Surface-expressed phosphoglycerate mutase of Candida albicans binds to salivary DMBT1.

Candida albicans colonizes oral tissues and causes infectious diseases. Colonization of C. albicans on the oral mucosa and tooth enamel surfaces is established via the interaction between C. albicans adhesins and salivary proteins, forming a film on the oral tissues. Deleted in malignant brain tumors 1 (DMBT1), also known as salivary agglutinin or gp-340, belongs to the scavenger receptor cysteine-rich (SRCR) superfamily. In the oral cavity, immobilized DMBT1 on oral tissues causes microbial adherence. Recently, we demonstrated that C. albicans binds to DMBT1 and isolated a 25-kDa C. albicans adhesin involved in the interaction with the binding domain of DMBT1, namely, SRCRP2. In the present study, we searched for additional DMBT1-binding adhesins in C. albicans. The component isolated here had a molecular mass of 29 kDa and was found to be phosphoglycerate mutase (Gpm1). Isolated Gpm1 inhibited C. albicans binding to SRCRP2 and directly bound to SRCRP2 in a dose-dependent manner. Gpm1 localization on the C. albicans cell wall surface was confirmed by immunostaining. These results suggest that surface-expressed Gpm1 functions as an adhesin for the establishment of C. albicans cells on the oral mucosa and tooth enamel by binding to DMBT1.

Evaluation of CSTB and DMBT1 expression in saliva of gastric cancer patients and controls.

BACKGROUND: Gastric cancer (GC) is the fifth most common cancer and the third cause of cancer deaths globally, with late diagnosis, low survival rate, and poor prognosis. This case-control study aimed to evaluate the expression of cystatin B (CSTB) and deleted in malignant brain tumor 1 (DMBT1) in the saliva of GC patients with healthy individuals to construct diagnostic algorithms using statistical analysis and machine learning methods. METHODS: Demographic data, clinical characteristics, and food intake habits of the case and control group were gathered through a standard checklist. Unstimulated whole saliva samples were taken from 31 healthy individuals and 31 GC patients. Through ELISA test and statistical analysis, the expression of salivary CSTB and DMBT1 proteins was evaluated. To construct diagnostic algorithms, we used the machine learning method. RESULTS: The mean salivary expression of CSTB in GC patients was significantly lower (115.55 ± 7.06, p = 0.001), and the mean salivary expression of DMBT1 in GC patients was significantly higher (171.88 ± 39.67, p = 0.002) than the control. Multiple linear regression analysis demonstrated that GC was significantly correlated with high levels of DMBT1 after controlling the effects of age of participants (R2 = 0.20, p < 0.001). Considering salivary CSTB greater than 119.06 ng/mL as an optimal cut-off value, the sensitivity and specificity of CSTB in the diagnosis of GC were 83.87 and 70.97%, respectively. The area under the ROC curve was calculated as 0.728. The optimal cut-off value of DMBT1 for differentiating GC patients from controls was greater than 146.33 ng/mL (sensitivity = 80.65% and specificity = 64.52%). The area under the ROC curve was up to 0.741. As a result of the machine learning method, the area under the receiver-operating characteristic curve for the diagnostic ability of CSTB, DMBT1, demographic data, clinical characteristics, and food intake habits was 0.95. The machine learning model's sensitivity, specificity, and accuracy were 100, 70.8, and 80.5%, respectively. CONCLUSION: Salivary levels of DMBT1 and CSTB may be accurate in diagnosing GCs. Machine learning analyses using salivary biomarkers, demographic, clinical, and nutrition habits data simultaneously could provide affordability models with acceptable accuracy for differentiation of GC by a cost-effective and non-invasive method.

Bronchial brush cytology, endobronchial biopsy, and SALSA immunohistochemistry in severe equine asthma.

Horses with severe equine asthma (SEA), also known as heaves and recurrent airway obstruction, have persistent neutrophilic inflammation of the lower airways. Cytologic evaluation of bronchoalveolar lavage (BAL) fluid is commonly used to confirm the clinical diagnosis of SEA. However, the utility of microscopic assessment of bronchial brushings, endobronchial biopsies, and immunohistochemical detection of disease-associated biomarkers for the diagnosis of SEA remain poorly characterized. Salivary scavenger and agglutinin (SALSA) has anti-inflammatory properties and downregulated gene expression in SEA; therefore, it was investigated as a tissue biomarker for airway and systemic inflammation. Six asthmatic and 6 non-asthmatic horses were exposed to an inhaled challenge. Before and after challenge, samples of BAL fluid, bronchial brushing, and endobronchial biopsy were collected. Location of SALSA in biopsies was determined, and immunohistochemical label intensity was computed using image analysis software. Serum amyloid A (SAA) was measured to assess systemic inflammation. After challenge, neutrophil proportions were significantly higher in asthmatic versus non-asthmatic horses in BAL fluid (least squares means, 95% confidence interval: 80.9%, 57.2% to 93.1%, vs 3.6%, 1.1% to 10.7%) and in brush cytology slides (39.5%, 7.7% to 83.6%, vs 0.2%, 0% to 2.3%), illustrating the potential of brush cytology as an alternate modality to BAL for assessing intraluminal inflammation. Bronchial histopathologic findings and intensity of SALSA immunolabeling in surface and glandular epithelium were similar in asthmatic and non-asthmatic horses, indicating limited changes in bronchial tissue from the inhaled challenge. Increases in SAA indicated systemic inflammation, but SALSA immunolabeling did not change significantly.

Isolation, identification, and characterization of the human airway ligand for the eosinophil and mast cell immunoinhibitory receptor Siglec-8.

BACKGROUND: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. OBJECTIVE: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. METHODS: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. RESULTS: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. CONCLUSION: A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.

Different Molecular Forms of TFF3 in the Human Respiratory Tract: Heterodimerization with IgG Fc Binding Protein (FCGBP) and Proteolytic Cleavage in Bronchial Secretions.

The polypeptide TFF3 belongs to the trefoil factor family (TFF) of lectins. TFF3 is typically secreted from mucous epithelia together with mucins. Both intestinal and salivary TFF3 mainly exist as disulfide-linked heterodimers with IgG Fc binding protein (FCGBP). Here, we investigated bronchial tissue specimens, bronchial secretions, and bronchoalveolar lavage (BAL) fluid from patients with a chronic obstructive pulmonary disease (COPD) background by fast protein liquid chromatography and proteomics. For the first time, we identified different molecular forms of TFF3 in the lung. The high-molecular mass form represents TFF3-FCGBP oligomers, whereas the low-molecular mass forms are homodimeric and monomeric TFF3 with possibly anti-apoptotic activities. In addition, disulfide-linked TFF3 heterodimers with an Mr of about 60k and 30k were detected in both bronchial secretions and BAL fluid. In these liquids, TFF3 is partly N-terminally truncated probably by neutrophil elastase cleavage. TFF3-FCGBP is likely involved in the mucosal innate immune defense against microbial infections. We discuss a hypothetical model how TFF3 might control FCGBP oligomerization. Furthermore, we did not find indications for interactions of TFF3-FCGBP with DMBT1gp340 or the mucin MUC5AC, glycoproteins involved in mucosal innate immunity. Surprisingly, bronchial MUC5AC appeared to be degraded when compared with gastric MUC5AC.

Intestinal SALSA/dmbt1 levels are decreased in prematurely born infants.

The first months of life represent a crucial time period for an infant. Alongside establishing the early microbiome, the mucosal immunological homeostasis is being developed. Both processes may be perturbed in prematurely born infants. The glycoprotein SALSA plays a role in mucosal inflammation and microbial clearance. It is one of the most abundant molecules on the intestinal mucosal surfaces in early life. SALSA binds to many types of microbes and host defence molecules like IgA, C1q and collectin molecules. We here describe the development in faecal SALSA levels during the first three months of life. During these 90 days, the median SALSA level in full-term babies decreased from 1100 µg/mL (range 49-17 000 µg/mL) to 450 µg/mL (range 33-1000 µg/mL). Lower levels of SALSA were observed in prematurely born infants in the same time period. Our novel observation thus indicates an impact of prematurity on an important component of the infant intestinal immune system. Changes in SALSA in early life may have an effect on the early establishment of the human microbiome.

Salivary Trefoil Factor Family (TFF) Peptides and Their Roles in Oral and Esophageal Protection: Therapeutic Potential.

Human saliva is a complex body fluid with more than 3000 different identified proteins. Besides rheological and lubricating properties, saliva supports wound healing and acts as an antimicrobial barrier. TFF peptides are secreted from the mucous acini of the major and minor salivary glands and are typical constituents of normal saliva; TFF3 being the predominant peptide compared with TFF1 and TFF2. Only TFF3 is easily detectable by Western blotting. It occurs in two forms, a disulfide-linked homodimer (Mr: 13k) and a high-molecular-mass heterodimer with IgG Fc binding protein (FCGBP). TFF peptides are secretory lectins known for their protective effects in mucous epithelia; the TFF3 dimer probably has wound-healing properties due to its weak motogenic effect. There are multiple indications that FCGBP and TFF3-FCGBP play a key role in the innate immune defense of mucous epithelia. In addition, homodimeric TFF3 interacts in vitro with the salivary agglutinin DMBT1gp340. Here, the protective roles of TFF peptides, FCGBP, and DMBT1gp340 in saliva are discussed. TFF peptides are also used to reduce radiotherapy- or chemotherapy-induced oral mucositis. Thus, TFF peptides, FCGBP, and DMBT1gp340 are promising candidates for better formulations of artificial saliva, particularly improving wound healing and antimicrobial effects even in the esophagus.

Germline Missense Mutation of Deleted in Malignant Brain Tumor 1 (DMBT1) in Familial Mediastinal Neuroendocrine Cancer and in vitro Effects in Thyroid Cancer Cells.

BACKGROUND: Neuroendocrine tumors (NETs) rarely occur in the mediastinum and their etiology and pathogenesis are still unclear. OBJECTIVES: This study assessed inherited or de novo mutations in familial mediastinal NETs. METHOD: DNA samples from 4 patients were subjected to the whole-exome sequencing, and Sanger sequencing was used to identify Deleted in malignant brain tumor 1 (DMBT1) mutations in all 45 family members. RESULTS: All patients showed a germline DMBT1 mutation at 4971C. Sanger sequencing data showed that 4 NETs and 2 carriers in the first patient's family and 2 NETs and 4 carriers in the second patient's family, respectively, had this DMBT1 mutation. The in vitro data showed that the ectopic expression of DMBT1 reduced tumor cell viability and migration by arresting the G1/S phase of the cell cycle. CONCLUSIONS: We identified a germline missense mutation in DMBT1D1657E as a susceptibility gene for familial mediastinal NETs.

DMBT1 suppresses progression of gallbladder carcinoma through PI3K/AKT signaling pathway by targeting PTEN.

Gallbladder carcinoma (GBC) is a highly lethal malignancy of the gastrointestinal tract. Despite extensive research, the underlying molecular mechanism of GBC remains largely unclear. Deleted in malignant brain tumors 1 (DMBT1) is low-expression during cancer progression and as a potential tumor-suppressor gene in various types of cancer. However, its role in Gallbladder cancer remains poorly understood. Here, we found that DMBT1 was significantly low-expression and deletion of copy number in GBC tissues by qRT-PCR and Western blot. Overexpression of DMBT1 impaired survival, promoted apoptosis in GBC cells in vitro, and inhibited tumor progression in vivo. Further study of underlying mechanisms demonstrated that DMBT1 combined with PTEN which could stabilize PTEN protein, resulting in inhibiting the activation of PI3K/AKT signaling pathway. Our study revealed a new sight of DMBT1 as a tumor-suppressor gene on the PI3K/AKT pathway in GBC, which may be a potential therapeutic target for improving treatment.

Different Forms of TFF3 in the Human Saliva: Heterodimerization with IgG Fc Binding Protein (FCGBP).

The peptide TFF3 is a member of a family of secretory lectins, and is typically synthesized by mucous epithelia together with mucins. It is mainly released from intestinal goblet cells as a high-molecular mass heterodimer with IgG Fc binding protein (FCGBP). Herein, we investigated human saliva by fast protein liquid chromatography (FPLC) and proteomics and identified high- and low-molecular-mass forms of TFF3. Whereas the high-molecular-mass forms represent a heterodimer with FCGBP, the low-molecular-mass forms represent homodimeric TFF3 forms. Proteomic analysis also revealed a C-terminally truncated form of TFF3. We hypothesize that salivary TFF3-FCGBP might play a role in the innate immune defense of the oral cavity and that TFF3 might also bind to microbial glycans. The known interaction of TFF3 with the agglutinin DMBT-1, a typical constituent of human saliva, further supports this protective role.

Sperm binding to porcine oviductal cells is mediated by SRCR domains contained in DMBT1.

The oviduct is an organ in which a subpopulation of sperm is stored in a reservoir, preserving its fertilizing potential. In porcine, two oviductal proteins have been identified in relation to sperm binding, Annexin A2 and Deleted in Malignant Brain Tumor 1 (DMBT1). DMBT1 is a multifunctional, multidomain glycoprotein, and the characteristics of all of its domains, as well as its carbohydrates, make them candidates for sperm binding. In this work, we challenge sperm for binding to pig oviductal cells on primary culture, after treatment with antibodies specific for the different domains present in DMBT1. Only anti-SRCR antibodies produced inhibition of sperm binding to cells. Thus, SRCR is the main domain in DMBT1 promoted sperm binding to form the reservoir in the oviduct, and this function is probably elicited through the polypeptide itself.

Silenced DMBT1 promotes nasal mucosa epithelial cell growth.

OBJECTIVE: The aim of this study was to investigate the role of the deleted in malignant brain tumors 1 (DMBT1) gene in the development of nasal polyps, as well as related mechanisms. METHODS: A stable human nasal mucosa epithelial cell (HNEpC) line with low expression of DMBT1 was generated. Three groups were established: a control group (HNEpCs without any treatment), a control short interference RNA (shRNA) group (HNEpCs transfected with an empty vector), and a DMBT1 shRNA group (HNEpCs with silenced DMBT1). Cell viability, apoptosis, and cell cycle distribution were measured after incubation. Expression of p53, signal transducer and activator of transcription 3 (STAT3), protein kinase B (AKT) and extracellular signal-regulated kinase1/2 (ERK1/2) was detected by western blotting. RESULTS: Compared with the control cell line, HNEpCs with silenced DMBT1 had increased viability and decreased apoptosis. Moreover, after DMBT1 silence, cell numbers were decreased significantly in the G1 phase and increased in the G2 and S phases. DMBT1 silence was associated with increased AKT expression and decreased p53 expression, but it did not alter expression of ERK1/2 or STAT3 (P > 0.05). Compared with the control cell line, HNEpCs transfected with an empty vector did not have altered cell viability, apoptosis, cell cycle distribution or expression of AKT, p53, ERK1/2, or STAT3 (P > 0.05). CONCLUSION: DMBT1 plays an important role in the growth and division of nasal epithelial cells. The possible mechanism might involve DMBT1 regulating the AKT-p53 pathway to promote cell viability and reduce apoptosis of nasal epithelial cells.

One-step FPLC-size-exclusion chromatography procedure for purification of rDMBT1 6 kb with increased biological activity.

Deleted in Malignant Brain Tumor 1 (DMBT1, alias SAG or gp340) is a pattern recognition receptor involved in immune defense, cell polarization, differentiation and regeneration. To investigate the role of the protein in physiological and pathological processes, the protein has often been isolated from saliva or produced in vitro and purified by a multistep affinity purification procedure using bacteria, followed by FPLC. Here, we compared a simple, one-step FPLC-SEC protocol for purification of recombinant DMBT1 6 kb, with that of the standard bacteria affinity purification-based protocol. Our data suggest that our FPLC-SEC protocol yields DMBT1 in a more native conformation.

Evolution of the rapidly mutating human salivary agglutinin gene (DMBT1) and population subsistence strategy.

The dietary change resulting from the domestication of plant and animal species and development of agriculture at different locations across the world was one of the most significant changes in human evolution. An increase in dietary carbohydrates caused an increase in dental caries following the development of agriculture, mediated by the cariogenic oral bacterium Streptococcus mutans. Salivary agglutinin [SAG, encoded by the deleted in malignant brain tumors 1 (DMBT1) gene] is an innate immune receptor glycoprotein that binds a variety of bacteria and viruses, and mediates attachment of S. mutans to hydroxyapatite on the surface of the tooth. In this study we show that multiallelic copy number variation (CNV) within DMBT1 is extensive across all populations and is predicted to result in between 7-20 scavenger-receptor cysteine-rich (SRCR) domains within each SAG molecule. Direct observation of de novo mutation in multigeneration families suggests these CNVs have a very high mutation rate for a protein-coding locus, with a mutation rate of up to 5% per gamete. Given that the SRCR domains bind S. mutans and hydroxyapatite in the tooth, we investigated the association of sequence diversity at the SAG-binding gene of S. mutans, and DMBT1 CNV. Furthermore, we show that DMBT1 CNV is also associated with a history of agriculture across global populations, suggesting that dietary change as a result of agriculture has shaped the pattern of CNV at DMBT1, and that the DMBT1-S. mutans interaction is a promising model of host-pathogen-culture coevolution in humans.

Single-nucleotide and copy-number variance related to severity of hypospadias.

BACKGROUND: The genetic association of hypospadias-risk studies has been conducted in Caucasians, Chinese-Han populations and few in Indian populations. However, no comprehensive approach has been followed to assess genetic involvement in the severity of the disorder. METHODS: The study evaluated to establish the correlation between genotyped single nucleotide and copy number variants (SNPs/CNVs) and severity of hypospadias by an association in a total 30 SNPs in genes related to sex hormone-biosynthesis and metabolism; embryonic-development and phospholipase-D-signalling pathways on 138 surgery-confirmed hypospadias-cases from North India (84 penile and 28 cases of penoscrotal-hypospadias as compared with 31 cases of glanular + coronal), and analyzed and identified CNVs in four familial cases (18 members) and three paired-sporadic cases (6 members) using array-based comparative-genomic-hybridization and validated in 32 hypospadias samples by TaqMan assay. RESULTS: Based on odds ratio at 95% CI, Z Statistic and Significance Levels, STS gene-rs17268974 was associated with Penile-Hypospadias and 9-SNPs [seven-SNPs (rs5934740; rs5934842; rs5934913; rs6639811; rs3923341; rs17268974; rs5934937)] of STS gene; rs7562326-SRD5A2 and rs1877031-STARD3 were associated with penoscrotal-hypospadias. On aggregate analysis with p < 0.001, we identified homozygous-loss of Ch7:q34 (PRSS3P2, PRSS2). On validation in previously CNV-characterized and new (32 hypospadias cases), we identified PRSS3P2-loss in most of the grade 3 and 4 hypospadias. Hence, Grade 1 and 2 (coronal and granular) show no-PRSS3P2-loss and no-association with SNPs in STS; SRD5A2; STARD3-gene but Grade 3 and 4 (Penile and Penoscrotal) show PRSS3P2-loss accompanied with the association of SNPs in STS; SRD5A2; STARD3. CONCLUSIONS: Hence, homozygous-loss of PRSS3P2 accompanied with the association of STS; SRD5A2; STARD3 may link to the severity of the disease.

DMBT1 promotes basal and meconium-induced nitric oxide production in human lung epithelial cells in vitro.

Meconium aspiration syndrome (MAS) is characterized by surfactant inactivation and inflammation. As lung epithelial cells up-regulate nitric oxide (NO) in response to inflammation, the NO production following meconium exposition was examined in relation to expression of Deleted in Malignant Brain Tumors 1 (DMBT1), a protein with functions in innate immunity and inflammatory regulation. Here, DMBT1 expression was analyzed by immunohistochemistry in postmortem lung sections from patients with MAS. The lung epithelial cell line A549, stably transfected with a DMBT1 (DMBT1+ cells) expression plasmid or with an empty expression plasmid (DMBT1- cells), was exposed to meconium. NO was determined in dependence of aminoguanidine (inducible NO synthase inhibitor), steroids and lipopolysaccharide (LPS). DMBT1 is highly expressed in lungs with MAS. In the absence of meconium, DMBT1+ cells showed a higher NO production than the DMBT1- cells (p = 0.0090). Meconium led in DMBT1- and DMBT1+ cells to elevated NO levels (p < 0.0001), but with a higher NO level in DMBT1+ cells (p < 0.0001). Aminoguanidine, an iNOS inhibitor, reduced the higher NO production in DMBT1+ cells (p = 0.0476), but NO levels remained above NO production from DMBT1- cells (p = 0.0289). Dexamethasone diminished NO production in DMBT1+ cells after meconium exposition (p = 0.0076). Combined addition of LPS and meconium significantly increased NO production in both cell types (p < 0.0001). In comparison to exposure with only meconium, the combined addition of LPS and meconium to the cells increased NO levels in both DMBT1- cells (p = 0.0030) and DMBT1+ cells (p = 0.0028). In conclusion, basal and meconium-induced NO production in lung epithelial cells is positively regulated by DMBT1.

DMBT1 expression in biliary carcinogenesis with correlation of clinicopathological data.

AIMS: Deleted in malignant brain tumours 1 (DMBT1) exerts functions in the regulation of epithelial differentiation and inflammation and has been proposed as a tumour suppressor. Because chronic inflammation is a hallmark of cholangiocarcinogenesis, the aim of this study was to investigate the expression of DMBT1 in biliary tract cancer (BTC) and to correlate this expression with clinicopathological data. METHODS AND RESULTS: The expression of DMBT1 protein was examined immunohistochemically in 157 BTC patients [41 intrahepatic (ICC), 60 extrahepatic cholangiocarcinomas (ECC) and 56 adenocarcinomas of the gallbladder (GBAC)]. Additionally, 56 samples of high-grade biliary intraepithelial neoplasia (BilIN 3) and 92 corresponding samples of histological non-neoplastic biliary tract tissues were included. DMBT1 expression was increased significantly in BilIN 3 compared to normal tissue (P < 0.0001) and BTC (P < 0.0001). BTC showed no significant difference in DMBT1 expression compared to non-neoplastic biliary tissue (P = 0.315). Absent DMBT1 expression in non-neoplastic biliary tissue of BTC patients was associated with poorer survival (P = 0.027). DMBT1 expression was correlated significantly with patients' age (P < 0.001). CONCLUSION: DMBT1 is expressed differently in cholangiocarcinogenesis and poorer patients' survival rates are associated with absent DMBT1 expression in non-neoplastic biliary tissue, suggesting a tumour-suppressive role of DMBT1 in early cholangiocarcinogenesis.

The evolution and functional impact of human deletion variants shared with archaic hominin genomes.

