Quantification of mixtures of analogues of illicit substances by benchtop NMR spectroscopy.

This paper investigates the possibility of using benchtop NMR spectroscopy for quantification of illicit drugs (methamphetamine) in binary and ternary mixtures with impurities and cutting agents (N-isopropylbenzylamine, phenethylamine and dimethylsulfone). To avoid handling regulated substances, methamphetamine in our experiments is substituted with amino-2-propanol, which has similar functional groups and chemical structure to methamphetamine and hence a related NMR spectrum. Binary and ternary mixtures at concentrations from 30 mmol/L up to 500 mmol/L for each of these species were measured using a 60 MHz benchtop spectrometer. The spectra were analysed using both integration and a model-based algorithm that relies on a full quantum mechanical description of the studied spin systems. Both techniques were able to quantify the composition of the mixtures. The root mean squared error in the measured concentration using the model-based algorithm was < 10 mmol/L, whereas the error using integration was typically > 20 mmol/L. Thus, we conclude benchtop NMR is viable for quantitative measurements of mixtures of illicit substances, particularly when coupled with a quantum mechanical model for the analysis.

Simultaneous analysis of 29 synthetic cannabinoids and metabolites, amphetamines, and cannabinoids in human whole blood by liquid chromatography-tandem mass spectrometry - A New Zealand perspective of use in 2018.

We describe the validation of a method for the simultaneous analysis of 29 synthetic cannabinoids (SCs) and metabolites, 4 amphetamines, and 2 cannabinoids in human whole blood. This method enables one analysis to cover what previously required multiple analyses for these classic and novel drugs-of-abuse with diverse physicochemical properties. The scope of targeted analytes was based on the most prevalent drugs-of-abuse and SCs encountered at the New Zealand border in 2017 and included parent compounds and metabolites belonging to the indole and indazole carboxamide, quinolinyl indole carboxylate, and naphthoylindole classifications. Samples were prepared by supported-liquid-extraction (SLE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis with positive electrospray ionization (ESI). The method was validated with respect to selectivity, matrix effects, process efficiency, sensitivity, repeatability, extract stability, and carryover for qualitative confirmation. Linearity as well as accuracy and precision data at target decision concentrations were also evaluated. The limits of detection and confirmation ranged from 0.1 to 6.0 ng/mL and 1.0 to 6.0 ng/mL, respectively. The described method was successfully applied to the analysis of 564 ante- and post-mortem blood samples in 2018. There were 132 cases (23%) with positive findings of at least one SC, with the five most commonly detected SCs being AMB-FUBINACA and/or acid (61%), 5F-ADB and/or acid (40%), ADB-FUBINACA (11%), 5F-MDMB-PICA acid (6%), and MDMB-FUBINACA acid (6%). The results also demonstrate the predominant presence of metabolites at higher levels than the unchanged parent SCs in blood, highlighting the need to maintain forensic screening methods capable of the simultaneous detection of both parent compounds and metabolites.

The Zygotic Transition Is Initiated in Unicellular Plant Zygotes with Asymmetric Activation of Parental Genomes.

The zygotic transition, from a fertilized egg to an embryo, is central to animal and plant reproduction. Animal embryos depend upon maternally provided factors until zygotic genome activation (ZGA). In plants, the timing and parental genome contributions to ZGA are unresolved. Here, we use the flowering plant Oryza sativa (rice) to characterize transcriptomes of time-staged isogenic and hybrid zygotes following fertilization. Large-scale transcriptomic changes were observed in unicellular zygotes, including upregulation of S-phase genes, a characteristic of ZGA. The parental contributions to ZGA were highly asymmetric. Zygotic transcription was primarily from the maternal genome and included genes for basic cellular processes. Transcription of the paternal genome was highly restricted but unexpectedly included genes encoding putative pluripotency factors expressed at the onset of ZGA. Thus, distinct transcriptional activities are exhibited by the parental genomes during the initiation of embryogenesis, which presumptively derive from divergent pre-zygotic transcriptional states established in the gametes.

Transcriptomes of isolated Oryza sativa gametes characterized by deep sequencing: evidence for distinct sex-dependent chromatin and epigenetic states before fertilization.

The formation of a zygote by the fusion of egg and sperm involves the two gametic transcriptomes. In flowering plants, the embryo sac embedded within the ovule contains the egg cell, whereas the pollen grain contains two sperm cells inside a supporting vegetative cell. The difficulties of collecting isolated gametes and consequent low recovery of RNA have restricted in-depth analysis of gametic transcriptomes in flowering plants. We isolated living egg cells, sperm cells and pollen vegetative cells from Oryza sativa (rice), and identified transcripts for approximately 36 000 genes by deep sequencing. The three transcriptomes are highly divergent, with about three-quarters of those genes differentially expressed in the different cell types. Distinctive expression profiles were observed for genes involved in chromatin conformation, including an unexpected expression in the sperm cell of genes associated with active chromatin. Furthermore, both the sperm cell and the pollen vegetative cell were deficient in expression of key RNAi components. Differences in gene expression were also observed for genes for hormonal signaling and cell cycle regulation. The egg cell and sperm cell transcriptomes reveal major differences in gene expression to be resolved in the zygote, including pathways affecting chromatin configuration, hormones and cell cycle. The sex-specific differences in the expression of RNAi components suggest that epigenetic silencing in the zygote might act predominantly through female-dependent pathways. More generally, this study provides a detailed gene expression landscape for flowering plant gametes, enabling the identification of specific gametic functions, and their contributions to zygote and seed development.

Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

Connexin43 gap junction protein is expressed in astrocytes and the vascular endothelium in the central nervous system. It is upregulated following central nervous system injury and is recognized as playing an important role in modulating the extent of damage. Studies that have transiently blocked connexin43 in spinal cord injury and central nervous system epileptic models have reported neuronal rescue. The purpose of this study was to investigate neuronal rescue following retinal ischaemia-reperfusion by transiently blocking connexin43 activity using a connexin43 mimetic peptide. A further aim was to evaluate the effect of transiently blocking connexin43 on vascular permeability as this is known to increase following central nervous system ischaemia. Adult male Wistar rats were exposed to 60 min of retinal ischaemia. Treatment groups consisted of no treatment, connexin43 mimetic peptide and scrambled peptide. Retinas were then evaluated at 1-2, 4, 8 and 24 h, and 7 and 21 days post-ischaemia. Evans blue dye leak from retinal blood vessels was used to assess vascular leakage. Blood vessel integrity was examined using isolectin-B4 labelling. Connexin43 levels and astrocyte activation (glial fibrillary acidic protein) were assessed using immunohistochemistry and western blot analysis. Retinal whole mounts and retinal ganglion cell counts were used to quantify neurodegeneration. An in vitro cell culture model of endothelial cell ischaemia was used to assess the effect of connexin43 mimetic peptide on endothelial cell survival and connexin43 hemichannel opening using propidium iodide dye uptake. We found that retinal ischaemia-reperfusion induced significant vascular leakage and disruption at 1-2, 4 and 24 h following injury with a peak at 4 h. Connexin43 immunoreactivity was significantly increased at 1-2, 4, 8 and 24 h post ischaemia-reperfusion injury co-localizing with activated astrocytes, Muller cells and vascular endothelial cells. Connexin43 mimetic peptide significantly reduced dye leak at 4 and 24 h. In vitro studies on endothelial cells demonstrate that endothelial cell death following hypoxia can be mediated directly by opening of connexin43 hemichannels in endothelial cells. Blocking connexin43 mediated vascular leakage using a connexin43 mimetic peptide led to increased retinal ganglion cell survival at 7 and 21 days to levels of uninjured retinas. Treatment with scrambled peptide did not result in retinal ganglion cell rescue. Pharmacological targeting of connexin43 gap junction protein by transiently blocking gap junction hemichannels following injury provides new opportunities for treatment of central nervous system ischaemia.

High pressure-induced retinal ischaemia reperfusion causes upregulation of gap junction protein connexin43 prior to retinal ganglion cell loss.

