hg19KIndel: ethnicity normalized human reference genome.

BACKGROUND: The most widely used human genome reference assembly hg19 harbors minor alleles at 2.18 million positions as revealed by 1000 Genome Phase 3 dataset. Although this is less than 2% of the 89 million variants reported, it has been shown that the minor alleles can result in 30% false positives in individual genomes, thus misleading and burdening downstream interpretation. More alarming is the fact that, significant percentage of variants that are homozygous recessive for these minor alleles, with potential disease implications, are masked from reporting. RESULTS: We have demonstrated that the false positives (FP) and false negatives (FN) can be corrected for by simply replacing nucleotides at the minor allele positions in hg19 with corresponding major allele. Here, we have effectively replaced 2.18 million minor alleles Single Nucleotide Polymorphism (SNPs), Insertion and Deletions (INDELs), Multiple Nucleotide Polymorphism (MNPs) in hg19 with the corresponding major alleles to create an ethnically normalized reference genome called hg19KIndel. In doing so, hg19KIndel has both corrected for sequencing errors acknowledged to be present in hg19 and has improved read alignment near the minor alleles in hg19. CONCLUSION: We have created and made available a new version human reference genome called hg19KIndel. It has been shown that variant calling using hg19KIndel, significantly reduces false positives calls, which in-turn reduces the burden from downstream analysis and validation. It also improved false negative variants call, which means that the variants which were getting missed due to the presence of minor alleles in hg19, will now be called using hg19KIndel. Using hg19KIndel, one even gets a better mapping percentage when compared to currently available human reference genome. hg19KIndel reference genome and its auxiliary datasets are available at https://doi.org/10.5281/zenodo.2638113.

Influence of punch geometry (head-flat diameter) and tooling type ('B' or 'D') on the physical-mechanical properties of formulation tablets.

The presented study assessed the influence of punch geometry (head-flat [HF] diameter) and tooling type ('B' or 'D') on the physical-mechanical properties of tablets prepared by direct-compression of two guaifenesin (25% or 40% w/w) formulations. Tablets of both formulations were prepared on instrumented, single-layer, rotary tablet press using 10 mm, flat-faced, 'B' or 'D'-type tooling with different HF diameters, and compression forces (CF) ranging from 5 to 25 kN with 5 kN increments. The tablets were evaluated for dimensions, weight variation, tensile strength (TS), friability, and capping index. In general, tablets prepared using 'D' tooling showed a significantly (p < 0.05) higher TS compared to those prepared using 'B' tooling, likely due to higher dwell-times associated with 'D' tooling. Formulations containing 25% w/w guaifenesin showed a significantly (p < 0.05) higher TS compared to those containing 40% w/w guaifenesin, at given compression CF, punch geometry, or tooling type. This could be due to the higher ratio of Prosolv® SMCC contributing to the compressibility. For both formulations compressed using 'B' tooling, differences in TS profiles were observed between different HF tooling. The TS of these tablets increased significantly with increasing HF diameter. For formulations compressed using 'D' tooling, this trend was observed only up to a CF of 15 kN, beyond which the TS plateaued, possibly due to work-hardening of the formulation at higher CF. These formulations also exhibited capping at CF above 15 kN and with higher HF diameters. The study showed a significant influence of punch geometry and tooling type on the physical properties of tablets.

Genetic variation in recalcitrant repetitive regions of the Drosophila melanogaster genome.

Many essential functions of organisms are encoded in highly repetitive genomic regions, including histones involved in DNA packaging, centromeres that are core components of chromosome segregation, ribosomal RNA comprising the protein translation machinery, telomeres that ensure chromosome integrity, piRNA clusters encoding host defenses against selfish elements, and virtually the entire Y chromosome. These regions, formed by highly similar tandem arrays, pose significant challenges for experimental and informatic study, impeding sequence-level descriptions essential for understanding genetic variation. Here, we report the assembly and variation analysis of such repetitive regions in Drosophila melanogaster, offering significant improvements to the existing community reference assembly. Our work successfully recovers previously elusive segments, including complete reconstructions of the histone locus and the pericentric heterochromatin of the X chromosome, spanning the Stellate locus to the distal flank of the rDNA cluster. To infer structural changes in these regions where alignments are often not practicable, we introduce landmark anchors based on unique variants that are putatively orthologous. These regions display considerable structural variation between different D. melanogaster strains, exhibiting differences in copy number and organization of homologous repeat units between haplotypes. In the histone cluster, although we observe minimal genetic exchange indicative of crossing over, the variation patterns suggest mechanisms such as unequal sister chromatid exchange. We also examine the prevalence and scale of concerted evolution in the histone and Stellate clusters and discuss the mechanisms underlying these observed patterns.

