Dates and rates in grape's plastomes: evolution in slow motion.

The family Vitaceae includes the domesticated grapevine (Vitis vinifera), one of the most economically important crops in the world. Despite the importance of Vitaceae, there is still considerable controversy surrounding their phylogenetic relationships and evolutionary timescales. Moreover, variation in rates of molecular evolution among Vitaceae remains mostly unexplored. The present research aims to fill these knowledge gaps through the analysis of plastome sequences. Thirteen newly sequenced grape plastomes are presented and their phylogenetic relationships examined. Divergence times and absolute substitution rates are inferred under different molecular clocks by the analysis of 95 non-coding plastid regions and 43 representative accessions of the major lineages of Vitaceae. Furthermore, the phylogenetic informativeness of non-coding plastid regions is investigated. We find strong evidence in favor of the random local clock model and rate heterogeneity within Vitaceae. Substitution rates decelerate in Ampelocissus, Ampelopsis, Nekemias, Parthenocissus, Rhoicissus, and Vitis, with genus Vitis showing the lowest values up to a minimum of ~ 4.65 × 10-11 s/s/y. We suggest that liana-like species of Vitaceae evolve slower than erect growth habit plants and we invoke the "rate of mitosis hypothesis" to explain the observed pattern of the substitution rates. We identify a reduced set of 20 non-coding regions able to accurately reconstruct the phylogeny of Vitaceae and we provide a detailed description of all 152 non-coding regions identified in the plastomes of subg. Vitis. These polymorphic regions will find their applications in phylogenetics, phylogeography, and population genetics as well in grapes identification through DNA barcoding techniques.

Microbial Diversity and Phage-Host Interactions in the Georgian Coastal Area of the Black Sea Revealed by Whole Genome Metagenomic Sequencing.

Viruses have the greatest abundance and highest genetic diversity in marine ecosystems. The interactions between viruses and their hosts is one of the hot spots of marine ecology. Besides their important role in various ecosystems, viruses, especially bacteriophages and their gene pool, are of enormous interest for the development of new gene products with high innovation value. Various studies have been conducted in diverse ecosystems to understand microbial diversity and phage-host interactions; however, the Black Sea, especially the Eastern coastal area, remains among the least studied ecosystems in this regard. This study was aimed at to fill this gap by analyzing microbial diversity and bacteriophage-host interactions in the waters of Eastern Black Sea using a metagenomic approach. To this end, prokaryotic and viral metagenomic DNA from two sampling sites, Poti and Gonio, were sequenced on the Illumina Miseq platform and taxonomic and functional profiles of the metagenomes were obtained using various bioinformatics tools. Our metagenomics analyses allowed us to identify the microbial communities, with Proteobacteria, Cyanobacteria, Actinibacteria, and Firmicutes found to be the most dominant bacterial phyla and Synechococcus and Candidatus Pelagibacter phages found to be the most dominant viral groups in the Black Sea. As minor groups, putative phages specific to human pathogens were identified in the metagenomes. We also characterized interactions between the phages and prokaryotic communities by determining clustered regularly interspaced short palindromic repeats (CRISPR), prophage-like sequences, and integrase/excisionase sequences in the metagenomes, along with identification of putative horizontally transferred genes in the viral contigs. In addition, in the viral contig sequences related to peptidoglycan lytic activity were identified as well. This is the first study on phage and prokaryote diversity and their interactions in the Eastern coastal area of the Black Sea using a metagenomic approach.

Associations between genetic variations and global motion perception.

The cholinergic system is known to strongly modulate perceptual and cognitive processes, and the alpha7 subunit of the cholinergic nicotinic receptor (CHRNA7) is broadly expressed within the visual system. Here, we assessed whether genetic variations of CHRNA7 affect coherent motion perception. Motion perception has been shown to decline with age, and it has previously been suggested that the effects of genetic variations are magnified by age. Therefore, we tested both older (n = 62) and younger adults (n = 63). We found that motion coherence thresholds were significantly higher for older compared to younger adults, which is in accordance with previous studies. Interestingly, there was a strong relationship between variants of the SNP rs2337980 of the CHRNA7 and motion direction discrimination. In particular, participants carrying the TC genotype had considerably lower motion coherence thresholds than CC carriers. The effect of genotype did not interact with age. Our results show that genetic variations are associated with perceptual performance, but are unlikely to explain age-related changes.

Intra-epidemic genome variation in highly pathogenic African swine fever virus (ASFV) from the country of Georgia.