We aimed to characterise the spatial and temporal expression of connexin43 (Cx43) following retinal ischaemia-reperfusion injury and to evaluate its relationship to retinal glial response and subsequent retinal ganglion cell loss. Unilateral retinal ischaemia-reperfusion injury was induced by elevating intraocular pressure to 120mmHg for 60 min and then normalized in Wistar rats. Retinas (n=110) were evaluated at 4, 8, and 24h, and 7, 14, and 21 days in 4 groups: ischaemic, contralateral, sham operated, and uninjured eyes. Immunohistochemistry was used to analyse the spatial and cell-specific expression of Cx43 protein, glial fibrillary acidic protein (astrocytes), glutamine synthetase (Muller cells), Isolectin B4 (vascular endothelium), DAPI (nuclear marker), and BRN3a (retinal ganglion cells). Retinal whole mounts were used to count retinal ganglion cells. Our results show that Cx43 immunoreactivity of the ischaemic eye is significantly increased in the ganglion cell layer and nerve fibre layer, colocalizing with activated retinal astrocytes and Muller cells at 8h. In the inner retinal layers Cx43 was also upregulated and colocalized with retinal vascular endothelium at 4, 8 and 24h post ischaemia. Notably, in the contralateral eye, Cx43 immunoreactivity was also significantly increased in the ganglion cell layer and nerve fibre layer at 8 and 24h, and at 4h in the inner layers. Sham operated controls did not show any change in Cx43 immunoreactivity. Subsequently a significant retinal ganglion cell loss was observed in the ischaemic eye at day 21 with a trend towards retinal ganglion cell loss in the contralateral eye. In conclusion, upregulation of Cx43 occurs in both the ischaemic and contralateral retinas although far more significantly in injured retinas. Cx43 colocalizes primarily with activated retinal astrocytes and Muller cells as well as vascular endothelium, suggesting that gap junction communication and/or hemichannel activity may be a mediator of inflammation, vascular permeability, and subsequently neuronal death.

Response of retinal Connexin43 to optic nerve injury.

PURPOSE: To characterize the spatial and temporal expression of Connexin43 (Cx43) after partial optic nerve transection and evaluate its relationship to retinal ganglion cell (RGC) loss and retinal glial response. METHODS: Partial, unilateral, superior optic nerve transection was performed in 150 Wistar rats. The retinas were evaluated at 8 and 24 hours and 3, 7, 14, 28, and 56 days after injury. Immunohistochemical analysis identified changes in several markers including Cx43 immunoreactivity (ir), RGC counts (Brn3a), and retinal astrocytes (GFAP). RESULTS: After injury, superior retinal Cx43-ir peaked at 3 days (192.1% of control; P = 0.0002) and 28 days (212.1% of control; P < 0.0001) and troughed at 14 days (73.8% of control; P = 0.0028) and 56 days (72.5% of control; P = 0.0232). Inferior retinal Cx43-ir was elevated at only 28 days (127.4% increase; P = 0.0481). Superior RGC loss began at 3 days (84.0% of control; P = 0.0454) and continued to decline by 56 days (18.8% of control; P < 0.0001). Inferior RGC loss began at 28 days (73.4% of control; P = 0.0021). An increase in GFAP-ir occurred in the superior retina from day 3 (153.7% of control; P = 0.0017) and from day 28 (186.7% of control; P = 0.0013) in the inferior retina, persisting in both the superior and inferior retina to 56 days (P = 0.0027). CONCLUSIONS: A biphasic upregulation of retinal Cx43 protein occurs in the superior retina with peaks at 3 and 28 days after injury, but at only 28 days in the inferior retina. There is an associated loss of RGCs and a retinal astrocytic inflammatory response.

Gap junction protein connexin43 (GJA1) in the human glaucomatous optic nerve head and retina.

Primary open angle glaucoma is characterised by the progressive and irreversible death of retinal ganglion cells. Experimental evidence suggests that the initial site of injury to the retinal ganglion cell is at or near the lamina cribrosa or in the peripapillary retina. However, the mediators of axonal injury remain poorly understood. The purpose of this study was to investigate the expression of the gap junction protein connexin43 (GJA1) in the human glaucomatous optic nerve head and retina as a potential mediator of axonal injury. Using affinity isolated polyclonal antibodies to the C-terminal segment of human connexin43, the expression of connexin43 was determined in post-mortem human eyes with primary open angle glaucoma and age-matched controls. In normal eyes, connexin43 was present on glial fibrillary acidic protein (GFAP)-positive astrocytes in the retinal ganglion cell layer and optic nerve head. In glaucomatous eyes, increased connexin43 immunoreactivity was observed at the level of the lamina cribrosa and in the peripapillary and mid-peripheral retina in association with glial activation. This novel finding may suggest that gap junction communication is a potential mediator of retinal ganglion cell injury in glaucoma.

Role of connexin43 in central nervous system injury.