Allele sharing between modern and archaic hominin genomes has been variously interpreted to have originated from ancestral genetic structure or through non-African introgression from archaic hominins. However, evolution of polymorphic human deletions that are shared with archaic hominin genomes has yet to be studied. We identified 427 polymorphic human deletions that are shared with archaic hominin genomes, approximately 87% of which originated before the Human-Neandertal divergence (ancient) and only approximately 9% of which have been introgressed from Neandertals (introgressed). Recurrence, incomplete lineage sorting between human and chimp lineages, and hominid-specific insertions constitute the remaining approximately 4% of allele sharing between humans and archaic hominins. We observed that ancient deletions correspond to more than 13% of all common (>5% allele frequency) deletion variation among modern humans. Our analyses indicate that the genomic landscapes of both ancient and introgressed deletion variants were primarily shaped by purifying selection, eliminating large and exonic variants. We found 17 exonic deletions that are shared with archaic hominin genomes, including those leading to three fusion transcripts. The affected genes are involved in metabolism of external and internal compounds, growth and sperm formation, as well as susceptibility to psoriasis and Crohn's disease. Our analyses suggest that these "exonic" deletion variants have evolved through different adaptive forces, including balancing and population-specific positive selection. Our findings reveal that genomic structural variants that are shared between humans and archaic hominin genomes are common among modern humans and can influence biomedically and evolutionarily important phenotypes.

Cloning, expression and purification of the SRCR domains of glycoprotein 340.

Glycoprotein 340 (gp340), an innate immunity molecule is secreted luminally by monolayered epithelia and associated glands within the human oral cavity. Gp340 contains 14 scavenger receptor cysteine rich (SRCR) domains, two CUB (C1r/C1s Uegf Bmp1) domains and one zona pellucida (ZP) domain. Oral streptococci are known to adhere to the tooth immobilized gp340 via its surface protein Antigen I/II (AgI/II), which is considered to be the critical first step in pathogenesis that eventually results in colonization and infection. In order to decipher the interactions between gp340's domains and oral streptococcal AgI/II domains, we undertook to express human gp340's first SRCR domain (SRCR1) and the first three tandem SRCR domains (SRCR123) in Drosophila S2 cells. While our initial attempts with human codons did not produce optimal results, codon-optimization for expression in Drosophila S2 cells and usage of inducible/secretory Drosophila expression system (DES) pMT/BiP/V5-HisA vector greatly enhanced the expression of the SRCR domains. Here we report the successful cloning, expression, and purification of the SRCR domains of gp340. Recognition of expressed SRCRs by the conformational dependent gp340 antibody indicate that these domains are appropriately folded and furthermore, surface plasmon resonance studies confirmed functional adherence of the SRCR domains to AgI/II.

Improvement of Gene Delivery by Minimal Bacteriophage Particles.

Direct delivery of therapeutic genes is a promising approach for treating cancers and other diseases. The current human viral vectors, however, suffer from several drawbacks, including poor cell-type specificity and difficult large-scale production. The M13 phage provides an alternative vehicle for gene therapy with engineerable specificity, but the low transduction efficiency seriously limits its translational application. In this work, we discovered important factors of cells and phages that greatly influence the phage transduction. The up-regulation of PrimPol or the down-regulation of DMBT1 in cells significantly enhanced the phage transduction efficiency. Furthermore, we found that the phage transduction efficiency was inversely correlated with the phage size. By carefully reconstructing the phage origin with the gene of interest, we designed "TransPhage" with a minimal length and maximal transduction efficiency. We showed that TransPhage successfully transduced the human cells with an excellent efficiency (up to 95%) comparable to or superior to that of the adeno-associated virus vectors. Moreover, we showed that TransPhage's tropism was specific to the cells that overexpress the target antigen, whereas adeno-associated viruses (AAVs) promiscuously infected many cell types. Using TransPhage as a gene therapy vehicle, we invented an NK-cell-mediated immunotherapy in which a membrane-bound fragment crystallizable region was introduced to cancer cells. We showed in vitro that the cancer cells expressing the membrane-bound fragment crystallizable (Fc) were effectively killed by CD16+ NK cells through an antibody-dependent cell-mediated cytotoxicity (ADCC)-like mechanism. In the xenograft mouse model, the administration of TransPhage carrying the membrane-bound Fc gene greatly suppressed tumor growth.