Molecular Weight Dependence of Exciton Diffusion in Poly(3‐hexylthiophene)

A joint experimental and theoretical study of singlet exciton diffusion in spin‐coated poly(3‐hexylthiophene) (P3HT) films and its dependence on molecular weight is presented. The results show that exciton diffusion is fast along the co‐facial π–π aggregates of polymer chromophores and about 100 times slower in the lateral direction between aggregates. Exciton hopping between aggregates is found to show a subtle dependence on interchain coupling, aggregate size, and Boltzmann statistics. Additionally, a clear correlation is observed between the effective exciton diffusion coefficient, the degree of aggregation of chromophores, and exciton delocalization along the polymer chain, which suggests that exciton diffusion length can be enhanced by tailored synthesis and processing conditions.     

Identification and Characterization of Type II Toxin-Antitoxin Systems in the Opportunistic Pathogen Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen that causes nosocomial infections. Due to the ability to persist in the clinical environment and rapidly acquire antibiotic resistance, multidrug-resistant A. baumannii clones have spread in medical units in many countries in the last decade. The molecular basis of the emergence and spread of the successful multidrug-resistant A. baumannii clones is not understood. Bacterial toxin-antitoxin (TA) systems are abundant genetic loci harbored in low-copy-number plasmids and chromosomes and have been proposed to fulfill numerous functions, from plasmid stabilization to regulation of growth and death under stress conditions. In this study, we have performed a thorough bioinformatic search for type II TA systems in genomes of A. baumannii strains and estimated at least 15 possible TA gene pairs, 5 of which have been shown to be functional TA systems. Three of them were orthologs of bacterial and archaeal RelB/RelE, HicA/HicB, and HigB/HigA systems, and others were the unique SplT/SplA and CheT/CheA TA modules. The toxins of all five TA systems, when expressed in Escherichia coli, inhibited translation, causing RNA degradation. The HigB/HigA and SplT/SplA TA pairs of plasmid origin were highly prevalent in clinical multidrug-resistant A. baumannii isolates from Lithuanian hospitals belonging to the international clonal lineages known as European clone I (ECI) and ECII.     

Specificity of the connexin W3/4 locus for functional gap junction formation

The N-terminal (NT) domain of the connexins forms an essential transjunctional voltage (V_j) sensor and pore-forming domain that when truncated, tagged, or mutated often leads to formation of a nonfunctional channel. The NT domain is relatively conserved among the connexins though the α- and δ-group connexins possess a G2 residue not found in the β- and γ-group connexins. Deletion of the connexin40 G2 residue (Cx40G2Δ) affected the V_j gating, increased the single channel conductance (γ_j), and decreased the relative K⁺/Cl^? permeability (P_K/P_Cl) ratio of the Cx40 gap junction channel. The conserved α/β-group connexin D2/3 and W3/4 loci are postulated to anchor the NT domain within the pore via hydrophilic and hydrophobic interactions with adjacent connexin T5 and M34 residues. Cx40D3N and D3R mutations produced limited function with progressive reductions in V_j gating and noisy low γ_j gap junction channels that reduced the γ_j of wild-type Cx40 channels from 150 pS to < 50 pS when coexpressed. Surprisingly, hydrophobic Cx40 W4F and W4Y substitution mutations were not compatible with function despite their ability to form gap junction plaques. These data are consistent with minor and major contributions of the G2 and D3 residues to the Cx40 channel pore structure, but not with the postulated hydrophobic W4 intermolecular interactions. Our results indicate an absolute requirement for an amphipathic W3/4 residue that is conserved among all α/β/δ/γ-group connexins. We alternatively hypothesize that the connexin D2/3-W3/4 locus interacts with the highly conserved FIFR M1 motif to stabilize the NT domain within the pore.     

Primary Cilium-Mediated Retinal Pigment Epithelium Maturation Is Disrupted in Ciliopathy Patient Cells

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Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.     

Autoreactive memory CD4+ T lymphocytes that mediate chronic uveitis reside in the bone marrow through STAT3-dependent mechanisms

Organ-specific autoimmune diseases are usually characterized by repeated cycles of remission and recurrent inflammation. However, where the autoreactive memory T cells reside in between episodes of recurrent inflammation is largely unknown. In this study, we have established a mouse model of chronic uveitis characterized by progressive photoreceptor cell loss, retinal degeneration, focal retinitis, retinal vasculitis, multifocal choroiditis, and choroidal neovascularization, providing for the first time to our knowledge a useful model for studying long-term pathological consequences of chronic inflammation of the neuroretina. We show that several months after inception of acute uveitis, autoreactive memory T cells specific to retinal autoantigen, interphotoreceptor retinoid-binding protein (IRBP), relocated to bone marrow (BM). The IRBP-specific memory T cells (IL-7Rα(High)Ly6C(High)CD4(+)) resided in BM in resting state but upon restimulation converted to IL-17/IFN-γ-expressing effectors (IL-7Rα(Low)Ly6C(Low)CD4(+)) that mediated uveitis. We further show that T cells from STAT3-deficient (CD4-STAT3KO) mice are defective in α4β1 and osteopontin expression, defects that correlated with inability of IRBP-specific memory CD4-STAT3KO T cells to traffic into BM. We adoptively transferred uveitis to naive mice using BM cells from wild-type mice with chronic uveitis but not BM cells from CD4-STAT3KO, providing direct evidence that memory T cells that mediate uveitis reside in BM and that STAT3-dependent mechanism may be required for migration into and retention of memory T cells in BM. Identifying BM as a survival niche for T cells that cause uveitis suggests that BM stromal cells that provide survival signals to autoreactive memory T cells and STAT3-dependent mechanisms that mediate their relocation into BM are attractive therapeutic targets that can be exploited to selectively deplete memory T cells that drive chronic inflammation.     

Crystallization and preliminary crystallographic studies of a bifunctional restriction endonuclease Eco57I

Restriction endonuclease Eco57I from Escherichia coli recognizes asymmetric DNA sequence 5'-CTGAAG and has both restriction (DNA cleavage a short distance away from the recognition site) and modification (methylation) activities residing in a single polypeptide chain. Single crystals of wild-type Eco57I ternary complexes with double-stranded DNA and sinefungin, a stimulator of endonuclease activity, were obtained by the vapor diffusion technique and characterized crystallographically for different variants of the DNA component. The best data for the complex with 25-mer DNA were collected to 4.2-A resolution at 100 K using synchrotron radiation. The crystals are orthorhombic, space group P2(1)2(1)2, with a=164.3, b=293.0, c=71.1 A, and contain two to four copies of the protein in the asymmetric unit.     

Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli

The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity.