The Direct Actions of GABA, 2’-Methoxy-6-Methylflavone and General Anaesthetics at β3γ2L GABAA Receptors: Evidence for Receptors with Different Subunit Stoichiometries

2’-Methoxy-6-methylflavone (2’MeO6MF) is an anxiolytic flavonoid which has been shown to display GABA_A receptor (GABA_AR) β2/3-subunit selectivity, a pharmacological profile similar to that of the general anaesthetic etomidate. Electrophysiological studies suggest that the full agonist action of 2’MeO6MF at α2β3γ2L GABA_ARs may mediate the flavonoid’s in vivo effects. However, we found variations in the relative efficacy of 2’MeO6MF (2’MeO6MF-elicited current responses normalised to the maximal GABA response) at α2β3γ2L GABA_ARs due to the presence of mixed receptor populations. To understand which receptor subpopulation(s) underlie the variations observed, we conducted a systematic investigation of 2’MeO6MF activity at all receptor combinations that could theoretically form (α2, β3, γ2L, α2β3, α2γ2L, β3γ2L and α2β3γ2L) in Xenopus oocytes using the two-electrode voltage clamp technique. We found that 2’MeO6MF activated non-α-containing β3γ2L receptors. In an attempt to establish the optimal conditions to express a uniform population of these receptors, we found that varying the relative amounts of β3:γ2L subunit mRNAs resulted in differences in the level of constitutive activity, the GABA concentration-response relationships, and the relative efficacy of 2’MeO6MF activation. Like 2’MeO6MF, general anaesthetics such as etomidate and propofol also showed distinct levels of relative efficacy across different injection ratios. Based on these results, we infer that β3γ2L receptors may form with different subunit stoichiometries, resulting in the complex pharmacology observed across different injection ratios. Moreover, the discovery that GABA and etomidate have direct actions at the α-lacking β3γ2L receptors raises questions about the structural requirements for their respective binding sites at GABA_ARs.     

Anaplastic thyroid cancer: outcomes of trimodal therapy

BackroundThe purpose of this study is to assess the impact of trimodal therapy [surgery, chemotherapy and external beam radiotherapy (EBRT)] in patients with anaplastic thyroid cancer (ATC) treated with curative intent.Materials and methodsRetrospective review of patients with ATC treated at a tertiary referral centre between January 2009 and June 2020. Data were collected regarding demographics, histology, staging, treatment and outcomes.ResultsSeven patients (4 female) were identified. Median age was 58 years (range 52–83 years). All patients received EBRT with concurrent doxorubicin. Six patients received surgery followed by chemoradiotherapy (CRT), and one underwent neoadjuvant CRT followed by surgery. Median radiological tumour size was 50mm (range 40–90 mm). Six patients had gross extrathyroidal extension and three had N1b disease. Prescribed radiotherapy schedules were 46.4 Gy in 29 bidaily fractions (n = 2, treated 2010), 60 Gy in 30 daily fractions (n = 2), 66 Gy in 30 fractions (n = 2) and 70 Gy in 35 fractions (n = 1; patient received neoadjuvant CRT). CRT was discontinued early for two patients due to toxicities. At median follow up of 5.8 months, 42.9% (3/7) patients were alive and disease-free. Only one patient developed a local failure. Three patients died from distant metastases without locoregional recurrence.ConclusionsDespite poor prognosis of ATC, selected patients with operable tumours may achieve high locoregional control rates with trimodal therapy, with possibility of long-term survival in select cases.     

Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions

High-throughput molecular technologies can profile microbial communities at high resolution even in complex environments like the intestinal microbiota. Recent improvements in next-generation sequencing technologies allow for even finer resolution. We compared phylogenetic profiling of both longer (454 Titanium) sequence reads with shorter, but more numerous, paired-end reads (Illumina). For both approaches, we targeted six tandem combinations of 16S rRNA gene variable regions, in microbial DNA extracted from a human faecal sample, in order to investigate their limitations and potentials. In silico evaluations predicted that the V3/V4 and V4/V5 regions would provide the highest classification accuracies for both technologies. However, experimental sequencing of the V3/V4 region revealed significant amplification bias compared to the other regions, emphasising the necessity for experimental validation of primer pairs. The latest developments of 454 and Illumina technologies offered higher resolution compared to their previous versions, and showed relative consistency with each other. However, the majority of the Illumina reads could not be classified down to genus level due to their shorter length and higher error rates beyond 60 nt. Nonetheless, with improved quality and longer reads, the far greater coverage of Illumina promises unparalleled insights into highly diverse and complex environments such as the human gut.     

