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Peptides present an alternative modality to immunoglobulin domains or small molecules for developing therapeutics to either agonize or antagonize cellular pathways associated with diseases. However, peptides often suffer from poor chemical and physical stability, limiting their therapeutic potential. Disulfide-constrained peptides (DCP) are naturally occurring and possess numerous desirable properties, such as high stability, that qualify them as drug-like scaffolds for peptide therapeutics. DCPs contain loop regions protruding from the core of the molecule that are amenable to peptide engineering via direct evolution by use of phage display technology. In this study, we have established a robust platform for the discovery of peptide therapeutics using various DCPs as scaffolds. We created diverse libraries comprising seven different DCP scaffolds, resulting in an overall diversity of 2 x 1011. The effectiveness of this platform for functional hit discovery has been extensively evaluated, demonstrating a hit rate comparable to that of synthetic antibody libraries. By utilizing chemically synthesized and in vitro folded peptides derived from selections of phage displayed DCP libraries, we have successfully generated functional inhibitors targeting the HtrA1 protease. Through affinity maturation strategies, we have transformed initially weak binders against Notch2 with micromolar Kd values to high-affinity ligands in the nanomolar range. This process highlights a viable hit-to-lead progression. Overall, our platform holds significant potential to greatly enhance the discovery of peptide therapeutics.

In this study, we identified and compared nucleotide-binding site (NBS) domain-containing genes from three Citrus genomes (C. clementina, C. sinensis from USA and C. sinensis from China). Phylogenetic analysis of all Citrus NBS genes across these three genomes revealed that there are three approximately evenly numbered groups: one group contains the Toll-Interleukin receptor (TIR) domain and two different Non-TIR groups in which most of proteins contain the Coiled Coil (CC) domain. Motif analysis confirmed that the two groups of CC-containing NBS genes are from different evolutionary origins. We partitioned NBS genes into clades using NBS domain sequence distances and found most clades include NBS genes from all three Citrus genomes. This suggests that three Citrus genomes have similar numbers and types of NBS genes. We also mapped the re-sequenced reads of three pomelo and three mandarin genomes onto the C. sinensis genome. We found that most NBS genes of the hybrid C. sinensis genome have corresponding homologous genes in both pomelo and mandarin genomes. The homologous NBS genes in pomelo and mandarin suggest that the parental species of C. sinensis may contain similar types of NBS genes. This explains why the hybrid C. sinensis and original C. clementina have similar types of NBS genes in this study. Furthermore, we found that sequence variation amongst Citrus NBS genes were shaped by multiple independent and shared accelerated mutation accumulation events among different groups of NBS genes and in different Citrus genomes. Our comparative analyses yield valuable insight into the structure, organization and evolution of NBS genes in Citrus genomes. Furthermore, our comprehensive analysis showed that the non-TIR NBS genes can be divided into two groups that come from different evolutionary origins. This provides new insights into non-TIR genes, which have not received much attention.

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases of citrus worldwide. HLB is associated with three species of 'Candidatus Liberibacter' with 'Ca. L. asiaticus' (Las) being the most widely distributed around the world, and the only species detected in Thailand. To understand the genetic diversity of Las bacteria in Thailand, we evaluated two closely-related effector genes, lasAI and lasAII, found within the Las prophages from 239 infected citrus and 55 infected psyllid samples collected from different provinces in Thailand. The results indicated that most of the Las-infected samples collected from Thailand contained at least one prophage sequence with 48.29% containing prophage 1 (FP1), 63.26% containing prophage 2 (FP2), and 19.38% containing both prophages. Interestingly, FP2 was found to be the predominant population in Las-infected citrus samples while Las-infected psyllids contained primarily FP1. The multiple banding patterns that resulted from amplification of lasAI imply extensive variation exists within the full and partial repeat sequence while the single band from lasAII indicates a low amount of variation within the repeat sequence. Phylogenetic analysis of Las-infected samples from 22 provinces in Thailand suggested that the bacterial pathogen may have been introduced to Thailand from China and the Philippines. This is the first report evaluating the genetic variation of a large population of Ca. L. asiaticus infected samples in Thailand using the two effector genes from Las prophage regions.

EML4-ALK is a new driver gene of non-small cell lung cancer and a target of crizotinib. The objectives of this study were to determine the frequency of ALK rearrangements in a large cohort of patients with primary lung adenocarcinoma and to analyze the association of ALK rearrangements with clinicopathological characteristics and clinical outcomes. The roles of fluorescence in situ hybridization (FISH), Ventana immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR) in the detection of ALK rearrangements were evaluated. The ALK rearrangement was detected in 430 specimens from individual patients with primary lung adenocarcinoma using FISH and Ventana IHC based on tissue microarrays. The EGFR status was detected in all of the specimens through DNA sequencing. An RT-PCR was performed on 200 of the specimens and confirmed by sequencing. Of the 430 patients, 46 (10.7%) harbored ALK rearrangements. The ALK rearrangements were associated with a younger age and the EGFR wild type in comparison with ALK-negative patients. The sensitivity and specificity of the Ventana IHC were 100% and 98.2%, respectively, and the concordance rate between the FISH and the Ventana IHC was 98.4%. The sensitivity and specificity of RT-PCR were 95.5% and 87.0%, respectively, and the concordance rate between the FISH and the RT-PCR was 89.0%. The Cox analysis indicated that an early stage and EGFR-activating mutations were independently associated with a longer OS. This study demonstrated that ALK rearrangements are associated with a younger age and the EGFR wild type rather than with other clinicopathological factors. Although the FISH and Ventana IHC have better concordance, and RT-PCR is a more sensitive method and can identify different variants or partners, the IHC and RT-PCR need to be further evaluated in clinical trials to identify their roles in guiding patients' targeted therapy using crizotinib.

'Candidatus Liberibacter asiaticus' (Las) is the primary causal agent of huanglongbing (HLB), the most devastating disease of citrus worldwide. There are three known insect vectors of the HLB-associated bacteria, and all are members of the Hemiptera: Diaphorina citri (Psyllidae), Trioza erytreae (Triozidae), and Cacopsylla (Psylla) citrisuga (Psyllidae). In this study, we found that another hemipteran, the striped mealybug Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae), was able to acquire and retain Las bacteria. The bacterial titers were positively correlated with the feeding acquisition time on Las-infected leaf discs, with a two-weeks feeding period resulting in Ct values ranging from 23.1 to 36.1 (8.24 × 10(7) to 1.07 × 10(4) Las cells per mealybug). We further discovered that the prophage/phage populations of Las in the mealybugs were different from those of Las in psyllids based on Las prophage-specific molecular markers: infected psyllids harbored the Las populations with prophage/phage FP1 and FP2, while infected mealybugs carried the Las populations with the iFP3 being the dominant prophage/phage. As in the psyllids, Las bacteria were shown to move through the insect gut wall to the salivary glands after being ingested by the mealybug based on a time-course quantitative polymerase chain reaction (qPCR) assay of the dissected digestive systems. However, Las populations transmitted by the mealybugs did not cause disease in host plants. This is the first evidence of genetic difference among Las populations harbored by different insect vectors and difference among Las populations with respect to whether or not they cause disease in host plants.

Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of Candidatus Liberibacter asiaticus' (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease development.

Proteins secreted by the type V secretion system (T5SS), known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. In this study, we characterized two novel autotransporter proteins of 'Candidatus Liberibacter asiaticus' (Las), and redesignated them as LasAI and LasAII in lieu of the previous names HyvI and HyvII. As a phloem-limited, intracellular bacterial pathogen, Las has a significantly reduced genome and causes huanglongbing (HLB), a devastating disease of citrus worldwide. Bioinformatic analyses revealed that LasAI and LasAII share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasAI C-terminus and the full length LasAII were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of a typical signal peptide, LasAI was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasAI protein. Its surface localization was also confirmed by the removal of the LasAI antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasAI translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria when expressed in the plants. These findings may lead to a better understanding of the pathogenesis of this intracellular bacterium.

Huanglongbing (HLB) is presently the most devastating citrus disease worldwide. As an intracellular plant pathogen and insect symbiont, the HLB bacterium, 'Candidatus Liberibacter asiaticus' (Las), retains the entire flagellum-encoding gene cluster in its significantly reduced genome. Las encodes a flagellin and hook-associated protein (Fla) of 452 amino acids that contains a conserved 22 amino acid domain (flg22) at positions 29 to 50 in the N-terminus. The phenotypic alteration in motility of a Sinorhizobium meliloti mutant lacking the fla genes was partially restored by constitutive expression of Fla(Las). Agrobacterium-mediated transient expression in planta revealed that Fla(Las) induced cell death and callose deposition in Nicotiana benthamiana, and that the transcription of BAK1 and SGT1, which are associated with plant innate immunity, was upregulated. Amino acid substitution experiments revealed that residues 38 (serine) and 39 (aspartate) of Fla(Las) were essential for callose induction. The synthetic flg22(Las) peptide could not induce plant cell death but retained the ability to induce callose deposition at a concentration of 20 µM or above. This demonstrated that the pathogen-associated molecular pattern (PAMP) activity of flg22 in Las was weaker than those in other well-studied plant pathogenic bacteria. These results indicate that Fla(Las) acts as a PAMP and may play an important role in triggering host plant resistance to the HLB bacteria.

