adjusted by false discovery rate. DEGs were selected based on the threshold P < 0.01 and |log2(fold change)| > 1.0 [P value herein was used to test if the gene was differentially expressed between the AR and the stable groups with the |fold change| > 2.0.We used the limma package of Bioconductor to analyze DEGs between BKVN and nonallograft injury patients in R project . Linear )| > 1.0 . The P vhttp://david.abcc.ncifcrf.gov/). The potential GO annotation and pathways were selected based on P < 0.01 and count ≥ 5 [By Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) in DEG enrichment analysis, we further investigated the potential mechanisms of AR. GO annotated genes by a defined, structured, and controlled vocabulary , includiount ≥ 5 .http://string-db.org/), which is the web source of biological database. According to the official explanation of STRING, the confidence score is the approximate probability that a predicted link exists between two proteins in the same metabolic map in the KEGG database (Getting Started in https://string-db.org/cgi/help.pl). Thus, PPIs of DEGs were selected with the threshold of score (high confidence) > 0.7 [http://www.cytoscape.org/). The proteins with higher degrees of interaction were considered as hub proteins [Connections and interaction networks of DEGs mean PPI. We uploaded the DEGs into the website of STRING  > 0.7 . Then thproteins .http://ci.smu.edu.cn/GenCLiP2.0/confirm_keywords.php), which is an online tool for literature mining of gene functions. In the website, biological keywords of hot proteins in previous literature database were analyzed by Gene Cluster with the Literature Profiles module with the threshold of P ≤ 1 × 10−6 and hits ≥ 6 [According to the analysis of STRING, hot proteins in the PPI were put into GenCLiP 2.0 | > 1.0, including 437 upregulated genes and 353 downregulated genes. The hierarchical cluster analysis was done to show the distribution of DEGs  were selected to construct the PPI network by Cytoscape . Top eigmembrane . InformaEach DEG was evaluated according to degree centrality, betweenness centrality, and subgraph centrality, respectively, in CytoNCA . LCK ranIn the present study, we aimed at investigating potential therapeutic targets for AR after kidney transplantation. First, 437 upregulated genes and 353 downregulated genes were selected as DEGs of AR compared with nonrejection recipients. According to gene enrichment, DEGs of AR are mainly located on membranes and participate in the TCR signaling pathway. We identified LCK as the potential key factor in the pathogenesis of AR.+ and CD8+ T cells by activating perforin/granzyme degranulation pathways [By GO annotation in DAVID, we further explored the biological functions of DEGs. The results of functional annotations helped us infer the possible effects of DEGs on how AR occurs. According to results of BP, CC, and MF ontologies, the majority of DEGs were enriched in functions of membrane receptors in the immune process. AR in kidney transplantation is generally considered as an alloimmune response process involving networks of interacting cells throughout the body . Our BP pathways . Regardlpathways . Meanwhipathways  implicatIn the cocitation network, CD3D, CD3E, and CD3G, which are compositions of CD3 complex of TCR, interacted with each other to affect the assembly of TCR membrane complex and disturb T-cell responsiveness , especiaThe strength of our study is the combined use of several bioinformatics methods including DEG analysis, GO, KEGG, literature mining, STRING, and the PPI analysis. These data mining methods may corroborate each other and make the results reliable. However, our study has limitations. The sample size is relatively small. Different from conventional method, however, the limma method is proven effective in microarray analysis , even foIn summary, we revealed the potential important role of LCK in the pathogenesis of AR. LCK interacts with other 27 proteins and is actively involved in TCR pathway to activate the T cells in AR. LCK might be a potential therapeutic target for AR.


Rehabilitation programmes are used to improve hip fracture outcomes. There is little published trial clinical trial or population-based data on the effects of the type or provider of rehabilitation treatments on hip fracture outcomes. We evaluated the associations of rehabilitation interventions with post-operative hip fracture outcomes.Cross-sectional (2013–2015) analysis of data from the English National Hip Fracture Database (NHFD) from all 191 English hospitals treating hip fractures. Of 62,844 NHFD patients, we included 17,708 patients with rehabilitation treatment and 30-day mobility data, and 34,142 patients with rehabilitation treatment and discharge destination data. The intervention was early mobilisation rehabilitation treatments delivered by a physiotherapist  or other clinical staff as identifiable in NHFD. We used ordinal logistic and propensity scoring regression models to adjust for confounding variables including age, sex, pre-fracture mobility, operative delay, and cognitive function and peri-operative risk scores.In both the adjusted multivariate and propensity-weighted analyses, mobilisation on the day or the day following surgery is associated with better mobility function 30 days after discharge. However patients mobilised by a PT did not have better mobility compared to mobilisation by other professionals. Patients who received a PT assessment were not protected from poorer mobility 30 days after discharge, compared with those who did not receive an assessment. The discharge destination outcome is also better in mobilised than unmobilised patients, whether done by a PT or another health professional, and the difference persists, slightly attenuated, after propensity weighting.In addition to the type of health professional initiating mobilisation, data on rehabilitation treatment activity and post-operative gait speed is needed to determine optimum rehabilitation dosage and functional outcome. After adjustment patients mobilised by non-PTs did as well as patients mobilised by PTs, suggesting that PTs’ current roles in very early rehabilitation should be reconsidered, with a view to redeploying them to more specialised later rehabilitation activity.The online version of this article (10.1186/s12891-018-2093-8) contains supplementary material, which is available to authorized users. Hip fractures in later life result in a high morbidity and mortality rate, with an often permanent decline in mobility, independence and quality of life . Over a Although surgery is generally successful, few people recover fully, and there is a significant impact on their quality of life. Most survivors fail to regain former levels of mobility and activity, many become more dependent, and around 10% are unable to return to their previous residence . Many olevaluate, after appropriate case-mix adjustment, the associations of rehabilitation (including physiotherapy) interventions with post-operative hip fracture mobilityevaluate the associations of rehabilitation interventions with postoperative discharge destination and return to the same setting as pre-fracture.A variety of post-operative rehabilitation programmes are used to improve mobility, maximise physical function and prevent or reverse physical deconditioning. A 2009 Cochrane review of inpatient multi-disciplinary rehabilitation found a lack of evidence regarding what components of rehabilitation were essential for mobility recovery . It founTo assess the data quality of relevant NHFD variablesTo assess the appropriateness of current NHFD variables to audit rehabilitation interventions and to make recommendations for improvementThe secondary objectives were:The NHFD is a clinically-led, web-based audit of hip fracture care and secondary prevention in England, Wales, Northern Ireland and the Channel Islands. All 253 eligible hospitals who treat hip fractures are registered with NHFD, and 98% participate by regularly uploading case records in a standard dataset format. All patients aged 60 and over with a hip fracture coded as S72.0, S72.1, S72.2 in ICD-10, including subtrochanteric fractures, are submitted, regardless of the fracture mechanism. The NHFD is approved by the NHS England Health Research Authority Confidentiality Advisory Group to collect patient data without consent under Section 251 exemption, so no ethical approval was required for this study. We carried out the same data quality checks used by NHFD on the 2013–15 data we received .To measure the effects of rehabilitation it would be most useful to have an activity type variable which described the type of rehabilitation delivered by specialist, mainly PT staff and the intensity e.g. number of sessions. As this is not part of the NHFD dataset we were forced to use proxies for rehabilitation activity or exposure. We used “mobilised on day of or day following surgery” as one measure of rehabilitation exposure. It is recognised that not all patients will be suitable for such mobilisation, and this field only seeks to capture how quickly individuals progress in initial physiotherapy. A patient would be described as ‘mobilised’ if they are able to sit or stand out of bed on the day of their return from operation, or on the following day. We used the variable “assessed by PT on day of or day after surgery” as another measure of rehabilitation exposure. Unfortunately these two variables are optional so some data is missing.We used demographic variables known to be associated with hip fracture outcomes, including age, sex, and delay from admission to operation. The Abbreviated Mental Test Score (AMTS), which is assessed pre-operatively, is a simple and robust 10 item screening tool for acute patients. In AMTS poor cognitive function = 0 and good cognitive function = 10. The American Society of Anaesthesiologists (ASA) score is a preoperative risk score based on the presence of co-morbidities at the time of surgery. An ASA score > 2 is associated with increased risk additional to that of classification of operation and duration of surgery.The first outcome variable was 30-day mobility score on a scale from 1 to 5, which counter-intuitively is higher for less mobile patients in NHFD. A new single mobility score has recently (2015) replaced all four measures previously used by the NHFD. In order to replace missing values of the single mobility score in older data, we used the translational matrix shown in Additional file The second outcome variable was discharge destination from acute orthopaedic ward in combination with the location from which the patient was admitted. For the preoperative setting we used “admitted from”. The method for combining the two variables is shown in Additional file Ordinal logistic regression models can be used where the outcome variable is nominal or ordinal i.e. with more than two levels , 18. In 2 tests and likelihood ratio tests). These variables were used to fit the ordinal logistic regression models. We used the predict command in Stata 14 to compute the ordinal-logistic