BACKGROUND: African swine fever virus (ASFV) causes an acute hemorrhagic infection in suids with a mortality rate of up to 100%. No vaccine is available and the potential for catastrophic disease in Europe remains elevated due to the ongoing ASF epidemic in Russia and Baltic countries. To date, intra-epidemic whole-genome variation for ASFV has not been reported. To provide a more comprehensive baseline for genetic variation early in the ASF outbreak, we sequenced two Georgian ASFV samples, G-2008/1 and G-2008/2, derived from domestic porcine blood collected in 2008. METHODS: Genomic DNA was extracted directly from low-volume ASFV PCR-positive porcine blood samples and subjected to next generation sequencing on the Illumina Miseq platform. De novo and mapped sequence assemblies were performed using CLCBio software. Genomic illustrations, sequence alignments and assembly figures were generated using Geneious v10.2.4. Sequence repeat architecture was analyzed using DNASTAR GeneQuest 14.1.0. RESULTS: The G-2008/1 and G-2008/2 genomes were distinguished from each other by coding changes in seven genes, including MGF 110-1 L, X69R, MGF 505-10R, EP364R, H233R, E199L, and MGF 360-21R in addition to eight homopolymer tract variations. The 2008/2 genome possessed a novel allele state at a previously undescribed intergenic repeat locus between genes C315R and C147L. The C315R/C147L locus represents the earliest observed variable repeat sequence polymorphism reported among isolates from this epidemic. No sequence variation was observed in conventional ASFV subtyping markers. The two genomes exhibited complete collinearity and identical gene content with the Georgia 2007/1 reference genome. Approximately 56 unique homopolymer A/T-tract variations were identified that were unique to the Georgia 2007/1 genome. In both 2008 genomes, within-sample sequence read heterogeneity was evident at six homopolymeric G/C-tracts confined to the known hypervariable ~ 7 kb region in the left terminal region of the genome. CONCLUSIONS: This is the first intra-epidemic comparative genomic analysis reported for ASFV and provides insight into the intra-epidemic microevolution of ASFV. The genomes reported here, in addition to the G-2007/1 genome, provide an early baseline for future genome-level comparisons and epidemiological tracing efforts.

Impact of hepatitis C virus recombinant form RF1_2k/1b on treatment outcomes within the Georgian national hepatitis C elimination program.

AIM: Hepatitis C virus (HCV) recombinant form RF1_2k/1b is common in ethnic Georgians. This chimera virus contains genomic fragments of genotype 2 and genotype 1 and is misclassified as genotype 2 by standard genotyping. We aimed to identify RF1_2k/1b strains among genotype 2 patients and assess its impact on treatment outcomes. METHODS: The study included 148 patients with HCV genotype 2 as determined by 5-untranslated region/core genotyping assay. RF1_2k/1b was identified by sequencing the non-structural protein 5B region. Patients were treated within the national hepatitis C elimination program with sofosbuvir/ribavirin (SOF/RBV), interferon (IFN)/SOF/RBV, or ledipasvir (LDV)/SOF/RBV. RESULTS: Of 148 patients, 103 (69.5%) had RF1_ 2k/1b. Sustained virologic response (SVR) data was available for 136 patients (RF1_ 2k/1b, n = 103; genotype 2, n = 33). Sustained virologic response was achieved in more genotype 2 patient than in RF1_2k/1b patients (97.0% vs. 76.7%, P = 0.009). Twelve weeks of LDV/SOF/RBV treatment was highly effective (100% SVR) in both genotypes. Among RF1_2k/1b patients, LDV/SOF/RBV for 12 weeks was superior (100% SVR) to SOF/RBV for 12 weeks (56.4%, P < 0.0001) or 20 weeks (79.2%, P = 0.05). Twelve weeks of IFN/SOF/RBV also showed better response than SOF/RBV for 12 weeks (88.9% vs. 56.4%, P = 0.02) in these patients. CONCLUSIONS: High prevalence of the RF1_2k/1b strain can significantly affect treatment outcomes. Treatment with IFN/SOF/RBV and especially LDV/SOF/RBV ensured significantly higher SVR in patients infected with RF1_2k/1b strain compared to standard HCV genotype 2 treatment with SOF/RBV. There is a need to reassess existing methods for the management of HCV genotype 2 infections, especially in areas with high prevalence of the RF1_2k/1b strain.

Complete chloroplast genomes of Aegilops tauschii Coss. and Ae. cylindrica Host sheds light on plasmon D evolution.