The reduced production or activity of the cysteine-rich glycoprotein progranulin is responsible for about 20% of cases of familial frontotemporal dementia. However, little is known about the molecular mechanisms that govern the level and secretion of progranulin. Here we show that progranulin is expressed in mouse cortical neurons and more prominently in mouse microglia in culture and is abundant in the endoplasmic reticulum (ER) and Golgi. Using chemical crosslinking, immunoprecipitation, and mass spectrometry, we found that progranulin is bound to a network of ER Ca(2+)-binding chaperones including BiP, calreticulin, GRP94, and four members of the protein disulfide isomerase (PDI) family. Loss of ERp57 inhibits progranulin secretion. Thus, progranulin is a novel substrate of several PDI family proteins and modulation of the ER chaperone network may be a therapeutic target for controlling progranulin secretion.

Zebra Chip (ZC) is an emerging plant disease that causes aboveground decline of potato shoots and generally results in unusable tubers. This disease has led to multi-million dollar losses for growers in the central and western United States over the past decade and impacts the livelihood of potato farmers in Mexico and New Zealand. ZC is associated with 'Candidatus Liberibacter solanacearum', a fastidious alpha-proteobacterium that is transmitted by a phloem-feeding psyllid vector, Bactericera cockerelli Sulc. Research on this disease has been hampered by a lack of robust culture methods and paucity of genome sequence information for 'Ca. L. solanacearum'. Here we present the sequence of the 1.26 Mbp metagenome of 'Ca. L. solanacearum', based on DNA isolated from potato psyllids. The coding inventory of the 'Ca. L. solanacearum' genome was analyzed and compared to related Rhizobiaceae to better understand 'Ca. L. solanacearum' physiology and identify potential targets to develop improved treatment strategies. This analysis revealed a number of unique transporters and pathways, all potentially contributing to ZC pathogenesis. Some of these factors may have been acquired through horizontal gene transfer. Taxonomically, 'Ca. L. solanacearum' is related to 'Ca. L. asiaticus', a suspected causative agent of citrus huanglongbing, yet many genome rearrangements and several gene gains/losses are evident when comparing these two Liberibacter. species. Relative to 'Ca. L. asiaticus', 'Ca. L. solanacearum' probably has reduced capacity for nucleic acid modification, increased amino acid and vitamin biosynthesis functionalities, and gained a high-affinity iron transport system characteristic of several pathogenic microbes.

Huanglongbing (HLB) is presently the most devastating citrus disease worldwide. As an intracellular plant pathogen and insect symbiont, the HLB bacterium, 'Candidatus Liberibacter asiaticus' (Las), retains the entire flagellum-encoding gene cluster in its significantly reduced genome. Las encodes a flagellin and hook-associated protein (Fla) of 452 amino acids that contains a conserved 22 amino acid domain (flg22) at positions 29 to 50 in the N-terminus. The phenotypic alteration in motility of a Sinorhizobium meliloti mutant lacking the fla genes was partially restored by constitutive expression of FlaLas. Agrobacterium-mediated transient expression in planta revealed that FlaLas induced cell death and callose deposition in Nicotiana benthamiana, and that the transcription of BAK1 and SGT1, which are associated with plant innate immunity, was upregulated. Amino acid substitution experiments revealed that residues 38 (serine) and 39 (aspartate) of FlaLas were essential for callose induction. The synthetic flg22Las peptide could not induce plant cell death but retained the ability to induce callose deposition at a concentration of 20 mM or above. This demonstrated that the pathogen-associated molecular pattern (PAMP) activity of flg22 in Las was weaker than those in other well-studied plant pathogenic bacteria. These results indicate that FlaLas acts as a PAMP and may play an important role in triggering host plant resistance to the HLB bacteria. [ABSTRACT FROM AUTHOR]