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Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS). They commonly comprise N -alkylated indole, indazole, or 7-azaindole scaffolds with amide-linked pendant amino acid groups. To explore the contribution of the amino acid side chain to the cannabinoid pharmacology of SCRA NPS, a systematic library of side chain-modified SCRAs was prepared based on the recent detections of amino acid derivatives 17 (5F-AB-PINACA), 18 (5F-ADB-PINACA), 15 (PX-1), 19 (PX-2), and 20 (NNL-1). In vitro binding affinities and functional activities at cannabinoid type 1 and 2 receptors (CB 1 and CB 2 , respectively) were determined for all the library members using radioligand competition experiments and a fluorescence-based membrane potential assay. Binding affinities and functional activities varied widely across compounds ( K i = 0.32 to >10 000 nM, EC 50 = 0.24-1259 nM), with several clear structure-activity relationships (SARs) emerging. Affinity and potency at CB 1 changed as a function of the heterocyclic core (indazole > indole > 7-azaindole) and the pendant amino acid side chain ( tert -butyl > iso -propyl > iso -butyl > benzyl > ethyl > methyl > hydrogen). Ensemble docking at CB 1 revealed a clear steric basis for observed SAR trends. Interestingly, although 15 (PX-1) and 19 (PX-2) have been detected in recreational drug markets, they failed to induce centrally CB 1 -mediated effects (e.g., hypothermia) in mice using radiobiotelemetry. Together, these data provide insights regarding structural contributions to the cannabimimetic profiles of 17 (5F-AB-PINACA), 18 (5F-ADB-PINACA), 15 (PX-1), 19 (PX-2), 20 (NNL-1), and other SCRA NPS.

Synthetic cannabinoid receptor agonists (SCRAs) remain one the most prevalent classes of new psychoactive substances (NPS) worldwide, and examples are generally poorly characterised at the time of first detection. We have synthesised a systematic library of amino acid-derived indole-, indazole-, and 7-azaindole-3-carboxamides related to recently detected drugs ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA, and characterised these ligands for in vitro binding and agonist activity at cannabinoid receptor subtypes 1 and 2 (CB 1 and CB 2 ), and in vivo cannabimimetic activity. All compounds showed high affinity for CB 1 ( K i 0.299-538 nM) and most at CB 2 ( K i = 0.912-2190 nM), and most functioned as high efficacy agonists of CB 1 and CB 2 in a fluorescence-based membrane potential assay and a βarr2 recruitment assay (NanoBiT®), with some compounds being partial agonists in the NanoBiT® assay. Key structure-activity relationships (SARs) were identified for CB 1 /CB 2 binding and CB 1 /CB 2 functional activities; (1) for a given core, affinities and potencies for tert -leucinamides (ADB-) > valinamides (AB-) ≫ phenylalaninamides (APP-); (2) for a given amino acid side-chain, affinities and potencies for indazoles > indoles ≫ 7-azaindoles. Radiobiotelemetric evaluation of ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA in mice demonstrated that ADB-BUTINACA and ADB-P7AICA were cannabimimetic at 0.1 mg kg -1 and 10 mg kg -1 doses, respectively, as measured by pronounced decreases in core body temperature. APP-BUTINACA failed to elicit any hypothermic response up to the maximally tested 10 mg kg -1 dose, yielding an in vivo potency ranking of ADB-BUTINACA > ADB-P7AICA > APP-BUTINACA., Competing Interests: None., (This journal is © The Royal Society of Chemistry.)