odds ratio. We computed the c-statistic for a predictive score to measure the performance of the model.We tested all the variables of interest in the NHFD dataset to check whether they were statistically associated with discharge destination .scenttest, [t-test between the mobilised and unmobilised scenarios. We carried out balance checks for the propensity score using the Stata add-on package somersd [We defined a propensity score for mobilisation treatment with respect to confounders, using a logistic regression model, then defined weights to estimate the average treatment effect (ATE),  equal toenttest,  which ca somersd .D suggested that our ATE weights had balanced the propensity score. We then used Poisson regression models and the Stata packages as for the mobility outcome.Discharge destination models were fitted as for Mobility. We defined a propensity score for mobilisation treatment with respect to confounders, using a multinomial logistic regression model. For each patient, we defined three propensity scores, one per mobilisation group, as the fitted probability of the patient being in the mobilisation group. We then defined weights for estimation of the ATE . TherefoPatient numbers are shown in the flowchart in Additional file Most of the baseline characteristics of the complete cases in the Mobility model population after data translation are shown in Table 2 tests . The Somers’ To further investigate the associations with who performs early mobilisation we then carried out a propensity-adjusted analysis comparing 30-day mobility score between mobilised  and unmobilised patients i.e. using three categories to separate the effects of PTs and other staff Fig. . The resAdditional file In summary, we investigated the associations of early mobilisation, as a proxy for rehabilitation, mainly delivered by PTs, with two outcome variables, both before and after adjustment for confounding variables. In both in the 30 day mobility and discharge destination outcome models, we confirmed that patients mobilised early had a better outcome than those not mobilised early, whether they were mobilised by PTs or by non-PTs. The unadjusted analyses suggest that patients mobilised by non-PTs were less immobile at 30 days, but after adjustment, patients mobilised by non-PTs did as well as patients mobilised by PTs. There are a number of possible explanations for this: there may be no actual difference, PTs could mobilise more vulnerable patients with a worse prognosis, or non-PTs such as nurses may deliver more rehabilitation sessions. However we also showed that 57% of patients are currently being mobilised early by a PT and only 3% by “Others”, presumably nurses. If our findings are confirmed, PTs’ roles in early rehabilitation should be reconsidered, with a view to redeploying them to more effective rehabilitation activity.There are few published cohort studies which have analysed rehabilitation exposures and outcomes, and in most instances they only measured effects of transfers to rehabilitation facilities. For example, in an Austrian cohort of 281 patients,  patientsWe used two methods and two outcomes and obtained similar results from both, suggesting that our findings are robust. However, both the models could be applied to only the sub-population of patients with complete exposure and outcome data.Unmeasured confounding is another limitation. Although we adjusted for five confounders, it is possible that other patient factors influenced outcomes. For example, a US study was assessed the relationship between self-reported disease burden and functional improvement during and after inpatient rehabilitation . CompareThe biggest limitation in our analysis is that we were forced to use binary variables for early mobilisation or PT assessment as a proxy for rehabilitation, because there is inadequate data about the process of rehabilitation in the NHFD. There is likely to be great variation in the amount of early mobilisation rehabilitation, and there is little published data on the dose-response relationship. The hip fracture guideline from the UK’s National Institute for Health & Care Excellence found only one, small randomised controlled trial with 60 patients of early versus delayed mobilisation, and three randomised studies with a total of 288 patients using three different types of PT programmes, which were not comparable and so could not be meta-analysed . The AmeIn the real world the effectiveness of interventions which were efficacious in clinical trials may be sub-optimal. Given appropriate measurement, observational research using large databases such as our study can provide valuable evidence of clinical effectiveness. We found that early mobilisation is associated with better mobility function. NHFD data collection could be improved to capture dosage of rehabilitation activity by PTs and other healthcare professionals. This should not be done by manual data entry into NHFD, but by linkage of electronic data from hospital and community-based information systems. Unfortunately there is no national dataset for paramedical activity delivered to inpatients, which would allow this data to be linked with NHFD. NHFD should ask NHS Digital to produce such a dataset. In the interim, adding two variables in NHFD to capture a meaningful numerical rehabilitation outcome such as gait speed, and rehabilitation activity such as number of sessions, should be considered. The latter would help to determine the dose-response relationship of rehabilitation.Future research and audit should aim to confirm our finding that, after adjustment, the results of early mobilisation by nurses seems to be similar to that achieved by PTs, although this could be a result of PTs treating patients with greater morbidity or frailty.Additional file 1:Table S1. Mobility variables translation matrix. Table S2. construction of admission/discharge destination outcome variable. Figure S1. Flowchart of mobility and discharge destination models. Table S3 all baseline characteristics of mobility outcome population (both complete and incomplete cases) before data translation. Table S4. Frequency of mobility categories before and after data translation. Table S5. all baseline characteristics of Mobility Model population after data translation, complete cases only. Table S6. Strength of association between patient characteristics used in the Mobility models and mobility 30 days after discharge. Figure S2 unadjusted and adjusted difference in average treatment effect (ATE) of no mobilisation (=no) or mobilisation (=yes) on day/day after surgery for 30-day mobility outcome. Table S7 baseline characteristics of the discharge destination model. Figure S3 chart of confounder categories vs Somers’ D score for two category mobility outcome. Figure S4. Somers’ D of propensity score and component covariates with respect to: mobilisation = No. Figure S5. Somers’ D of propensity score and component covariates with respect to: mobilisation==No. (DOCX 1030 kb)