The Zn2+ Complex of 1,4,7,10-Tetraazacyclododecane as an Artificial Nucleobase

{2-Deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4′-dimethoxytrityl)-α-D- erythro-pentofuranosyl}-N-{2-[4,7,10-tris(2,2,2-trifluoroacetyl)-1,4,7,10-tetraazacyclododecan-1- yl]ethyl}acetamide (1) was prepared and incorporated into a 2′-O-methyl oligoribonucleotide. The hybridization of this oligonucleotide with complementary 2′-O-methyl oligoribonucleotides incorporating one to five uracil bases opposite to the azacrown structure was studied in the absence and presence of Zn²⁺. Introduction of Zn²⁺ moderately stabilized the duplex with U-bulged targets.     

Mammalian and Malaria Parasite Cyclase-associated Proteins Catalyze Nucleotide Exchange on G-actin through a Conserved Mechanism

Cyclase-associated proteins (CAPs) are among the most highly conserved regulators of actin dynamics, being present in organisms from mammals to apicomplexan parasites. Yeast, plant, and mammalian CAPs are large multidomain proteins, which catalyze nucleotide exchange on actin monomers from ADP to ATP and recycle actin monomers from actin-depolymerizing factor (ADF)/cofilin for new rounds of filament assembly. However, the mechanism by which CAPs promote nucleotide exchange is not known. Furthermore, how apicomplexan CAPs, which lack many domains present in yeast and mammalian CAPs, contribute to actin dynamics is not understood. We show that, like yeast Srv2/CAP, mouse CAP1 interacts with ADF/cofilin and ADP-G-actin through its N-terminal α-helical and C-terminal β-strand domains, respectively. However, in the variation to yeast Srv2/CAP, mouse CAP1 has two adjacent profilin-binding sites, and it interacts with ATP-actin monomers with high affinity through its WH2 domain. Importantly, we revealed that the C-terminal β-sheet domain of mouse CAP1 is essential and sufficient for catalyzing nucleotide exchange on actin monomers, although the adjacent WH2 domain is not required for this function. Supporting these data, we show that the malaria parasite Plasmodium falciparum CAP, which is entirely composed of the β-sheet domain, efficiently promotes nucleotide exchange on actin monomers. Collectively, this study provides evidence that catalyzing nucleotide exchange on actin monomers via the β-sheet domain is the most highly conserved function of CAPs from mammals to apicomplexan parasites. Other functions, including interactions with profilin and ADF/cofilin, evolved in more complex organisms to adjust the specific role of CAPs in actin dynamics.     

Matrix Metalloproteinases -8 and -9 and Tissue Inhibitor of Metalloproteinase-1 in Burn Patients. A Prospective Observational Study

IntroductionMatrix metalloproteinases (MMPs) -8 and -9 are released from neutrophils in acute inflammation and may contribute to permeability changes in burn injury. In retrospective studies on sepsis, levels of MMP-8, MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) differed from those of healthy controls, and TIMP-1 showed an association with outcome. Our objective was to investigate the relationship between these proteins and disease severity and outcome in burn patients.MethodsIn this prospective, observational, two-center study, we collected plasma samples from admission to day 21 post-burn, and burn blister fluid samples on admission. We compared MMP-8, -9, and TIMP-1 levels between TBSA<20% (N = 19) and TBSA>20% (N = 30) injured patients and healthy controls, and between 90-day survivors and non-survivors. MMP-8, -9, and TIMP-1 levels at 24-48 hours from injury, their maximal levels, and their time-adjusted means were compared between groups. Correlations with clinical parameters and the extent of burn were analyzed. MMP-8, -9, and TIMP-1 levels in burn blister fluids were also studied.ResultsPlasma MMP-8 and -9 were higher in patients than in healthy controls (P<0.001 and P = 0.016), but only MMP-8 differed between the TBSA<20% and TBSA>20% groups. MMP-8 and -9 were not associated with clinical severity or outcome measures. TIMP-1 differed significantly between patients and controls (P<0.001) and between TBSA<20% and TBSA>20% groups (P<0.002). TIMP-1 was associated with 90-day mortality and correlated with the extent of injury and clinical measures of disease severity. TIMP-1 may serve as a new biomarker in outcome prognostication of burn patients.     

Spring development of a Chlamydomonas population in Lake Nimetön,a small humic forest lake in southern Finland

Biomass development and vertical distribution of a Chlamydomonas population in a small humic forest lake was followed by daily sampling in May-June, 1984. Chlamydomonas dominated the phytoplankton spring bloom, forming 71% of the maximum phytoplankton biomass on 18 May. In early May the outflow rate was high and during the 24 hour period when the maximum rate of surface runoff was recorded (8–9 May), 43% of the Chlamydomonas biomass was flushed out of the lake, which delayed the onset of biomass increase. When surface runoff had slowed down Chlamydomonas biomass started increasing and during wax of the population most cells were < 10 µm in diameter. Population maximum lasted for one day (18 May) and there-after Chlamydomonas biomass decreased towards the end of the study. During wane of the population most cells were > 10 µm in diameter.     

Thieno[3,4‐c]Pyrrole‐4,6‐Dione‐Based Polymer Acceptors for High Open‐Circuit Voltage All‐Polymer Solar Cells

While polymer acceptors are promising fullerene alternatives in the fabrication of efficient bulk heterojunction (BHJ) solar cells, the range of efficient material systems relevant to the “all‐polymer” BHJ concept remains narrow, and currently limits the perspectives to meet the 10% efficiency threshold in all‐polymer solar cells. This report examines two polymer acceptor analogs composed of thieno[3,4‐c ]pyrrole‐4,6‐dione (TPD) and 3,4‐difluorothiophene ([2F]T) motifs, and their BHJ solar cell performance pattern with a low‐bandgap polymer donor commonly used with fullerenes (PBDT‐TS1; taken as a model system). In this material set, the introduction of a third electron‐deficient motif, namely 2,1,3‐benzothiadiazole (BT), is shown to (i) significantly narrow the optical gap (E _opt) of the corresponding polymer (by ≈0.2 eV) and (ii) improve the electron mobility of the polymer by over two orders of magnitude in BHJ solar cells. In turn, the narrow‐gap P2TPDBT[2F]T analog (E _opt = 1.7 eV) used as fullerene alternative yields high open‐circuit voltages (V _OC) of ≈1.0 V, notable short‐circuit current values (J _SC) of ≈11.0 mA cm⁻², and power conversion efficiencies (PCEs) nearing 5% in all‐polymer BHJ solar cells. P2TPDBT[2F]T paves the way to a new, promising class of polymer acceptor candidates.     

Effects of streptozotocin-induced diabetes and physical training on gene expression of titin-based stretch-sensing complexes in mouse striated muscle

In striated muscle, a sarcomeric noncontractile protein, titin, is proposed to form the backbone of the stress- and strain-sensing structures. We investigated the effects of diabetes, physical training, and their combination on the gene expression of proteins of putative titin stretch-sensing complexes in skeletal and cardiac muscle. Mice were divided into control (C), training (T), streptozotocin-induced diabetic (D), and diabetic training (DT) groups. Training groups performed for 1, 3, or 5 wk of endurance training on a motor-driven treadmill. Muscle samples from T and DT groups together with respective controls were collected 24 h after the last training session. Gene expression of calf muscles (soleus, gastrocnemius, and plantaris) and cardiac muscle were analyzed using microarray and quantitative PCR. Diabetes induced changes in mRNA expression of the proteins of titin stretch-sensing complexes in Z-disc (MLP, myostatin), I-band (CARP, Ankrd2), and M-line (titin kinase signaling). Training alleviated diabetes-induced changes in most affected mRNA levels in skeletal muscle but only one change in cardiac muscle. In conclusion, we showed diabetes-induced changes in mRNA levels of several fiber-type-biased proteins (MLP, myostatin, Ankrd2) in skeletal muscle. These results are consistent with previous observations of diabetes-induced atrophy leading to slower fiber type composition. The ability of exercise to alleviate diabetes-induced changes may indicate slower transition of fiber type.     

Plaque2.0—A High-Throughput Analysis Framework to Score Virus-Cell Transmission and Clonal Cell Expansion

Classical plaque assay measures the propagation of infectious agents across a monolayer of cells. It is dependent on cell lysis, and limited by user-specific settings and low throughput. Here, we developed Plaque2.0, a broadly applicable, fluorescence microscopy-based high-throughput method to mine patho-biological clonal cell features. Plaque2.0 is an open source framework to extract information from chemically fixed cells by immuno-histochemistry or RNA in situ hybridization, or from live cells expressing GFP transgene. Multi-parametric measurements include infection density, intensity, area, shape or location information at single plaque or population levels. Plaque2.0 distinguishes lytic and non-lytic spread of a variety of DNA and RNA viruses, including vaccinia virus, adenovirus and rhinovirus, and can be used to visualize simultaneous plaque formation from co-infecting viruses. Plaque2.0 also analyzes clonal growth of cancer cells, which is relevant for cell migration and metastatic invasion studies. Plaque2.0 is suitable to quantitatively analyze virus infections, vector properties, or cancer cell phenotypes.     

SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.