Intracellular Accumulation of High Levels of γ-Aminobutyrate by Listeria monocytogenes 10403S in Response to Low pH: Uncoupling of γ-Aminobutyrate Synthesis from Efflux in a Chemically Defined Medium

It is well established that the glutamate decarboxylase (GAD) system is central to the survival of Listeria monocytogenes at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular γ-aminobutyrate (GABA_i). The glutamate is then decarboxylated to GABA_i, a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when L. monocytogenes 10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABA_e) in response to acidification. In addition, high levels of GABA_i (>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of gadD1T1 and gadD3 occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABA_i in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABA_i. These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.The capacity to produce γ-aminobutyric acid (GABA) through glutamate decarboxylation is commonly found in both Gram-negative and Gram-positive bacterial genera (10, 12). In several cases, this reaction has been shown to be critical for bacteria to survive potentially lethal acidic environments (15, 18, 20). It is generally held that the hydrogen ion consumed during the decarboxylation reaction helps to prevent excessive acidification of the cytoplasm, thereby protecting the cells against acidic environments. The GABA produced in the reaction is removed from the cell through the activity of an antiporter that exchanges a GABA molecule for an extracellular glutamate (Glu) molecule (6, 12).In Listeria monocytogenes, the Gram-positive food-borne pathogen that was the focus of the present study, the glutamate decarboxylase (GAD) system has been shown to play an essential role in acid tolerance (8, 9). Mutants compromised in their ability to catalyze this decarboxylation reaction survive poorly both in acidic foods (8) and gastric juice (9). The GAD system in most L. monocytogenes strains is encoded by a total of five genes. There are three genes, designated gadD1, gadD2, and gadD3, that encode distinct glutamate decarboxylase enzymes and two genes, designated gadT1 and gadT2, that encode two Glu-GABA antiporters. These genes are organized at three separate genetic loci: gadD1T1, gadT2D2, and gadD3 (11). The decarboxylase/antiporter system encoded by gadT2D2 plays a central role in allowing survival under extreme acidic conditions; mutants lacking either the GadT2 antiporter or the GadD2 decarboxylase are highly sensitive to low pH (9, 11). In contrast, the GadD1/GadT1 decarboxylase/antiporter system appears to be more important for growth under moderately acidic conditions (11). The genes encoding this system are absent from most serotype 4 strains, and this generally correlates with a reduced ability of these strains to grow well at low pH (11). The role of GadD3 is less clear since it has not been possible to generate a deletion mutant lacking the corresponding gene (9).Although the activity of the decarboxylase is generally thought to be coupled directly to the antiporter activity (i.e., the efflux of GABA is coupled to the supply of Glu) there is little direct evidence for this, even in bacteria where the system has been very well characterized. Most studies of the bacterial GAD system have used complex growth media when studying acid tolerance and GABA production (7, 8, 15). In the present study, we sought to determine whether extracellular Glu is a requirement for the production of GABA in L. monocytogenes. To do this, we have used a chemically defined growth medium (DM) that supports the growth of L. monocytogenes but does not include Glu. The results indicate that cells cultured in this medium do not produce extracellular GABA (GABA_e) in response to low pH but are capable of accumulating substantial pools of intracellular GABA (GABA_i). We establish that some component of complex medium is indispensable for efficient efflux of GABA. Surprisingly, supplementation of the DM with Glu failed to stimulate the extracellular release of GABA. We show that the GadD2/GadT2 decarboxylase/antiporter system is not transcribed when cells are grown in DM and suggest that this accounts for much of the difference in GABA production between cells cultured in DM and complex growth medium.     

Resuspending ram spermatozoa in seminal plasma after cryopreservation does not improve pregnancy rate in cervically inseminated ewes

