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The use of lectin microarray for assessing glycosylation of therapeutic proteins

ABSTRACT Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins.

Introduction
Glycosylation of proteins is a complex post-translational modification that attaches carbohydrates or named glycans at specific sites on a protein backbone, most commonly at Asn (N-linked) or Ser/Thr (O-linked) residues. 1 The N-linked glycosylation occurs at the consensus sequence of Asn-X-Ser/Thr (where X is any amino acid except proline), whereas O-linked glycans are usually attached to Ser or Thr residues. Both Nand O-glycosylation involve a series of enzymatic reactions catalyzed by glycan-processing enzymes, which are responsible for trimming and modifications of glycan epitopes, resulting in diverse N-glycan structures (e.g., high-mannose, complex, and hybrid glycans) and O-glycan variants containing up to 8 O-GalNAc glycan core structures. To add complexity, protein glycosylation is influenced by the type of host cells and fluctuations in fermentation conditions (e.g., media, pH, temperature, agitation). 2 For instance, therapeutic glycoproteins produced by mammalian cells such as Chinese hamster ovary (CHO) cells usually contain human-like glycans. By contrast, proteins expressed by yeast strains usually contain high levels of mannose (up to 100 units). Other hosts including engineered plant cells and genetically modified animals may produce proteins with non-human glycan variants such as xylose, N-glycolylneuraminic acid (Neu5Gc) or terminal a-galactose (a-Gal), which are known to be immunogenic. As a result, a glycoprotein produced by living cell systems usually contains a mixture of different glycoforms. These protein variants share an identical peptide backbone, but may differ in glycosylation properties such as glycosylation site, glycan structure and content.
Glycans attached to a therapeutic protein can directly affect product quality, safety and efficacy. It is well documented that glycans attached to a protein affect protein solubility and stability, [3][4][5] pharmacokinetics/pharmacodynamics (PK/PD), 3,6-8 and immunogenicity. 2,9 In the latter, non-human glycans attached onto a therapeutic protein such as Neu5Gc and terminal a-Gal epitopes could cause immunogenic responses. For many monoclonal antibodies (mAbs), proper glycosylation of the crystallizable fragment (Fc) is essential to IgG antibody effector functions. 10 Therefore, glycan moieties of therapeutic proteins must be adequately characterized and controlled throughout product life cycle. The commonly used methods include highperformance liquid chromatography (HPLC), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), mass spectrometry (MS) and capillary electrophoresis (CE), [11][12][13][14][15][16] which provide information on glycosylation sites, site occupancy, and contents of glycan variants attached to glycoproteins.
There is growing interest in the development of high throughput platforms for assessing protein glycan profiles. Lectins are glycan binding proteins (GBPs) that selectively recognize glycan epitopes of free carbohydrates or glycoproteins. 17 Lectin-based microarrays have been used to analyze glycan profiles of purified glycoproteins or cell surface proteins. [18][19][20][21][22][23] In this study, we evaluated the potential utility of a lectin microarray for characterization of therapeutic glycoproteins. Using commercial lectin chips containing 45 distinct lectins, we tested a panel of 15 therapeutic proteins for their glycan profiles. Our data show that the lectin microarray is robust in generating glycan profiles that are generally consistent with the known glycan characteristics of an individual glycoprotein.

Results
The utility of a lectin microarray in determining glycan profiles of therapeutic mAbs We assessed the utility of a lectin microarray in profiling glycan variants of therapeutic proteins using commercial lectin microchips printed with 45 distinct lectin proteins (Fig. 1A). These include lectins that selectively bind core-fucose, sialic acids, Nacetyl-D-lactosamine (Galb1-4GlcNAc), mannose, or N-acetylglucosamine (GlcNAc) oligomers, respectively (Table 1). 18 These glycan structures are commonly found in recombinant glycoproteins. We tested a panel of 15 distinct proteins, including 8 therapeutic mAbs, one Fc-fusion protein, 4 recombinant therapeutic cytokines and enzymes, and 2 different versions of human transferrin proteins (Table 2). To facilitate microarray analysis, protein samples were fluorescently labeled with Cy3 followed by incubation onto lectin-coated chips. We first performed dose-titrations and identified the optimal concentrations of Cy3-labeled protein sample that fell within the linear response ranges for most lectin spots (Supplement I). We also tested the effect of incubation time on lectin binding signals, showing that overnight incubation is required to obtain optimal binding between a Cy3-labeled glycoprotein and the lectins printed on chips (Supplement II). In the following experiments, a fixed concentration of protein samples (125 ng/mL) was applied to lectin chips and incubated overnight then lectin binding signals were detected. Bound glycoprotein signals were determined using evanescent-field fluorescence scanner, which allows a direct measure of glycans bound onto lectins without the need of washing procedures to remove unbound species (Fig. 1B). Using lectin chips from different batches, we determined the assay reproducibility to be < 10% CV for most lectin-glycan binding signals of the samples tested.
We first tested a panel of 6 therapeutic mAbs, including bevacizumab (Avastin Ò ), trastuzumab (Herceptin Ò ), adalimumab (Humira Ò ), infliximab (Remicade Ò ), rituximab (Rituxan Ò ) and omalizumab (Xolair Ò ). These products are known to be IgG1 isotypes with glycosylation exclusively occurring in the Fc. [24][25][26] All these IgG1 mAbs showed similar lectin binding patterns ( Fig  interactions (Table 1), the lectin binding patterns indicate the presence of core fucose, outer galactose, mannose, and GlcNAc oligomer, and the absence of sialic acids and tri/tetra-antennary complex-type N-glycans. Overall, the glycan profiles derived from lectin microarray are consistent with the reported glycans in IgG1 Fc that are known to contain principally bi-antennary non-sialylated complex-type N-glycans with little or no highmannose type or sialylation (Fig. 2C). 27 Using the specified lectin chips, we detected similar lectin binding patterns for IgG1 mAbs containing only Fc glycosylation (Fig. 2B). Compared to the other 5 mAbs, rituximab appeared to display relative higher binding signals at AOL/AAL (core fucose), RCA120 (terminal galactose), and GNA (high mannose). Panitumumab (Vectibix Ò ), an IgG2 isotype, displayed much weaker signal intensities across the lectin chip despite a similar lectin binding pattern as observed for IgG1 mAbs ( Fig. 2A). This data is consistent with the reported lower level of glycan content in the IgG2 antibodies in comparison with IgG1 antibodies. 28,29 Next, we tested 2 other therapeutic glycoproteins that are associated with more complex glycosylation patterns. Cetuximab (Erbitux Ò ) was chosen because it contains N-glycosylation sites in both the antigen-binding fragment (Fab) and Fc of the molecule. 30 In contrast to glycan profiles for IgG1 mAbs (Fig. 2), cetuximab showed unique binding patterns at lectins (SNA, SSA and TJA-I), which are known to bind a2-6-linked sialic acids (Fig. 3A & B). A binding signal was also detected at  Overall, this data is consistent with the known glycan patterns of cetuximab Fab, which include an abundant N-linked sialic acid (Neu5Gc) and terminal a-Gal variants (Fig. 3C). 30 Another sample tested was etanercept (Enbrel Ò ), a homodimer of Fc-fusion protein consisting of TNF-a receptor and an IgG1 Fc portion, which was reported to contain 3 N-linked and 13 O-linked glycosylation sites. 33 Etanercept displayed distinct lectin binding patterns compared to IgG1 mAbs. For example, strong binding signals were detected at MAL-I and ACG, which are known to selectively bind a2-3 linked sialic acid epitope. No signals were detected at lectins SNA, SSA or TJA-1, showing the lack of a2-6 sialylation in the protein sample. 34 This data confirms the presence of complex glycans in etanercept, including a2-3-sialic acids and abundant bi-antennary neutral glycans. Compared to other IgG1 mAbs, etanercept displayed a strong and novel signal at MPA, a lectin that is known to selectively bind Galb1-3GalNAc and aGalNAc. 35 This data is consistent with the reported abundance of O-glycans onto etanercept. 33 To support the selectivity of lectin binding signals, we tested the Fab and Fc purified from rituximab and cetuximab, respectively. Rituximab is known to contain only one N-glycosylation site in its Fc. 26 In the lectin microarray, the isolated rituximab Fab showed little or no signals across the lectin chip whereas the rituximab Fc displayed a similar lectin profile as intact rituximab (Fig. 4A). By contrast, cetuximab contains 2 N-glycans, one located within its Fc portion and another in the Fab. 30 The uncommon a-Gal and Neu5Gc epitopes were reported to be solely in the Fab (Fig. 3C). 30 The cetuximab Fc expressed a typical lectin profile for IgG1 Fc glycans (bi-antennary G0F, G1F, and G2F) (Fig. 4B). The lectin signals of SNA, SSA and TJA-1, which indicate expression of a2-6-sialylation, were only present in intact cetuximab and cetuximab Fab profiles, but were absent in the Fc profile. GSL-I signal also indicated the presence of a-Gal structure in the Fab, but not in the Fc (Fig. 4B). These data not only confirm the proper locations of glycosylation sites in Fc or Fab portions, but also support the selectivity of lectin bindings toward particular glycans in a testing sample. As noted, the rituximab Cy3-labeled Fab showed no lectin-binding signals across the lectin chips, confirming no or little interaction between the protein backbone and the lectins.
The utility of lectin microarray in glycan profiling of proteins produced by different host cell systems We assessed whether lectin microarray is capable of profiling glycan variants of therapeutic proteins that are produced by different host cell systems such as mammalian cells, yeast strains and bacterial strains. These cell systems are different in their glycosylation machinery, which produce proteins with distinct glycan patterns. For example, glycoproteins expressed by yeast strains usually contain high-mannose structures whereas Escherichia coli (E. coli) proteins are all non-glycosylated due to the lack of glycosylation machinery in natural bacterial. 2 We evaluated a panel of 6 proteins, including 2 therapeutic proteins produced by CHO cells (darbepoetin alfa (Aranesp Ò ) and dornase alfa (Pulmozyme Ò )), one therapeutic protein produced by yeast (rasburicase (Elitek Ò )), human transferrin proteins expressed by recombinant rice strain or isolated from human plasma, and filgrastim (Neupogen Ò ) produced by E. coli ( Fig. 5A & B). Darbepoetin alfa showed strong signals at MAL-I, demonstrating the presence of a2-3-sialylation structures. Moreover, darbepoetin alfa displayed strong signals at PHALcoated spots, which are known to be selective for tri-/tetraantennary N-glycan structures. Such a signal was not detected in other proteins, confirming the absence of tri-/tetra-antennary glycans in these samples. This pattern was consistent with the reported data that darbepoetin alfa contains high levels of sialylation and abundant tri-or tetra-antennary structures (Fig. 5C). 36 Darbepoetin displayed relative week binding signals at other lectin spots compared to MAL-I and PHAL, raising a possibility that those other glycan species (e.g., Galb1-4GlcNAc and mannose oligomers) might be "capped" by the outermost galactose and sialic acid. Dornase alfa (Pulmozyme Ò ), a recombinant enzyme expressed by CHO cells, displayed a simpler lectin binding pattern compared to the above IgG1 and IgG2 mAbs ( Fig. 5A and  5B). The sample tested showed unique binding signals at SNA/ SSA for a2-6-sialylation, RCA120 for Galb1-4GlcNAc, DSA for GlcNAc oligomer and/or Galb1-4GlcNAc, 37,38 ConA for mannose, and LEL/STL for GlcNAc oligomers. The spectrum of selective binding signals suggests the presence of complextype glycans with a2-6-sialylation in dornase alfa molecules. By contrast, rasburicase (Elitek Ò ), a therapeutic glycoprotein produced by yeast strains, displayed distinctively different lectin profiles compared to the above described products produced by mammalian cells. Rasburicase showed relatively weak binding signals across the lectin chips, which is consistent with its known low level of glycosylation. 39 Despite the overall weak binding signals, rasburicase appeared to interact exclusively with mannose binding lectins (NPA, ConA, and GNA) and GlcNAc oligomer binding lectins (STL and UDA). This data confirms the presence of high-mannose carbohydrates that are mainly attached onto glycoproteins produced by yeast strains. 40 No binding signals were detected at sialic acid-binding lectins (e.g., MAL_I, SNA, SSA, and TJA-I), fucose-binding lectins (e.g., PSA and LCA) or galactose-binding lectins (e.g., RCA 120 and PHAE), even when the protein concentration of rasburicase was enhanced to 500 ng/mL (data not shown), demonstrating the absence of the relevant glycan species in rasburicase. The two versions of human transferrin proteins also showed distinct glycan patterns in which the recombinant human transferrin expressed in rice (transferrin-rice) showed binding signals primarily at mannose-binding lectin (NPA) and GlcNAc oligomer-binding lectins (LEL, STL and UDA). The DSA signal indicated the presence of either GlcNAc oligomer or Galb1-4GlcNAc. By contrast, transferrin proteins isolated from human plasma showed additional signals at a2-6-sialic acid-binding lectins (SNA, SSA, and TJA-I) and galactose-binding lectins (RCA120 and PHAE). As expected, no lectin binding signals were detected for filgrastim (Neupogen Ò ) that is produced by E. coli as a non-glycosylated protein. 41 The utility of lectin microarray in monitoring terminal galactosylation and sialylation of glycoproteins To further evaluate the utility of lectin microarray in glycan profiling, we prepared protein variants with defined galactosylation and sialylation modifications. This was achieved through in vitro enzymatic glycoengineering of rituximab using commercially available galactosyltransferase and sialyltransferase. b1-4-galactosyltransferase (b1-4GalT) catalyzes the transfer of galactose from donor substrate UDP-galactose (UDP-Gal) to GlcNAcb1-2Man units of glycoproteins to form a b1-4-galactosylation linkage, while a2-6-sialyltransferase (a2-6SiaT) facilitates sialylation by adding sialic acids to terminal Galb1-4GlcNAc units. Modified rituximab protein variants were purified and then characterized using mass spectrometry (MS), revealing distinct deconvoluted MS spectra for the light chain and heavy chain (Fig. 6A). The light chain fragments resolved as a single species at an average mass of 23036 Da, corresponding to the theoretical mass of rituximab light chain. 42,43 Consistent with the lack of glycosylation sites within the rituximab light chains, the mass of light chain remained unchanged after treatments of rituximab with b1-4GalT or further with a2-6SiaT. The other 3 major mass species at 50507, 50669, 50832 Da correspond to the heavy chains of rituximab containing G0F, G1F or G2F glycoforms, respectively (Fig. 2C). 42,43 Treatment of rituximab with b1-4GalT resulted in a mass shift from G0F and G1F to G2F, indicating galactosylation reactions were effectively accomplished. When the galactosylated rituximab mixture was sequentially treated with a2-6SiaT, the final product showed a further mass shift from 50832 Da (G2F) to 51122 Da (C290) and 51414 Da (C582). These mass shifts corresponded to an addition of one or two Neu5Ac residues, indicating that G2F glycoform was effectively converted to primarily mono-sialylated species (S1G2F) and a small portion of di-sialylated species (S2G2F). These data indicate that the in vitro glycan engineering reactions produced rituximab variants containing the desired modifications (e.g., galactosylation and sialylation). The engineered rituximab samples with defined glycan variations were then analyzed by lectin microarray (Fig. 6B). The reaction buffer alone had no effect on lectin binding profile of rituximab. The sample resulting from b1-4GalT reaction (rituximab C b1-4GalT), which was confirmed by MS to majorly contain G2F galactosylation (Fig. 6A), displayed strong binding signals at positions of ECA and RCA120. Both lectins are known to bind N-glycan Galb1-4GlcNAc, and therefore the incurred ECA lectin-binding signal and the significantly enhanced RCA120 signal were indicative of the increased galactose species in the samples. Notably, there was a concomitant decrease in the binding signals at ABA, a lectin with dual binding affinity toward Gal-exposed O-glycans and GlcNAcexposed N-glycans. 44 Because rituximab was not reported to undergo O-glycosylation, the ABA binding signal detected for native rituximab was likely due to GlcNAc-exposed N-glycans. Such GlcNAc-exposed N-glycans appeared to be occupied upon galactosylation catalyzed in the reactions with b1-4GalT. Samples of native rituximab and galactosylated species showed no detectable signals at the lectins SNA, SSA, TJA-I that are known to bind sialic acids. By contrast, strong binding signals at these lectins were detected for the samples derived from the sequential reactions with b1-4GalT and a2-6SiaT enzymes. No signal was detected at MAL-I (a lectin selective to a2-3-sialylation), demonstrating not only the specificity of glycan engineering but also the utility of the lectin microarray in distinguishing different terminal sialic acid linkages.

Discussion
The complexity of glycosylation poses an analytical challenge in the development of therapeutic glycoproteins. The methods most commonly used for analysis include MS, HPLC, HPAEC-PAD, and CE. Among these methods, MS remains a powerful tool in the characterization of glycosylation site(s) occupancy and carbohydrate structures. MS-based methods involve enzymatic digestion of a glycoprotein into peptide fragments and separation by liquid chromatography. HPLC-, HPAEC-and CE-based methods usually require the release of glycans from a glycoprotein through enzymatic or chemical reactions. As such, an accurate assessment of glycosylation requires a complete release of all glycans that are present in a glycoprotein being tested. By contrast, lectin microarray directly measures glycan profiles on an intact protein without the need for enzymatic digestion or clipping glycans from the protein backbone. Such a platform is unique in increasing the likelihood of full coverage of all glycan variants of a glycoprotein. Using the commercial lectin chips, we were able to determine glycan profiles for a panel of therapeutic proteins that were generally consistent with their known glycosylation properties (Figs. [2][3][4][5]. Notably the lectin microarray was highly sensitive to alterations in the terminal glycan structures, i.e., galactosylation vs.  Figure 6. Assessing glycan variants in glycan-engineered rituximab protein samples. Rituximab was incubated with a reaction buffer alone (rituximab C buffer), b1-4-galactosyltransferase (rituximab C b1-4GalT), and b1-4-galactosyltransferase followed by a2-6-sialyltransferase (rituximab C b1-4GalT C a2-6SiaT) (see detail in Materials and Methods). After affinity purification, the resulting samples were analyzed using mass spectrometry and lectin microarray, respectively. Shown are representatives of deconvoluted mass spectra (A) and corresponding lectin binding profiles (B) for the samples produced under the indicated conditions. sialyation (Fig. 6). Lectin microarray can effectively distinguish glycan isomers containing different sialic acid linkages. Our data demonstrate a usefulness of the lectin microarray in screening glycan patterns of protein samples. As noted, the commercial lectin chips employed in this study was not designed specifically for assessing therapeutic mAbs. In principle, assay performance could be further improved through the use of lectins with improved selectivity and binding affinity to distinct glycan species. To analyze a specific glycoprotein, lectin chips can be customized to include the lectins that are relevant to the glycan species that are possibly present in the testing sample. Upon optimization of lectin chips, lectin microarray platform could be adopted as a complementary tool for high throughput screening of glycan profiles of therapeutic glycoproteins.

Therapeutic proteins and reagents
All therapeutic proteins were purchased from the Division of Veterinary Resources pharmacy, Office of Research Services, National Institute of Health, and handled according to respective package inserts. Recombinant human transferrin expressed by rice and transferrin purified from human blood plasma were purchased from Sigma-Aldrich. Cy3 mono-reactive dye was obtained from GE Healthcare. Zeba spin desalting columns (7K MWCO) were purchased from Thermo Scientific. Lectin chips coated with 45 distinct lectin proteins and the probing solutions were obtained from GlycoTechnica. Glycosyltransferases b1-4-galactosyltransferase (b1-4GalT) and a2-6-sialyltransferase (a2-6SiaT) were purchased from Prozyme. Micro BCA Protein Assay Kit, NAb Protein A Plus Spin Kit and Pierce Fab micro preparation kit were obtained from Thermo Scientific. NuPAGE 4-12% Bis-Tris gels and Coomassie stain (SimplyBlue SafeStain) were purchased from Life Technologies.

Lectin microarray analysis
Protein samples were diluted to 50 mg/mL in phosphate-buffered saline (PBS). Aliquots (20 mL, 1 mg protein) were mixed with 100 mg Cy3 mono-reactive dye, and incubated at room temperature for 2 h. The unbound Cy3 dye was removed using Zeba spin desalting columns (7K MWCO). Cy3-labeled protein samples were diluted to 125 ng/mL in probing solution, and an aliquot of 100 mL was applied onto a lectin chip, and incubated at room temperature on an orbital shaker for 18 hours in dark. The resulting lectin chips were scanned for fluorescence intensities on each lectin-coated spot using an evanescent-field fluorescence scanner GlycoStation Reader 1200 (GlycoTechnica, Japan).

Preparation of IgG Fab and Fc
The Fab and Fc of therapeutic mAbs were prepared using Pierce Fab micro preparation kit following the vendor's protocol (Thermo Scientific). Briefly, immobilized papain (settled resin) was located in a 0.8 mL spin column and equilibrated using Fab digestion buffer. Antibody samples were diluted to 0.4 mg/mL using Fab digestion buffer, and aliquot of 120 mL was added to the spin column containing equilibrated immobilized papain. The digestion reaction was incubated overnight at 37 C. Digested samples were collected by column centrifugation and applied onto NAb Protein A Plus Spin Column to separate the Fab and Fc. Protein concentrations of the resulting samples were determined using Micro BCA Protein Assay Kit (Thermo Scientific).

Liquid chromatography-mass spectrometry analysis
Liquid chromatography-mass spectrometry analyses were conducted on an Agilent 1260 HPLC-Chip nano-electrospray-ionization 6520 Q-TOF MS system. Solvent-A was 0.1% formic acid in water and solvent-B was 0.1% formic acid in 95% acetonitrile. Mass correction was enabled during the run using internal reference ions with masses of 299.2945 and 1221.9906 Da. Intact protein mass measurement was performed using an Agilent 43 mm 300 A C8 chip with a 40 nL trap column (G4240-63001 SPQ105). All rituximab samples were reduced in 10 mM DTT for 1 hour at 37 C, and then formic acid was added to reach a final concentration of 0.1% (V/V). The samples were diluted to 150 ng/mL in 0.1% formic acid, and 1 mL (1 pmol) or larger volume was injected onto the trap column in the C8 chip at a flow rate of 2.5 mL/min in 100% A, then eluted at 0.5 mL/ min with a linear gradient of 10-100% B in 18 min and held for additional 4 min. Q-TOF VCap, fragmentor, and skimmer settings were 1,890 eV, 225 eV, and 65 eV, respectively. HPLC-Chip gas temperature and drying gas flow rate were 350 C and 9 L/min, respectively. The data were analyzed using Agilent MassHunter (version B.05.00) Qualitative Analysis software and deconvoluted with Agilent MassHunter Bioconfirm software.


s2fieldsofstudy: Biology Medicine

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Exome sequencing identifies a mutation in TMC1 as a novel cause of autosomal recessive nonsyndromic hearing loss

Background Autosomal recessive non-syndromic hearing loss (ARNSHL) is highly heterogeneous, and mutations in the gene encoding transmembrane channel-like 1 (TMC1) have been implicated in its development. To date, 35 homozygous mutations in TMC1, identified in over 60 families worldwide, have been shown to be associated with ARNSHL. However, few of these mutations were detected in the Chinese population. In this study, we describe a pathogenic missense mutation located in the T5–T6 domain of TMC1 in a three-generation Chinese family with 14 members. Methods Whole exome sequencing was performed using samples from one unaffected individual and two affected individuals to systematically search for deafness susceptibility genes. Candidate mutations and cosegregation of the phenotype were verified by polymerase chain reaction and Sanger sequencing in all of the family members. Results We identified a novel TMC1 mutation in exon 20, c.1979C>T, p.P660L, which segregated with prelingual autosomal recessive sensorineural hearing loss. Conclusions We found a new missense mutation in the T5–T6 domain of TMC1, which is highly conserved in many species. These data support the potential conserved role of p.P660L in human TMC1 function. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0780-5) contains supplementary material, which is available to authorized users.

Background
Hearing loss (HL) is one of the most prevalent human birth defects, affecting one in 1000 individuals. Most congenital cases of HL have a genetic etiology, and nonsyndromic hearing loss (NSHL) accounts for approximately 80 % of genetic deafness [1]. To date, a total of 87 NSHL genes have been identified (http://hereditaryhearingloss. org), and most hearing impairments originating from these gene mutations are inherited in an autosomal recessive pattern. Genetic HL exhibits marked heterogeneity, which can be explained by the complexity of auditory system [2]. The proteins encoded by the identified deafness genes are involved in different functions, including gene regulation, embryonic development, ionic and osmotic homeostasis, synaptic transmission, generation of endocochlear potential, hair cell bundle morphology, and mechano-electrical transduction (MET) [3].
The gene encoding transmembrane channel-like 1 (TMC1) has been implicated in the pathogenesis of both dominant and recessive nonsyndromic HL, DFNA36 and DFNB7/11, respectively [4]. Although TMC1 is expressed in cochlear hair cells and is involved in hair-cell function [5], the expression and function of the TMC1 protein in the inner ear are unclear. Deafness (dn) and Beethoven (Bth) mutant mice carrying recessive (Tmc1dn) and dominant (Tmc1Mhdabth) TMC1 mutations show several physiological deficits in hair cell maturation, and recent studies have indicated that hair cells from Tmc1-knockout mice lack mechanosensory potential [5]. Chatzigeorgiou et al. [6] demonstrated that TMC1 from Caenorhabditis elegans encodes a sodium-sensitive ion channel; however, this study lacked a mutant control, and the observations presented in this study have not been replicated for vertebrate TMC1. Another study found that TMC1 is a component of hair cell transduction channels [7], although these results are controversial. Taken together, the results of many studies have supported that TMC1 expression is required for conventional mechanotransduction in auditory and vestibular hair cells [7][8][9].
To date, 35 homozygous mutations in TMC1, identified in 60 families worldwide, have been shown to be associated with ARNSHL (Additional file 1: Table S1). However, these mutations have rarely been found in the Chinese population [2]. Almost all reported recessive cases have been shown to exhibit a similar phenotype characterized by prelingual severe-to-profound HL [3]. The most common recessive mutation in TMC1 reported to affect HL is the nonsense mutation p.R34X, which accounts for over 30 % of mutant alleles of TMC1 in Asian and North African populations [10].
In this study, we identified a novel homozygous mutation in TMC1 located in exon 20, c.1979C>T, p.P660L in a three-generation, 14-member family (SH-02) presenting segregating ARNSHL with whole exome sequencing (WES). We further sequenced 500 ethnically unrelated healthy individuals and 300 sporadic deafness cases; none of these individuals carried the P660L mutation. Therefore, we concluded that the HL in this family was caused by novel homozygous mutation in TMC1.

Family recruitment and clinical evaluations
A three-generation, non-consanguineous 14-member family (SH-02) presenting with segregating ARNSHL was identified by the Department of Otolaryngology, Head and Neck Surgery of Shanghai East Hospital, Tongji University, Shanghai, China (Fig. 1). All clinical information was collected at the Department of Otolaryngology and Head and Neck Surgery, Shanghai East Hospital, Tongji University, Shanghai, China. Medical histories were obtained using a questionnaire covering the following issues: subjective degree of HL, age at onset, progression, symmetry of hearing impairment, use of hearing aids, presence of tinnitus and vertigo, medication, noise and ototoxic drug exposure, pathological changes in the ear, and other relevant clinical manifestations. Systemic medical examinations and approximate intelligence assessments were also performed for all affected individuals.
Another 300 individuals with sporadic HL participated in this study, in addition to 500 ethnically matched healthy controls. All procedures were approved by the Ethics Committee of Shanghai East Hospital, which is associated with Tongji University, and were carried out only after written informed consent had been obtained from all study participants and from the parents of participants younger than 18 years of age. Participants were informed that all data collected would be used only for scientific research and not for any commercial purposes.

Whole exome sequencing
Genomic DNA was extracted from whole blood samples using a blood DNA kit (Qiagen, Germany), and 1 μg of TruSeq DNA LT/HT Sample Prep Kit and TruSeq Exome Enrichment Kit were used to enrich exomes. Equal amounts of library samples were pooled and then hybridized to the customized capture array, including exons, splicing sites, and immediate flanking intron sequences. Sequencing was performed on an Illumina HiSeq 2500 to generate paired end reads. Reads that aligned to exon regions were collected for mutation identification and subsequent analysis. Samtools mpileup was used for variant calling and to identify SNPs and indels. ANNOVAR was used for annotating the genes.

Sanger sequencing
Samples from all available members from the SH-02 family were subjected to Sanger sequencing to determine whether the potential mutations in causative genes, particularly the homozygous mutation in TMC1, cosegregated with the disease phenotype in this family. The polymerase chain reaction (PCR) products were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA, USA) and analyzed using an ABI 3700XL Genetic Analyzer. And the primers for the TMC1 sequencing were as follows: TMC1-Forward: AGGATGGGCTCCTTCTTTGC; TMC1-Reverse: ACGAGGTTTCACCGTGTTGG.

In silico analysis
In the present study, we used SIFT [11], Polyphen2 [12], and MutationTaster [13] software to determine possible changes in the protein structure that may affect the phenotype. Clustal X1.83 software was used to compare the human wild-type TMC1 protein sequence with orthologs from Mus musculus, Physeter catodon, Macaca fiscicularis, Ailuropoda melanoleuca, Octodon degus, and Lipotes vexillifer and to examine evolutionary conservation and structural prediction for this protein. Sequences were obtained from http://www.ensembl.org/.

Clinical description
The proband and his sister were congenital deaf-mute, while all their parents and grandparents had normal auditory and verbal functions. The two patients were delivered full-term after normal deliveries, and none of the parents had any history of constant exposure to noise or ototoxic drugs or a history of serious infection during pregnancy. Bone and air conduction of pure tone audiometry displayed no auditory reaction at any frequency. Tympanometry indicated a type A curve for the tympanogram, demonstrating that the tympanic cavity exhibited normal function, and acoustic reflex could not be induced in either ear (the threshold of the acoustic reflex was 100 dBHL; Fig. 2a). Otoacoustic emission could not be induced at any frequency. Upon ABR examination, well-differentiated wave profiles and regular latency could not be induced by click stimuli presented at 100 dBnHL in both ears, indicating that there was no effective acoustic information generated or sent to the brain by mechanosensory hair cells in the organ of Corti. CT scans and MRI data indicated that the mastoid process, cochlea, internal auditory meatus, and membranous labyrinth were all well developed, as was the ossicular chain (Fig. 2b, c). Clinical and instrumental evaluations did not reveal any evidence of syndromic features, such as cardiovascular diseases, diabetes, visual problems, or neurological disorders. The younger brother and his elder sister both displayed normal intelligence (Table 1).

Whole exome sequencing
To systematically search for deafness susceptibility genes, we performed WES of samples from one unaffected (II-3) and two affected (III-4 and III-5) individuals from the SH-02 family pedigree. An average of 4.67 billion bases of high-quality sequence was generated per individual, with an average sequencing depth of approximately 97 in the target region; this satisfied the requirements for calling single nucleotide polymorphisms (SNPs) and indels. The sequencing data were aligned to the NCBI human reference genome and compared with dbSNP138, which contains pilot data from the 1000 Genomes Project, from eight sequenced HapMap individuals and from ten individuals in the YH database.
In total, 7898 variants were identified in the two patients; 5760 of these were nonsynonymous variants, including splice acceptor and donor site mutations and coding indels, which were more likely to be pathogenic mutations. Next, these variants were prioritized for further evaluation using two filtering criteria: (1) variants within the allele frequency cutoff (less than 0.01 in the dbSNP138, HapMap, 1000 Genomes, and local datasets); and (2) variant found in all the affected individuals but not in the unaffected individual; variants meeting these criteria were retained for further analysis. These filtering criteria reduced the list of candidate variants to 25 nonsynonymous homozygous variations.
Then, we screened 25 variations found among the pedigree samples by Sanger sequencing and found a missense variant, c.1979C>T, p.P660L, in exon 20 of TMC1 [NM_138691, (MIM # 600974)], which co-segregated with the disease (Fig. 3a). This novel TMC1 mutation was exclusively identified in all two affected patients but was not found in the 12 unaffected family members. To assess the possibility that P660L is a disease-causing mutation, we further sequenced 500 ethnically unrelated healthy individuals and 300 sporadic deafness cases, and confirmed that none of these people carried the P660L mutation. Thus, our data suggested that P660L was a disease-causing mutation in the Chinese pedigree (SH-02) with NSHL.

In silico analysis
To determine the potential effects of the p.P660L missense mutation on TMC1 function, we further performed in silico analyses. This mutation was predicted to be "Damaging", "Probably Damaging", and "Disease-causing" by SIFT, Polyphen2, and MutationTaster, respectively ( Table 2). The conservation analysis indicated that the Pro residue at 660 in the TMC1 protein was conserved across Homo sapiens, Mus musculus, Physeter catodon, Macaca fiscicularis, Ailuropoda melanoleuca, Octodon degus, and Lipotes vexillifer (Fig. 3b). This finding indicated that this novel mutation may be the cause of the observed HL in this Chinese family.

Discussion
The World Health Organization has estimated that 360 million people worldwide have disabling HL (http://www. who.int/mediacentre/factsheets/fs300/en/) and that as the population ages, the global burden of diseases attributable to deafness will increase [14]. Linkage analysis was once regarded as the most powerful and widely used Fig. 2 The SH-02 audiometric phenotype. a Pure-tone bone and air conduction thresholds are presented for the right ears of SH-02 family patients. b Based on the CT scan and MRI data of the proband (III5) and his sister (III4), the mastoid process and cochlea were well developed, and the ossicular chain was intact. Additionally, both the internal auditory meatus and the membranous labyrinth were well developed Hu et al. J Transl Med (2016) 14:29 method for linking critical intervals to identify diseasecausing genes in large pedigrees; however, this method is not appropriate for small families. Recently, with the marked advancements in sequencing technology, WES, which involves targeted sequencing of the protein-coding subset of the human genome, has been used as a convenient, rapid method for identification of new genes. Nextgeneration sequencing has advantages of small samples  quantity, minimal cost, high-throughput sequencing, and low requirement for family size [15]. In this study, WES was used to find the disease-causing gene of a Chinese family with HL, and we identified a novel TMC1 missense mutation in exon 20, c.1979C>T, p.P660L. TMC1 on chromosome 9q21 contains 24 exons that make up a coding region of 2283 nucleotides. The gene sequence is highly conserved, which suggests strong selective pressure throughout animal evolution. Mutations in TMC1 are a common cause of autosomal recessive nonsyndromic deafness, particularly in India, Pakistani, Turkish, and Tunisian families [2]. Thirty-five reported homozygous recessive mutations in TMC1, found in over 60 families worldwide, have been identified in different parts of TMC1 and cause different structural and functional disparity in intracellular and extracellular domains (Fig. 4).  . 4 Location of the p.P660L mutation and other reported exonic mutations in TMC1. The red rhombus represents a missense or nonsense mutation; the green triangle represents a deletion mutation; and the green arrow/yellow star represents the p.P660L mutation located within a predicted third extracellular loop found between the fifth and sixth transmembrane domains TMC1 topology is predicted to include six membranespanning domains with three extracellular loops, a large intracellular loop settled between transmembrane (TM) domains four and five, a long intracellular N-terminus, and a short intracellular C-terminus. The structure suggests that the protein may function as a receptor, transporter, pump, or channel [16]. The mutation found in this study, p.P660L, was located within a predicted third extracellular loop situated between the fifth and sixth TM domains (Fig. 4). Pro660 is located in a highly conserved glycosyltransferase domain, which is highly conserved among many species. To the best of our knowledge, this is the first report describing a missense mutation in the glycosyltransferase domain of TMC1, supporting the importance of the conserved role of P660L in human TMC1 function.
TMC1 expression is constant in mature cochlear and vestibular hair cells, as shown in dn and Bth mutant mice carrying recessive (Tmc1dn) and dominant (Tmc1bth) TMC1 alleles. In the DFNA36 models, heterozygous mice (Tmc1 Bth/+ ) showed progressive hair cell degeneration [17], while homozygous mice (Tmc1 Bth/Bth ) exhibited profound deafness [5]. Models of human recessive deafness, i.e., DFNB7/11, TMC1 dn/dn mice, do not have cochlear responses to sound stimuli and show several physiological deficits in hair cell maturation [5]. Kim et al. [9] concluded that TMC1/2 double mutant mice lacked conventional mechanotransduction, leading them to hypothesize that TMC1 is required for targeting the MET channel to the tips of the stereocilia, where they can interact with other constituents of the transduction complex, including the tip link. Although the specific function of TMC1 is unknown, recent studies have revealed that TMC1 is necessary for MET in cochlear and vestibular hair cells. Additionally, TMC1 is thought to be a component of the mechanotransduction channel in hair cells of the mammalian inner ear [3].
Caenorhabditis elegans tmc1 has been shown to encode a sodium-sensitive ion channel. The predicted structure of TMC1 is similar to that of the α-subunit of voltagedependent K + channels, which have six TM segments and intracellular N-and C-termini [18], and TMC1 has been predicted to function as an ion channel or transporter mediating K + homeostasis in the inner ear [19]. Ion channels serve many functions, including the transport of ions and water, control of electrical excitability, and regulation of ion homeostasis. The first four TM domains of the K + channel α-subunit act as voltage sensors for activation gating [20], whereas the intervening segment between TM5 and TM6 appears to confer channel function [18]. TMC1 c.1979C>T, p.P660L is located in a predicted third extracellular loop located extracellularly between TM5 and TM6, and mutations in this loop may affect ion channel function in hair cells.

Conclusion
In this report, we described a novel missense mutation in the highly conserved T5-T6 domain of TMC1, suggesting the potentially conserved role of Pro660 in the human amino acid sequence. This is only the second homozygous mutation in TMC1 reported in the Chinese population. Moreover, this novel mutation expands the mutational spectrum of TMC1, which will contribute to the clinical understanding of HL caused by mutations in this gene.


extfieldsofstudy: Chemistry Medicine

text:
Glycine triggers a non-ionotropic activity of GluN2A-containing NMDA receptors to confer neuroprotection

Ionotropic activation of NMDA receptors (NMDARs) requires agonist glutamate and co-agonist glycine. Here we show that glycine enhances the activation of cell survival-promoting kinase Akt in cultured cortical neurons in which both the channel activity of NMDARs and the glycine receptors are pre-inhibited. The effect of glycine is reduced by shRNA-mediated knockdown of GluN2A subunit-containing NMDARs (GluN2ARs), suggesting that a non-ionotropic activity of GluN2ARs mediates glycine-induced Akt activation. In support of this finding, glycine enhances Akt activation in HEK293 cells over-expressing GluN2ARs. The effect of glycine on Akt activation is sensitive to the antagonist of glycine-GluN1 binding site. As a functional consequence, glycine protects against excitotoxicity-induced neuronal death through the non-ionotropic activity of GluN2ARs and the neuroprotective effect is attenuated by Akt inhibition. Thus, this study reveals an unexpected role of glycine in eliciting a non-ionotropic activity of GluN2ARs to confer neuroprotection via Akt activation.

Glycine increases Akt phosphorylation independent of Ca 2+ influx through NMDAR channels.
To test the effect of glycine on the activation of cell survival-promoting kinase Akt (protein kinase B) in cultured mouse cortical neurons where no Ca 2+ pass through the NMDAR channels, we treated the neurons with glycine (100 μ M) for 30 min in a modified extracellular solution (5.0 mM EGTA, 137 mM NaCl, 5.4 mM KCl, 1.0 mM MgCl 2 , 25 mM HEPES, 33 mM Glucose, titrated to pH 7.4 with osmolarity of 300-320 mOsm) in which Ca 2+ was not included but with the addition of 5.0 mM EGTA to chelate the residual Ca 2+ . The activation of Akt was quantified by measuring Akt phosphorylation (p-Akt) on Ser473 in western blot assay 26,27 . The levels of p-Akt were quantified by calculating the ratio of p-Akt to total Akt (t-Akt). Our results showed that treatment of glycine (100 μ M) for 30 min increased Akt phosphorylation in the cortical neurons in which there were no Ca 2+ influx into the NMDAR channels (Fig. 1A).
To exclude the possibility that residual Ca 2+ in the extracellular solution might pass through NMDAR channels, we treated the neurons with the extracellular solution described above but with the addition of non-competitive NMDAR antagonist MK-801 28,29 . We named this specific Ca 2+ -free extracellular solution as ECS-1 (10 μ M MK-801, 5.0 mM EGTA, 137 mM NaCl, 5.4 mM KCl, 1.0 mM MgCl 2 , 25 mM HEPES, 33 mM Glucose, titrated to pH 7.4 with osmolarity of 300-320 mOsm). Since MK-801 is a use-dependent pharmacological agent, we first treated the neurons with NMDA (1.0 μ M) and glycine (1.0 μ M) for 1.0 min that opened the NMDAR channels and allowed MK-801 in the ECS-1 to fully block NMDARs [28][29][30] . The cultured neurons were then washed with ECS-1 for three times (10 min wash/each). As this procedure eliminates the possibility of Ca 2+ in activation of NMDAR channels, in this study we referred to this as NMDAR channel inactivation procedure (Fig. 1B). As shown in Fig. 1C, glycine (100 μ M) treatment increased Akt phosphorylation in the cortical neurons where the NMDAR channel activity was inhibited by the NMDAR channel inactivation procedure. In the same experimental conditions, the effect of glycine on Akt phosphorylation was found to be dose-dependent (Fig. 1D). These data indicate the possibility that glycine-induced Akt activation is independent of the channel activity of NMDARs.
To provide further evidence that the effect of glycine on Akt phosphorylation was independent of extracellular Ca 2+ , we tested the effect of BAPTA, a Ca 2+ chelator that has faster calcium-binding kinetics than EGTA 31 . The experimental condition was same as that in Fig. 1C. But BAPTA (0.1, 1.0 or 5.0 mM) was included in the ECS-1 in the + BAP groups (Fig. 1E). BAPTA was included both during the NMDAR channel inactivation procedure and during treatment with glycine. Compared with the group without BAPTA treatment, BAPTA treatment did not interfere with glycine-induced elevation of Akt phosphorylation (Fig. 1E), suggesting that Ca 2+ -mediated channel activity of NMDARs does not contribute to the observed effect of glycine in cortical neurons. Elevation of Akt phosphorylation by glycine does not depend on the activation of glycine receptors. Glycine is the agonist for strychnine-sensitive glycine receptors. Glycine receptors are not significantly expressed in the mature but expressed in the developing cortex 32,33 . To exclude the possible effect of glycine receptors on the observed Akt activation by glycine, we used the same experimental design as that in Fig. 1C. But strychnine was added into the ECS-1 for all the treatment in both -Gly and + Gly group ( Fig. 2A). Our data showed that strychnine (10 μ M) failed to block the enhancement of Akt phosphorylation by glycine (100 μ M) in cortical neurons subjected to NMDAR channel inactivation procedure ( Fig. 2A). Thus, glycine-induced enhancement of Akt phosphorylation does not depend on the activation of strychnine-sensitive glycine receptors.
Glycine does not affect p38-MAPK activation in cortical neurons where NMDAR channel activities and glycine receptors are inhibited. The p38-MAPK is implicated in NMDAR-dependent LTD 34 , and was shown to be activated by non-ionotropic NMDAR signaling after chemical LTD induction 20,24 . The activation of p38-MAPK is also involved in excitotoxicity 35 . To determine whether glycine also altered p38-MAPK signaling independent of glycine receptors and the activation of NMDAR channels, we tested the effect of glycine on p38-MAPK phosphorylation in cortical neurons following the experimental procedure described in Fig. 2A. Our results showed that glycine (100 μ M) had no significant effect on p38-MAPK phosphorylation in our experimental conditions (Fig. 2B), suggesting that a specific activation of Akt but not p38-MAPK by glycine occurs in the condition in which NMDAR channel activities and glycine receptors were suppressed.
Glycine enhances Akt activation through a non-ionotropic activation of GluN2ARs. Our results thus far suggest a non-ionotropic activity of NMDAR to mediate the potentiation of Akt activation by glycine. To provide direct evidence for this possibility, we measured the effects of glycine on Akt phosphorylation in HEK293 cells transiently over-expressing NMDARs. The cDNAs of GluN1, GluN2A and/or GluN2B subunits were transfected in various combinations into the HEK293 cells 36 . The Ca 2+ -mediated channel activities of NMDARs expressed in the transfected cells were inhibited by the NMDAR channel inactivation procedure. Treatment of glycine (100 μ M) for 30 min had no effect on Akt phosphorylation in both non-transfected HEK293 cells and the cells transfected with cDNAs of green fluorescence protein (GFP) (Fig. 3A). However, glycine increased Akt phosphorylation in HEK293 cells transfected with cDNAs of GluN1 + GluN2A following the NMDAR channel inactivation procedure (Fig. 3B), but not in cells transfected with cDNAs of GluN1 + GluN2B (Fig. 3C). We also found that glycine did not increase Akt phosphorylation in HEK293 cells transfected with cDNAs of GluN1, GluN2A and GluN2B, respectively (Fig. 3D). Together, these results indicate that a non-ionotropic activity of GluN2ARs mediates the elevation of Akt phosphorylation by glycine.
To validate the role of a non-ionotropic activity of GluN2AR in mediating the enhancement of Akt activation by glycine in cortical neurons, we applied a GluN2A knockdown approach. The GluN2A protein expression was suppressed in the cultured cortical neurons transducted with GluN2A shRNA lentiviral particles (Fig. 4A). The same experimental design as that in Fig. 1C was applied to inhibit NMDARs. As shown in Fig. 4B, glycine (100 μ M) increased Akt phosphorylation in neurons transducted with lentiviral shRNA control, but the effect of glycine was significantly reduced in neurons transducted with lentiviral GluN2A shRNA. As another control of GluN2A shRNA against GluN2A, the GluN2B shRNA had no influence on the observed effect of glycine (Fig. 4C,D). These results lead us to conclude that glycine promotes Akt activation through a non-ionotropic activity of GluN2ARs in cortical neurons.
The glycine-GluN1 binding site mediates the non-ionotropic activation of GluN2ARs. To determine how glycine exerts its effect through the non-ionotropic activation of GluN2ARs, we tested the effect of glycine-GluN1 binding site antagonist L-689560 on glycine-induced Akt activation after Ca 2+ -mediated channel activities of NMDARs were inhibited [40][41][42][43] . The L-689560 was included in the ECS-1 in the wash step of the NMDAR channel inactivation procedure and in the step of glycine treatment (Fig. 1B). The cultures were then treated with ECS-1 containing glycine (100 μ M) and L-689560 for 30 min. We showed that after the channel activities of NMDARs were inhibited, L-689560 (50 μ M) blocked glycine-induced Akt phosphorylation in the cultured neurons and in the HEK293 cells transfected with GluN1 + GluN2A (Fig. 5A,B). These data suggest that the glycine-GluN1 binding is required for the non-ionotropic activation of GluN2ARs.
As a control study, we also tested the effect GluN2B antagonist Ro 25-6981 (5.0 μ M) 42,43 . Our data showed that Ro 25-6981 had no significant effect on glycine-induced Akt activation in the neurons and the HEK293 cells transfected with GluN1 + GluN2A + GluN2B after Ca 2+ -mediated NMDAR channel activity was inhibited (Fig. 5C,D). D-serine is the endogenous agonist of glycine-GluN1 binding site 44 . As a further support for the role of glycine-GluN1 binding in mediating the effect of non-ionotropic GluN2ARs, we tested the role of D-serine in Akt phosphorylation in the cortical neurons and the HEK293 cells transfected with GluN1 + GluN2A or GluN1 + GluN2B following NMDAR channel inactivation procedure. D-serine increased Akt phosphorylation in both cortical neurons and HEK293 cells transfected with cDNAs of GluN1 + GluN2A but not with those of GluN1 + GluN2B (Fig. 5E-G).
Glycine prevents glutamate neurotoxicity-induced neuronal death through non-ionotropic activation of GluN2ARs. As Akt is a survival-promoting kinase that plays a crucial role in preventing neuronal death 26 47 . The levels of lactate dehydrogenase (LDH) released from injured neurons was also measured to quantify the neuronal damage 27 . Our data showed that after glutamate neurotoxicity insult, glycine (100 μ M) treatment protected against the death of cortical neurons in which Ca 2+ -mediated NMDAR channel activity was inhibited (Fig. 6B-D).
Our results further demonstrated that the neuroprotective effect of glycine (100 μ M) was reduced in injured cortical neurons in which the GluN2A expression was suppressed by lentiviral GluN2A shRNA (Fig. 6E,F). The neurons in both shRNA control and GluN2A shRNA groups were subjected to the same experimental procedures described in Fig. 6B-D. We conclude that the neuroprotective effect of glycine is at least in part mediated through non-ionotropic activation of GluN2ARs in glutamate neurotoxicity-induced neuronal injury.
To determine the roles of Akt activation and glycine-GluN1 binding in glycine-induced neuroprotection, we tested the effect of Akt inhibitor IV and glycine-GluN1 binding antagonist L-689560 in our experimental model. The experimental condition was the same as that described in Fig. 6A-D. The Akt inhibitor IV and L-689560 were included in both wash and treatment steps. We found that both Akt inhibitor IV (1.0 μ M) and L-689560 (50 μ M) significantly reduced glycine-induced neuroprotective effect in neurons where Ca 2+ -mediated channel activity of NMDARs were inhibited (Fig. 6G,H). Thus, Akt activation and glycine-GluN1 binding mediate glycine-induced neuroprotection. Together, these results provide functional evidence for the role of non-ionotropic activity of GluN2ARs in mediating the neuroprotective effect of glycine.

Discussion
Using a Ca 2+ -free ECS-based procedure to inactivate the channel activity of NMDARs in cultured cortical neurons and HEK293 cells expressing GluN2ARs, we tested the effect of glycine on Akt phosphorylation, a cellular process playing important role in neuronal survival. We provided the first evidence that glycine induced a potentiation of Akt phosphorylation independent of the channel activity of NMDARs. We confirmed that glycine-induced non-ionotropic activation of GluN2ARs, but not GluN2BRs, mediated the enhancement of Akt activation. Thus, our study identified a non-ionotropic function of GluN2ARs. To ensure no Ca 2+ -mediated channel activities of NMDARs contributing to glycine-induced effect in our study, we established a NMDAR channel inactivation procedure in which Ca 2+ was not included in the ECS but with the addition of Ca 2+ chelator EGTA and the use-dependent open channel blocker MK-801. To allow MK-801 to fully block the channels  of NMDARs, we pretreated the cells with NMDA and glycine to open the NMDAR channels. Thus, Ca 2+ influx through NMDARs would not likely occur in our experimental conditions.
Increasing evidence supports the non-ionotropic function of NMDARs [19][20][21][22][23][24][25] . It has been recently shown that a non-ionotropic activation of NMDAR was insensitive to the glycine-GluN1 site antagonist 20,21,24 . However, our study shows that the glycine-GluN1 binding is required to activate the non-ionotropic activity of GluN2ARs. These findings suggest that glycine triggers a non-ionotropic activity of GluN2ARs through the glycine-GluN1 binding site. By testing the effects of glycine in HEK293 transfected with different combinations of NMDARs, we were able to obtain direct evidence to reveal that a non-ionotropic activation of GluN2ARs but not GluN2BRs mediates the enhancement of Akt activation.
The novel non-ionotropic NMDAR may have functional significance. NMDAR-mediated neurotoxicity induces neuronal death and neurodegeneration in various CNS disorders including ischemic stroke, traumatic brain injury and neurodegenerative diseases [48][49][50] . However, the use of NMDAR antagonists as neuroprotective agents was disappointing in clinical trials [51][52][53] . A simple possibility is that these antagonists, while suppressing NMDAR-mediated neurotoxicity, block the biological and/or neural survival-promoting effects of NMDARs 1,16-18 . Thus, identification of molecular mechanisms by which specific NMDAR subtype selectively exerts its effect on neuronal survival or death would provide a critical basis for the development of potent therapy for CNS injuries and neurodegenerative diseases.
GluN2ARs and GluN2BRs play different role in neuronal survival or death 14,16 . But the underlying molecular mechanism remains unclear. It is recently reported that a non-ionotropic function of NMDARs was required for β -amyloid-induced synaptic depression and synaptic loss [21][22][23] , providing new evidence for the involvement of GluN2BRs in neurotoxicity. Our observation for a non-ionotropic activation of GluN2ARs by glycine explains in part why GluN2AR plays a different role than GluN2BR in neuronal survival.
The molecular mechanism underlying glycine-induced non-ionotropic NMDAR activation is unclear. It is possible that glycine binds to GluN1 and induces conformational changes in GluN2A. If this is the case, why it cannot change the conformation of GluN2B? The different structure between GluN1-GluN2A coupling and GluN1-GLuN2B coupling may be the possible reason. Future study is required to reveal the mechanisms.
How non-ionotropic activity of GluN2ARs enhances Akt activation is unclear. It is likely that the C-terminal domain of GluN2A may mediate the effect of glycine on Akt activation. Akt deactivation is believed to be a causal mediator of cell death 26,45,46 . Enhancement of Akt activity exerts pro-survival effect in neuronal injury and neurodegenerative diseases 26,45,46 . In this study, we identify Akt as a downstream neuroprotective signal of glycine that activates non-ionotropic GluN2ARs. We provide evidence that non-ionotropic activation of GluN2ARs by glycine reduces glutamate neurotoxicity-induced Akt deactivation and thus prevents cortical neuronal death. Akt is known to influence neuronal survival through activation or inhibition of substrates 26,45,46 . For example, activated Akt promotes survival through phosphorylation of transcription factors forkhead/FOXO, NF-κ B and mdm2 or through phosphorylation of Bcl-2 family members Bad and Bim 26,45,46 . Further study is needed to determine which Akt-dependent signal pathway mediates the activation of non-ionotropic GluN2ARs by glycine.
It would be important to determine where is glycine coming from considering the brain does not normally have glycinergic transmissions. Glial cells would be the source of glycine release, especially during the ischemia injury process. In this study, we show that D-serine has similar effect to glycine, suggesting that D-serine is also the endogenous agonist for this non-ionotropic NMDA receptor activity.

Experimental Procedures
General methods. Randomization was used to assign samples to the experimental groups, and to collect and process data. All animal experiments were approved and carried out in compliance with the IACUC guidelines of University of Nevada and Wuhan University School of Medicine. All experimental protocols were approved by the Animal Care and Ethics Committee of University of Nevada and Wuhan University School of Medicine.
Neuronal culture. The cortical neuronal cultures were prepared from female C57BL/6 mice at gestation day 17 as described 27,54 . Briefly, dissociated neurons were suspended in plating medium (Neurobasal medium, 2% B-27 supplement, 0.5% FBS, 0.5 μ M L-glutamine, and 25 μ M glutamic acid) and plated on poly-D-lysine coated Petri dishes. After 1 day in culture, half of the plating medium was removed and replaced with maintenance medium (Neurobasal medium, 2% B-27 supplement, and 0.5 μ M L-glutamine). Thereafter, maintenance medium was changed in the same manner every 3 days. The cultured neurons were used for experiments at 12 days after plating.
Scientific RepoRts | 6:34459 | DOI: 10.1038/srep34459 transfected in cultured HEK293 cells 36 . Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as described in our previous studies 15,27 . DNA-Lipofectamine complexes were made in serum-free medium Opti-MEM. To prevent NMDAR-induced cell death, the transfected HEK293 cells were treated with 1.0 mM DAPV to prevent NMDAR-induced excitotoxicity 55 . The transduction of GluN2A and GluN2B shRNA lentiviral particles were performed in cultured cortical neurons based on the manufacturer's instructions.
Western blotting. Western blotting assay was performed as described previously 15,27 . For the detection of phospho-Akt, the samples prepared in the same day were used. The polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) was incubated with primary antibody against phospho-Akt (Ser473) (Cell Signaling Technology, Beverly, MA), Akt (Cell Signaling Technology, Beverly, MA), phospho-p38-MAPK (Cell Signaling Technology, Beverly, MA), p38-MAPK (Cell Signaling Technology, Beverly, MA), β -actin (Santa Cruz Biotech), GluN2A (Santa Cruz Biotech), or GluN2B (Santa Cruz Biotech). Primary antibodies were labeled with horseradish peroxidase-conjugated secondary antibody, and protein bands were imaged using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA). The EC3 Imaging System (UVP, LLC, Upland, CA) was used to obtained blot images directly from the polyvinylidene difluoride membrane. For the detection of total Akt, the same polyvinylidene difluoride membrane was stripped and then re-incubated with primary antibody against total Akt (Cell Signaling Technology). The quantification of Western blot data was performed using ImageJ software.
Neuronal viability assays. Double staining of propidium iodide (PI) and fluorescein diacetate (FDA) was performed to detect neuronal viability using a modified procedure 47 . Briefly, cultures were rinsed with extracellular solution and incubated with FDA (5 μ M) and PI (2 μ M) for 30 min. The cultures were washed with extracellular solution and then viewed on an Olympus fluorescent microscope (IX51, Olympus). Neuronal viability was determined by calculating the number of PI-labeled cells over FDA-labeled cells. The investigator for the cell count was blinded to the experimental treatment.
The lactate dehydrogenase (LDH) is a cytoplasmic enzyme retained by viable cells with intact plasma membranes and released from cells with damaged membranes. The LDH release was measured using CytoTox 96 Cytotoxicity kit based on the manufacturer's instructions (Promega, Madison, WI) 27 . The levels of maximal LDH release were measured by treating the cultures with 10× lysis solution (provided by the manufacturer) to yield complete lysis of the cells. Absorbance data were obtained using a 96-well plate reader (Molecular Devices, Palo Alto, CA) at 490 nm. According to the manufacturer's instructions, the LDH release (%) was calculated by calculating the ratio of experimental LDH release to maximal LDH release.
Statistics. Student's t test or ANOVA test was used where appropriate to examine the statistical significance of the differences between groups of data. Newman-Keuls tests were used for post-hoc comparisons when appropriate. All results are presented as mean ± SE. Significance was placed at p < 0.05.


In vitro Antifungal activity of leaf extracts from Aloe secundiflora, Bulbine frutescens, Vernonia lasiopus and Tagetes minuta against Candida albicans

Copyright: © 2016 Rachuonyo HO, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In vitro Antifungal activity of leaf extracts from Aloe secundiflora, Bulbine frutescens, Vernonia lasiopus and Tagetes minuta against Candida albicans

Introduction
The use of medicinal plants (herbs) to treat diseases is almost universal among non-industrialized societies, and is often more affordable than purchasing expensive conventional drugs [1]. Medicinal plants are used by almost 80% of the world's population for their basic health care because of their low cost and ease in availability [2]. The use of herbal medicinal plants has always played a positive role in the control or prevention of diseases such as diabetes, heart disorders and various cancers [3]. Medicinal plants have been used in production of various drugs singly or in combination and even as principal raw material for the production of other conventional medicines [4]. Candida albicans is a normal micro biota mainly found in the mucosal cavity, vagina and gastrointestinal tract of an individual [5]. They are yeast like fungus that are commensals in healthy humans but can cause systemic infections in immunocompromised individuals [6]. Incidence of fungal infections has led to increased antimicrobial resistance hence making a few antifungal agents active [7,8]. There has been an increase in resistance by Candida albicans to conventionally produced antimicrobials recently, leading to the search of a new antifungal agent [9,10]. Herbal extracts from medicinal plants have been used to effectively treat infectious diseases such as candidiasis in developing nations [11][12][13]. Tagetes minuta L. is also known as Southern Cone Marigold, Stinking Roger or black mint [14]. The genus comprises of 56 species which grow either annually or perennially and mostly herbaceous plants [15]. Tagetes minuta extracts have been used as medicinal tea in some areas [15]. The total extracts from leaves, flowers, stem and other parts of the plant have shown antibacterial activity as well as antifungal properties [16]. Extracts from the other common species have also been used as medicine in treating various illnesses such as stomach problems and intestinal disorders [17]. The genus Aloe belongs to Aloeaceae (liliaceae) family which has around 360 to 400 different species [18]. Aloe secundiflora leaf components have been found to contain a credited for antibacterial, antifungal, antiviral, antihelmintic and immunomodulatory medicinal properties [19,20]. Its crude extracts have also shown some level of antifungal activity against candida albicans [21]. Bulbine is a genus of plants in the family xanthorrhoeaceae and sub family asphodeloideae and its members are well known for their medicinal value [22]. The most common species

Abstract
Crude leaf extracts from medicinal plants were screened for in vitro antifungal properties against clinical isolate of Candida albicans. The antifungal test was carried out using the Kirby Bauer method. All the extracts from the plants tested showed antifungal activity against clinical isolate of Candida albicans with the extract from Vernonia lasiopus being more active as compared to the others. The minimum inhibitory concentration ranged from 4.0 to 8.9 mg/ml. The standard antifungal used as positive control was fluconazole (15 µg/ml) whereas dimethyl sulphoxide and distilled water were used as negative control. Preliminary screening for the presence of phytochemicals was also done to determine if there was presence of any, flavonoids, saponins, tannins and alkaloids.
is Bulbine frutescens which is popularly grown in flower gardens [23]. Many species have bulb shaped tuber and they are chiefly found in South Africa have been found to contain antibacterial and antifungal properties and are used for blood cleansing, treatment of ringworms and gravel rush by some local communities such as the Xhosa [24]. A decoction of bulbs and roots of some of the species has been used in the treatment of some of the venereal diseases in women and stomach upsets [23]. Vernonieae is a tribe which has about 1300 species and in the family Asteraceae (Compositae) which mostly contains herbaceous plants [25]. Vernonia lasiopus decoctions from the stems and leaves have been traditionally been found to contain antifungal, anthelminthic, antiparasitic and antibacterial activity and has been used by herbalists in East Africa to treat, malaria, worms and gastrointestinal problems [26]. The main aim of the study was to establish the antifungal activity of the crude extracts from the medicinal plants against Candida albicans.

Plant material collection
The fresh plant material of Aloe secundiflora, Bulbine frutescens, Vernonia lasiopus and Tagetes minuta were collected at Kenyatta University Arboretum. Voucher specimens were prepared and deposited in the university herbarium in Plant Sciences Department for future reference. The plants were brought to the laboratory and thoroughly washed in running water to remove debris and dust particles and then rinsed using distilled water and finally air dried.

Preparation of plant extract
The air dried plant materials were tause into powder and soaked in methanol for 72 hours, placed in a Gallenkamp shaker at 65 revolutions per minute. The contents were homogenized and filtered using whatman filter paper no. 1. The filtrate was then poured into a round bottom flask and concentrated using a vacuum evaporator and stored in a labelled amber glass bottle at room temperature away from light and heat before being used for antifungal efficacy test.

Antifungal susceptibility testing
Clinical isolate of Candida albicans obtained from Kenyatta University Health Centre Laboratory, Nairobi was used for the susceptibility test. It was tested against methanolic extracts of Tagetes minuta, Aloe secundiflora, Bulbine frutescens and Vernonia lasiopus. The pure culture of clinical isolate Candida albicans was concentrated by comparing it with a 0.5 McFarland standard. Discs of 6 milliliters were prepared from whatman no.1 filter paper. The discs were sterilized by autoclaving. After sterilization the moisture discs were dried on hot air oven at 50°C [27]. The various solvent extracts discs were prepared were impregnated with the extracts from the highest concentration of 1000 mg/ml to the lowest concentration of 1 mg/ml [2]. The antimicrobial efficacy test was carried out using Kirby Bauer method [18]. Potato dextrose agar (PDA) was used in the spread plate technique where the clinical isolate of Candida albicans was spread using sterilized cotton wool swabs and exposed to extracts impregnated discs in milligrams per microliter from Aloe secundiflora, Tagetes minuta, Vernonia lasiopus and Bulbine frutescens. The discs were placed with equal distance between them on agar plates inoculated with Candida albicans. Positive control discs containing fluconazole were used as well as negative control discs impregnated with dimethyl sulphoxide (DMSO) and distilled water. The plates were incubated at 37°C for 24 hours. Zones of inhibition were measured in millimetres and their average determined. The experiment was carried in duplicates and the diameter of zones of inhibition formed measured. Minimal inhibitory concentration (MIC) was determined using the broth tube method [28]. 100 µl of 250 mg/ml of methanol extract was added to 100 µl of sterile peptone water in the first well of the 96 well micro plate and mixed well with a micropipette. 100 µl of this dilution was transferred subsequently to wells two folding each dilution of the original extract. This was done to the extracts of Aloe secundiflora, Bulbine frutescens, Vernonia lasiopus, and Tagetes minuta. An inoculum of 100 µl (0.5 McFarland standard) of overnight clinical culture of Candida albicans was added in each of the wells. Triplicate of each micro plate was made and the procedure repeated. The plates were then incubated at 37ºC for 24 hours. After incubation 40 µl of 0.2 mg/µl of INT were added in each of the wells and the plates examined after an additional 60 minutes of incubation. Growth was indicated by a red colour (conversion of INT to formazan). The lowest concentration at which the colour was apparently invisible as compared to the next dilution was taken as the minimum inhibitory concentration [29]. Minimum fungicidal concentration (MFC) was determined according whereby; 100 µl of suspension was taken from micro plate wells that demonstrated no growth and inoculated on agar plates. The plates were incubated at 37°C for 24 hours. In the case where there was fungal growth and also not greater than the minimum inhibitory concentration was used to determine maximum fungicidal concentration [29].

Phytochemical analysis
Presence of saponins, tannins, flavonoids and alkaloids in the crude extract were determined according to the method defined [30].
Tannins: Each of the extracts was weighed to 0.5 mg and dissolved in 1 ml of distilled water. Filtration was carried out after 2 ml of FeCl 3 was added. If there was presence of a blue or black precipitate then it indicated the presence of tannins.
Flavonoids: Each of the extracts was weighed to 0.5 mg and dissolved in 1 ml of ethanol and filtered. 2 ml of 1% HCl and magnesium ribbon was added to the filtrate. If there was formation of a pink or red colour it indicated the presence flavonoids.
Alkaloids: Each of the extracts was weighed to 0.5 mg and dissolved in 1 ml of methanol and filtered. 1% HCL was added to the filtrate and the solution heated. Mayor`s reagent was added drop wise and if there was formation of any colored precipitate it indicated the presence of alkaloids.
Saponins: Each of the extracts was weighed to 0.5 mg and dissolved in 1 ml of methanol and filtered. Distilled water was added and shaking done for a few minutes. If there was persistence frothing then it indicated the presence of saponins.

Results and Discussion
All the extracts from the plant leaves showed a considerable level of antifungal activity against clinical isolate of Candida albicans [21,31,32]. The plant extract from Vernonia lasiopus proved to be more active against Candida albicans in low concentrations whereas Aloe secundiflora was less active. The standard antifungal control used (fluconazole 15 µg/ml) formed a desirable zone of inhibition of 28 mm. Among the plant extracts Vernonia lasiopus produced the highest zone of inhibition against Candida albicans approximately 20 ± 1.86 mm in diameter. There were no zones of inhibitions formed by the negative controls used (dimethyl sulphoxide and distilled water) ( Table 1). The results obtained from the study showed that the antifungal activity against Candida albicans by the plants extracts may be due to the presence of phytochemicals in the extracts namely flavonoid, tannins, saponins and alkaloids (Table 2). Tannins bioactive components such as catechin and pyrogallol have been known to contain antifungal capabilities and they are toxic to most microbes [33]. The flavonoids are also known for their antimicrobial activity of inhibiting the synthesis of the nucleic acids, tempering with integrity of the cytoplasmic membrane function and the energy metabolism process [34].

Conclusion
In conclusion, the extracts from the plants proved to be active against Candida albicans. This shows that the extracts from the plants could be used as an antifungal agent against Candida albicans and other fungal pathogens. The bioactive components of the screened phytochemicals need to be determining so as to know the compounds responsible for the antifungal activities. This will aid as a natural source of treating fungal infections and also provide raw materials that can be used in the manufacturing of conventionally used antifungal agents.


extfieldsofstudy:
Biology
Medicine
Computer Science

text:
OGEE v2: an update of the online gene essentiality database with special focus on differentially essential genes in human cancer cell lines

OGEE is an Online GEne Essentiality database. To enhance our understanding of the essentiality of genes, in OGEE we collected experimentally tested essential and non-essential genes, as well as associated gene properties known to contribute to gene essentiality. We focus on large-scale experiments, and complement our data with text-mining results. We organized tested genes into data sets according to their sources, and tagged those with variable essentiality statuses across data sets as conditionally essential genes, intending to highlight the complex interplay between gene functions and environments/experimental perturbations. Developments since the last public release include increased numbers of species and gene essentiality data sets, inclusion of non-coding essential sequences and genes with intermediate essentiality statuses. In addition, we included 16 essentiality data sets from cancer cell lines, corresponding to 9 human cancers; with OGEE, users can easily explore the shared and differentially essential genes within and between cancer types. These genes, especially those derived from cell lines that are similar to tumor samples, could reveal the oncogenic drivers, paralogous gene expression pattern and chromosomal structure of the corresponding cancer types, and can be further screened to identify targets for cancer therapy and/or new drug development. OGEE is freely available at http://ogee.medgenius.info.

Being essential is not an intrinsic property of a gene; rather, it is highly dependent on a variety of factors including the function and expression pattern of the gene, the genetic background of the host, the environment and other settings. For example, genes coding for proteins involved in the biosynthesis of amino acids, nucleic acids and vitamins are essential for cell survival in minimal media, but not in rich media where the corresponding metabolites are supplied (20). In addition, different experimental methods may generate different results. For example, CRISPR-based methods could identify more essential genes than siRNAbased methods (21) tion of essential genes than in vivo if the same multi-cellular organism is used (22). Genes with variable essentiality statuses under different circumstances are referred to 'conditionally essential genes (CEGs)' or 'differentially essential genes (DEGs)' (14,22). CEG is a biologically meaningful and very important concept; e.g. genes that are essential in a cancer cell line but are non-essential in human tissues can reveal the oncogenic drivers, paralogous gene expression pattern and chromosomal structure of the corresponding cancer type (14).
In 2012, we introduced OGEE v1 (22) to promote the concept of 'conditional essentiality', which had not been widely adopted by existing essential gene databases at the time, and to advance our understanding on gene essentiality. We did so by including not only essential and non-essential genes, but also associated gene properties that are known to affect gene essentiality; we provided tools that allow users to compare gene essentiality among different gene groups, or compare properties of essential genes to non-essential genes. In addition, we organized experimentally tested genes into data sets according to their sources and tagged those with variable essentiality statuses across data sets as CEGs.
In this study we introduce an updated version of OGEE. In this new version we added new species and new data sets; we added genes with intermediate essentiality statuses (fitness genes) and non-coding essential genes. In addition, we re-organized the 16 gene essentiality data sets from human cancer cell lines corresponding to nine cancer types in order to help users to explore the shared and differentially essential genes within and between cancer types, because these genes, especially those derived from cell lines that are similar to tumor samples, could be further screened to identify targets for cancer therapy and/or new drug development.

Collection and organization of genes tested for essentiality
We collected 99 large-scale gene essentiality experiments (data sets) for 48 species, including 34 data sets for 9 eukaryotes and 65 data sets for 39 prokaryotes. We added 1609 noncoding genes and 122 non-transcribed genomic regions from 10 species. In addition to essential and nonessential genes, we also included 1911 fitness genes from 10 species, and 37 growth-advanced genes from two species. Fitness genes are defined as those whose removal is not lethal but could result in significantly decreased fitness, while growthadvanced genes are defined as genes whose removal lead to significantly increased fitness. In the statistics below, fitness genes are counted as non-essential genes.
In sum, our database contains 167 799 genes tested for essentiality from 48 species, increased significantly from the 91 436 genes and 24 species respectively from the last version (22). In total 43 961 genes are covered by multiple data sets (in each species the text-mining results are considered as a data set), representing ∼26.2% of all collected genes; among which 13 397 genes are CEGs, accounting for ∼30.5% of those covered by multiple data sets. The proportion of conditionally essential genes (P CEG ) in species having more than 200 genes covered by two or more data sets ranges from 9.2% in Staphylococcus aureus subsp. aureus NCTC 8325 to 41.6% in Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344, as shown in Table 1. The number of data sets does not seem to contribute significantly to P CEG (Pearson's correlation coefficient = −0.24, P-value = 0.36). Species having at least 200 genes covered by multiple data sets are listed here; the species are ordered first by the kingdom they are in (the 1st column) and then by the number of genes covered by multiple data sets (the 5th column). Essential genes are those that are essential in any collected data sets, i.e. genes that are essential in one data set but non-essential in others are also counted. The proportion of conditionally essential genes (P CEG , percentage in parentheses of the last column) is calculated as the ratio between the 'conditionally essential genes' (the last column) and the 'genes covered by multiple data sets' (the 5th column). Please note that text-mining results, if available, will be counted as one data set in a species; please consult the 'Browse' page of the database for a complete and interactive version of the table.

Collection of gene properties influencing gene essentiality
We also collected several gene properties that are known to influence gene essentiality, including duplication status (23), the number of homologous genes (family size) in the same genome, connectivity in protein-protein interaction (PPI) networks (defined as the number of direct neighbors) (24), functional category of a gene (25) and the earliest expression stage during embryonic development. We used the BLAST tool (26) to search for duplicated genes within each genome using parameters and cutoffs described previously (27), and calculated the family size for each duplicated gens accordingly. We also calculated evolutionary measurements for each duplicated gene and its best BLAST hit, including Ka, Ks and Ka/Ks ratio using KaKs Calculator (v2.0) (28). We obtained the PPI data from STRING v10.0 (29), the functional category data from Gene Ontology (30) and the expression data (for multi-cellular organisms only) from NCBI UniGene database (31). For more information, please consult the 'Help' page of the database.

BUILT-IN TOOLS FOR ANALYZING COLLECTED GENE PROPERTIES
We also provided integrated tools in the 'Analyze' page for users to analyze the impact of the collected gene properties on gene essentiality: users can divide genes into distinct groups according to one of the available properties, calculate the proportion of essential genes (P E ) in each group, and then plot the results as bar-chart. To illustrate this feature, we plotted in Figure 1 the P E values of different groups of mouse genes as functions of their involvement in development ( Figure 1A) and duplication status ( Figure 1B). These results showed that that developmental genes are more essential than non-developmental genes, while singletons are more essential than duplicated genes, consisting to previous results (23,25); these trends are generally true in other species.

RE-ORGANIZATION OF ESSENTIAL GENES FROM HUMAN CANCER CELL LINES
In recent years, 'conditional essentiality' or 'differential essentiality' has been increasingly used as a tool for researchers to interrogate genes that are essential under spe-cific conditions and search for genes required by the survival of human cancer cell lines (7,(9)(10)(11)14,16). In OGEE we collected in total 16 such data sets and re-organized them into 9 groups according to their cancers of origin, including breast cancer, Burkitt's lymphoma, chronic myelogenous leukemia (CML), colon cancer, esophageal squamous carcinoma, glioblastoma (GBM), non-small cell lung cancer (NSCLC), ovarian cancer and pancreatic cancer. Shared and differentially essential genes within and between cancer types were pre-calculated. Shown in Table 2 are the brief summary on the nine cancers, including the number of data sets for each cancer, the number of total essential genes and the number of uniquely essential genes. Here, the 'uniquely essential genes' are defined as those that are non-essential in any other human data sets available in OGEE. An up-todate version of this table and additional results can be found in the 'Cancer' page of our website. Lineage-specific essential genes, i.e. those that are essential only in a particular cancer type, are important targets for cancer therapies; because they are likely the results of the unique mutational profile and subsequent functional consequences of the cell line, targeting these genes in cancer therapies will achieve high efficiency and specificity. Cancer cell lines are often used as models for cancer research. However, recent studies suggest that although some cell lines are indeed good models for cancers, some other cancer cell lines could have pronounced differences as compared to tumor samples of the same origin in terms of copynumber changes, key mutations and mRNA expression profiles, due in part to ambiguity in classification and annotation (32,33). Thus, in the future, we will exclude cell lines that are remarkably different from cancers of the same origin, and keep only the good ones, should such information are reliable and easily accessible.

DATA ACCESS
All data are freely accessible to all academic users. This work is licensed under a Creative Commons Attribution 3.0 Unported License (CC BY 3.0). Users can download combined data from the 'Downloads' page. Users can also download individual data sets or combined data sets for individual species in the 'Browse' page.
Nucleic Acids Research, 2017, Vol. 45, Database issue D943 Table 2. Summary of the 16 gene essentiality data sets from 9 human cancers collected in OGEE   Cancer  Data sets  Essential genes  Uniquely essential genes  breast  1  146  67  Burkitt's lymphoma  2  1897  198  CML  4  3210  1324  colon  2  1394  899  esophageal squamous  1  41  34  GBM  1  21  14  NSCLC  1  28  20  ovarian  2  130  87  pancreatic  2  199  126 'Essential genes' (the 3rd column) are genes that are essential in any of the data set(s) of a particular cancer type; 'Uniquely essential genes' (the last column) are genes that are subset of 'Essential genes' but non-essential in any other collected human data sets. An up-to-date version of this table can be found at http://ogee.medgenius.info/cancer/.

CONCLUSIONS
In this article, we introduced OGEE v2, an online gene essentiality database. Updates since the last updated version include increased numbers of species and gene essentiality data sets, inclusion of non-coding essential sequences and fitness genes. We also re-organize the essentiality data sets from nine human cancers so that our users can easily explore the shared and differentially essential genes within and between cancer types. As compared with existing gene essentiality databases such as DEG (34), OGEE provides several unique features. For example, (i) OGEE provides both essential and non-essential genes from large-scale as well as small-scale studies; (ii) OGEE introduces 'conditional essentiality' to reflect the complexity of biological systems and the interplay between gene functions, genetic backgrounds and environments; (iii) OGEE lists a variety of gene properties known to influence gene essentiality; (iv) OGEE provides a set of online tools to explore and analyze the data and to visualize the results. We thus believe that OGEE should be highly useful to biologists and bioinformaticians studying gene essentiality, whether focusing on individual genes or on genome-wide analyses. In the future, we aim to update OGEE regularly in order to provide upto-date contents to our users.

FUNDING
Funding for open access charge: startup funding for Chen's lab at the Huazhong University of Science and Technology, China Conflict of interest statement. None declared.