Uncultivated Microbial Eukaryotic Diversity: A Method to Link ssu rRNA Gene Sequences with Morphology

Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA “phylotypes” from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent in situ hybridization (FISH) with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-α-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker® or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked Naegleria spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar Colpoda-like ciliates using tubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of novel lineages, identified in diverse environments

Selective Cyclooxygenase-2 Inhibitor Suppresses Renal Thromboxane Production but Not Proliferative Lesions in the MRL/lpr Murine Model of Lupus Nephritis

BACKGROUND: Proliferative lupus nephritis (LN) is marked by increased renal thromboxane (TXA(2)) production. Targeting the TXA(2) receptor or TXA(2) synthase effectively improves renal function in humans with LN and improves glomerular pathology in murine LN. This study was designed to address the following hypotheses: 1) TXA(2) production in the MRL/MpJ-Tnfrsf6(lpr)/J (MRL/lpr) model of proliferative lupus nephritis is COX2-dependent, and 2) COX2 inhibitor therapy improves glomerular filtration rate (GFR), proteinuria, markers of innate immune response, and glomerular pathology. METHODS: 20 female MRL/lpr and 20 BALB/cJ mice were divided into two equal treatment groups: 1) SC-236, a moderately selective COX2 inhibitor, or 2) vehicle. After treatment from 10 to 20 weeks of age, the effectiveness of inhibition of TXA(2) was determined by measuring urine TXB(2). Response endpoints measured at 20 weeks of age were renal function (GFR), proteinuria, urine nitrate + nitrite (NO(X)), and glomerular histopathology. RESULTS: SC236 therapy reduced surrogate markers of renal TXA(2) production during early, active glomerulonephritis. When this pharmacodynamic endpoint was reached, therapy improved GFR. Parallel reductions in markers of the innate immune response (urine NO(X)) during therapy were observed. However, the beneficial effect of SC236 therapy on GFR was only transient, and renal histopathology was not improved in late disease. CONCLUSIONS: These data demonstrate that renal TXA(2) production is COX2-dependent in murine LN and suggest that NO production is directly or indirectly COX2-dependent. However, COX2 inhibitor therapy in this model failed to improve renal pathology, making COX2 inhibition a less attractive approach for treating LN

Co-nonsolvency in concentrated aqueous solutions of PNIPAM: effect of methanol on the collective and the chain dynamics

The polymer dynamics in concentrated solutions of poly(N-isopropyl acrylamide) (PNIPAM) in D2O/CD3OD mixtures is investigated in the one-phase region. Two polymer concentrations (9 and 25 wt%) and CD3OD contents in the solvent mixture of 0, 10 and 15 vol% are chosen. Temperature-resolved dynamic light scattering (DLS) reveals the collective dynamics. Two modes are observed, namely the fast relaxation of polymer segments within the blobs and the slow collective relaxation of the blobs. As the cloud point is approached, the correlation length related to the fast mode increases with CD3OD content. It features critical scaling behavior, which is consistent with mean-field behavior for the 9 wt% PNIPAM solution in pure D2O and with 3D Ising behavior for all other solutions. While the slow mode is not very strong in the 9 wt% PNIPAM solution in pure D2O, it is significantly more prominent as CD3OD is added and at all CD3OD contents in the 25 wt% solution, which may be attributed to enhanced interaction between the polymers. Neutron spin-echo spectroscopy (NSE) reveals a decay in the intermediate structure factor which indicates a diffusive process. For the polymer concentration of 9 wt%, the diffusion coefficients from NSE are similar to the ones from the fast relaxation observed in DLS. In contrast, they are significantly lower for the solutions having a polymer concentration of 25 wt%, which is attributed to the influence of the dominant large-scale dynamic heterogeneities. To summarize, addition of cosolvent leads to enhanced large-scale heterogeneities, which are reflected in the dynamic behavior at small length scales