FAN1 Removes Triplet Repeat Extrusions via a PCNA- And RFC-Dependent Mechanism

Human genome-wide association studies have identified FAN1 and several DNA mismatch repair (MMR) genes as modifiers of Huntington’s disease age of onset. In animal models, FAN1 prevents somatic expansion of CAG triplet repeats, whereas MMR proteins promote this process. To understand the molecular basis of these opposing effects, we evaluated FAN1 nuclease function on DNA extrahelical extrusions that represent key intermediates in triplet repeat expansion. Here, we describe a strand-directed, extrusion-provoked nuclease function of FAN1 that is activated by RFC, PCNA, and ATP at physiological ionic strength. Activation of FAN1 in this manner results in DNA cleavage in the vicinity of triplet repeat extrahelical extrusions thereby leading to their removal in human cell extracts. The role of PCNA and RFC is to confer strand directionality to the FAN1 nuclease, and this reaction requires a physical interaction between PCNA and FAN1. Using cell extracts, we show that FAN1-dependent CAG extrusion removal relies on a very short patch excision-repair mechanism that competes with MutSβ-dependent MMR which is characterized by longer excision tracts. These results provide a mechanistic basis for the role of FAN1 in preventing repeat expansion and could explain the antagonistic effects of MMR and FAN1 in disease onset/progression

Frequency and phenotype of natural killer cells and natural killer cell subsets in bovine lymphoid compartments and blood

Natural killer (NK) cells are widely distributed in lymphoid and non‐lymphoid tissues, but little is known about the recirculation of NK cells between blood and tissues. This is relevant to understanding recirculation in the steady‐state and also for determining the roles for NK cells in vaccine‐induced immunity and responses to infection. Therefore, the percentage of NK cells and their phenotype across peripheral blood, afferent lymph and lymph nodes in steady‐state conditions was investigated in cattle using the pseudo‐afferent lymphatic cannulation model. CD2(+) CD25(lo) NK cells were the predominant subset of NK cells within the blood. In contrast, CD2(−) CD25(hi) NK cells were the main subset present within the skin‐draining afferent lymphatic vessels and lymph nodes, indicating that CD2(−) NK cells are the principal NK cell subset trafficking to lymph nodes via the afferent lymphatic vessel. Furthermore, a low percentage of NK cells were present in efferent lymph, which were predominantly of the CD2(−) subset, indicating that NK cells can egress from lymph nodes and return to circulation in steady‐state conditions. These compartmentalization data indicate that NK cells represent a population of recirculating lymphocytes in steady‐state conditions and therefore may be important during immune responses to vaccination or infection

A citizen science project: Monitoring seagrass in the Pensacola Bay System

Seagrasses play a critical role in estuaries; thus monitoring is crucial. UWF and Florida Sea Grant have a partnership supporting citizen science in Big Lagoon, Santa Rosa Sound, Predido Bay and urban bayous

Molecular diversity time series database for northern Gulf of Mexico

While microorganisms are rather small, they encompass a large percentage of the biomass and diversity seen in marine environments. They are the building blocks that makeup the foundations of critical marine food webs. Cataloging and monitoring these organisms is important to understand their vital roles in food webs and nutrient cycling (Azam et al. 1983). It is crucial to establish baseline community dynamics in an aquatic system over an extended period. Once a baseline has been established. we can explore how or why microbial communities change. Through the continuation of this seasona study, we have collected water samples from the Pensacola Beach Pier for over six years, providing a wealth of experience and many molecular diversity samples to be processed

Continuation of Time Series Database for Pensacola Beach microbial ecology

The ocean has some of the most diverse and productive ecosystem communities In the world, specifically microbial communities. • Although quite small, microorganism play important ecological roles in terrestrial, aquatic, and marine environments • Understanding microbial diversity and how these communities change when faced with environmental stressors is important as humans continue to alter the environment. • Baseline datasets provide valuable opportunities to compare and understand the effects of anthropogenic activities on any given environment • By creating a time series database study for microbial communities at Pensacola Beach, seasonal changes in biogeochemical processes, diversity, and community structure could be easily detected and studied

Subunit-dependent interaction of the general anaesthetic etomidate with the γ-aminobutyric acid type A receptor

1. The GABA modulating and GABA-mimetic actions of the general anaesthetic etomidate were examined in voltage-clamp recordings performed on Xenopus laevis oocytes induced, by cRNA injection, to express human recombinant γ-aminobutyric acid(A) (GABA(A)) receptor subunits. 2. Currents mediated by recombinant receptors with the ternary subunit composition α(x)β(y)γ(2L) (where x=1,2,3 or 6 and y=1 or 2), in response to GABA applied at the appropriate EC(10), were enhanced by etomidate in a manner that was dependent upon the identity of both the α and β subunit isoforms. 3. For the β(2)-subunit containing receptors tested, the EC(50) for the potentiation of GABA-evoked currents by etomidate (range 0.6 to 1.2 μM) was little affected by the nature of the α subunit present within the hetero-oligomeric complex. However, replacement of the β(2) by the β(1) subunit produced a 9–12 fold increase in the etomidate EC(50) (6 to 11 μM) for all α-isoforms tested. 4. For α(1), α(2) and α(6), but not α(3)-subunit containing receptors, the maximal potentiation of GABA-evoked currents by etomidate was greater for β(2)- than for β(1)-subunit containing receptors. This was most clearly exemplified by receptors composed of α(6)β(1)γ(2L) compared to α(6)β(2)γ(2L) subunits, where a maximally effective concentration of etomidate potentiated currents evoked by GABA at EC(10) to 28±2% and 169±4% of the maximal GABA response, respectively. 5. For α(1) subunit-containing receptors, the potency and maximal potentiating effect of either pentobarbitone or propofol was essentially unaffected by the β subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5α-pregnan-3α-ol-20-one was marginally higher for β(1) rather than the β(2) subunit-containing receptor, although its maximal effect was similar at the two receptor isoforms. 6. The GABA-mimetic action of etomidate was supported by β(2)- but not β(1)-subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either β isoform. For β(2)-subunit containing receptors, both the agonist EC(50) and the maximal current produced by etomidate were additionally influenced by the α isoform. 7. It is concluded that the subtype of β-subunit influences the potency with which etomidate potentiates GABA-evoked currents and that the β isoform is a crucial determinant of the GABA-mimetic activity of this compound. The nature of the α-subunit also impacts upon the maximal potentiation and activation that the compound may elicit. Such pronounced influences may aid the identification of the site that recognises etomidate. More generally, these results provide a clear example of structural specificity in anaesthetic action