Gap junctions are specialized cell-to-cell contacts that provide direct intercellular communication. In the central nervous system (CNS), gap junction coupling occurs between both neurons and glial cells. One of the most abundant gap junction proteins in the CNS is connexin43 (Cx43). The functional syncytium formed by astrocytes via Cx43 gap junction intercellular communication has, for example, been implicated in maintaining the homeostasis of the extracellular milieu of neurons. In particular, astrocytes are involved in the spatial buffering of many ions, signalling molecules and energy sources. In this review, the role of Cx43 following CNS injury is examined by combining evidence surrounding the response of Cx43 to CNS injury and the effects of Cx43 gap junction blockade on neuronal survival in various models of injury. Combined evidence suggests that transient blockade targeting the window of initial Cx43 upregulation observed following injury is potentially therapeutic.

Immunolocalization of gap junction protein connexin43 (GJA1) in the human retina and optic nerve.

PURPOSE: Gap junctions are intercellular channels that have been implicated in the pathogenesis of neuronal death after central nervous system injury. This study determines the expression pattern of gap junction protein connexin43 in the human retina and optic nerve. METHODS: An affinity-isolated polyclonal antibody to the C-terminal segment of the cytoplasmic domain of human connexin43 was used to determine connexin43 localization. Postmortem human eyes were examined by immunohistochemical staining of frozen sections using antibodies to connexin43. Antibody binding was detected using confocal microscopy and fluorochrome-conjugated secondary antibodies. Double-label immunohistochemistry identified the cell types expressing connexin43. RESULTS: Connexin43 immunoreactivity was detected in the human retina on glial fibrillary acidic protein (GFAP)-positive astrocytes in the retinal ganglion cell layer and, to a lesser extent, on the processes of glutamine synthetase-labeled Müller cells. The retinal and choroidal circulations showed strong connexin43 immunolabeling. Dense connexin43 immunoreactivity was present between adjacent cells of the retinal pigment epithelium, and there was diffuse connexin43 immunoreactivity on GFAP-positive astrocytes in the optic nerve. CONCLUSIONS: In the human retina and optic nerve, connexin43 is present on glia, blood vessels, and epithelial cells. An understanding of the distribution of connexin43 in the normal retina and optic nerve may be used to evaluate changes associated with retinal and optic nerve disease.

Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis.

The plant life cycle involves an alternation of generations between sporophyte and gametophyte. Currently, the genes and pathways involved in gametophytic development and function in flowering plants remain largely unknown. A large-scale mutant screen of Ds transposon insertion lines was employed to identify 130 mutants of Arabidopsis thaliana with defects in female gametophyte development and function. A wide variety of mutant phenotypes were observed, ranging from defects in different stages of early embryo sac development to mutants with apparently normal embryo sacs, but exhibiting defects in processes such as pollen tube guidance, fertilization or early embryo development. Unexpectedly, nearly half of the mutants isolated in this study were found to be primarily defective in post-fertilization processes dependent on the maternal allele, suggesting that genes expressed from the female gametophyte or the maternal genome play a major role in the early development of plant embryos. Sequence identification of the genes disrupted in the mutants revealed genes involved in protein degradation, cell death, signal transduction and transcriptional regulation required for embryo sac development, fertilization and early embryogenesis. These results provide a first comprehensive overview of the genes and gene products involved in female gametophyte development and function within a flowering plant.

TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor.

Mutants of a new gene, TRANSPARENT TESTA GLABRA2 (TTG2), show disruptions to trichome development and to tannin and mucilage production in the seed coat. The gene was tagged by the endogenous transposon Tag1 and shown to encode a WRKY transcription factor. It is the first member of this large, plant-specific family known to control morphogenesis. The functions of all other WRKY genes revealed to date involve responses to pathogen attack, mechanical stress, and senescence. TTG2 is strongly expressed in trichomes throughout their development, in the endothelium of developing seeds (in which tannin is later generated) and subsequently in other layers of the seed coat, and in the atrichoblasts of developing roots. TTG2 acts downstream of the trichome initiation genes TTG1 and GLABROUS1, although trichome expression of TTG2 continues to occur if they are inactivated. Later, TTG2 shares functions with GLABRA2 in controlling trichome outgrowth. In the seed coat, TTG2 expression requires TTG1 function in the production of tannin. Finally, TTG2 also may be involved in specifying atrichoblasts in roots redundantly with other gene(s) but independently of TTG1 and GLABRA2.