Reinvestigation of aminoacyl-TRNA synthetase core complex by affinity purification-mass spectrometry reveals TARSL2 as a potential member of the complex

10.1371/journal.pone.0081734PLoS ONE812-POLN

Developmental seizures and mortality result from reducing GABAᴀ receptor α2-subunit interaction with collybistin

Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development

Single-cell analysis tools for drug discovery and development

The genetic, functional or compositional heterogeneity of healthy and diseased tissues presents major challenges in drug discovery and development. Such heterogeneity hinders the design of accurate disease models and can confound the interpretation of biomarker levels and of patient responses to specific therapies. The complex nature of virtually all tissues has motivated the development of tools for single-cell genomic, transcriptomic and multiplex proteomic analyses. Here, we review these tools and assess their advantages and limitations. Emerging applications of single cell analysis tools in drug discovery and development, particularly in the field of oncology, are discussed

Identification of a Core Amino Acid Motif within the α Subunit of GABAᴀRs that Promotes Inhibitory Synaptogenesis and Resilience to Seizures

The fidelity of inhibitory neurotransmission is dependent on the accumulation of γ-aminobutyric acid type A receptors (GABAARs) at the appropriate synaptic sites. Synaptic GABAARs are constructed from α(1-3), β(1-3), and γ2 subunits, and neurons can target these subtypes to specific synapses. Here, we identify a 15-amino acid inhibitory synapse targeting motif (ISTM) within the α2 subunit that promotes the association between GABAARs and the inhibitory scaffold proteins collybistin and gephyrin. Using mice in which the ISTM has been introduced into the α1 subunit (Gabra1-2 mice), we show that the ISTM is critical for axo-axonic synapse formation, the efficacy of GABAergic neurotransmission, and seizure sensitivity. The Gabra1-2 mutation rescues seizure-induced lethality in Gabra2-1 mice, which lack axo-axonic synapses due to the deletion of the ISTM from the α2 subunit. Taken together, our data demonstrate that the ISTM plays a critical role in promoting inhibitory synapse formation, both in the axonic and somatodendritic compartments

New Policies, New Technologies: Modelling the Potential for Improved Smear Microscopy Services in Malawi

Background To quantify the likely impact of recent WHO policy recommendations regarding smear microscopy and the introduction of appropriate low-cost fluorescence microscopy on a) case detection and b) laboratory workload.Methodology/Principal Findings An audit of the laboratory register in an urban hospital, Lilongwe, Malawi, and the application of a simple modelling framework. The adoption of the new definition of a smear-positive case could directly increase case detection by up to 28%. Examining Ziehl-Neelsen (ZN) sputum smears for up to 10 minutes before declaring them negative has previously been shown to increase case detection (over and above that gained by the adoption of the new case definition) by 70% compared with examination times in routine practice. Three times the number of staff would be required to adequately examine the current workload of smears using ZN microscopy. Through implementing new policy recommendations and LED-based fluorescence microscopy the current laboratory staff complement could investigate the same number of patients, examining auramine-stained smears to an extent that is equivalent to a 10 minutes ZN smear examination.Conclusions/Significance Combined implementation of the new WHO recommendations on smear microscopy and LED-based fluorescence microscopy could result in substantial increases in smear positive case-detection using existing human resources and minimal additional equipment

Kin-cohort estimates for familial breast cancer risk in relation to variants in DNA base excision repair, BRCA1 interacting and growth factor genes

BACKGROUND: Subtle functional deficiencies in highly conserved DNA repair or growth regulatory processes resulting from polymorphic variation may increase genetic susceptibility to breast cancer. Polymorphisms in DNA repair genes can impact protein function leading to genomic instability facilitated by growth stimulation and increased cancer risk. Thus, 19 single nucleotide polymorphisms (SNPs) in eight genes involved in base excision repair (XRCC1, APEX, POLD1), BRCA1 protein interaction (BRIP1, ZNF350, BRCA2), and growth regulation (TGFß1, IGFBP3) were evaluated. METHODS: Genomic DNA samples were used in Taqman 5'-nuclease assays for most SNPs. Breast cancer risk to ages 50 and 70 were estimated using the kin-cohort method in which genotypes of relatives are inferred based on the known genotype of the index subject and Mendelian inheritance patterns. Family cancer history data was collected from a series of genotyped breast cancer cases (N = 748) identified within a cohort of female US radiologic technologists. Among 2,430 female first-degree relatives of cases, 190 breast cancers were reported. RESULTS: Genotypes associated with increased risk were: XRCC1 R194W (WW and RW vs. RR, cumulative risk up to age 70, risk ratio (RR) = 2.3; 95% CI 1.3–3.8); XRCC1 R399Q (QQ vs. RR, cumulative risk up to age 70, RR = 1.9; 1.1–3.9); and BRIP1 (or BACH1) P919S (SS vs. PP, cumulative risk up to age 50, RR = 6.9; 1.6–29.3). The risk for those heterozygous for BRCA2 N372H and APEX D148E were significantly lower than risks for homozygotes of either allele, and these were the only two results that remained significant after adjusting for multiple comparisons. No associations with breast cancer were observed for: APEX Q51H; XRCC1 R280H; IGFPB3 -202A>C; TGFß1 L10P, P25R, and T263I; BRCA2 N289H and T1915M; BRIP1 -64A>C; and ZNF350 (or ZBRK1) 1845C>T, L66P, R501S, and S472P. CONCLUSION: Some variants in genes within the base-excision repair pathway (XRCC1) and BRCA1 interacting proteins (BRIP1) may play a role as low penetrance breast cancer risk alleles. Previous association studies of breast cancer and BRCA2 N372H and functional observations for APEX D148E ran counter to our findings of decreased risks. Due to the many comparisons, cautious interpretation and replication of these relationships are warranted

Application of prolonged microdialysis sampling in carboplatin-treated cancer patients

Purpose: To better understand the mechanisms underlying (in)sensitivity of tumors to anticancer drugs, assessing intra-tumor drug pharmacokinetics (PKs) could be important. We explored the feasibility of microdialysis in tumor tissue for multiple days in a clinical setting, using carboplatin as model drug. Methods: Plasma and microdialysate samples from tumor and adipose normal tissues were collected up to 47 h after dosing in eight carboplatin-treated patients with an accessible (sub)cutaneous tumor. Results: Pharmacokinetics were evaluable in tumor tissue in 6/8 patients and in adipose normal tissue in 3/8 patients. Concentration-time curves of unbound platinum in both the tissues followed the pattern of the curves in plasma, with exposure ratios of tissue versus plasma ranging from 0.64 to 1.46. Conclusions: Microdialysis can be successfully employed in ambulant patients for multiple days, which enables one to study tissue PK of anticancer drugs in normal and malignant tissues in more detail

Rapid and Efficient Clearance of Blood-borne Virus by Liver Sinusoidal Endothelium

The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (∼90%), finding ∼10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity