Macrophages are the primary effector cells of the innate immune system and are also activated in response to tissue injury. The avian cochlea contains a population of resident macrophages, but the precise function of those cells is not known. The present study characterized the behavior of cochlear macrophages after aminoglycoside ototoxicity and also examined the possible role of macrophages in sensory regeneration. We found that the undamaged chick cochlea contains a large resting population of macrophages that reside in the hyaline cell region, immediately outside the abneural (inferior) border of the sensory epithelium. Following ototoxic injury, macrophages appear to migrate out of the hyaline cell region and towards the basilar membrane, congregating immediately below the lesioned sensory epithelium. In order to determine whether recruited macrophages contribute to the regeneration of sensory receptors, we quantified supporting cell proliferation and hair cell recovery after the elimination of most resident macrophages via application of liposomally-encapsulated clodronate. Examination of macrophage-depleted specimens at two days following ototoxic injury revealed no deficits in hair cell clearance, when compared to normal controls. In addition, we found that elimination of macrophages did not affect either regenerative proliferation of supporting cells or the production of replacement hair cells. However, we did find that macrophage-depleted cochleae contained reduced numbers of proliferative mesothelial cells below the basilar membrane. Our data suggest that macrophages are not required for normal debris clearance and regeneration, but that they may play a role in the maintenance of the basilar membrane. Sensory transduction in the inner ear is mediated by hair cells, which detect sound vibrations and head movements, and provide synaptic input to afferents of the eighth cranial nerve. Hair cells – along with their accompanying supporting cells - reside in epithelial sheets that form barriers between the two fluid spaces of the inner ear (perilymph and endolymph). Cells within these sensory epithelia can be injured or killed by acoustic trauma, treatment with ototoxic drugs, or as a consequence of aging. In order to preserve the integrity of the inner ear fluid chambers, it is essential that cellular debris be quickly removed after injury or apoptosis. Epithelial debris clearance can be mediated by several distinct mechanisms. Cell corpses may be actively extruded from the epithelium in vitroIn light of the uncertain role of macrophages in the inner ear, the aims of the present study were to determine whether macrophages are required for removal of hair cell debris after ototoxic injury and to test the hypothesis that resident macrophages promote hair cell regeneration. Experiments were conducted on organotypic cultures of the chick cochlea, which retains its regenerative ability when maintained Fertilized chicken eggs were obtained from Charles River  and incubated until hatching. Chicks were then housed in heated brooders for 10–15 days, prior to use in experiments. All protocols were approved by the Animal Studies Committee of Washington University.in vivo through systemic treatment with streptomycin sulfate. Streptomycin was dissolved in 0.9% NaCl and chicks were given intramuscular injections of 1,200 mg/kg. Injections were given at ∼12∶00 PM for three consecutive days. At 24 hr after the final injection, chicks were euthanized via CO2 asphyxiation and cochleae were removed, fixed 30 min in 4% paraformaldehyde (in 0.1 M phosphate buffer) and processed for immunohistochemistry.Cochlear hair cells were lesioned 2 asphyxiation and decapitated. Following removal of the skin and mandible, heads were placed in 70% EtOH for 5–10 min., in order to kill surface pathogens. The temporal bones were opened and cochleae were quickly explanted and placed in chilled Medium 199 (M199) with Hank’s salts and HEPES buffer. Fine forceps were used to remove the tegmentum vasculosum from each cochlea, and the lagena was also cut away using iridectomy scissors. However, the tectorial membranes were not removed from any of the specimens, either before placement in organotypic culture or prior to immunohistochemical processing. Individual dissected cochleae were transferred into culture wells (MatTek) that contained 100 µl of M199 with Earle’s salts 2,200 mg/l sodium bicarbonate, 0.69 mM L-glutamine, 25 mM HEPES, supplemented with 1% FBS. Cochleae were initially incubated in medium that contained 1 mM streptomycin sulfate. After 24 hr of streptomycin treatment, all specimens were rinsed 3× in fresh medium and maintained for an additional 2–7 days in streptomycin-free medium. Cultured specimens were maintained at 37°C in a 5%CO2/95% air environment, and were fed fresh medium at two-day intervals.Cultures of the cochlea (basilar papilla) were prepared following previously described methods Liposomally-encapsulated clodronate (∼18 mg/ml clodronate Proliferating (S-phase) cells were labeled by the addition of bromodeoxyuridine  to culture medium. Specimens were incubated with BrdU for four days; BrdU-containing medium was replaced with fresh medium every 48 hr.All cultures were fixed for 30 min with 4% paraformaldehyde . Specimens were then thoroughly rinsed with PBS and nonspecific antibody binding was blocked by incubation for 2 hr in PBS with 5% normal horse serum (NHS) and 0.2% Triton X-100. Hair cells were identified with an antibody against myosin VIIA . Macrophages were labeled with the KUL01 antibody  was used for all imaging. Image stacks were reconstructed and processed using Volocity software (PerkinElmer) and stored as TIFF files. All quantification was conducted offline, from stored images. Macrophages within the sensory and hyaline/cuboidal cell regions were counted from 100 µm-wide strips, beginning ∼1 mm from the distal tip of the cochlea and proceeding toward the proximal end see . SurviviIn initial studies, we used the KUL01 antibody to characterize the distribution of macrophages in the normal (undamaged) chick cochlea. Surface views revealed moderate numbers of macrophages associated with the sensory region . HoweverWe next examined changes in the numbers and distribution of cochlear macrophages following aminoglycoside ototoxicity. Chicks were given three injections of streptomycin , which killed nearly all hair cells in the proximal ∼50% of the sensory epithelium. Surface views of cochleae fixed one day after streptomycin injections revealed enhanced numbers of macrophages within the lesioned sensory regions . Notablyin vivo and in vitro) have employed liposomally-encapsulated clodronate to reduce macrophage numbers 2, while comparable regions of control cochleae contained 74.0±8.2 hair cells/10,000 µm2 (n = 4 samples from each specimen). These observations suggest clodronate can eliminate macrophages from the chick cochlea without affecting hair cell survival.The processes of hair cell injury, macrophage recruitment, and hair cell regeneration can be readily observed in organotypic cultures of the chick cochlea  [4], [7]in vitro for an additional 48 hr . Specimens were then fixed and labeled with phalloidin and immunolabeled for myosin VIIA. We observed widespread myosin VIIA immunoreactivity in all cochleae, which co-localized with hair cell debris  were placed in organotypic culture; ten specimens received liposomal clodronate while the remaining cochleae served as untreated controls. After 24 hours, all specimens were rinsed 3× and then treated for 24 hr in 1 mM streptomycin, in order to kill hair cells. Following ototoxic injury, the specimens were thoroughly rinsed and maintained l debris . Using cl debris . It shoul debris . Quantif2, which is an approximately15-fold increase over the hair cell density observed at 48 hr after ototoxic injury  clodronate-containing liposomes, (2) ‘empty’ (PBS-containing) liposomes, or (3) normal medium without liposomes. Each treatment group consisted of 10 cochleae. After these treatments, all specimens were thoroughly rinsed with fresh medium, incubated for 24 hr in medium that contained 1 mM streptomycin, and then thoroughly rinsed in fresh medium and maintained in culture for seven days . After fixation, the specimens were immunolabeled for myosin VIIA and the numbers of hair cells were quantified from five 100×100 µm regions in the central region of each specimen . Regardl compare . This reThe avian cochlea can regenerate hair cells through two distinct mechanisms: (1) the evoked proliferation of supporting cell precursors, or (2) direct transdifferentiation of supporting cells into replacement hair cells entire depth of the sensory region, which contained both epithelial supporting cells as well as the basilar membrane and associated mesothelial cells . However, our BrdU data indicate that clodronate treatment reduced proliferation in these regions by ∼12 cells/10,000 µm2  region of the avian cochlea hosts a large population of resting macrophages. This epithelial region is comprised of so-called hyaline and cuboidal cells, which form the inferior boundary of the sensory region and connect the basilar membrane to the surrounding cartilage. Hyaline cells receive extensive efferent innervation One established function of tissue macrophages is to quickly remove the debris of apoptotic cells, in order to preserve epithelial integrity and to prevent an inflammatory response. Although it is reasonable to suspect that cochlear macrophages might contribute to the clearance of debris after hair cell injury, definitive evidence remains lacking. Prior studies of the avian ear have revealed at least two distinct mechanisms of ‘corpse’ removal after hair cell death. In the cochlea, most dying hair cells appear to be directly extruded from the apical surface of the sensory epithelium in vitro, we cannot rule out the possibly that cochlear injury in vivo results in the recruitment of additional macrophages from circulation, which may then enhance regeneration. However, regenerative proliferation in the chick cochlea begins within 24 hr of hair cell death The ears of nonmammalian vertebrates are capable of regenerating hair cells after acoustic trauma or aminoglycoside ototoxicity Although the depletion of cochlear macrophages did not affect the regenerative proliferation of supporting cells, we did find that clodronate treatment reduced cell proliferation below the basilar membrane . Some ofin vivo experiments only characterized the redistribution of macrophages within 24 hours after ototoxic injury (e.g., Finally, our results point to some clear differences in the behavior of both resting and recruited macrophages in the cochleae of birds and mammals. The mouse organ of Corti contains relatively few resident macrophages, but increased numbers of those cells are recruited into the cochlea following acoustic trauma or ototoxic injury ry e.g., , and we


NOG) is a responsible gene for multiple synostosis syndrome (SYNS1) and proximal symphalangism (SYM1), two conditions that are recently known to be within a wider range of clinical manifestations of stapes ankylosis with symphalangism. This study was performed to determine the range of phenotype caused by NOG mutations, using Japanese patients with various phenotypes including sporadic inherited SYM1, dominantly inherited SYM1, stapes ankylosis with broad thumb and toes (Teunissen and Cremer syndrome). In addition, 33 patients with typical otosclerosis  were studied. Direct sequencing analysis disclosed three novel mutations of the NOG gene in three SYM1 families. None of the otosclerosis patients without symphalangism had NOG mutations, indicating that NOG mutations may be restrictively found within patients with various skeletal abnormalities. These results together with the literature review indicated that there are no clear genotype–phenotype correlations for NOG mutations. With regard to surgical outcome, most of the patients in these three families with NOG mutations showed remarkable air–bone gap recovery after stapes surgery. Molecular genetic testing is useful to differentiate syndromic stapes ankylosis from otosclerosis, and even mild skeletal anomalies can be a diagnostic indicator of NOG-associated disease.Human noggin ( NOG) is a responsible gene for a wide range of clinical manifestations of stapes ankylosis with symphalangism. Proximal symphalangism (SYM1: MIM #185800) Human noggin (Otosclerosis (MIM#166800) is known as the single most common cause of progressive conductive hearing loss, characterized by abnormal bone remodeling in the otic capsule. Although there are a small number of familial cases that are likely to be monogenic, the majority of cases are sporadic. A series of studies has suggested that this condition involves both genetic and environmental factors NOG mutations. We thought this may be true because (i) within SYM1, NOG mutations were found in patients with minor skeletal anomalies without symphalangism +/− mice Typical otosclerosis was included in this study because it is an interesting question as to whether some of the typical otosclerosis is a continuum of a category of disease caused by NOG mutations in the stapes ankylosis patients with symphalangism in Asian populations. Therefore this study was undertaken to address whether NOG mutations are also causative and commonly found in those populations and if so, whether there is a different mutation spectrum.To date, no detailed survey was available for NOG mutations were reviewed to determine their spectrum as well as whether there are any particular genotype–phenotype correlations caused by NOG mutations.In addition, previously reported NOG gene. Their clinical symptoms, including ages at surgery (36–77 years old: average 54.4 years old), onset age (15–57 years old: average 37.3 years old), gender , laterality , and hearing threshold (average 63.1 dB), are summarized in We ascertained three Japanese families to be associated with conductive hearing loss and symphalangism, including an autosomal dominant SYM1 family, a sporadic SYM1 case with normal parents, and an autosomal dominant stapes ankylosis with broad thumb and toes (Teunissen and Cremer syndrome) family. Thirty-three Japanese otosclerosis patients, who underwent stapes surgery, were also screened for mutations in the We obtained informed consent for participation in this project from each subject and also from 192 normal control subjects who were unrelated Japanese individuals without any noticeable hearing loss evaluated by auditory testing. Otologic examination, audiometric analysis, and radiologic imaging were carried out for each patient.As shown in the pedigree , patient #991 was diagnosed with symmetric conductive hearing loss of 50 dB  at the aAs shown in the pedigree , a girl and her father (patients #3925 and #3926) visited our hospital due to bilateral hearing loss. Audiograms indicated bilateral mixed hearing loss . AnamnesThe pedigree shows hearing loss was inherited in four generations, indicating autosomal dominant inheritance . In addition to conductive hearing loss , the famNOG gene coding is constituted of one single exon, in which an open reading frame of 696 nucleotides encodes a NOG polypeptide of 232 amino acids. A sequence obtained from GeneBank U31202 was used to design primers containing the entire coding region of NOG. Two fragments to entirely cover the coding region of NOG were amplified with polymerase chain reaction (PCR) and two specific primer pairs, as follows: F1, 5′-CTTGTGTGCCTTTCTTCCGC-3′; R1, 5′-TACTGGATGGGAATCCAGCC-3′; and F2, 5′-TACGACCCAGGCTTCATGGC-3′; R2, 5′-TAGCACGAGCACTTGCACTC-3′.Human PCR reactions were carried out in 25 µl total volume containing 40 ng of genomic DNA, 10 pmol of each primer, 2 mM dNTPs, ×10 PCR buffer and 0.2 U of ExTaq polymerase . PCR conditions were denaturing at 94°C for 2 min; 35 cycles at 96°C for 30 s, 60°C for 30 s, 72°C for 1 min extension, with a final extension step at 72°C for 5 min in a Perkin-Elmer 9600 thermal cycler . PCR products were purified with a Suprec filter (Takara) and sequenced directly, using four primers  and ABI BigDye terminators, on an ABI 3100 sequencer (Perkin-Elmer).NOG gene were found by direct sequencing analysis in three families, whose common clinical features were compatible with SYM1, i.e. immobility of the PIP joints of the hands and toes, and congenital conductive hearing loss due to stapes ankylosis. Patient #991 of family 1 had a heterozygous G>T transversion at nucleotide 551 , predicting a cysteine (C) for phenylalanine (F) substitution at amino acid 184 (C184F) in the coding region of NOG. Since the C184F mutation was not found in either parent and was found only in the proband (patient #991), it was suggested that the mutation arose de novo in only the affected individual. Patients #3925 and #3926 of family 2 had a heterozygous T>A transversion at nucleotide 463 , predicting a cysteine (C) for serine (S) substitution at amino acid 155 (C155F) in the coding region of NOG. Patients #4106 and #4351 had a heterozygous C215X mutation.Three novel mutations of the NOG mutations. These three mutations were not observed in any of the other family members nor in the 192 unrelated Japanese controls (384 chromosomes).None of the otosclerosis patients had NOG gene in families with symphalangism, being consistent with the previous work showing that NOG is the responsible gene for SYM1 and stapes ankylosis with broad thumb and toes/Teunissen and Cremer syndrome. One mutation was a nonsense mutation (C215X), leading to a truncated protein, and was likely to be a pathologic mutation. The other two mutations are also likely to be pathologic rather than functionally neutral polymorphic changes because: (i) none were found in any of the controls, (ii) the alignment of NOG sequences from human, mouse, chicken, Xenopus laevis and zebrafish showed that C155 and C184 are well-conserved amino acids in all species (data not shown), and (iii) all affected subjects showed similar phenotypes.This study identified three novel mutations in the NOG mutations have been reported in SYM1, SYNS1, TCC, BDB2 and TCS families  have minor skeletal abnormalities such as broad thumbs and great toes Whether NOG mutations are located in the evolutionally well conserved and therefore functionally critical region  had hearing deterioration 3–10 years after the initial surgery, in accordance with a previous report With regard to surgical outcome, stapes surgery for conductive hearing loss due to The identification of the causative genes responsible for various middle/inner ear diseases will enable us to classify new congenital deafness groups in the future, and lead to clinical application in the diagnosis of middle ear disorders and better counseling for the selection of ideal intervention.The authors declare no conflict of interest.


