Novel missense variants cause intermediate phenotypes in the phenotypic spectrum of SLC5A6-related disorders.

SLC5A6 encodes the sodium-dependent multivitamin transporter, a transmembrane protein that uptakes biotin, pantothenic acid, and lipoic acid. Biallelic SLC5A6 variants cause sodium-dependent multivitamin transporter deficiency (SMVTD) and childhood-onset biotin-responsive peripheral motor neuropathy (COMNB), which both respond well to replacement therapy with the above three nutrients. SMVTD usually presents with various symptoms in multiple organs, such as gastrointestinal hemorrhage, brain atrophy, and global developmental delay, at birth or in infancy. Without nutrient replacement therapy, SMVTD can be lethal in early childhood. COMNB is clinically milder and has a later onset than SMVTD, at approximately 10 years of age. COMNB symptoms are mostly limited to peripheral motor neuropathy. Here we report three patients from one Japanese family harboring novel compound heterozygous missense variants in SLC5A6, namely NM_021095.4:c.[221C>T];[642G>C] p.[(Ser74Phe)];[(Gln214His)]. Both variants were predicted to be deleterious through multiple lines of evidence, including amino acid conservation, in silico predictions of pathogenicity, and protein structure considerations. Drosophila analysis also showed c.221C>T to be pathogenic. All three patients had congenital brain cysts on neonatal cranial imaging, but no other morphological abnormalities. They also had a mild motor developmental delay that almost completely resolved despite no treatment. In terms of severity, their phenotypes were intermediate between SMVTD and COMNB. From these findings we propose a new SLC5A6-related disorder, spontaneously remitting developmental delay with brain cysts (SRDDBC) whose phenotypic severity is between that of SMVTD and COMNB. Further clinical and genetic evidence is needed to support our suggestion.

Circular RNA hsa_circ_0000915 promotes propranolol resistance of hemangioma stem cells in infantile haemangiomas.

BACKGROUND: Propranolol is a first-line clinical drug for infantile haemangiomas (IH) therapy. Nevertheless, resistance to propranolol is observed in some patients with IH. Circular RNAs (circRNAs) has been increasingly reported to act as a pivotal regulator in tumor progression. However, the underlying mechanism of circRNAs in IH remains unclear. METHODS: Quantitative real-time polymerase chain reaction was performed to detect Circ_0000915, miR-890 and RNF187 expression. Protein levels were determined using western blot. CCK-8 assay was used to measure cell proliferation. Caspase-3 activity assay and flow cytometry were conducted to determine cell apoptosis. Luciferase reporter assay was carried out to assess the interaction between miR-890 and Circ_0000915 or RNF187. Chromatin immunoprecipitation assay was performed to detect the interaction between STAT3 and Circ_0000915 promoter. Biotin pull-down assay was used to detect the direct interaction between miR-890 and Circ_0000915. In vivo experiments were performed to measure tumor formation. RESULTS: Here, we discovered depletion of Circ_0000915 increased propranolol sensitivity of haemangioma derived stem cells (HemSCs) both in vitro and in vivo, whereas forced expression of Circ_0000915 exhibited opposite effects. Mechanistically, Circ_0000915, transcriptionally induced by IL-6/STAT3 pathway, competed with RNF187 for the biding site in miR-890, led to upregulation of RNF187 by acting as a miR-890 "sponge". Furthermore, silence of miR-890 reversed increased propranolol sensitivity of HemSCs due to Circ_0000915 ablation. Moreover, increased Circ_0000915 and RNF187 levels were observed in IH tissues and positively associated with propranolol resistance, miR-890 exhibited an inverse expression pattern. CONCLUSION: We thereby uncover the activation of IL-6/STAT3/Circ_0000915/miR-890/RNF187 axis in propranolol resistance of IH, and provide therapeutic implications for patients of IH with propranolol resistance.

Comprehensive analysis of transcriptome and metabolome analysis reveal new targets of Glaesserella parasuis glucose-specific enzyme IIBC (PtsG).