Hexaploid wheat (Triticum aestivum L., genomes AABBDD) originated in South Caucasus by allopolyploidization of the cultivated Emmer wheat T. dicoccum (genomes AABB) with the Caucasian Ae. tauschii ssp strangulata (genomes DD). Genetic variation of Ae. tauschii is an important natural resource, that is why it is of particular importance to investigate how this variation was formed during Ae. tauschii evolutionary history and how it is presented through the species area. The D genome is also found in tetraploid Ae. cylindrica Host (2n = 28, CCDD). The plasmon diversity that exists in Triticum and Aegilops species is of great significance for understanding the evolution of these genera. In the present investigation the complete nucleotide sequence of plasmon D (chloroplast DNA) of nine accessions of Ae. tauschii and two accessions of Ae. cylindrica are presented. Twenty-eight SNPs are characteristic for both TauL1 and TauL2 accessions of Ae. tauschii using TauL3 as a reference. Four SNPs are additionally observed for TauL2 lineage. The longest (27 bp) indel is located in the intergenic spacer Rps15-ndhF of SSC. This indel can be used for simple determination of TauL3 lineage among Ae. tauschii accessions. In the case of Ae. cylindrica additionally 7 SNPs were observed. The phylogeny tree shows that chloroplast DNA of TauL1 and TauL2 diverged from the TauL3 lineage. TauL1 lineage is relatively older then TauL2. The position of Ae. cylindrica accessions on Ae. tauschii phylogeny tree constructed on chloroplast DNA variation data is intermediate between TauL1 and TauL2. The complete nucleotide sequence of chloroplast DNA of Ae. tauschii and Ae. cylindrica allows to refine the origin and evolution of D plasmon of genus Aegilops.

Whole genome sequence comparison of ten diagnostic brucellaphages propagated on two Brucella abortus hosts.

BACKGROUND: Recently the genome sequences of two brucellaphages, isolated in Georgia (Tb) and Mexico (Pr) were reported revealing pronounced sequence homogeneity and the presence of two major indels discriminating the two phages. Subsequent genome sequencing of six diagnostic brucellaphages: Tbilisi (Tb), Firenze (Fz), Weybridge (Wb), S708, Berkeley (Bk) and R/C phages identified three major genetic groups. However, the propensity for fine-scale genetic variability of diverse brucellaphages grown on multiple hosts within a single Brucella species remains unknown. METHODS: We sequenced the complete genomes of ten brucellaphages following initial propagation on B. abortus strain 141 and after subsequent propagation on B. abortus strain S19. All brucellaphages were isolated and propagated at the Eliava Institute including the original Tb phage. Genomic libraries were quantified using the Qbit and sheared on the Covaris M220. QC for fragmentation was performed on the BioAnalyzer 2100. DNA libraries were prepared using an Illumina Paired-End protocol and sequenced on the Illumina MiSeq. Sequence analysis was performed using Geneious and MEGA software. RESULTS: Comparative whole genome sequence analysis revealed genetic homogeneity consistent with previously published data as well as multiple nucleotide variations. Genomic changes as a result of passages were observed in similar genes and predominantly occurred at identical sites in separate phages. Multiple instances of within-sample genetic heterogeneity were observed often at shared genomics positions across phages. Positive selection was detected in the tail collar protein gene. We also identified a Staphylothermus marinus F1-like CRISPR spacer and sequences orthologous to both prophage antirepressors of Brucella spp. and intergenic sequences encoded by Ochrobactrum anthropi. CONCLUSION: We surveyed whole genome level diversity in phage lytic for B. abortus as they are propagated on alternate vaccine strains within the species. Our data extend previous results indicating select variable hotspots and broad genomic homogeneity as well as multiple common polymorphisms and within-sample variation. These data also provide additional genomes for future reference in comparative studies involving the molecular evolution and host specificity of brucellaphages.

A nonaggregating surfactant protein C mutant is misdirected to early endosomes and disrupts phospholipid recycling.

Interstitial lung disease in both children and adults has been linked to mutations in the lung-specific surfactant protein C (SFTPC) gene. Among these, the missense mutation [isoleucine to threonine at codon 73 = human surfactant protein C (hSP-C(I73T) )] accounts for ∼30% of all described SFTPC mutations. We reported previously that unlike the BRICHOS misfolding SFTPC mutants, expression of hSP-C(I73T) induces lung remodeling and alveolar lipoproteinosis without a substantial Endoplasmic Reticulum (ER) stress response or ER-mediated intrinsic apoptosis. We show here that, in contrast to its wild-type counterpart that is directly routed to lysosomal-like organelles for processing, SP-C(I73T) is misdirected to the plasma membrane and subsequently internalized to the endocytic pathway via early endosomes, leading to the accumulation of abnormally processed proSP-C isoforms. Functionally, cells expressing hSP-C(I73T) demonstrated both impaired uptake and degradation of surfactant phospholipid, thus providing a molecular mechanism for the observed lipid accumulation in patients expressing hSP-C(I73T) through the disruption of normal phospholipid recycling. Our data provide evidence for a novel cellular mechanism for conformational protein-associated diseases and suggest a paradigm for mistargeted proteins involved in the disruption of the endosomal/lysosomal sorting machinery.

Phenotypic and Genetic Characterization of Aeromonas hydrophila Phage AhMtk13a and Evaluation of Its Therapeutic Potential on Simulated Aeromonas Infection in Danio rerio.

Phage therapy can be an effective alternative to standard antimicrobial chemotherapy for control of Aeromonas hydrophila infections in aquaculture. Aeromonas hydrophila-specific phages AhMtk13a and AhMtk13b were studied for basic biological properties and genome characteristics. Phage AhMtk13a (Myovirus, 163,879 bp genome, 41.21% CG content) was selected based on broad lytic spectrum and physiologic parameters indicating its lytic nature. The therapeutic potential of phage AhMtk13a was evaluated in experimental studies in zebrafish challenged with A. hydrophila GW3-10 via intraperitoneal injection and passive immersion in aquaria water. In experimental series 1 with single introduction of AhMtk13a phage to aquaria water at phage-bacteria ratio 10:1, cumulative mortality 44% and 62% was registered in fish exposed to phage immediately and in 4 h after bacterial challenge, correspondingly, compared to 78% mortality in the group with no added phage. In experimental series 2 with triple application of AhMtk13a phage at ratio 100:1, the mortality comprised 15% in phage-treated group compared to the 55% in the control group. Aeromonas hydrophila GW3-10 was not detectable in aquaria water from day 9 but still present in fish at low concentration. AhMtk13a phage was maintained in fish and water throughout the experiment at the higher concentration in infected fish.

Phylogeny of caucasian rock lizards (Darevskia) and other true lizards based on mitogenome analysis: Optimisation of the algorithms and gene selection.

We generated a phylogeny for Caucasian rock lizards (Darevskia), and included six other families of true lizards (Lacertini), based on complete mitochondrial genome analysis. Next-generation sequencing (NGS) of genomic DNA was used to obtain 16 new mitogenomes of Darevskia. These, along with 35 sequences downloaded from GenBank: genera Darevskia, Zootoca, Podarcis, Phoenicolacerta, Takydromus, Lacerta, and Eremias-were used in the analysis. All four analytical methods (Bayesian Inference, BI; Maximum Likelihood, ML; Maximum Parsimony, MP; and Neighbor-Joining, NJ) showed almost congruent intra-generic topologies for Darevskia and other lizard genera. However, ML and NJ methods on one side, and BI and MP methods on the other harvested conflicting phylogenies. The ML/NJ topology supports earlier published separation of Darevskia into three mitochondrial clades (Murphy, Fu, Macculloch, Darevsky, and Kupinova, 2000), but BI and MP topologies support that the basal branching occurred between D. parvula from the western Lesser Caucasus and the rest of Darevskia. All topologies altered the phylogenetic position of some individual species, including D. daghestanica, D. derjugini, and D. chlorogaster. Reanalysis after excluding four saturated genes from the data set, and excluding genus Eremias gives fully convergent topologies. The most basal branching for true lizards was between Far Eastern Takydromus and the Western Eurasian genera (BI). Comparing phylogenetic performance of individual genes relative to whole mitogenome data, concatenated 16S RNA (the least saturated gene in our analyses) and Cytochrome b genes generate a robust phylogeny that is fully congruent with that based on the complete mitogenome.

Comparative genomic analysis of four multidrug-resistant isolates of Acinetobacter baumannii from Georgia.

OBJECTIVES: This study reports the draft genomes of four newly isolated multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) isolates (0830, 0365, 4022, and 2846) from western Georgia to identify putative antimicrobial resistance genes (ARGs) and to determine the clonal subtypes of local clinical isolates. METHODS: An Illumina MiSeq sequencer was used to perform whole-genome sequencing (WGS). The Vitek 2 automated system was used for microbial identification and antimicrobial resistance profiling. RESULTS: Taxonomical identification as A. baumannii was confirmed by WGS. In silico analyses resolved their ARG content and clonal relatedness using the Oxford (Oxf) and Pasteur (Pas) multi-locus sequence typing schemes. Isolates 0365 and 4022 displayed similar allelic profiles corresponding to ST944Oxf/ST78Pas. Isolate 2846 displayed a different allelic profile consistent with ST19Pas/IC 1 (International or European Clone I) and exhibited a novel Oxford ST that was designated as 1868. Isolate 0830 displayed the ST78Pas allelic profile, similar to isolates 0365 and 4022, and also possessed a single allelic mismatch in the gpi gene, resulting in an ST1104Oxf allele profile in the Oxford typing scheme. CONCLUSION: Circulating MDR A. baumannii exhibited genetic heterogeneity with variations in the structure and content of genomic A. baumannii resistance islands and encoded multiple putative ARGs. This report represents the first clonal subtype information and genomic characterization of MDR A. baumannii in Georgia and may inform future epidemiological investigations.

The identification of novel single nucleotide polymorphisms to assist in mapping the spread of Bacillus anthracis across the Southern Caucasus.

Anthrax is common as a zoonotic disease in the southern Caucasus area including parts of Turkey and Georgia. In this region, population genetics of the etiological agent Bacillus anthracis comprises, where known, the major canonical single nucleotide polymorphism (canSNP) groups A.Br.Aust94 and A.Br.008/009 of the pathogen's global phylogeny, respectively. Previously, isolates of B. anthracis from Turkey have been genotyped predominantly by multi locus variable number of tandem repeat analysis (MLVA) or canSNP typing. While whole genome sequencing is the future gold standard, it is currently still costly. For that reason we were interested in identifying novel SNPs which could assist in further distinguishing closely related isolates using low cost assay platforms. In this study we sequenced the genomes of seven B. anthracis strains collected from the Kars province of Eastern Anatolia in Turkey and discovered new SNPs which allowed us to assign these and other geographically related strains to three novel branches of the major A-branch canSNP-group (A.Br.) Aust94. These new branches were named Kafkas-Geo 1-3 and comprised isolates from the Kars region and the neighboring republic of Georgia suggesting a common ancestry. The novel SNPs identified in this study connect the population genetics of B. anthracis in the South Caucasus and Turkey and will likely assist efforts to map the spread of the pathogen across this region.

Three Draft Genome Sequences of the Bacterial Plant Pathogen Ralstonia solanacearum, Isolated in Georgia.

Ralstonia solanacearum, the causative agent of bacterial wilt, is a devastating bacterial plant pathogen with a wide range of hosts. We report here the first draft genome sequences for three strains of Ralstonia solanacearum isolated from infected potato, tomato, and pepper plants in Georgia.

Genome Sequence of the Soviet/Russian Bacillus anthracis Vaccine Strain 55-VNIIVViM.

Bacillus anthracis strain 55-VNIIVViM is a live-attenuated nonencapsulated Soviet/Russian veterinary anthrax vaccine strain. We report here the genome of 55-VNIIVViM and confirm its phylogenetic placement in the global population structure of B. anthracis.

Comparative whole genome analysis of six diagnostic brucellaphages.

Whole genome sequencing of six diagnostic brucellaphages, Tbilisi (Tb), Firenze (Fz), Weybridge (Wb), S708, Berkeley (Bk) and R/C, was followed with genomic comparisons including recently described genomes of the Tb phage from Mexico (TbM) and Pr phage to elucidate genomic diversity and candidate host range determinants. Comparative whole genome analysis revealed high sequence homogeneity among these brucellaphage genomes and resolved three genetic groups consistent with defined host range phenotypes. Group I was composed of Tb and Fz phages that are predominantly lytic for Brucella abortus and Brucella neotomae; Group II included Bk, R/C, and Pr phages that are lytic mainly for B. abortus, Brucella melitensis and Brucella suis; Group III was composed of Wb and S708 phages that are lytic for B. suis, B. abortus and B. neotomae. We found that the putative phage collar protein is a variable locus with features that may be contributing to the host specificities exhibited by different brucellaphage groups. The presence of several candidate host range determinants is illustrated herein for future dissection of the differential host specificity observed among these phages.

Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.

BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. PRINCIPAL FINDINGS: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and -RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. SIGNIFICANCE: We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies.

Anterograde transport of surfactant protein C proprotein to distal processing compartments requires PPDY-mediated association with Nedd4 ubiquitin ligases.

Biosynthesis of surfactant protein C (SP-C) by alveolar type 2 cells requires proteolytic processing of a 21-kDa propeptide (proSP-C21) in post-Golgi compartments to yield a 3.7-kDa mature form. Scanning alanine mutagenesis, binding assays, and co-immunoprecipitation were used to characterize the proSP-C targeting domain. Delivery of proSP-C21 to distal processing organelles is dependent upon the NH2-terminal cytoplasmic SP-C propeptide, which contains a conserved PPDY motif. In A549 cells, transfection of EGFP/proSP-C21 constructs containing polyalanine substitution for Glu11-Thr18, 13PPDY16, or 14P,16Y produced endoplasmic reticulum retention of the fusion proteins. Protein-protein interactions of proSP-C with known WW domains were screened using a solid-phase array that revealed binding of the proSP-C NH2 terminus to several WW domains found in the Nedd4 family of E3 ligases. Specificity of the interaction was confirmed by co-immunoprecipitation of proSP-C and Nedd4 or Nedd4-2 in epithelial cell lines. By Western blotting and reverse transcription-PCR, both forms were detected in primary human type 2 cells. Knockdown of Nedd4-2 by small interference RNA transfection of cultured human type 2 cells blocked processing of 35S-labeled proSP-C21. Mutagenesis of potential acceptor sites for ubiquitination in the cytosolic domain of proSP-C (Lys6, Lys34, or both) failed to inhibit trafficking of EGFP/proSP-C21. These results indicate that PPDY-mediated interaction with Nedd4 E3-ligases is required for trafficking of proSP-C. We speculate that the Nedd4/proSP-C tandem is part of a larger protein complex containing a ubiquitinated component that further directs its transport.

Misfolded BRICHOS SP-C mutant proteins induce apoptosis via caspase-4- and cytochrome c-related mechanisms.

Several mutations within the BRICHOS domain of surfactant protein C (SP-C) have been linked to interstitial lung disease. Recent studies have suggested that these mutations cause misfolding of the proprotein (proSP-C), which initiates the unfolded protein response to resolve improper folding or promote protein degradation. We have reported that in vitro expression of one of these proteins, the exon 4 deletion mutant (hSP-C(Deltaexon4)), causes endoplasmic reticulum (ER) stress, inhibits proteasome function, and activates caspase-3-mediated apoptosis. To further elucidate mechanisms and common pathways for cellular dysfunction, various assays were performed by transiently expressing two SP-C BRICHOS domain mutant (BRISPC) proteins (hSP-C(Deltaexon4), hSP-C(L188Q)) and control proteins in lung epithelium-derived A549 and kidney epithelium-derived (HEK-293) GFP(u)-1 cell lines. Compared with controls, cells expressing either BRICHOS mutant protein consistently exhibited increased formation of insoluble aggregates, enhanced promotion of inositol-requiring enzyme 1-dependent splicing of X-box binding protein-1 (XBP-1), significant inhibition of proteasome activity, enhanced induction of mitochondrial cytochrome c release, and increased activations of caspase-4 and caspase-3, leading to apoptosis. These results suggest common cellular responses, including initiation of cell-death signaling pathways, to these lung disease-associated BRISPC proteins.

Ca2+/calmodulin protein kinase II and memory: learning-related changes in a localized region of the domestic chick brain.

The role of calcium/calmodulin-dependent protein kinase II (CaMKII) in the recognition memory of visual imprinting was investigated. Domestic chicks were exposed to a training stimulus and learning strength measured. Trained chicks, together with untrained chicks, were killed either 1 h or 24 h after training. The intermediate and medial hyperstriatum ventrale/mesopallium (IMHV/IMM), a forebrain memory storage site, was removed together with a control brain region, the posterior pole of the neostriatum/nidopallium (PPN). Amounts of membrane total alphaCaMKII (tCaMKII) and Thr286-autophosphorylated alphaCaMKII (apCAMKII) were measured. For the IMHV/IMM 1 h group, apCaMKII amount and apCAMKII/tCaMKII increased as chicks learned. The magnitude of the molecular changes were positively correlated with learning strength. No learning-related effects were observed in PPN, or in either region at 24 h. These results suggest that CaMKII is involved in the formation of memory but not in its maintenance.