The role of seminal plasma (SP) components on the maintenance of motility, viability and fertilising ability of frozen-thawed spermatozoa is of considerable interest. However, differences observed in constituents of SP among males could explain differences in fertility obtained in vivo. Two experiments were designed to examine the effects of seminal plasma on fertility from cervically inseminated frozen-thawed semen. The objective of Experiment 1 was to investigate if source or type of SP influences pregnancy rate. Seminal plasma was collected from rams previously classified as having either High (HSP; n=3) or Low (LSP; n=3) fertility in vivo. Artificial SP (fructose/sodium solution with 10% BSA; ASP) was made. Frozen semen from the same 6 rams was thawed and inseminated (Control) or resuspended either in HSP, LSP or ASP (20% in semen) prior to insemination of ewes (n=284, over 2 farms). The overall pregnancy rate was 28.1%. Treatments (Control, ASP, HSP and LSP) were not significantly different (P>0.3). There was no difference between HSP and LSP (P>0.5), and no effect of using ASP compared to ram SP (P>0.7), on pregnancy rate. As there was no effect of SP on pregnancy rate a repeat experiment (Experiment 2) was designed to test the effect of washing and selecting motile sperm prior to resuspending in phosphate-buffered saline (PBS) containing SP on pregnancy rate. Frozen-thawed semen from each of 2 rams was centrifuged through a density gradient, pellets were centrifuged through a wash medium and the sperm concentration/ram was counted. Sperm cells were resuspended in: (1) control PBS, (2) PBS containing 30% HSP or (3) PBS containing 30% LSP to give 100 x 10(6) motile sperm in 0.25 mL. Control straws were thawed and inseminated directly. Ewes (n=223 over 2 farms) were inseminated 57 h post-sponge withdrawal and those not returning to oestrus were slaughtered 29-50 days post-insemination for pregnancy determination. In Experiment 2, the pregnancy rate for Control, PBS, HSP and LSP were 15.4%, 2.3%, 0% and 0%, respectively, for Farm 1 (P>0.05) and 17.8%, 11.0%, 3.9% and 12.4%, respectively, for Farm 2. Under the conditions of the current study, addition of SP from different donors of either High or Low fertility status to frozen-thawed ram semen post-thawing did not improve pregnancy rate in ewes. ASP had no effect on pregnancy rate in ewes when added to frozen-thawed semen. Washing and selection of motile sperm prior to resuspension in PBS with or without SP (30%) before insemination had a negative effect on pregnancy rate in cervically inseminated ewes. Hence, the addition of seminal plasma or some of its constituents to semen does not appear to improve pregnancy rate in cervically inseminated ewes.     

Being Muslim and being Irish after 9/11: self-conceptions of place in Irish society

There has been a significant, well-established if somewhat invisible Muslim population in Ireland since the 1950s. An increase in immigration during the Celtic tiger years along with the hysteria of 9/11 caused a rapid visibilization of this population. Muslims became synonymous with extremism and terrorism, but also fell victim to racist constructions emerging from the economic decline. The experience of Muslim youth since 9/11 has been well researched in the UK and Europe, however there has been little empirical work conducted with Ireland’s Muslim communities. In the literature, it is assumed that the British experience is replicated for Muslim youth in Ireland – this is not the case. This paper examines the lived experience of Irish Muslim youth given the visibilization of Islam after 9/11 and their ensuing experiences of Irishness. An analysis of ethnographic data reveals the idiosyncratic experiences of these youth growing up in the shadow of a discriminatory and Islamophobic narrative on extremism and terrorism and an evolving immigrant landscape.     

Platelet adhesion and degranulation induce pro-survival and pro-angiogenic signalling in ovarian cancer cells

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.     

Further Identification of Novel Lantibiotic Operons Using LanM-Based Genome Mining

Lantibiotics are gene-encoded antimicrobial peptides that are distinguished by the presence of the unusual structures, lanthionine and β-methyllanthionine, which are introduced through enzyme-catalysed post-translational modification. Lantibiotics can be subdivided on the basis of the nature of the enzyme(s) which catalyse this reaction. Lantibiotic synthetases, generically designated LanM, which catalyse the dehydration of serines (and threonines) followed by the formation lanthionine (and β-methyllanthioine), are responsible for the synthesis of the largest subdivision, type 2. One can take advantage of the conserved nature of LanM proteins to screen for and bioinformatically characterize novel lantibiotic-encoding operons in genome-sequenced microorganisms. Having employed this strategy with success previously, here we update the investigation to reveal the existence of 124 LanM homologs encoded within genome-sequenced microbes. Further analysis focussed specifically on 9 novel lantibiotic gene clusters in Anaerocellum thermophilum DSM 6725, Anaerococcus tetradius ATCC 35098, Corynebacterium matruchotti ATCC 33806, Streptococcus suis 98HAH33, Geobacillus sp. G11MC16, Nostoc punctiforme PCC 73102 (× 2; one on plasmid and one on the chromosome) and Streptococcus pneumoniae CDC 0288-04 and TIGR4. Furthermore, screening of metagenomic datasets revealed 11 additional LanM-encoding genes from a variety of environments. The alignment of these LanM proteins facilitated a detailed investigation of conserved domains and led to the design of an improved set of degenerate primers which can be employed in the laboratory to identify strains containing type 2 lantibiotic gene clusters.