The ptsG (hpIIBCGlc) gene, belonging to the glucose-specific phosphotransferase system, encodes the bacterial glucose-specific enzyme IIBC. In this study, the effects of a deletion of the ptsG gene were investigated by metabolome and transcriptome analyses. At the transcriptional level, we identified 970 differentially expressed genes between ΔptsG and sc1401 (Padj<0.05) and 2072 co-expressed genes. Among these genes, those involved in methane metabolism, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, pyruvate metabolism, phosphotransferase system (PTS), biotin metabolism, Two-component system and Terpenoid backbone biosynthesis showed significant changes in the ΔptsG mutant strain. Metabolome analysis revealed that a total of 310 metabolites were identified, including 20 different metabolites (p < 0.05). Among them, 15 metabolites were upregulated and 5 were downregulated in ΔptsG mutant strain. Statistical analysis revealed there were 115 individual metabolites having correlation, of which 89 were positive and 26 negative. These metabolites include amino acids, phosphates, amines, esters, nucleotides, benzoic acid and adenosine, among which amino acids and phosphate metabolites dominate. However, not all of these changes were attributable to changes in mRNA levels and must also be caused by post-transcriptional regulatory processes. The knowledge gained from this lays the foundation for further study on the role of ptsG in the pathogenic process of Glaesserella parasuis (G.parasuis).

NEAT1 variant 1 weakens the genome-wide effect of miR-3122 on blocking H3K79me3 in bladder cancer.

Nuclear-enriched abundant transcript 1 (NEAT1) is one of the most well-studied long non-coding RNAs (lncRNAs) in multiple human carcinoma. Two distinct variants of NEAT1, however, are never illuminated their specific functions and mechanisms underlying carcinogenesis. In this study, biotin-labelled NEAT1 variants were generated to incubate with cell lysate of bladder cancer cell T24 cells, and fished a batch of RNA substances. Here, we observed that NEAT1.1 (the short transcript) could capture 122 microRNAs (miRNAs), 36 small nucleolar RNAs (snoRNAs), 55 lncRNAs and 38 mRNAs while NEAT1.2 (the long transcript) could obtain 142 miRNAs, 51 snoRNAs, 72 lncRNAs and 41 mRNAs. Furthermore, we also found that the distinctions of RNA binding substances between these two variants were mainly expressed in nucleus rather than cytoplasm. GO analysis indicated that these non-coding RNAs governed histone modification, nucleosome assembly and chromosome organization. We picked up miRNA miR-3122, which substantially interacted with NEAT1.1, and found that histone H3K79me3 was reduced in bladder cancer T24, BIU-87 and EJ-1 cells after miR-3122 overexpression, and rescued by NEAT1.1 additional compensation. Nonetheless, we failed to find that miR-3122 could interfere with expression of H3K79 methyltransferase disruptor of telomeric silencing-1 like (DOT1L). Interestingly, we harvested histone 3 fished by biotin-labelled miR-3122, and validated this intercrossing using RNA immunoprecipitation. Taken together, we demonstrated that NEAT1.1 weakened the effect of miR-3122 on H3K79me3 suppression in bladder cancer.

Hydroxymethylation-Specific Ligation-Mediated Single Quantum Dot-Based Nanosensors for Sensitive Detection of 5-Hydroxymethylcytosine in Cancer Cells.

5-Hydroxymethylcytosine (5hmC) modification is a key epigenetic regulator of cellular processes in mammalian cells, and its misregulation may lead to various diseases. Herein, we develop a hydroxymethylation-specific ligation-mediated single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor for sensitive quantification of 5hmC modification in cancer cells. We design a Cy5-modified signal probe and a biotinylated capture probe for the recognition of specific 5hmC-containing genes. 5hmC in target DNA can be selectively converted by T4 ß-glucosyltransferase to produce a glycosyl-modified 5hmC, which cannot be cleaved by methylation-insensitive restriction enzyme MspI. The glycosylated 5hmC DNA may act as a template to ligate a signal probe and a capture probe, initiating hydroxymethylation-specific ligation to generate large amounts of biotin-/Cy5-modified single-stranded DNAs (ssDNAs). The assembly of biotin-/Cy5-modified ssDNAs onto a single QD through streptavidin-biotin interaction results in FRET and consequently the generation of a Cy5 signal. The nanosensor is very simple without the need for bisulfite treatment, radioactive reagents, and 5hmC-specific antibodies. Owing to excellent specificity and high amplification efficiency of hydroxymethylation-specific ligation and near-zero background of a single QD-based FRET, this nanosensor can quantify 5hmC DNA with a limit of detection of 33.61 aM and a wider linear range of 7 orders of magnitude, and it may discriminate the single-nucleotide difference among 5hmC, 5-methylcytosine, and unmodified cytosine. Moreover, this nanosensor can distinguish as low as a 0.001% 5hmC DNA in complex mixtures, and it can monitor the cellular 5hmC level and discriminate cancer cells from normal cells, holding great potential in biomedical research and clinical diagnostics.