Seasonal dynamics in the vertical distribution of leaf area index (LAI) may impact the seasonality of forest productivity in Amazonian forests. However, until recently, fine-scale observations critical to revealing ecological mechanisms underlying these changes have been lacking. To investigate fine-scale variation in leaf area with seasonality and drought we conducted monthly ground-based LiDAR surveys over 4 yr at an Amazon forest site. We analysed temporal changes in vertically structured LAI along axes of both canopy height and light environments. Upper canopy LAI increased during the dry season, whereas lower canopy LAI decreased. The low canopy decrease was driven by highly illuminated leaves of smaller trees in gaps. By contrast, understory LAI increased concurrently with the upper canopy. Hence, tree phenological strategies were stratified by height and light environments. Trends were amplified during a 2015-2016 severe El Niño drought. Leaf area low in the canopy exhibited behaviour consistent with water limitation. Leaf loss from short trees in high light during drought may be associated with strategies to tolerate limited access to deep soil water and stressful leaf environments. Vertically and environmentally structured phenological processes suggest a critical role of canopy structural heterogeneity in seasonal changes in Amazon ecosystem function., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Objective: To analyze the clinical and molecular features of a child with carnitine palmitoyltransferase 1A (CPT1A) deficiency., Methods: Clinical data of the child was collected. Blood acylcarnitine was determined with tandem mass spectrometry. DNA was extracted from the child and his parents. All exons and flanking regions of the CPT1A gene were analyzed by PCR and Sanger sequencing., Results: Analysis showed that the patient carried compound heterozygous mutations c.1787T>C and c.2201T>C of the CPT1A gene, which derived his father and mother, respectively. Both mutations were verified as novel through the retrieval of dbSNP, HGMD and 1000 genome databases. Bioinformatic analysis suggested that the mutations can affect protein function., Conclusion: Acyl carnitine analysis has been the main method for the diagnosis of CPT1A deficiency. The c.1787T>C and c.2201T>C mutations of the CPT1A gene probably underlie the disease in this patient. Gene testing can provide important clues for genetic counseling and prenatal diagnosis.

Objective: To analyze the clinical features and gene mutations in an adolescent patient affected with late-onset multiple aeyl-CoA dehydrogenase deficiency (MADD) with severe fatty liver., Methods: Potential mutations of the ETFDH gene were detected with polymerase chain reaction (PCR) and DNA sequencing., Results: The 13-year-and-10-month girl has presented with weakness without any other special manifestation. Laboratory tests demonstrated an elevation of myocardial enzyme levels, total cholesterol, lactic acid and abnormal serum free fatty acids. H magnetic resonance spectroscopy revealed severe fatty liver. An increase in multiple plasma acyl-carnitines was detected by gas chromatography/mass spectrometry and isobutyrylglycine in urine by screening with tandem mass spectrometry. Genetic analysis demonstrated 2 heterozygous missense mutations c.250G>A (p.Ala84Thr) and c.353G>T (p.Cys118Phe) in the ETFDH gene. The diagnosis of MADD was confirmed. The patient was given large dose of vitamin B2, which resulted in rapid clinical and biochemical improvement., Conclusion: A common mutation c.250G>A and a novel mutation c.353G>T in the ETFDH gene were identified in the patient. The pathogenic role of c.353G>T (p.Cys118Phe) deserves further study. Early diagnosis of MADD and appropriate therapy is crucial for the prognosis.

Objective: To analyze PCCA and PCCB gene mutations in 10 Chinese patients with propionic acidemia(PA)., Methods: Genomic DNA was extracted from peripheral blood leukocytes. The 39 exons and flanking sequences of the PCCA and PCCB genes were amplified with polymerase chain reaction and subjected to direct DNA sequencing., Results: DNA sequencing has revealed that 7 patients have carried a PCCA gene mutation, 2 patients carried PCCB gene mutation and 1 patient carried mutations in both PCCA and PCCB genes. Ten PA mutations were confirmed, including 8 affecting the PCCA gene and 2 affecting the PCCB gene. Three PCCA mutations c.245G>A, IVS15+5del5, c.1288C>T and 2 PCCB mutations c.838insC, c.1087T>C were found for the first time., Conclusion: Among Chinese patients with propionic acidemia patients, their genetic mutations are mainly found on the PCCA gene.

Objective: To identify the types of OTC gene mutations in three male patients with late onset ornithine transcarbamylase deficiency (OTCD, MIM #311250)., Methods: Genomic DNA was extracted from peripheral blood leukocytes. The 10 exons and their flanking sequences of the OTC gene were amplified with polymerase chain reaction and subjected to direct DNA sequencing., Results: Based on DNA sequence analysis, all of the three patients have carried OTC gene mutations. Patients 1 and 2 were both hemizygous for mutation c.586G> A(p.D196N). A novel mutation c.800G> C(p.S267T) were confirmed in patient 3., Conclusion: p.S267T mutation has affected the conserved amino acid motif of the OTC protein, and is therefore a pathogenic mutation.

Objective: To detect potential mutations of Y9ASS1, ASL and SLC25A13 genes in four patients manifesting citrullinemia., Methods: Genomic DNA was extracted from peripheral blood leukocytes. Exons and their flanking sequences of the three genes were amplified with polymerase chain reaction and subjected to direct DNA sequencing., Results: Based on DNA sequence analysis, one case was diagnosed with argininosuccinate synthetase deficiency, and the mutation type (ASS1 gene) was c.236C>T (p.S79F) + c.431C>G (p.P144R). Two cases were diagnosed with argininosuccinic aciduria (ASL gene), and their gene mutations were c.434A>G (p.D145G) + c.1366C>T (p.R456W) and c.331C>T (p.R111W) + IVS8+2insT, respectively. A thirteen months boy who carried a heterozygous 851del4 mutation (SLC25A13 gene) was diagnosed with citrullinemia adult-onset type II., Conclusion: Through analysis of relevant pathogenic genes, four patients have been diagnosed.

CD9, a member of the tetraspanin family, plays important roles in a variety of cell activities. Fibrosarcoma is a malignant tumor that arises from fibroblasts. Low CD9 expression is found in fibrosarcoma tumor, but function of CD9 in fibrosarcoma has been rarely studied. In this study, stable cell lines for CD9 overexpression and vector were generated in HT1080, a human fibroscarcoma cell line, and cellular functions were widely investigated. In CD9-HT1080 cells, CD9 mainly localized in the membrane and co-localized with F-actin in the filopodia of cell surface. In functional assays, we demonstrated that CD9 could up-regulate total and active caspase-3 expression and induce cell apoptosis, but cell proliferation remained unchanged. CD9 overexpression inhibited HT1080 cell adhesion to FN but promoted cell spreading on FN. We also observed CD9 reduced cell migration using FN a chemoattractant and inhibited cell colony formation in soft agar medium. To explore the biochemical mechanism for functional changes, we investigated the effects of CD9 overexpression on cellular pathways and protein association. CD9 overexpression induced Akt phosphorylation on FN but did not change total Akt expression. Phosphorylation of p38 but not ERK was increased by CD9 overexpression, total p38 and ERK were not affected. CD9 overexpression did not affect the expression of TGFα, EGFR, β1, and EWI-2, but EWI-F expression was up-regulated. Moreover, CD9 could associate with TGFα, EGFR, β1, EWI-2, and EWI-F in HT1080 cell line. Take together, CD9 overexpression had promoting effects on cell apoptosis and cell spreading, but had inhibitory effects on cell adhesion, migration, and cell colony formation. These effects might be ascribed to CD9 associations with EWI-2/EWI-F/β1 complex and EGFR pathway, and the activation of Akt and p38 signalings as well.

The ortho palladation of prochiral (di-tert-butyl)(diphenylmethyl)phosphine proceeded readily to give rise to the dimeric complex, di-mu-chlorobis{[(phenyl)(di-tert-butylphosphino)methyl]phenyl-C2, P}dipalladium(II). The (S,S)-(+)-dimer was subsequently obtained by optical resolution with sodium (S)-prolinate. The absolute configuration of the optically resolved (+)-dimer was concluded from the X-ray diffraction studies of the derivatized O,O-acetylacetonate complex. The availability of the (+)-dimer is crucial to the study of the properties of the Pd-C bond. The phosphapalladacycle Pd-C bond exhibited a remarkable thermodynamic stability. It could not be permanently ruptured to give rise to the eta1-P monodentate even in a refluxing acetone solution containing concentrated hydrochloric acid. Instead, the phosphine was noted to fluctuate between the ring closed and opened states via the reversible Pd-C bond cleavage/formation under this condition. Inevitably, this resulted in the racemization of the five-membered organopalladium ring structure. In contrast, such bond cleavage was not observed at room temperature in the absence of HCl. In fact, the phosphine was observed to readily ortho palladate even under conditions not favorable to cyclopalladation. Indeed, the difficulty of isolating the phosphine as a simple eta1-P monodentate coordination complex was further noted by its lack of reactivity toward the N,N-dimethyl-1-(1'naphthyl)ethylaminate palladacycle mu-chloro dimer. Only by enhancing the Lewis acidity of the palladacycle in the form of the positively charged bis(acetonitrile) complex could the phosphine be encouraged to participate in monodentate eta1-P bonding. Even then, this form of coordination was weak and was only observed by NMR spectroscopy.