The thermodynamics of the opening/closure process of a GC base pair located at the stem-loop junction of the SL1 sequence from HIV-1(Lai) genomic RNA was investigated in the context of a loop-loop homodimer (or kissing complex) using molecular dynamics simulation. The free energy, enthalpy and entropy changes for the closing reaction are 0 kcal x mol(-1), -11 kcal x mol(-1) and -0.037 kcal x mol(-1) x K(-1) at 300 degrees K respectively. Furthermore it is found that the free energy change is the same for the formation of a 11 nucleotide loop closed with UG and for the formation of a 9 nucleotide loop closed with GC. Our study evidences the high flexibility of the nucleotides at the stem-loop junction explaining the weak stability of this structure.

An important characteristic of the transcription of a ribosomal RNA gene (rDNA) mediated by DNA-dependent RNA polymerase (Pol) I is its stringent species specificity. SL1/TIF-IB is a key complex for species specificity, but its functional complex has not been reconstituted. Here, we established a novel and highly sensitive monitoring system for Pol I transcription to reconstitute the SL1 activity in which a transcript harboring a reporter gene synthesized by Pol I is amplified and converted into translatable mRNA by the influenza virus RNA-dependent RNA polymerase. Using this monitoring system, we reconstituted Pol I transcription from the human rDNA promoter in mouse cells by expressing four human TATA-binding protein (TBP)-associated factors (TAFIs) in the SL1 complex. The reconstituted SL1 also re-activated human rDNA transcription in mouse A9 cells carrying an inactive human chromosome 21 that contains the rDNA cluster. Chimeric SL1 complexes containing human and mouse TAFIs could be formed, but these complexes were inactive for human rDNA transcription. We conclude that four human TAFIs are necessary and sufficient to overcome the barrier of species specificity for human rDNA transcription in mouse cells.

OBJECTIVE

To check the efficiency of shock waves in the treatment of induratio penis plastica. The Minilith SL1, successfully used in orthopedic or salivary stones because of its lithotriptic power, can be used to break plaques in Peyronie's disease.

METHODS

A total of 130 patients affected with Peyronie's disease were entered into a prospective trial. Patients with completely calcified plaques as determined by ultrasound evaluation were excluded. We divided the patients into three treatment groups: (A) shock waves alone in 21 patients; (B) a combination of shock waves and verapamil (perilesional injection) in 36 patients, and (C) verapamil alone in 73 patients. First, we treated all groups A and B patients 3 times, 20 min each time, with a Minilith SL1, and then only the patients of the second group received a complete cycle of twelve injections of verapamil (10 mg) every 2 weeks for 6 months. The group of 73 patients (group C) treated during the previous 2 years with a medical therapy (only injection of verapamil) was used as a control group.

RESULTS

Ultrasound evaluation showed a reduction of plaque in 11/21 group A patients and 7/36 group B patients. The treatment was tolerated very well and only 11 petechiae in some patients were noticed after ESW treatment.

CONCLUSIONS

The therapeutic association of shock waves with verapamil injection is an effective nonoperative treatment for the stabilization of Peyronie's disease.

Glutathione metabolism represents a potential target for anti-parasite drug design. The central role of glutathione reductase (GR) in maintenance of the thiol redox state and in anti-oxidative defence has to be evaluated in more detail in order to establish the essential function of this enzyme for the survival of the filarial parasite Onchocerca volvulus. The O. volvulus GR (OvGR) gene was cloned and sequenced. The gene is composed of 13 exons and 12 introns and spans 4065 bp. The first intron is located within the 5'-untranslated region of the gene, 16 nucleotides upstream of the translation initiation codon. Southern-blot analysis and structural characterization of the genomic sequence indicate that OvGR is encoded by a single-copy gene. Isolation of various cDNA clones revealed a polymorphism of polyadenylation initiation with no consensus polyadenylation sites in any of the cDNAs analysed. The entire cDNA is 1977 bp long and carries the nematode-specific spliced leader sequence SL1 at its 5' end, 236 nucleotides upstream of the first in-frame methionine. The cDNA codes for a polypeptide of 462 amino acids with 53.5% sequence identity with human GR (HsGR). A total of 18 out of 19 residues contributing to glutathione binding are identical in OvGR and HsGR. However, one of the arginine residues (Arg-224 in HsGR) involved in discrimination between NADPH and NADH in all known GRs is substituted by tryptophan (Trp-207 in OvGR). The coding region of OvGR was expressed in Escherichia coli as a histidine-fusion protein, and it was established that the parasite protein still favours the binding of NADPH (Km 10.9 microM) over NADH (Km 108 microM). The histidine-fusion protein has a subunit size of 54 kDa and is active as a homodimer of 110 kDa.

BACKGROUND

Providing adequate transfusion support for alloimmunised patients for whom antigen negative blood is not readily available is hampered by the risk of a haemolytic reaction. The monocyte monolayer assay (MMA) has shown good correlation between the antibody clinical significance and the fate of antigen positive blood.

METHODS

From 2006 to 2013, the clinical significance of red cell alloantibodies produced by 61 patients was evaluated using a MMA; and antigen positive blood offering the best survival advantage was selected for transfusion following a secondary MMA crossmatch. Post-transfusion, patients were evaluated for clinical signs of haemolysis.

RESULTS

Overall, 19 of 61 (31·1%) of our antibodies were potentially clinically significant, with a monocyte index (MI)>> 5%. There was no correlation between the clinical significance as showed by the MMA, and the specificity of the antibody or the strength of reactivity at antihuman globulin (AHG) phase. Using the MMA as a secondary crossmatch method, 31 alloimmunised patients (including: eight anti-hr(B), four anti-Yt(a), one each anti-Rg1, -Co(a), Er(a), Le(b), -LW, -Sl1) received 103 antigen positive blood units with no clinical sign of a post-transfusion reaction. For three patients (one each anti-Jo(a), -AnWj, unidentified 'HTLA'), initial MMA was performed as part of an investigation of a suspected haemolytic reaction. In each case, the MMA accurately identified the unit responsible for the reaction.

CONCLUSIONS

Used as a crossmatch surrogate, the MMA provided valuable information in the decision of transfusing antigen positive blood to alloimmunised patients, avoiding delay because of the search of rare antigen negative units.

BACKGROUND

The KN blood group system, which consists of nine antigen specificities, is located on complement receptor Type 1 (CR1/CD35). CR1, a complement regulatory protein, acts as a vehicle for immune complex clearance. CR1 exhibits a red blood cell (RBC) density polymorphism. CR1 sites on RBCs in normal individuals range from 150 to 1200 molecules per cell. CR1 density polymorphism is regulated by HindIII restriction fragment length polymorphism and Q981H and P1786R polymorphisms in Caucasians. Yet, the role of the different polymorphisms in determining the CR1 density on RBCs remains unknown. The "null" serologic KN phenotype, known as Helgeson phenotype, was reported to be related with a very low CR1 density, less than 150 molecules per cell.

METHODS

The aim of this work was to investigate whether the KN-negative phenotype displayed by 60 individuals was related to the CR1 density by performing the phenotypic and genetic analysis of CR1 and to investigate the molecular background associated with the KN system.

RESULTS

We showed that the Helgeson-like phenotype had a prevalence of 12% in this population. The overall genotype/phenotype concordance was 90%. Among individuals with a KN-negative phenotype, the prevalences of Kn(a-), McC(a-), Sl1-negative, Sl3-negative, and KCAM-negative deduced phenotype were 37, 12, 29, 7, and 24%, respectively.

CONCLUSIONS

From our data, we suggest that the definition of the Helgeson phenotype must be revised, since the latter may be due not only to a very low CR1 density on RBCs, but also to the absence of expression of a high-prevalence KN antigen.

The aim of this prospective study was to assess the efficacy of shockwave (SW) therapy in the management of complex regional pain syndrome (CRPS). In this study, 30 patients (pts) who were affected by CRPS of the medial femoral condyle and unresponsive to previous standard physiotherapeutic and pharmacological treatment underwent 3 SW sessions at 72-h intervals, each consisting of 4000 shocks emitted by a MiniLith SL1 Storz electromagnetic generator. An energy flux density (EFD) of 0.035 or 0.09 mJ/mm(2) was used, depending on how well the patient endured the pain during the treatment. Satisfactory results were observed in 76.7% of the cases (23 pts) at the 2-month follow-up (FU) visit, and in 80% (24 pts) at the 6-month FU visit. The therapeutic effects of SW were caused by decreasing pain. The significant improvements we obtained bear witness to the potential value of SW therapy in the management of CRPS.

The repeated ACCA or AC-rich sequence and structural (SL1) elements in the 5' non-translated region (NTR) of the Potato virus X (PVX) RNA play vital roles in the PVX life cycle by controlling translation, RNA replication, movement, and assembly. It has already been shown that the repeated ACCA or AC-rich sequence affect both gRNA and sgRNA accumulation, while not affecting minus-strand RNA accumulation, and are also required for host protein binding. The functional significance of the repeated ACCA sequence elements in the 5' NTR region was investigated by analyzing the effects of deletion and site-directed mutations on PVX replication in Nicotiana benthamiana plants and NT1 protoplasts. Substitution (ACCA into AAAA or UUUU) mutations introduced in the first (nt 10-13) element in the 5' NTR of the PVX RNA significantly affected viral replication, while mutations introduced in the second (nt 17-20) and third (nt 20-23) elements did not. The fourth (nt 29-32) ACCA element weakly affected virus replication, whereas mutations in the fifth (nt 38-41) significantly reduced virus replication due to the structure disruption of SL1 by AAAA and/or UUUU substitutions. Further characterization of the first ACCA element indicated that duplication of ACCA at nt 10-13 (nt 10-17, ACCAACCA) caused severe symptom development as compared to that of wild type, while deletion of the single element (nt 10-13), DeltaACCA) or tripling of this element caused reduced symptom development. Single- and double-nucleotide substitutions introduced into the first ACCA element revealed the importance of CC located at nt positions 11 and 12. Altogether, these results indicate that the first ACCA element is important for PVX replication.

The type 1 human immunodeficiency virus (HIV-1), like all retroviruses, contains two copies of the RNA genome as a dimer. A dimer initially forms via a self-complementary sequence in the dimer initiation site (DIS) of the genomic RNA, but that dimer is converted to a mature dimer in a process generally promoted by the viral nucleocapsid (NC) protein. Formation of the mature dimer is correlated with infectivity. Study of genomic dimerization has been facilitated by discovery of short RNA transcripts containing the DIS stem-loop 1 (SL1), which can dimerize spontaneously without any protein factors in vitro as well as via the NC protein. On the basis of the palindromic nature of the apical loop of SL1, a kissing loop model has been proposed. First, a metastable kissing dimer is formed via a loop-loop interaction and then converted into a more stable extended dimer by the NC protein. This dimerization process in vitro is believed to mimic the in vivo RNA maturation. During experimental screening of potential inhibitors, we discovered a small molecule, Lys-Ala-7-amido-4-methylcoumarin (KA-AMC), which facilitates the in vitro conversion from kissing dimer to extended dimer. Here we report the structure-activity relationship for KA-AMC for promoting dimer maturation. Guanidino groups and increasing positive charge on the side chain enhance activity. For activity, the charged side chain is preferred on the benzene ring, and O 1 in the coumarin scaffold is essential. NMR studies show that the coumarin derivatives stack with aromatic bases of the RNA. The coumarin derivatives may aid in the investigation of some aspects of dimer maturation and serve as a scaffold for design of maturation inhibitors or of activators of premature maturation, either of which can lead to a potential HIV therapeutic.

Nanospray-FT-ICR has been employed to investigate the processes of genome dimerization, selection, and packaging in human immunodifficiency virus type 1, which are mediated by specific interactions between the nucleocapsid protein (NC) and the structural elements formed by the genome's packaging signal [Psi- ribonucleic acid (RNA)]. This analytical platform allowed for the unambiguous characterization of all the non-covalent complexes formed in vitro by simultaneous RNARNA and proteinRNA binding equilibria. Competitive binding experiments involving the isolated RNA elements were completed to evaluate their ability to sustain specific protein interactions. In similar fashion, ad hoc RNA mutants were used to locate two distinct binding sites on the apical loop and stem-bulge of the monomeric stemloop 1 (SL1) domain, which is responsible for initiating the dimerization process. The stem-bulge motifs provided viable binding sites in both the kissing-loop (KL) and the extended duplex forms of dimeric SL1, whereas the latter included additional sites corresponding to the A- bulge motifs that flank the annealed palindromes. A cross-linking approach using pre-derivatized, photo-cross- linkable NC demonstrated that the SL3 domain was the preferred site for protein binding in the context of full-length Psi-RNA. This concerted strategy is expected to provide new valuable insight into the effects induced by the global folding of Psi-RNA on its ability to interact with the NC protein during genome dimerization, selection and packaging.

We have developed a sensitive and specific PCR method for detecting plankton DNA in cases of death by drowning. However, this PCR method could not be used for cases of drowning in water containing no plankton. Bacteria species are normally localized in the throat and trachea and they may invade into blood through the respiratory tract in people who have drowned as well as species localized in water. The aim of this study was to establish a novel and expedient PCR method for detecting bacterial genes in samples from drowning cases. We designed primer pairs for Streptococcus salivarius (SL1) and Streptococcus sanguinis (SN1), which are common species in the throat, and for Aeromonas hydrophila (AH1), which has been found in various water samples. With SL1, SN1, and AH1, we detected 10, 0.1, and 1 pg of target DNA, respectively. Among 19 drowned cases within 3 days postmortem, SL-DNA was detected in all of the blood samples from hearts with SL1 and AH-DNA was detected in several samples with AH1. In a case of drowning in a bathtub, use of the conventional acid digestion method for diatom analyses and the PCR method for identifying plankton DNA revealed no plankton, but our PCR method for detecting bacterial DNA showed a positive result for SL-DNA in a blood sample from the heart. In conclusion, our novel PCR method is highly specific and sensitive for detecting bacterial DNA and is useful for cases of death by drowning in water containing no plankton.

The current experiment was designed to examine if a compensatory feed regimen influenced storage of glycogen forms, activity of proteolytic systems, and meat quality. Female pigs (Large White × Landrace × Duroc cross) with an initial age of 74 d were allocated to 6 feeding treatment groups (n=8 for each group). Groups then consumed feed ad libitum for 40 (A40), 42 (A42), or 82 d (A82). The compensatory growth groups were fed 0.70 of ad libitum intake for 40 d (R40) followed by refeeding for ad libitum intake for 2 (R40A2) or 42 d (R40A42). Pigs were slaughtered at the end of the restriction period (SL1), then after refeeding for 2 (SL2) and 42 d (SL3). The feeding regimen caused restricted animals at SL2 to have a decreased BW (P=0.039), with the refed animals undergoing compensatory growth by SL3 so BW was not different (P=0.829). At SL1 there was a trend for the R40 pigs to have less intramuscular fat than A40 (P=0.084). There was a trend for macroglycogen (MG; P=0.051) and a significant effect for proglycogen (ProG; P=0.014) to be greater at slaughter in R40 than A40, along with a greater postmortem decline in both MG (P=0.033) and ProG (P=0.022) over the first 2 h in R40, which was associated with the R40 having a lower pH at 24 h postmortem (P=0.043). After refeeding for 2 d (SL2), only MG of R40A2 was greater (P=0.030) than A42 and had a trend for a greater difference of decline at 24 h postmortem (P=0.091), which was associated with lower pH at 24 h (P=0.012). The data suggest that the concentrations of ProG are more labile and recovered to the concentrations of pigs fed for ad libitum intake sooner than MG. After full compensation in SL3, there was no difference for MG content (at 0 h, P=0.721; at 2 h, P=0.987; at 24 h, P=0.343), ProG content (at 0 h, P=0.879; at 2 h, P=0.946; at 24 h, P=0.459), and muscle pH (at 45 min, P=0.373; at 24 h, P=0.226). At all slaughter points, there was no difference in shear force (at SL1, P=0.101; at SL2, P=0.420; at SL3, P=0.167). There were no significant effects of the feeding regimen on micro- and milli-calpain large subunit gene expression (for micro-calpain at SL1, P=0.450; at SL2, P=0.171; at SL3, P=0.281; for milli-calpain at SL1, P=0.666; at SL2, P=0.123; at SL3, P=0.617) or the activity of the 2 proteolytic enzymes at any of the slaughter dates (for micro-calpain at SL1, P=0.238; at SL2, P =0.238; at SL3, P=0.222; for milli-calpain at SL1, P=0.296; at SL2, P=0.230; at SL3, P=0.615). In R40 there was a trend (P=0.070) for greater gene expression of caspase 3, whereas in R40A2 the increase was significant (P=0.009) relative to pigs consuming feed ad libitum. However, gene expression of the E3 ligase, MuRF1, at SL3 was less in R40A42 (P=0.019). Although compensatory growth does appear to influence the expression of various proteolytic systems, the changes do not appear to be associated with meat quality as measured by shear force.

Microbial biosurfactants, produced by fungi, yeast, and bacteria, are surface-active compounds with emulsifying properties that have a number of known activities, including the solubilization of microbial biofilms. In an on-going survey to uncover new or enhanced antimicrobial metabolite-producing microbes from harsh environments, such as oil-rich niches, 123 bacterial strains were isolated from three oil batteries in the region of Chauvin, Alberta, and characterized by 16S rRNA gene sequencing. Based on their nucleotide sequences, the strains are associated with 3 phyla (Actinobacteria, Proteobacteria and Firmicutes), as well as 17 other discrete genera that shared high homology with known sequences, with the majority of these strains identified to the species level. The most prevalent strains associated with the three oil wells belonged to the Bacillus genus. Thirty-four of the 123 strains were identified as biosurfactant-producers, among which Bacillus methylotrophicus strain OB9 exhibited the highest biosurfactant activity based on multiple screening methods and a comparative analysis with the commercially available biosurfactant, Tween 20. B. methylotrophicus OB9 was selected for further antimicrobial analysis and addition of live cultures of B. methylotrophicus OB9 (or partially purified biosurfactant fractions thereof) were highly effective on biofilm disruption in agar diffusion assays against several Gram-negative food-borne bacteria and plant pathogens. Upon co-culturing with B. methylotrophicus OB9, the number of either Salmonella enterica subsp. enterica Newport SL1 or Xanthomonas campestris B07.007 cells significantly decreased after 6 h and were not retrieved from co-cultures following 12 h exposure. These results also translated to studies on plants, where bacterized tomato seedlings with OB9 significantly protected the tomato leaves from Salmonella enterica Newport SL1 contamination, as evidenced by a 40% reduction of log₁₀ CFU of Salmonella/mg leaf tissue compared to non-bacterized tomato leaves. When B. methylotrophicus 0B9 was used for bacterized lettuce, the growth of X. campestris B07.007, the causal agent of bacterial leaf spot of lettuce, was completely inhibited. While limited, these studies are noteworthy as they demonstrate the inhibition spectrum of B. methylotrophicus 0B9 against both human and plant pathogens; thereby making this bacterium attractive for agricultural and food safety applications in a climate where microbial-biofilm persistence is an increasing problem.

We use continuous wavelet analysis (WA) of Laser Doppler Flux (LDF) time series measured in basal cell carcinomas (BCC) and plaque psoriasis (PP) in order to investigate the rhythmical behavior of blood flow in tumor or inflammatory associated neoangiogenesis.A total of 68 patients with primary BCCs and 40 patients with PP were included in the study. LDF time series were separated in four scaling levels corresponding to the influences of sympathetic activity (SL1), myogenic activity in the vessel wall (SL2), respiration (SL3) and heart beat (SL4).In BCC, SL1 decreased compared to healthy skin. In all other scaling levels, we found a statistically significant increase of the SLs compared to healthy skin. These increases were not found in PP.Rhythmical behavior of blood flow in malignant tumors is totally different from that in regions with inflammation. In BCCs, thermoregulatory processes, ascribed to sympathetic activity, decrease statistically significant. In contrast, inflammatory processes in PP do not substantially change sympathetic activity. WA of tumor perfusion could open a new noninvasive monitor system for controlling tumor therapy.

The E3 ubiquitin ligases participate in the degradation of plant proteins and play a regulatory role in stress response. However, the role of tomato E3 ubiquitin ligase genes in plant response to heavy metal stress remains elusive. Here, we identified 17 tomato E3 ubiquitin ligase genes using blast analysis of highly expressed E3 ubiquitin ligase genes of Arabidopsis thaliana. Through organ expression analysis, three E3 ubiquitin ligase genes with higher expression levels in roots were further screened out, and they were named Sl1, SlRHE1, and SlRING1. Among these three genes, SlRING1 expression was the highest in response to cadmium (Cd) stress. Silencing SlRING1 significantly decreased chlorophyll content, Fv/Fm, photosynthetic rate, and biomass accumulation under Cd stress. The levels of H₂O₂, electrolyte leakage, and malondialdehyde significantly increased in SlRING1-silenced plants under Cd stress compared with that in non-silenced tomato plants. Cd stress-induced increases in the transcript levels of antioxidant and detoxification genes such as CAT, DHAR, MDHAR, GSH, and PCS were compromised by SlRING1 silencing. Moreover, Cd accumulation in shoots and roots significantly increased in SlRING1-silenced plants compared with non-silenced tomato plants. These findings suggest that SlRING1 plays a positive role in plant tolerance to Cd stress in tomato.

Keywords: Cadmium; E3 ubiquitin ligase; oxidative stress; protein degradation; tomato.

Spliced leader trans-splicing (SLTS) plays a part in the maturation of pre-mRNAs in select species across multiple phyla but is particularly prevalent in Nematoda. The role of spliced leaders (SL) within the cell is unclear and an accurate assessment of SL occurrence within an organism is possible only after extensive sequencing data are available, which is not currently the case for many nematode species. SL discovery is further complicated by an absence of SL sequences from high-throughput sequencing results due to incomplete sequencing of the 5'-ends of transcripts during RNA-seq library preparation, known as 5'-bias. Existing datasets and novel methodology were used to identify both conserved SLs and unique hypervariable SLs within Heterodera glycines, the soybean cyst nematode. In H. glycines, twenty-one distinct SL sequences were found on 2,532 unique H. glycines transcripts. The SL sequences identified on the H. glycines transcripts demonstrated a high level of promiscuity, meaning that some transcripts produced as many as nine different individual SL-transcript combinations. Most uniquely, transcriptome analysis revealed that H. glycines is the first nematode to demonstrate a higher SL trans-splicing rate using a species-specific SL over well-conserved Caenorhabditis elegans SL-like sequences.

Discovery of trans-splicing in multiple metazoan lineages led to the identification of operon-like gene organization in diverse organisms, including trypanosomes, tunicates, and nematodes, but the functional significance of such operons is not completely understood. To see whether the content or organization of operons serves similar roles across species, we experimentally defined operons in the nematode model Pristionchus pacificus. We performed affinity capture experiments on mRNA pools to specifically enrich for transcripts that are trans-spliced to either the SL1- or SL2-spliced leader, using spliced leader-specific probes. We obtained distinct trans-splicing patterns from the analysis of three mRNA pools (total mRNA, SL1 and SL2 fraction) by RNA-seq. This information was combined with a genome-wide analysis of gene orientation and spacing. We could confirm 2219 operons by RNA-seq data out of 6709 candidate operons, which were predicted by sequence information alone. Our gene order comparison of the Caenorhabditis elegans and P. pacificus genomes shows major changes in operon organization in the two species. Notably, only 128 out of 1288 operons in C. elegans are conserved in P. pacificus. However, analysis of gene-expression profiles identified conserved functions such as an enrichment of germline-expressed genes and higher expression levels of operonic genes during recovery from dauer arrest in both species. These results provide support for the model that a necessity for increased transcriptional efficiency in the context of certain developmental processes could be a selective constraint for operon evolution in metazoans. Our method is generally applicable to other metazoans to see if similar functional constraints regulate gene organization into operons.

EMG responses to toe-up tilt perturbations on a movable platform system were analysed in 86 children between the age of 12 months and 13 years. To assess the relative contribution of peripheral and central nerve conduction properties, a concomitant recording of the fastest efferent pathways in the central and peripheral motor system was made using non-invasive transcranial magnetic stimulation of motor cortex and peripheral nerve roots. This allowed the determination of the fastest downstream efferent connection times from motor cortex to lumbar motor neuron pools and to measure the fastest efferent conduction from these motor neuron pools to effector muscles in the lower leg. The sequence observed for stance stabilizing EMG responses was similar to that obtained in earlier studies with short latency (SL) and middle latency (ML) companents occurring in the stretched triceps surae muscle and long latency (LL) responses occurring in the non-stretched tibialis anterior muscle. Homologous responses were also obtained in upper leg muscles, being recruited consistently later than those in lower leg muscles across all age groups. In the short latency range two different SL1- and SL2-responses were obtained in children of all age groups as well as in adult controls. Both the SL1- and the SL2-responses showed a flat developmental profile, reaching adult values between 20 and 30 months of age which correlated with that of the fastest efferents from lumbar motor neuron pools to leg muscles, i.e. the final motor path. ML-responses showed a steeper developmental profile.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial transcription termination factors (mTERFs) are highly conserved proteins in metazoans. Plants have many more mTERF proteins than animals. The functions and the underlying mechanisms of plants' mTERFs remain largely unknown. In plants, mTERF family proteins are present in both mitochondria and plastids and are involved in gene expression in these organelles through different mechanisms. In this study, we screened Arabidopsis mutants with pigment-defective phenotypes and isolated a T-DNA insertion mutant exhibiting seedling-lethal and albino phenotypes [seedling lethal 1 (sl1)]. The SL1 gene encodes an mTERF protein localized in the chloroplast stroma. The sl1 mutant showed severe defects in chloroplast development, photosystem assembly, and the accumulation of photosynthetic proteins. Furthermore, the transcript levels of some plastid-encoded proteins were significantly reduced in the mutant, suggesting that SL1/mTERF3 may function in the chloroplast gene expression. Indeed, SL1/mTERF3 interacted with PAP12/PTAC7, PAP5/PTAC12, and PAP7/PTAC14 in the subgroup of DNA/RNA metabolism in the plastid-encoded RNA polymerase (PEP) complex. Taken together, the characterization of the plant chloroplast mTERF protein, SL1/mTERF3, that associated with PEP complex proteins provided new insights into RNA transcription in the chloroplast.

Keywords: Arabidopsis; chloroplast; mitochondrial transcription termination factor; nucleoid; plastid-encoded RNA polymerase.

In Saccharomyces cerevisiae, Core Factor (CF) is a key evolutionarily conserved transcription initiation factor that helps recruit RNA polymerase I (Pol I) to the ribosomal DNA (rDNA) promoter. Upregulated Pol I transcription has been linked to many cancers, and targeting Pol I is an attractive and emerging anti-cancer strategy. Using yeast as a model system, we characterized how CF binds to the Pol I promoter by electrophoretic mobility shift assays (EMSA). Synthetic DNA competitors along with anti-tumor drugs and nucleic acid stains that act as DNA groove blockers were used to discover the binding preference of yeast CF. Our results show that CF employs a unique binding mechanism where it prefers the GC-rich minor groove within the rDNA promoter. In addition, we show that yeast CF is able to bind to the human rDNA promoter sequence that is divergent in DNA sequence and demonstrate CF sensitivity to the human specific Pol I inhibitor, CX-5461. Finally, we show that the human Core Promoter Element (CPE) can functionally replace the yeast Core Element (CE) in vivo when aligned by conserved DNA structural features rather than DNA sequence. Together, these findings suggest that the yeast CF and the human ortholog Selectivity Factor 1 (SL1) use an evolutionarily conserved, structure-based mechanism to target DNA. Their shared mechanism may offer a new avenue in using yeast to explore current and future Pol I anti-cancer compounds.

The literature confirms that individuals with autism spectrum disorder (ASD) have planning deficits. However, few interventions have targeted these deficits. The aims of this study were to: (1) show that the mixed methods approach can be useful in studying planning skills of children with ASD during and after an educational intervention; (2) assess whether the planning skills of two groups of children with ASD improved during the intervention and if this progress was maintained 1 month after completing the intervention. The groups were formed depending on each child's severity level (SL) of ASD according to DSM-5: SL1 (requiring support) and SL2 (requiring substantial support). Each group was composed of four children. In the framework of mixed methods, we used observational methodology, which is considered as mixed methods in itself because it integrates qualitative and quantitative elements. A nomothetic/follow-up/multidimensional observational design was used. Planning skills manifested by children during the intervention were codified, as well as the scaffolding behaviors provided by the educational specialist. These skills and behaviors were also coded in one session, which took place 1 month after the intervention. Coded data of each group were submitted to prospective and retrospective lag sequential analysis. This informed of the sequential structure of planning skills performed by children in interaction with the educational specialist at the beginning and at the end of the intervention, as well as 1 month later after the intervention. The comparison of the patterns obtained in these three temporal moments allowed us to know the improvement of the two groups in the use of planning skills. Results showed that both groups improved their autonomous use of planning skills. However, SL1 group used successfully and autonomously complex planning skills, while SL2 group were unable to achieve this gain. SL2 group progressed in autonomy, but only using basic planning skills. Both groups can further improve their use of planning skills; therefore, the intervention should be adjusted to their characteristics and temporarily extended. These findings contribute to the, as yet, little studied field of intervention and assessment of planning skills in children with ASD using a mixed methods approach.

A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy.

BACKGROUND

The dimer initiation site/dimer linkage sequence (DIS/DLS) region of HIV is located on the 5' end of the viral genome and suggested to form complex secondary/tertiary structures. Within this structure, stem-loop 1 (SL1) is believed to be most important and an essential key to dimerization, since the sequence and predicted secondary structure of SL1 are highly stable and conserved among various virus subtypes. In particular, a six-base palindromic sequence is always present at the hairpin loop of SL1 and the formation of kissing-loop structure at this position between the two strands of genomic RNA is suggested to trigger dimerization. Although the higher-order structure model of SL1 is well accepted and perhaps even undoubted lately, there could be stillroom for consideration to depict the functional SL1 structure while in vivo (in virion or cell).

RESULTS

In this study, we performed several analyses to identify the nucleotides and/or basepairing within SL1 which are necessary for HIV-1 genome dimerization, encapsidation, recombination and infectivity. We unexpectedly found that some nucleotides that are believed to contribute the formation of the stem do not impact dimerization or infectivity. On the other hand, we found that one G-C basepair involved in stem formation may serve as an alternative dimer interactive site. We also report on our further investigation of the roles of the palindromic sequences on viral replication. Collectively, we aim to assemble a more-comprehensive functional map of SL1 on the HIV-1 viral life cycle.

CONCLUSIONS

We discovered several possibilities for a novel structure of SL1 in HIV-1 DLS. The newly proposed structure model suggested that the hairpin loop of SL1 appeared larger, and genome dimerization process might consist of more complicated mechanism than previously understood. Further investigations would be still required to fully understand the genome packaging and dimerization of HIV.

Melatonin is a well-known bioactive molecule with an array of health-promoting properties. Here, we detected the physiological function of melatonin in transgenic switchgrass overexpressing the homologous sheep arylalkylamine N-acetyltransferase and hydroxyindole O-methyltransferase genes, which catalyze the last two steps of melatonin synthesis. Compared to the wild-type (WT) and transgenic control (EV, expressing the empty vector only) plants, the transgenic switchgrass showed higher melatonin levels. Melatonin was detected in almost all switchgrass tissues, and relatively higher levels were detected in the roots and stems. Besides, melatonin showed diurnal or circadian rhythms in switchgrass similar to that in other species. Furthermore, we also found that melatonin positively affected switchgrass growth, flowering and salt tolerance. The genes related to flowering (APL3, SL1, FT1, FLP3, MADS6 and MADS15) and salt stress resistance (PvNHX1) in transgenic switchgrass exhibited a different expression profiles when compared to the control plants. Our study provided valuable findings that melatonin functions as a promoter in the regulation of switchgrass growth, flowering and salt tolerance.