Antibody mimetic drug conjugate manufactured by high-yield Escherichia coli expression and non-covalent binding system.

Antibody-drug conjugates (ADCs) are a major therapeutic tool for the treatment of advanced cancer. Malignant cells in advanced cancer often display multiple genetic mutations and become resistant to monotherapy. Therefore, a therapeutic regimen that simultaneously targets multiple molecules with multiple payloads is desirable. However, the development of ADCs is hampered by issues in biopharmaceutical manufacturing and the complexity of the conjugation process of low-molecular-weight payloads to biologicals. Here, we report antibody mimetic-drug conjugates (AMDCs) developed by exploiting the non-covalent binding property of payloads based on high-affinity binding of mutated streptavidin and modified iminobiotin. Miniprotein antibodies were fused to a low immunogenic streptavidin variant, which was then expressed in Escherichia coli inclusion bodies, solubilized, and refolded into functional tetramers. The AMDC developed against human epidermal growth factor receptor 2 (HER2) effectively killed cultured cancer cells using bis-iminobiotin conjugated to photo-activating silicon phthalocyanine. The HER2-targeting AMDC was also effective in vivo against a mouse KPL-4 xenograft model. This AMDC platform provides rapid, stable, and high-yield therapeutics against multiple targets.

Application of Skyline for Analysis of Protein-Protein Interactions In Vivo.

Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein-protein interactions (PPIs) in cells is also important since these interactions are the bases of many processes, such as the cell cycle and signaling pathways. This paper describes the application of Skyline software for the identification and quantification of the biotinylated form of the biotin acceptor peptide (BAP) tag, which is a marker of in vivo PPIs. The tag was used in the Proximity Utilizing Biotinylation (PUB) method, which is based on the co-expression of BAP-X and BirA-Y in mammalian cells, where X or Y are interacting proteins of interest. A high level of biotinylation was detected in the model experiments where X and Y were pluripotency transcription factors Sox2 and Oct4, or heterochromatin protein HP1γ. MRM data processed by Skyline were normalized and recalculated. Ratios of biotinylation levels in experiment versus controls were 86 ± 6 (3 h biotinylation time) and 71 ± 5 (9 h biotinylation time) for BAP-Sox2 + BirA-Oct4 and 32 ± 3 (4 h biotinylation time) for BAP-HP1γ + BirA-HP1γ experiments. Skyline can also be applied for the analysis and identification of PPIs from shotgun proteomics data downloaded from publicly available datasets and repositories.

Fungal biotin homeostasis is essential for immune evasion after macrophage phagocytosis and virulence.

Biotin is an important cofactor for multiple enzymes in central metabolic processes. While many bacteria and most fungi are able to synthesise biotin de novo, Candida spp. are auxotrophic for this vitamin and thus require efficient uptake systems to facilitate biotin acquisition during infection. Here we show that Candida glabrata and Candida albicans use a largely conserved system for biotin uptake and regulation, consisting of the high-affinity biotin transporter Vht1 and the transcription factor Vhr1. Both species induce expression of biotin-metabolic genes upon in vitro biotin depletion and following phagocytosis by macrophages, indicating low biotin levels in the Candida-containing phagosome. In line with this, we observed reduced intracellular proliferation of both Candida cells pre-starved of biotin and deletion mutants lacking VHR1 or VHT1 genes. VHT1 was essential for the full virulence of C. albicans during systemic mouse infections, and the lack of VHT1 led to reduced fungal burden in C. glabrata-infected brains and C. albicans-infected brains and kidneys. Together, our data suggest a critical role of Vht1-mediated biotin acquisition for C. glabrata and C. albicans during intracellular growth in macrophages and systemic infections.

Multi-level metabolic engineering of Pseudomonas mutabilis ATCC31014 for efficient production of biotin.

Biotin (Vitamin H or B7) is one of the most important cofactors involved in central metabolism of pro- and eukaryotic cells. Currently, chemical synthesis is the only route for commercial production. This study reports efficient microbial production of biotin in Pseudomonas mutabilis via multi-level metabolic engineering strategies: Level 1, overexpressing rate-limiting enzyme encoding genes involved in biotin synthesis (i.e. promoter and ribosome binding site engineering); Level 2, deregulating biotin biosynthesis (i.e. deletion of the negative regulator and the biotin importer genes); Level 3, enhancing the supply of co-factors (i.e. S-adenosyl-L-methionine and [Fe-S] cluster) for biotin biosynthesis; Level 4, increasing the availability of the precursor pimelate thioester (i.e. introduction of the BioW-BioI pathway from Bacillus subtilis). The combination of these interventions resulted in the establishment of a biotin overproducing strain, with the secretion of biotin increased for more than 460-fold. In combination with bioprocess engineering efforts, biotin was produced at a final titer of 87.17 mg/L in a shake flask and 271.88 mg/L in a fed-batch fermenter with glycerol as the carbon source. This is the highest biotin titer ever reported so far using rationally engineered microbial cell factories.