Sorcerer II sailing vessel from the southern Indian Ocean in an effort to more thoroughly understand the lifestyle strategies of the microbial inhabitants of this ultra-oligotrophic region. No investigations of whole virioplankton assemblages have been conducted on waters collected from the Indian Ocean or across multiple size fractions thus far. Therefore, the goals of this study were to examine the effect of size fractionation on viral consortia structure and function and understand the diversity and functional potential of the Indian Ocean virome. Five samples were selected for comprehensive metagenomic exploration; and sequencing was performed on the microbes captured on 3.0-, 0.8- and 0.1 µm membrane filters as well as the viral fraction (<0.1 µm). Phylogenetic approaches were also used to identify predicted proteins of viral origin in the larger fractions of data from all Indian Ocean samples, which were included in subsequent metagenomic analyses. Taxonomic profiling of viral sequences suggested that size fractionation of marine microbial communities enriches for specific groups of viruses within the different size classes and functional characterization further substantiated this observation. Functional analyses also revealed a relative enrichment for metabolic proteins of viral origin that potentially reflect the physiological condition of host cells in the Indian Ocean including those involved in nitrogen metabolism and oxidative phosphorylation. A novel classification method, MGTAXA, was used to assess virus-host relationships in the Indian Ocean by predicting the taxonomy of putative host genera, with Prochlorococcus, Acanthochlois and members of the SAR86 cluster comprising the most abundant predictions. This is the first study to holistically explore virioplankton dynamics across multiple size classes and provides unprecedented insight into virus diversity, metabolic potential and virus-host interactions.The characterization of global marine microbial taxonomic and functional diversity is a primary goal of the Global Ocean Sampling Expedition. As part of this study, 19 water samples were collected aboard the  Viruses (predominantly bacteriophages) are the most abundant biological components of marine ecosystems, generally outnumbering their microbial hosts by an order of magnitude To date, no investigations of whole virioplankton assemblages have been conducted on waters collected from the Indian Ocean, however, targeted studies of cyanophage ; Table S1). The microbial community was pre-filtered using 20 µm mesh Nytex net and then size fractionated by serial filtration through 3.0-, 0.8-, and 0.1 µm membrane filters. The viral fraction of water samples (i.e. <0.1 µm) was concentrated and purified as described previously Surface water samples (∼400 L) were collected from 17 sites from the tropical Indian Ocean between August and October 2005 aboard the S/V Sorcerer II. Two additional sites were sampled off the island of Zanzibar, Tanzania using alternate vessels . The five samples that were comprehensively sequenced were selected based on sufficient quantity and quality of DNA across all size fractions. A total of approximately 228K Sanger and 2.2M 454 Titanium sequence reads were produced from the five viral libraries, resulting in a combined 2.7M predicted ORFs  (Table S2), representing 2.8% of the total predicted proteins from the Indian Ocean microbial dataset . This estimate is almost identical to a previous study which found that viral sequences accounted for ∼3% of GOS Phase I microbial data collected between Nova Scotia and French Polynesia The identification of viral sequences within the metagenomic data from larger size fractions was accomplished using a phylogenomic approach, the Automated Phylogenetic Inference System (APIS), that was designed for annotation of genomic , , and Table S3). Estimates of genotype richness varied widely between the Sanger and most of the 454 categories, but the relative ranking was conserved. On average, the most diverse site among all assemblies tested was GSIOVIR117, which also had the largest number of genotypes. The least diverse site, on average, was GSIOVIR122, which also had the lowest number of genotypes  tool To account for the possibility of varying genome size, diversity estimates were also obtained on the sub-sampled 454 datasets using a range of average viral genome sizes Table 3.To the best of our knowledge, this is the first study to compare PHACCS-based estimations of viral diversity using sequence information produced from two different platforms (Sanger and 454), yet assembled using the same methods. The number of predicted viral genotypes (richness) in a sample was the most variable with respect to the type of data produced . Sub-sampling of datasets is recommended to reduce coverage and improve the quality of PHACCS estimations.This comparison demonstrated that estimations of viral diversity using PHACCS, particularly the number of predicted genotypes, may be influenced by the nature of viral data and their subsequent assembly; yet most estimates fell within the range of previously published reports Sequences originating from discrete viral fractions (VFs) and the larger fractions (LFs) were taxonomically characterized using two approaches. One approach was based on a BLASTP comparison against the NCBI non-redundant (nr) protein database, which does not include predicted protein sequences from viral metagenomes. The other was a phylogenomic approach using APIS. Examination of metagenomic data using APIS resulted in ∼20% of sequences from the VFs producing phylogentic trees, and thus taxonomic classification Table 1.Caudoviridae family, demonstrating the utility of including viral metagenomes in search databases  and Synechococcus (S-PM2 and the Syn group of phages) (Table S4).According to phylogenetic characterization, the largest proportion of sequences from VF in the dsDNA virus category were most similar to sequences derived from environmental samples, including marine planktonic (∼95%) and marine sediment viral sequences (∼4%) Table 4.The VF and LF also differed with respect to the relative distribution of nucleocytoplasmic large DNA viruses (NCLDV), a group that includes the phycodna- and Mimi-viridae families that appear to infect eukaryotic organisms Table 4.Prochlorococcus and Synechococcus cyanophage reference sequences in addition to environmental viral sequences, which could represent uncultivated cyanophage. The VF group, corresponding to cluster B, was more phylogenetically diverse, characterized by multiple well-defined clades containing mixtures of reference phage and environmental viral sequences. The reference phage in the cluster B group belonged to all three of the tailed phage families rather than just described T4-like myoviruses present in the LF group, similar to the taxonomic distribution of Indian Ocean VF sequences . A heatmap of functional categories  further demonstrated the partitioning of VF and LF sequences as observed through PCA with differential clustering of the VF and LF. The vast majority of VF (∼79%) and LF (∼80%) sequences within the genetic information processing pathway were categorized as putative DNA replication, recombination and repair proteins (Table S5).Protein clusters containing viral sequences originating from the VF and LF were also categorized in the context of KEGG pathways, with the addition of a virus structure category, in order to assess the functional potential of Indian Ocean virioplankton. The majority of protein sequences from VF and LF were not mapped to a pathway and remained uncharacterized . This level of functional novelty was not unexpected due to high abundance of hypothetical proteins in each category . Smaller proportions of data were considered poorly characterized  or were not specific to a particular pathway . The remaining sequences were mapped primarily to the Virus Structure, Metabolism and Genetic Information Processing categories (Table S5). A relatively small proportion of these sequences from the VF and LF were classified into lower categories including nitrogen metabolism and oxidative phosphorylation. Although nitrogen metabolism genes have been previously documented in viral metagenomes created from a variety of environmental settings Table S1). The presence of glutamate synthase genes suggest that viruses may play a role in nitrogen modulation and assimilation during the infection of host cells. Proteins involved in oxidative phosphorylation (OP) pathway were much more abundant than photosynthesis-related proteins, comprising ∼30% of VF and ∼11% of LF sequences within the energy metabolism category (Table S5); with 466 VF and 25 LF sequences possessing EC numbers. NADH dehydrogenase I subunit and inorganic diphosphatase were represented in both the VF (n = 255 and 53 respectively) and LF (n = 7 and 18 respectively) while the cbb3-type cytochrome C oxidase subunit I was only detected in the VF (n = 158). To the best of our knowledge, this is the first report of viral cytochrome C oxidase and inorganic diphosphatase genes in the marine environment. NADH dehydrogenase and cytochrome C oxidase are both components of the electron transport chain in bacteria, which is ultimately used to produce ATP. Viral type I NADH dehydrogenase genes were first reported by Alperovitch-Lavy and colleagues Prochlorococcus and Synechococcus (PSI) (Table S6) did reveal the presence of additional viral PS genes. The PSII genes psbA and psbD  far outnumbered the PSI genes that were previously noted in the marine environment including psaA, psaB, psaC, psaD psaE and psaK  (Table S7). Viral PSI genes were also noted in previous analyses of GOS microbial metagenomic data including 17 samples collected from the Indian Ocean The largest proportion of viral sequences within the Metabolism pathway was attributed to energy metabolism, with a slight enrichment in the VF (Carbonic anhydrase (CA) enzymes were also present in the viral data . CA is responsible for catalyzing the conversion of carbonic acid to CO2 and is utilized by a diverse group of marine phytoplankton as part of their inorganic carbon concentration mechanism (CCM) to support photosynthetic carbon fixation There is a growing body of evidence based on analyses of fully sequenced viral genomes that phage tend to mimic the polynucleotide sequence composition of their hosts Two approaches were taken to predict the bacterial hosts: 1) direct assignment – the ICMs trained on RefSeq (NCBI) genomes and 2) a transitive assignment – the hosts were predicted using ICMs trained on large metagenomic bacterial scaffolds (>100 kb) from the cellular fraction, to which a bacterial taxonomy was in turn assigned based on scoring against RefSeq ICMs (see Prochlorococcus and Idiomarina. Assignments to NCBI unclassified Gammaproteobacteria (which encompasses diverse marine isolates including the uncultivated SAR86 cluster), and to a lesser degree, the SAR86 cluster itself were also prevalent Table S8) such as the SAR86 cluster (119 scaffolds) and Rhodobacterales HTCC2255 (33 scaffolds). This result was expected by the design of the transitive assignment methodology. However, this trend was not universal, as demonstrated by the number of Prochlorococcus assignments for which 38 bacterial scaffolds were assigned . The viral contigs that were assigned to these putative host taxa by the direct method were largely reassigned to several different bacterial genera by the transitive method (Table S9). Transitive assignments to Candidatus Pelagibacter and the SAR11 cluster were also sparse despite the prevalence of bacterial scaffolds attributed to these organisms (87 and 33 respectively) (Table S8). The top predicted host for Indian Ocean viral assemblages using transitive methodology was the SAR86 cluster. The SAR86 cluster encompasses a group of uncultivated, proteorhodopsin-containing Gammaproteobacteria d rather than ChlaThe method was subsequently applied to all 5 Kb or longer contigs assembled from the Indian Ocean 454 viral libraries. Procholorococcus represent the dominant hosts for viruses in the Indian Ocean, providing insight into the types of viruses that putatively possess these metabolic capabilities.This is the first study to examine the Indian Ocean virome using holistic metagenomic approaches. Since our analyses were not constrained to the “viral fraction” of samples, we were able to gain a much more comprehensive understanding of virus diversity, total gene complement and functional potential as well as virus-host relationships. Significant taxonomic differences were evident between viruses represented in the VF versus the LF. An enrichment of cyano-myoviruses and viruses that likely infect eukaryotic phytoplankton or heterotrophic protists was found in the LF; while podo- and siphoviruses were prevalent in the VF. Similarly, notable differences in functional potential were evident by the distribution of abundances within metabolic pathways. The presence of putative viral genes potentially involved in nitrogen metabolism, carbon fixation and oxidative phosphorylation suggests that viruses infecting autotrophic and heterotrophic microbes may influence host cell physiology through diverse mechanisms in the Indian Ocean. Predicted virus-host relationships suggest that members of the SAR86 cluster and the cyanobacteria Acaryochloris and Surface water samples (∼400 L) were collected from 17 sites from the tropical Indian Ocean between August and October, 2005 aboard the S/V Sorcerer II. Two additional sites, GS148 and GS149 (∼200 L), were sampled off the island of Zanzibar, Tanzania using alternate vessels. All field studies conducted within the EEZ of foreign nations received Marine Research Permits as required under the U.N. Convention on Law of the Sea, and as required, separate agreements to access genetic resources. The locations were not privately owned or protected in any way and the field studies did not involve endangered or protected species. A YSI (model 6600) was used to measure the physical environmental parameters including water temperature, salinity, dissolved oxygen and sample depth. Sub-samples were collected for dissolved nutrient analyses as described previously E. coli cells) and negative (DEPC water) samples. Gel electrophoresis was performed on 2 µl  on a 0.8% agarose gel stained with SybrGold (Invitrogen). If no discernible 16S rRNA gene product (∼1500 bp) was visualized the samples were further processed through DNA extraction.The viral fraction of water samples (i.e. <0.1 µm) was concentrated using tangential flow filtration (TFF) as described previously et al., 2007. Viral DNA was extracted from purified VCs and modified linker amplified shotgun libraries (LASLs) were constructed as described previously Methods describing DNA extraction from filters, construction of clone libraries, template preparation, and automated cycle sequencing can be found in Rusch The Automated Phylogenetic Inference System (APIS) was used for the taxonomic classification of viral predicted proteins as well as the identification of viral proteins within the larger size classes of data A BLAST-based approach was used, which is part of the Viral Metagenome Annotation Pipeline (VMGAP) ab initio gene finder program. The predicted protein coding sequences were then annotated using the Viral Metagenome Annotation Pipeline (VMGAP), described by Lorenzi and coworkers Open reading frames were predicted as described previously 3. For those protein sequences that did not produce any significant hit against the PSSM database, a BLASTP search was conducted against a database of proteins belonging to clusters containing fewer than 20 members. Proteins were then assigned to clusters based on the hit having the lowest e-value with at least 40% identity and 70% coverage. Protein sequences that did not result in any significant hit remained unassigned.ORFs predicted on 454 sequence reads were mapped to protein clusters as follows. RPS-BLAST was first used to compare each protein sequence against a database of Position Specific Scoring Matrices (PSSM) representing clusters containing more than 20 proteins. Then, proteins were assigned to clusters based on the PSSM that gave the highest bit score with an e-value ≤1×10−Annotation was performed on all protein clusters http://hmmer.org/). The multiple alignments were processed through a gap-filtering step to remove sequences that contain 60% or more gaps. Phylogenetic trees were constructed using the program FastTree http://phylosoft.org/archaeopteryx/).Environmental sequences and references were retrieved from two proteins clusters, both containing putative genes encoding terminase enzymes. The average length of all sequences was calculated and sequences below 40% of the average were removed. Non-viral GOS Phase I sequences were reduced through clustering and only the representative sequence used in subsequent steps. Existing hidden Markov model (HMM) profiles, including fragment HMMs, obtained in cluster annotation or peptide annotation were identified and the HMM that accounted for the majority of viral sequences was selected for alignment using HMMER  and Canonical Component Analysis (CCA) were performed using the R statistical program. For PCA, library proportional abundances of viral sequences for each protein cluster were calculated and used to build the centered matrix. For CCA, oceanographic metadata was included to assess correlation between these and protein cluster proportional abundances.The Sanger and 454 data from each viral library were assembled independently using the Newbler GS De Novo Assembler, version 2.3, with a minimum identity threshold of 86%. In addition to virus-specific assemblies, a comprehensive global assembly was also conducted on all Sanger reads produced from Phase I of GOS and all Indian Ocean size fractions as described by Rusch et al. 2007 using the Celera Assembler version 5.3 with a minimum identity threshold of 86%. A minimum overlap length of 40 bp was used for all assemblies.A local command line version of the PHACCS program As this is the first report of an alignment-independent method that aims to predict the virus∶host relationships in the metagenomic datasets, detailed methodology and benchmarking is reported in All Sanger-generated viral data was submitted to the NCBI Trace Archives. These include the raw reads from viral metagenomes as well as the predicted protein sequences extracted from the larger size fractions of data. All 454-generated viral data was submitted to the NCBI Short Read Archive (SRA), corresponding to accession numbers SRX096024, SRX096023, SRX096025, and SRX096299. The microbial and viral assemblies used for the analysis of virus-host classification were submitted under NCBI's Project ID 19733.Methods S1Additional methods including, i) Alternative cluster annotation, ii) Identification of viral photosynthesis genes, iii) Classification of Virus∶Host relationships.(DOCX)Click here for additional data file.Figure S1Heatmap showing delineation of viral sequences from large vs. viral fraction. Abundance of sequences from each site within selected functional groups at the level-2 classification of Gene Ontology (GO) was used to generate heatmap. Hierarchical clustering of sites indicates a grouping of large (blue) versus viral (green) fraction. Functional groups are color-coded as follows: cellular processes (purple), cellular processes and signaling (blue), environmental information processing (green), genetic information processing (yellow), metabolism (orange), phage structure (red).(TIF)Click here for additional data file.Table S1Indian Ocean sample characteristics and oceanographic metadata.(XLSX)Click here for additional data file.Table S2Viral sequences retrieved from the larger size classes.(XLSX)Click here for additional data file.Table S3Assembly statistics for Indian Ocean viral metagenomic libraries.(XLSX)Click here for additional data file.Table S4Top five most abundant cyanophage homologs based on taxonomic assignments.(XLSX)Click here for additional data file.Table S5Functional characterization of VF and LF viral sequences by KEGG category.(XLSX)Click here for additional data file.Table S6List of reference sequences and genomes used to identify Indian Ocean viral photosystem genes.(XLSX)Click here for additional data file.Table S7Viral photosystem genes present in the Indian Ocean metagenomic data.(XLSX)Click here for additional data file.Table S8GOS Bacterial Scaffold Taxonomic Assignments based on MGTAXA.(XLSX)Click here for additional data file.Table S9Re-distribution of viral contigs to bacterial taxa from direct to transitive assignments.(XLSX)Click here for additional data file.Table S10NCBI RefSeq prokaryotic genomes used for building reference models.(XLS)Click here for additional data file.Table S11Benchmarking accuracy for predicting the host taxonomy.(XLSX)Click here for additional data file.Table S12Benchmarking set of virus∶host pairs compiled from NCBI RefSeq.(XLS)Click here for additional data file.Table S13Summary counts for the benchmark virus∶host pairs in (XLSX)Click here for additional data file.


Microarray mRNA data indicated that smoking promoted an “inverse” M1 mRNA expression program, defined by decreased expression of M1-induced transcripts and increased expression of M1-repressed transcripts with few changes in M2-regulated transcripts. RT-PCR arrays identified altered expression of many miRNAs in alveolar macrophages of smokers and a decrease in global miRNA abundance. Stratification of human subjects suggested that the magnitude of the global decrease in miRNA abundance was associated with smoking history. We found that many of the miRNAs with reduced expression in alveolar macrophages of smokers were predicted to target mRNAs upregulated in alveolar macrophages of smokers. For example, miR-452 is predicted to target the transcript encoding MMP12, an important effector of smoking-related diseases. Experimental antagonism of miR-452 in differentiated monocytic cells resulted in increased expression of MMP12. The comprehensive mRNA and miRNA expression profiles described here provide insight into gene expression regulation that may underlie the adverse effects cigarette smoking has on alveolar macrophages.Human alveolar macrophages are critical components of the innate immune system. Cigarette smoking-induced changes in alveolar macrophage gene expression are linked to reduced resistance to pulmonary infections and to the development of emphysema/COPD. We hypothesized that microRNAs (miRNAs) could control, in part, the unique messenger RNA (mRNA) expression profiles found in alveolar macrophages of cigarette smokers. Activation of macrophages with different stimuli  Cigarette smoking is a prominent risk factor for many respiratory diseases including emphysema/COPD, respiratory bronchiolitis, interstitial lung disease, and desquamative interstitial pneumonitis Alveolar macrophages are essential immune effector cells in the lung with functions that include pathogen clearance and responses to inhaled environmental exposures in vitro identified unique macrophage gene expression programs and the associated activation phenotypes. For example, exposure to inflammatory stimuli such as IFNγ and LPS polarizes macrophages toward an M1 activated phenotype that is associated with microbicidal activity Macrophage gene expression programs are altered in response to local environmental cues. These changes may underlie the regulatory role macrophages play in many disease processes Transcriptional profiles of human alveolar macrophages directly isolated from nonsmokers and active smokers have shown that cigarette smoke exposure alters macrophage gene expression MicroRNAs (miRNAs) are small, noncoding RNAs that have an important regulatory role in gene expression programs In vitro experiments showed a direct link between low expression of miR-452 and increased expression of MMP12 mRNA. These results are consistent with the hypothesis that miRNAs play a role in regulating gene expression in alveolar macrophages of smokers, and possibly a corresponding role in disease pathogenesis.An initial study on the effect of smoking on human miRNA expression was reported by Schembri et al All procedures and protocols described in this communication were approved by the University of Iowa Institutional Review Board. Written informed consent was obtained and all clinical investigation has been conducted according to the principles expressed in the Declaration of Helsinki.Subjects were recruited from the community by the Iowa Institute for Clinical and Translational Science (ICTS) Clinical Core via advertisements and word-of-mouth. Inclusion criteria for case subjects required at least a 10 pack-year history of smoking, while the nonsmoker control subjects were self-reported never smokers. Subjects were excluded if they had any significant co-morbid conditions such as pregnancy or other acute or chronic disease such as pre-existing asthma, interstitial lung disease or cardiovascular disease. Subjects were also excluded if a baseline spirometry revealed the forced expiratory volume in the first second was less than 60% of the predicted value based on National Health and Nutrition Examination Survey III data set.The first cohort of alveolar macrophage donors consisted of 4 nonsmokers and 4 active smokers with 31±14 pack-year histories. All subjects were Caucasian. The nonsmoker group had 3 males and 1 female with a mean group age of 26±9 years. The smoker group had 2 males and 2 females with a mean group age of 51±8 years.The second cohort included 4 nonsmokers and 4 smokers with 31±3 pack-year histories. All subjects were Caucasian except for 1 African American in the smoker group. The nonsmoker group had 2 females and 2 males with a mean group age of 31±6 years. The smoker group had 2 males and 2 females with a mean age of 47±years.The third cohort included 4 nonsmokers, 4 light smokers (12±2 pack-year histories), and 4 heavy smokers (33±6 pack-year histories). All subjects were Caucasian except for 1 African American in each of the smoker groups. Each of the three groups had 2 male and 2 female donors. The mean age for the groups was 35±12 years for the nonsmokers, 36±7 years for the light smokers, and 51±7 years for the heavy smokers.6 cells were stored at −80°C until RNA isolation procedure was performed. Cell yields from bronchoscopy in cohort 1 averaged 25±3×106 cells for the nonsmokers and 67±4×106 cells for the active smokers. In all three cohorts the procedure generated a relatively pure population of alveolar macrophages with fewer than 5% neutrophils or lymphocytes in the bronchoalveolar lavage fluid.After informed consent was obtained, subjects underwent standard flexible bronchoscopy RNA was isolated from alveolar macrophages or PMA-differentiated THP-1 cells using the mirVana miRNA Isolation kit (Applied Biosystems (ABI)). The quantity and quality of the RNA samples was assessed using an Experion Automated Electrophoresis Station (Bio-Rad). The RNA quality indicator was above 8 for all samples where values of greater than 8 indicate primarily intact RNA on a scale of 1–10. After preparation, RNA samples were stored at −80°C until use.Measurements of genome-wide macrophage mRNA expression were conducted using the GeneChip Human Exon 1.0 ST Arrays (Affymetrix). Generation of labeled cDNA, hybridizations, and scanning of the microarray were performed under contract by the University of Iowa DNA facility. The resulting data were analyzed using the Partek Genomics Suite version 6.5 (Partek). The data were assessed for quality and subjected to robust multiarray averaging (RMA) normalization. The normalized data were then analyzed using an ANOVA model with linear contrasts to calculate p-values and smoker-to-nonsmoker expression ratios. The false discovery rate (FDR) step-up method RNA from alveolar macrophages of nonsmokers and active smokers was reverse transcribed with MultiScribe Reverse Transcriptase (ABI) using Megaplex Primers version 2.0 (ABI). Changes in miRNA expression was then determined using human TaqMan Low Density Arrays version 2.0 (ABI). Ct values calculated using SDS version 2.4 (ABI) were exported to the Partek Genomics Suite to calculate smoker-to-nonsmoker expression ratios. RMA-normalized data were subjected to an ANOVA model with linear contrasts to calculate p-values.http://discover.nci.nih.gov/cimminer/).Principle component analysis (PCA) was performed using standard functions built in MATLAB software version 7.9 (MathWorks). This analysis identified a representative miRNA within each cluster with the highest Pearson correlation between its expression profile and the first principal component from our PCA analysis Validation studies were performed using alveolar macrophage RNA collected from the initial array group (cohort 1) and a non-redundant sample set (cohort 2). Individual TaqMan MicroRNA Assays corresponding to assays including in the TLDA version 2.0 assays (ABI) were used to document abundance of the mature forms of three of the downregulated miRNAs. Briefly, 10 ng RNA was reversed transcribed with MultiScribe RT (ABI) using a miRNA-specific stem loop primer. Then, PCR with TaqMan Universal PCR Master Mix (ABI) was performed using miRNA-specific real time primers. Expression levels were defined as a ratio between the Ct values of the indicated miRNA and the endogenous control, RNU48.Using the Partek Genomics Suite software, each indicated miRNA was used to query the TargetScan THP-1 cells were maintained in RPMI 1640 (Gibco) supplemented with fetal bovine serum . Cells were incubated with PMA  for 18 hours to induce differentiation toward a macrophage phenotype Cigarette smoking has been shown to alter the transcriptional profile of human alveolar macrophages in a consistent manner as reported by two independent groups th column of rd and 4th columns within rd and 5th columns within in vitro M2a-polarizing conditions.To further evaluate the extent to which the gene expression profile in alveolar macrophages from smokers contrasts with an M1 profile, the highly regulated mRNAs listed in A global mRNA abundance increase was reported in lung tissue from a rat model of cigarette smoking To address our hypothesis that miRNAs have a role in maintaining the unique smoking-associated gene expression profile, we next obtained miRNA expression profiles. TaqMan Low Density Array (TLDA) assays were used to measure miRNA expression in the same RNA samples from alveolar macrophages of smokers and nonsmokers that had been used to analyze mRNA expression (cohort 1). TLDA assays use quantitative RT-PCR to specifically measure the abundance of 667 mature human miRNAs. The total miRNA abundance appeared to be lower in alveolar macrophages from cigarette smokers compared to nonsmokers, with a median expression ratio of 0.63. Therefore, the majority (75.7%) of miRNA smoker-to-nonsmoker expression ratios was less than 1 . InteresThe expression of miRNAs was determined using a small nucleolar RNA, RNU48, as an endogenous control. Three other small nucleolar RNAs  were included in the TLDA assays as alternative endogenous controls. The expression of all three of the additional controls was unchanged when calculated using RNU48 as an endogenous control . The genThe overall repression of miRNA expression in alveolar macrophages of smokers compared to nonsmokers was also observed when comparing the number of miRNAs with statistically significant (p<0.05) abundance differences. For example, when using a 2-fold cut-off, 11 miRNAs have increased abundance and 43 miRNAs have decreased abundance . GeneralIndividual RT-PCR miRNA assays were used to confirm the observations from the TLDA assays. Three differentially expressed miRNAs  were validated using the same RNA samples from cohort 1 analyzed in the TLDA assays . FurtherTLDA assays were used to evaluate miRNA expression profiles in a third set of alveolar macrophages (cohort 3). Four donors each were categorized as nonsmokers (no smoking history), light smokers, and heavy smokers. Light smoking resulted in a modest reduction in miRNA expression compared to nonsmokers with 54.2% of miRNAs have an expression ratio less than 1 relative to nonsmokers. The decrease in global miRNA expression was more pronounced in heavy smokers with expression ratios of less than 1 for 72.6% of the miRNAs . The genMost mRNAs targeted for repression by miRNAs are degraded To test our hypothesis that miRNAs influence the mRNA expression profiles in alveolar macrophages of cigarette smokers, we evaluated whether antagonizing the function of a specific miRNA would lead to increased mRNA expression of the predicted target. We were particularly interested in whether the highly downregulated miRNA, miR-452, influenced the expression of MMP12, a protease relevant to smoking-related diseases that is highly upregulated in alveolar macrophages of smokers. Transfecting in an inhibitor of miR-452 resulted in elevated expression of MMP12 transcripts, but had no effect on another predicted target of miR-452, TM7SF4 .in vitro system to support our hypothesis that miRNAs influence the expression of an important macrophage product.This study reports on miRNA and mRNA expression in alveolar macrophages from nonsmokers and active cigarette smokers. Significant differences in both miRNA and mRNA expression were found in alveolar macrophages obtained from nonsmokers and smokers. We identified a smoking history-dependent decrease in global miRNA abundance. Importantly, we describe many examples of inverse relationships between miRNAs and their predicted mRNA targets and used an In vitro polarization of monocyte-derived macrophages (MDMs) leads to distinct phenotypes that have been categorized as M1, M2a, M2b, and M2c in vivo has been difficult to determine, partly because purification of human macrophages from the tissues in which they are embedded is usually not possible. Human alveolar macrophages are unique in this aspect since relatively pure populations can be obtained from bronchoalveolar lavage fluid. A consistent alteration in gene expression profiles of human alveolar macrophages from cigarette smokers compared to nonsmokers has been reported by two independent groups of investigators in vitro is accomplished by treatment with two stimuli, typically IFNγ and either TNFα or another molecule that promotes TNFα production such as LPS Since cigarette smoking is associated with increased risk of pulmonary infections We hypothesized that miRNAs are at least partly responsible for regulating the unique gene expression profile in alveolar macrophages of smokers. The RNA samples collected from cohort 1 for mRNA expression profiling of smoker and nonsmoker alveolar macrophages were also used in TLDA miRNA profile assays to compare the abundance of 667 human miRNAs. This global approach to evaluating miRNA expression identified 54 miRNAs with significantly altered expression. Among the 10 miRNAs that were significantly upregulated by >2-fold, 7 are located on two polycistronic pri-miRNAs, the miR-132/212 cluster and the miR-221/222 cluster. Therefore, increased transcription of these two polycistronic pri-miRNAs could explain almost all of the highly upregulated miRNAs in smoker alveolar macrophages. Similarly, transcriptional regulation may also be important for many of the significantly repressed miRNAs in smoker alveolar macrophages because ten downregulated miRNAs were processed from four polycistronic pri-miRNAs .An important observation from the miRNA profiling experiments was that the majority of miRNAs with altered expression in alveolar macrophages of smokers compared to nonsmokers were downregulated. Functionally, this should lead to increased mRNA expression in targeted genes. Using both the TargetScan and MicroCosm target prediction algorithms to analyze the results from cohort 1, we found that 30 of the 70 statistically significant mRNAs upregulated by >2-fold were putative targets of miRNAs that were significantly downregulated by >2-fold. The most strongly downregulated miRNA in smokers, miR-452, had the most predicted targets with upregulated expression. In experiments utilizing a miR-452 inhibitor, we show that inhibition of this miRNA in differentiated THP-1 cells resulted in elevated MMP12 transcript expression.It is likely that miRNAs are not the only mechanism that could contribute to the inverse M1 alveolar macrophage phenotype. For example, soluble TNF receptor type II is increased in the sputum of smokers with COPD There was a striking ∼50–60% reduction in total miRNA abundance in alveolar macrophages of smokers relative to nonsmokers. Global repression of miRNA expression that was also noted using non-TLDA miRNA profiling platforms in epithelial cells of smokers and in lung tissue of rats in a model of cigarette smoking The reduced global expression of miRNAs reported here in alveolar macrophages of smokers has also been reported for many cancers. For example, total miRNA abundance is lower in tumors and tumor-derived cell lines relative to corresponding normal tissue The impact that global miRNA expression levels has on cancer progression was addressed by experimentally knocking down the expression of Drosha, DCGR8, and Dicer In summary, this is the first study to analyze miRNA expression in human alveolar macrophages from nonsmokers and active smokers. The data show a global repression of miRNA levels in smokers. In addition, many of the downregulated miRNAs are predicted to target mRNAs that had increased expression in alveolar macrophages of smokers. This supports a role for miRNA expression in regulating disease-relevant changes in gene expression in smoker alveolar macrophages.


Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors.Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both  Glioblastoma multiforme (GBM) is the most common and lethal primary brain tumor in adults, and therefore, there is an urgent need to develop novel therapeutic strategies that effectively target therapy-resistant GBM cells. Among heterogeneous GBM cells glioma stem cells (GSCs) represent a subpopulation of highly tumorigenic cells that possess stem cell characteristics. While our understanding of GSCs is evolving, there is a great deal of evidence supporting the hypothesis that GSCs drive GBM propagation and promote resistance to conventional therapies such as radiation and chemotherapy in vitro and tumor growth in vivo, suggesting that MELK is a clinically relevant molecular target for GBM therapy Maternal embryonic leucine zipper kinase (MELK) is a serine/threonine kinase that is highly expressed in various organ-specific stem cells and cancers Experiments using de-identified human tissue-derived materials were carried out under the approved Institutional Review Board at University of California, Los Angeles (UCLA) or Ohio State University (OSU). Microarray studies were carried out at UCLA. Primary samples collected at UCLA were de-identified and sent to OSU for further studies. The OSU Institutional Review Board approved this research study and waived the need for further written informed consent from the participants. The name of this protocol is Investigating Novel Therapeutic Strategies for Brain Tumor Treatment and the approval number at OSU is 2005C0075. I Nakano serves as the Principal Investigator for this approved protocol. All animal experimentation was performed at OSU with the approval of the OSU Animal Research Committee, following NIH guidelines.Cells derived from 3 samples of GBM surgical tissues were established in Dr. Harley Kornblum’s laboratory at UCLA and were cultured as previously described www.ingenuity.com). The GEO submission number for this microarray is GSE50227.RNA was extracted from GBM sphere samples  treated with 1 μM Siomycin A or control (DMSO) for 24 hours with RNeasy Mini Kit according to the manufactur’s protocol (Qiagen). RNA samples were subjected to cluster (A) and canonical pathway analyses (D) by Ingenuity software  and CD133(−) cells from GBM spheres were performed using CD133 antibody (clone: AC133) according to manufacturer’s protocol  and as described previously d = 18 μM), which showed no or minimal activity to the other kinases. Similarity search to the Chemical Abstracts Service database was performed in order to check the novelty of this computationally discovered MELK inhibitor candidate.Using the structure-based virtual screening method, C1 was identified as a potential MELK inhibitor from readily available half a million commercial compounds. C1 compound was subsequently validated via experimental enzyme assay as previously described 2. After incubation we confirmed that the tumor slice cultures retain the histopathological characteristics of GBM. These treated tissues were fixed with 10 mL of 10% v/v formalin for 24 hours and processed for paraffin-embedded sections (4 μm thickness) for immunohistochemistry.GBM surgical tissues of 2 patients were received immediately after surgery from the Department of Pathology at OSU and they were histopathologically diagnosed as GBM by the assigned neuro-pathologists. Serial sections of the surgical specimens were cut to create tumor blocks (10 mm in diameter) and these blocks were transferred into 6 well plates as described previously Ten thousand GBM157 sphere cells in 5 μl of phosphate buffered saline (PBS) were injected intracranially into immunocompromised mice (n = 16)  according to the methods described previously Studies were performed as described previously 2) for 22 to 24 hours. One μM of C1 or DMSO was added to the cell culture just before imaging. To avoid drug combinations, cells were only synchronized by trypsination around 24 hours before imaging. Images were acquired on a Zeiss dynascope confocal microscope (LSM 710) equipped for alive cells  by using a Plan-Apochromat 40 X-water immersion objective. The positions of the mitotic cells on the stage were registered and therefore, these cells were imaged continuously. None of the followed mitotic cells divided in two daughter cells. Control and treated cells were imaged simultaneously. Then, the Lab-Teck was kept under normal culture conditions and cells were fixed upon 72 hours of treatment by 4% paraformaldehyde and nuclei stained by Hoechst 3342. Images were recorded with the same equipment as above but at room temperature and analyzed with the Zen software provided by Zeiss. Only one cell line was imaged since the behavior of the cells upon C1 treatment is similar to what was previously observed in HeLa, Hek or H358 cells U251 cells were transfected with the vector E-GFP-N1 using lipofectamine (Invitrogen) according to the manufacturer’s protocol. Cells were selected by gentamicin (100 μg/ml), seeded on a 2-wells Lab-Tek chambered coverglass , and maintained under standard culture conditions  using the Apoptosis Detection Kit  according to the manufacturer’s instructions. Data was confirmed by 3 independent experiments.Neurosphere formation assay and radiosensitivity assay were performed as described previously Statistical analysis was performed using the SPSS17 Statistics software  using one-way ANOVA and student’s T test. A probability of p<0.05 was considered to be significant. All the data are shown in mean ± standard error of the mean (SEM).in vitro and in vivoPreviously we demonstrated that the thiazole antibiotic Siomycin A attenuates a MELK-mediated signaling, thereby diminishing GSC growth Next we investigated whether the function of MELK in GSCs specifically depends on its kinase activity. To address this question, we combined lentiviral infection of MELK shRNA vector, to downregulate endogenous MELK protein, with overexpression of MELK wild type or the kinase dead version of MELK (MELK D150A) in silico screening of small molecules and identified a benzo[e]pyridoindole, C1 . These data suggest that C1 has a potent inhibitory effect on the GBM cell proliferation and GSC growth under the condition of a relatively preserved microenvironment for GBM tumors.Organotypic slice culture of tumor tissues is one experimental model readily available to assess the influence of drug treatment on tumor cells in vitro and ex vivo, we next investigated the effect of C1 treatment on mouse models of GBM in vivo. Following transplantation of GBM157 cells into immunocompromised mouse brains, we injected C1 at day 7 with quantities of 2.5 pmol, 25 pmol, and 250 pmol into the tumor cavities , indicating that C1 treatment sensitizes GSCs to radiation-induced cell death in vitro.Irradiation is the current first line post-surgical therapy for GBM patients. Nonetheless, the survival benefit for GBM patients of radiation treatment is no greater than 3 months in vitro, but also inhibited the growth of GSC-derived tumors in vivo. Finally, we found that C1 treatment sensitizes GSCs to radiation-induced cell death, supporting C1 as an attractive molecule-targeting therapy to combine with current standard protocols of GBM treatment.In this study, we demonstrated that MELK acts on GSC survival through its kinase activity. We performed the computational structure analysis of MELK protein to determine the ATP binding region of this kinase. Using this information, we identified C1 as a kinase inhibitor with substantial potency against MELK. C1 treatment not only induced G2/M arrest and subsequent mitotic catastrophe of GSCs in vivo using a small molecule kinase inhibitor. Previously, we found that the oxo-group of C1 is necessary for kinase inhibitory activity in silico model predicted that the oxo-group of C1 forms a strong hydrogen bond with the Tyr88 residue of the ATP-binding triplet domain, confirming our earlier report Our study contributes several novel findings to the current understanding of the GBM pathophysiology. To our knowledge, we present the first pre-clinical data for MELK kinase inhibition-mediated suppression of GBM cell growth Given that mutations in the kinase domain or in other molecular elements of MELK do not appear to be a frequent event in cancers, the distinct function of MELK in normal and oncogenic stem cells is likely to be epigenetic. Several recent studies, including our own, have implicated MELK in cell cycle regulation in vitro. Recently, Golding et al. reported that ATM inhibition effectively radiosensitizes GBM cells without harming normal neural progenitor cells et al. demonstrated that radiosensization through ATM inhibition occurs preferentially in GSCs but not in non-GSCs in vivo without causing a noticeable harmful effect on normal brain cells The potent radioresistance of GSCs has been partly attributed to upregulation of the ATM/ATR DNA damage response pathway in vivo, it is attempted to evaluate the permeability of the BBB and bioavailablity/stability of C1 in vivo.Regarding the clinical application of C1 for GBM therapeutics, some open questions remain. In fact, the efficacy of chemotherapy of brain malignancies is often hampered by the presence of the blood-brain barrier (BBB). From the point of molecular weight, the size calculated from the structure of C1 is 293 Da, which is presumably small enough to penetrate the BBB. However, the permeability of the BBB is not solely dependent on the molecular size but also affected by several kinds of drug property and circumstances. Given the potent effect of C1 treatment on mouse GBM-like tumor models In conclusion, our data indicate that C1 is a novel inhibitor for protein kinases with substantial inhibitory effect on MELK. This study suggests that pharmacological inhibition of MELK kinase activity represents an attractive therapeutic approach for GBM that may overcome the resistance seen after current, standard treatment protocols. We postulate that C1 may also effectively treat a variety of cancers with elevated activation of MELK.


In vitro effects of EGCg and the antibiotic colistin (COL) on growth inhibition, survival, and also against young and mature biofilms of S. maltophilia were determined. Qualitative and quantitative changes on the biofilms were assessed by confocal laser scanning microscopy (CLSM). Further, in vivo effects of nebulized EGCg in C57BL/6 and Cftr mutant mice during acute Sm lung infection were evaluated. Subinhibitory concentrations of EGCg significantly reduced not only biofilm formation, but also the quantity of viable cells in young and mature biofilms. CLSM showed that EGCg-exposed biofilms exhibited either a change in total biofilm biovolume or an increase of the fraction of dead cells contained within the biofilm in a dose depended manner. Sm infected wild-type and Cftr mutant mice treated with 1,024 mg/L EGCg by inhalation exhibited significantly lower bacterial counts than those undergoing no treatment or treated with COL. EGCg displayed promising inhibitory and anti-biofilm properties against CF Sm isolates in vitro and significantly reduced Sm bacterial counts in an acute infection model with wild type and CF mice. This natural compound may represent a novel therapeutic agent against Sm infection in CF.We investigated the  Bacterial pathogens are progressively reported as an important cause of high morbidity and mortality rates among patients with cystic fibrosis (CF) S. maltophilia with increased risk of developing pulmonary exacerbation, lung transplantation and death Although studies have shown associations between infections caused by Camellia sinesis). Notably, in vitro studies have shown that EGCg is an effective antimicrobial compound against a variety of Gram-positive and Gram-negative bacterial, as well as fungal pathogens Pseudomonas aeruginosa and Staphylococcus aureus, two of the most relevant pathogens in patients with CF Cftr mutant mice. Further, we determine EGCg effects on biofilms in comparison to that of COL.Epigallocatechin-3-gallate (EGCg) is the most abundant polyphenol found in green tea  or aqua (Cftr mutant mice experiments) at room temperature.EGCg and COL were obtained from Sigma . Stock solutions  of EGCg were freshly prepared and diluted in Mueller-Hinton broth  containing 1% (v/v) dimethyl sulfoxide (DMSO). COL stock solutions  were also dissolved and diluted in MHB including 1% DMSO. COL stock solution was stored at −20°C until use. For in vitro activity of EGCg. Sputum samples were collected from the patients as part of standard care. Bacterial cultures are collected as part of our epidemiological surveillance. EGCg susceptibility profiles of the clinical isolates and reference strain (ATCC13637) were determined using broth microdilution guidelines proposed by the Clinical and Laboratory Standards Institute (CLSI) A collection of 60 different clonal Sm isolates from CF patients were selected for investigation of the The kinetics of the bactericidal effect of EGCg on Sm was investigated against the strain ATCC 13637 (control) and two CF clinical isolates  in a microtiter plate assay as previously described Cftr mutant mice B6.192P2 (CF/3)-CftrTgH (neoim)Hgu (abbreviated Cftr-/-) at the age of 12 to 14 weeks. Cftr mutant mice) were nebulised either with sterile distilled water (n = 10) to prevent any potential mucus clearance or 1,024 mg/L EGCg (n = 10). All mice were maintained in isolated cages to provide a pathogen-free environment at the Central Laboratory Animal Facility of the University Hospital Essen, Essen, Germany.All animal experiments were approved by the ethical committee of Landesumweltamt Nordrhein-Westfalen (Q1299/12), Germany. We investigated the antimicrobial effects of EGCg in comparison to COL in female C57BL/6 specific-pathogen-free mice purchased from Harlan Laboratories  at 6 to 8 weeks of age. C57BL/6 mice were nebulised for 5 min with 1×PBS (n = 13), 128 mg/L COL (n = 12) or 1,024 mg/L EGCg (n = 11). Infection with Sm1 was performed as previously described see also  [24]. OnC), and isolates were classified as follows: no biofilm producer (OD≤ODC), weak biofilm producer (ODC≤OD≤2×ODC), moderate biofilm producer (2×ODC≤OD≤4×ODC), and strong biofilm producer (4×ODC<OD) (23).The biofilm assay was performed as previously described The effects of EGCg and COL on Sm biofilm formation were determined by the method previously described in vitro effect of EGCg on the viability of Sm biofilm was plotted as the ratio of viability (cells with active metabolism) in treated samples to viability in untreated samples. The effects of COL on mature biofilms were determined as described for EGCg.To evaluate the reduction of cell viability of EGCg, we cultivated Sm biofilms  in a 96-well microtiter plate assay as previously described BacLight viability staining and automated confocal laser scanning microscopy (CLSM), as previously described 3] calculated from the image stacks with a customer-designed solution in the software Developer XD (Definiens). The negative controls were biofilms treated with Luria Bertani (LB) medium supplemented with 1% (v/v) DMSO, and the positive controls (killing control) were treated with formalin at final concentration of 1% (v/v) formalin. Visualization of biofilm sections was performed with the software IMARIS (Bitplane). Data are expressed as means of two independent experiments. Experiments were carried out in duplicates.Qualitative and quantitative microscopic evaluations of the biofilms were carried out through a combination of the LIVE/DEAD P<0.05.All assays were performed in triplicate, and the results are shown as means ± SDs. A one-way analysis of variance (ANOVA), followed by the Dunnett test, was used to detect differences in activity against biofilm formation (spectrophotometry OD readings) and biovolume/bioviability of young and mature biofilms between isolates exposed to EGCg or COL for 24 h. The Mann-Whitney U test was used to address the differences among bacterial counts in mice nebulized with EGCg, COL, PBS and aqua. Significance was set at in vitro against 60 CF Sm isolates. EGCg was active against all isolates; MIC values ranged from 64 to 512 mg/L, and MBC values ranged from 64 to 1024 mg/L  against acute pulmonary infection caused by Sm1 strain in wild type mice in comparison to COL (P = 0.0127) in the lungs compared to untreated (but infected controls) and COL-treated (P = 0.0106). Infected mice receiving COL did not show significantly lower CFU's in the lungs in comparison to controls (P = 0.4964). Interestingly, experiments with C. elegans, considered a versatile platform for drug discovery, have shown similar results when carried out under the same conditions  adhered to and formed biofilm on the polystyrene plates; only 10.0% were considered not to produce biofilm. The isolates were classified as weak (11.7%), moderate (15.0%), or strong (63.3%) biofilm producers. Strains ATCC 13637, Sm1 and Sm2 were considered strong biofilm producers.The activity of EGCg and COL was then examined at 0.25×MIC and 0.5×MIC to ascertain if these compounds have an inhibitory effect on biofilm development from the three tested strains in the growth medium TSB . In compin vitro dynamics of mature biofilms exposed to EGCg or COL  on Sm S. maltophilia50/90 = 256 mg/L) and the time-kill results obtained from CF S. maltophilia isolates were similar to those obtained by Gordon and Wareham First, we examined the antibacterial activity of EGCg. Previous studies have demonstrated that EGCg exerts antimicrobial activity against a variety of organisms, including in vivo, we performed intratracheal instillation infection in wild type and CF mice. Our results showed that EGCg significantly reduced the bacterial counts in the lungs  of the CF Sm isolates produced biofilm on polystyrene surfaces. These findings are in line with the results obtained by Di Bonaventura and co-workers, who found that the ability of Sm strains to form biofilm is influenced by environmental conditions  We verified that COL and EGCg at subinhibitory concentrations noticeably decreased biofilm formation and cell viability as well. The strongest effect was observed with COL and EGCg at 0.5×MIC. It has been shown that quinolones also exhibit anti-biofilm properties against Sm at sub-MIC concentrations in vitro. Interestingly, when biofilm susceptibility testing was performed and estimated by CLSM, stronger effects were observed after EGCg treatment both with regard to morphology and viability. COL only reduced the total biovolume of both clinical isolates which, however, was already observed by crystal violet staining in We examined for the first time whether bactericidal and/or subinhibitory concentrations of COL and EGCg were capable of damaging young and mature biofilms. COL and EGCg reduced 24-h- and 7-day-old biofilms biovolume at various concentrations Streptococcus mutans, Staphylococcus aureus, and Candida albicansStaphylococcus aureus from the upper respiratory tract Since no anti-biofilm therapies are clearly established, there is a great need to develop effective strategies for preventing or controlling biofilm-associated bacterial infections. Green tea is not only consumed as a beverage but is also considered a millenary method of traditional medicine in most of Asia; interest in its medicinal properties is increasing in the Western world 2O2) The precise mechanisms involved in the activity of EGCg against bacterial growth are still poorly defined. For instance, it has been suggested that EGCg's mechanism of action on Sm is associated with its antifolate activity, which will consequently lead to disruption of DNA synthesis in vivo were only evaluated simultaneously. Thus, further studies should aim to analyse whether EGCg alone could work as either a prophylactic or therapeutic treatment. Due to the scope for this research, we were unable to verify the antimicrobial effects of EGCg on in vivo biofilm. The assessment of chronic bacterial infections in vivo can differ from data obtained from in vitro experiments. In vivo experiments can provide a better insight to the microenvironmental circumstances associated with biofilm and the defence mechanisms exhibited by the host Although this work shows clearly that EGCg has beneficial effects, future research needs to solve the current limitations. Prophylactic and therapeutic effects of EGCg in vitro and in vivo effects of EGCg on CF Sm isolates. Our results revealed important insights into the antibacterial properties of EGCg and support its future use in the prevention of biofilm formation and possible treatment of Sm biofilms in CF patients. EGCg shows promise as a novel therapeutic compound against Sm colonization and infection in patients with CF.In summary, this study is the first to evaluate the Figure S1Percentage mortality of wild-type C. elegans exposed during 48 h to diverse concentrations of EGCg . Data express the mean values of two independent experiments performed in triplicated, SDs are shown.(TIF)Click here for additional data file.Figure S2EGCg enhances the survival of C. elegans infected with S. maltophilia clinical isolate (Sm1). Results are shown as mean values of three independent experiments performed in triplicated, SDs are shown.(TIF)Click here for additional data file.File S1Detailed description of material and methods.(DOC)Click here for additional data file.