The pilot-scale preparation of the SA-hGM-CSF bi-functional fusion protein.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) can be used to induce a powerful immune response. Based on the specific binding of biotin and streptavidin, SA-hGM-CSF was anchored on the surface of biotinylated tumor cells, which could enhance the anti-tumor effect of tumor cell vaccines in our previous reports, suggesting it would have potential clinical value. Preparation of the biologically active proteins in large-scale production is the basis of clinical application, however, only a small amount of biologically active protein was obtained according to previous studies. In this study, we researched the effects of various factors on the purification and simultaneous renaturation of SA-hGM-CSF fusion protein by single factor experiment and orthogonal experiment. Here, we developed a viable pilot-scale trial in the fermentation, purification, refolding and freeze-drying of SA-hGM-CSF proteins in order to efficiently obtain more biologically active proteins with high purity, which will lay the foundation for industrial production.

Crystal structure and acetylation of BioQ suggests a novel regulatory switch for biotin biosynthesis in Mycobacterium smegmatis.

Biotin (vitamin B7), a sulfur-containing fatty acid derivative, is a nutritional virulence factor in certain mycobacterial species. Tight regulation of biotin biosynthesis is important because production of biotin is an energetically expensive process requiring 15-20 equivalents of ATP. The Escherichia coli bifunctional BirA is a prototypical biotin regulatory system. In contrast, mycobacterial BirA is an unusual biotin protein ligase without DNA-binding domain. Recently, we established a novel two-protein paradigm of BioQ-BirA. However, structural and molecular mechanism for BioQ is poorly understood. Here, we report crystal structure of the M. smegmatis BioQ at 1.9 Å resolution. Structure-guided functional mapping defined a seven residues-requiring motif for DNA-binding activity. Western blot and MALDI-TOF MS allowed us to unexpectedly discover that the K47 acetylation activates crosstalking of BioQ to its cognate DNA. More intriguingly, excess of biotin augments the acetylation status of BioQ in M. smegmatis. It seems likely that BioQ acetylation proceeds via a non-enzymatic mechanism. Mutation of this acetylation site K47 in BioQ significantly impairs its regulatory role in vivo. This explains in part (if not all) why BioQ has no detectable requirement of the presumable bio-5'-AMP effecter, which is a well-known ligand for the paradigm E. coli BirA regulator system. Unlike the scenario seen with E. coli carrying a single biotinylated protein, AccB, genome-wide search and Streptavidin blot revealed that no less than seven proteins require the rare post-translational modification, biotinylation in M. smegmatis, validating its physiological demand for biotin at relatively high level. Taken together, our finding defines a novel biotin regulatory machinery by BioQ, posing a possibility that development of new antibiotics targets biotin, the limited nutritional virulence factor in certain pathogenic mycobacterial species.

Digestion-ligation-only Hi-C is an efficient and cost-effective method for chromosome conformation capture.

Chromosome conformation capture (3C) technologies can be used to investigate 3D genomic structures. However, high background noise, high costs, and a lack of straightforward noise evaluation in current methods impede the advancement of 3D genomic research. Here we developed a simple digestion-ligation-only Hi-C (DLO Hi-C) technology to explore the 3D landscape of the genome. This method requires only two rounds of digestion and ligation, without the need for biotin labeling and pulldown. Non-ligated DNA was efficiently removed in a cost-effective step by purifying specific linker-ligated DNA fragments. Notably, random ligation could be quickly evaluated in an early quality-control step before sequencing. Moreover, an in situ version of DLO Hi-C using a four-cutter restriction enzyme has been developed. We applied DLO Hi-C to delineate the genomic architecture of THP-1 and K562 cells and uncovered chromosomal translocations. This technology may facilitate investigation of genomic organization, gene regulation, and (meta)genome assembly.

Long-term drug modification to the surface of mesenchymal stem cells by the avidin-biotin complex method.

Mesenchymal stem cells (MSCs) have various functions, making a significant contribution to tissue repair. On the other hand, the viability and function of MSCs are not lasting after an in vivo transplant, and the therapeutic effects of MSCs are limited. Although various chemical modification methods have been applied to MSCs to improve their viability and function, most of conventional drug modification methods are short-term and unstable and cause cytotoxicity. In this study, we developed a method for long-term drug modification to C3H10T1/2 cells, murine mesenchymal stem cells, without any damage, using the avidin-biotin complex method (ABC method). The modification of NanoLuc luciferase (Nluc), a reporter protein, to C3H10T1/2 cells by the ABC method lasted for at least 14 days in vitro without major effects on the cellular characteristics (cell viability, cell proliferation, migration ability, and differentiation ability). Moreover, in vivo, the surface Nluc modification to C3H10T1/2 cells by the ABC method lasted for at least 7 days. Therefore, these results indicate that the ABC method may be useful for long-term surface modification of drugs and for effective MSC-based therapy.

Identification of the SOX2 Interactome by BioID Reveals EP300 as a Mediator of SOX2-dependent Squamous Differentiation and Lung Squamous Cell Carcinoma Growth.

Lung cancer is the leading cause of cancer mortality worldwide, with squamous cell carcinoma (SQCC) being the second most common form. SQCCs are thought to originate in bronchial basal cells through an injury response to smoking, which results in this stem cell population committing to hyperplastic squamous rather than mucinous and ciliated fates. Copy number gains in SOX2 in the region of 3q26-28 occur in 94% of SQCCs, and appear to act both early and late in disease progression by stabilizing the initial squamous injury response in stem cells and promoting growth of invasive carcinoma. Thus, anti-SOX2 targeting strategies could help treat early and/or advanced disease. Because SOX2 itself is not readily druggable, we sought to characterize SOX2 binding partners, with the hope of identifying new strategies to indirectly interfere with SOX2 activity. We now report the first use of proximity-dependent biotin labeling (BioID) to characterize the SOX2 interactome in vivo We identified 82 high confidence SOX2-interacting partners. An interaction with the coactivator EP300 was subsequently validated in both basal cells and SQCCs, and we demonstrate that EP300 is necessary for SOX2 activity in basal cells, including for induction of the squamous fate. We also report that EP300 copy number gains are common in SQCCs and that growth of lung cancer cell lines with 3q gains, including SQCC cells, is dependent on EP300. Finally, we show that EP300 inhibitors can be combined with other targeted therapeutics to achieve more effective growth suppression. Our work supports the use of BioID to identify interacting protein partners of nondruggable oncoproteins such as SOX2, as an effective strategy to discover biologically relevant, druggable targets.

Long Distance Modulation of Disorder-to-Order Transitions in Protein Allostery.

Elucidation of the molecular details of allosteric communication between distant sites in a protein is key to understanding and manipulating many biological regulatory processes. Although protein disorder is acknowledged to play an important thermodynamic role in allostery, the molecular mechanisms by which this disorder is harnessed for long distance communication are known for a limited number of systems. Transcription repression by the Escherichia coli biotin repressor, BirA, is allosterically activated by binding of the small molecule effector biotinoyl-5'-AMP. The effector acts by promoting BirA dimerization, which is a prerequisite for sequence-specific binding to the biotin biosynthetic operon operator sequence. A 30 Å distance separates the effector binding and dimerization surfaces in BirA, and previous studies indicate that allostery is mediated, in part, by disorder-to-order transitions on the two coupled sites. In this work, combined experimental and computational methods have been applied to investigate the molecular basis of allosteric communication in BirA. Double-mutant cycle analysis coupled with thermodynamic measurements indicates functional coupling between residues in disordered loops on the two distant surfaces. All atom molecular dynamics simulations reveal that this coupling occurs through long distance reciprocal modulation of the structure and dynamics of disorder-to-order transitions on the two surfaces.

The Staphylococcus aureus group II biotin protein ligase BirA is an effective regulator of biotin operon transcription and requires the DNA binding domain for full enzymatic activity.

Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis and transport. The Staphylococcus aureus Group II BPL which is called BirA has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins. However, the S. aureus BirA was reported to dimerize and bind DNA in the absence of biotin or biotinoyl-AMP (Soares da Costa et al. (2014) Mol Microbiol 91: 110-120). These in vitro results argued that the protein would be unable to respond to the levels of biotin or acceptor proteins and thus would lack the regulatory properties of the other characterized BirA proteins. We tested the regulatory function of the protein using an in vivo model system and examined its DNA binding properties in vitro using electrophoretic mobility shift and fluorescence anisotropy analyses. We report that the S. aureus BirA is an effective regulator of biotin operon transcription and that the prior data can be attributed to artifacts of mobility shift analyses. We also report that deletion of the DNA binding domain of the S. aureus BirA results in loss of virtually all of its ligation activity.

Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect L-Lysine Production in Corynebacterium glutamicum.

Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in L-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport--NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885--were also expressed at significantly higher levels in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, L-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.

A SNARE-Like Protein and Biotin Are Implicated in Soybean Cyst Nematode Virulence.

Phytoparasitic nematodes that are able to infect and reproduce on plants that are considered resistant are referred to as virulent. The mechanism(s) that virulent nematodes employ to evade or suppress host plant defenses are not well understood. Here we report the use of a genetic strategy (allelic imbalance analysis) to associate single nucleotide polymorphisms (SNPs) with nematode virulence genes in Heterodera glycines, the soybean cyst nematode (SCN). To accomplish this analysis, a custom SCN SNP array was developed and used to genotype SCN F3-derived populations grown on resistant and susceptible soybean plants. Three SNPs reproducibly showed allele imbalances between nematodes grown on resistant and susceptible plants. Two candidate SCN virulence genes that were tightly linked to the SNPs were identified. One SCN gene encoded biotin synthase (HgBioB), and the other encoded a bacterial-like protein containing a putative SNARE domain (HgSLP-1). The two genes mapped to two different linkage groups. HgBioB contained sequence polymorphisms between avirulent and virulent nematodes. However, the gene encoding HgSLP-1 had reduced copy number in virulent nematode populations and appears to produce multiple forms of the protein via intron retention and alternative splicing. We show that HgSLP-1 is an esophageal-gland protein that is secreted by the nematode during plant parasitism. Furthermore, in bacterial co-expression experiments, HgSLP-1 co-purified with the SCN resistance protein Rhg1 α-SNAP, suggesting that these two proteins physically interact. Collectively our data suggest that multiple SCN genes are involved in SCN virulence, and that HgSLP-1 may function as an avirulence protein and when absent it helps SCN evade host defenses.

Novel roles of holocarboxylase synthetase in gene regulation and intermediary metabolism.

The role of holocarboxylase synthetase (HLCS) in catalyzing the covalent binding of biotin to the five biotin-dependent carboxylases in humans is well established, as are the essential roles of these carboxylases in the metabolism of fatty acids, the catabolism of leucine, and gluconeogenesis. This review examines recent discoveries regarding the roles of HLCS in assembling a multiprotein gene repression complex in chromatin. In addition, emerging evidence suggests that the number of biotinylated proteins is far larger than previously assumed and includes members of the heat-shock superfamily of proteins and proteins coded by the ENO1 gene. Evidence is presented linking biotinylation of heat-shock proteins HSP60 and HSP72 with redox biology and immune function, respectively, and biotinylation of the two ENO1 gene products MBP-1 and ENO1 with tumor suppression and glycolysis, respectively.

Development of streptavidin-based nanocomplex for siRNA delivery.

In our previous study, we have identified a PCBP2 siRNA that exhibits antifibrotic activity in rat hepatic stellate cells (HSCs) by inhibition of αCP2, a protein responsible for stabilization of the collagen α1 (I) mRNA in alcoholic liver fibrosis. This study aims to develop a streptavidin-based nanocomplex that can efficiently deliver the PCBP2 siRNA to HSCs. Biotin-siRNA and biotin-cholesterol were mixed with streptavidin to form the streptavidin-biotin complex, which was further condensed electrostatically with positively charged protamine to form the final multicomponent siRNA nanocomplex in the size range of 150-250 nm. The siRNA nanocomplex does not induce cytotoxicity in rat HSCs as compared to commercially available transfection agents. The cellular uptake efficiency of the siRNA nanocomplex is higher in rat HSCs than other cell lines, such as Caco-2 and PC-3, indicating that receptor-mediated endocytosis mainly contributes to the cellular uptake of the siRNA nanocomplex. The siRNA nanocomplex exhibits more than 85% silencing effect on the PCBP2 mRNA in HSCs. Stability study indicates that the nanocomplex can efficiently protect siRNA from degradation in the serum. The streptavidin-based multicomponent siRNA nanocomplex provides a promising strategy to deliver the PCBP2 siRNA to HSCs. Moreover, the nanocomplex can be used as a platform for other diseases by changing the siRNA sequence and targeting ligand.