Assessment of Salivary Mutans Streptococci Counts to Atraumatic Restorative Treatment Among Children: A Randomised Controlled Trial.

Introduction Besides the survival rate of restorations, the effect of atraumatic restorative treatment (ART) on bacterial count is also important. After ART restoration, the bacterial count drops due to the fluoride's antibacterial activity and hence it can decrease the chances of further decay. The present study assessed salivary mutans streptococci counts for six months of evaluations subsequent to ART among 12-15-year-old children attending schools in Piparia village, Vadodara district. Methods ART was performed on 32 children and followed up after six months to evaluate the success rate of ART. Saliva collection and microbial analysis were performed five times from every participant, before ART restoration placement, seven days, 30 days (one month), 90 days (three months) and 180 days (six months) post-restoration. Results At baseline, the mean colony forming units (CFU) was 48.30 ± 46.58, which reduced to 32.64 ± 30.40 at one week, which showed a 32% reduction in colony counts. This further reduced to 18.60 ± 20.81 at one month, marking a further 43% reduction in colony counts. This again reduced to 13.63 ± 18.04 at three months, which showed a 27% reduction in colony counts. CFU came to 16.23 ± 23.34 at six months, which showed a 19% increase in colony counts. Comparison of baseline mean CFU (48.30 ± 46.58) to six months mean CFU of streptococcus mutans (SM) (16.23 ± 23.34) showed a 66% reduction in colony counts. A statistically significant difference was found between numerous intervals of time of CFU of streptococcus mutans. Conclusion The findings reveal that ART is a clinical treatment that can be used to treat caries in young children, and it helped significantly reduce Streptococcus Mutans levels in saliva.

Understanding the Impact of Multi-factorial Composition on Efficient Loading of the Stable Ketoprofen Nanoparticles on Orodispersible Films Using Box-Behnken Design.

The purpose of the present study was to prepare Orodispersible films (ODFs) loaded with ketoprofen nanoparticles (KT-NP). The Box-Behnken design was constructed in developing and optimizing the KTF-NP-ODFs. The effect of independent variables: Soluplus® concentration (X1, stabilizer), Tween 80 concentration (X2, surfactant), and KTF concentration (X3, drug) were studied on the dependent variables: particle size (PS, Y1), zeta potential (ZP, Y2), and the polydispersity index (PDI, Y3) of the NPs, as well as on the tensile strength (TS, Y4) and permeability coefficient (PC, Y5) of the KTF-NP-ODFs. Hydroxypropyl methylcellulose (HPMC E15) and polyethylene glycol (PEG 400) were used as the film former polymer and plasticizer, respectively, and their concentrations were kept constant for all formulations. KTF-NPs were prepared by antisolvent precipitation technology. This was followed by the addition of HPMC E15 and PEG 400 to prepare the ODFs using the solvent-casting method. The PS, PDI, and ZP for all the formulations were found in the range of 94 nm to 350 nm, 0.09 to 0.438, and -21.83 mV to -8.03 mV, respectively. The TS and PC of the prepared KTF-NP-ODFs were found between 1.21 MPa to 3.93 MPa and 3.12 × 10-4 cm/h to 34.23 × 10-4 cm/h, respectively. The amorphous nature of the KTF-NP in the ODFs was confirmed by the absence of characteristic crystalline peaks and endothermic events of KTF in X-ray diffraction (XRD) and modulated differential scanning calorimetry (mDSC), respectively. The optimized formulation showed Ì´ 4 times higher permeability as compared to the pure KTF. In addition, the dissolution of pure KTF and the optimized KTF-NP-ODF in pH 1.2 at the end of 60 min was found to be Ì´ 30% and Ì´ 95%, respectively. Conclusively, KTF-NP-ODFs can be a promising drug delivery system to counter the issues related to dysphagia and bypass the common side effects, such as the gastric irritation associated with NSAIDs like KTF.

Oncolytic virus therapy of multiple tumors in the brain requires suppression of innate and elicited antiviral responses.

The occurrence of multiple tumors in an organ heralds a rapidly fatal course. Although intravascular administration may deliver oncolytic viruses/vectors to each of these tumors, its efficiency is impeded by an antiviral activity present in complement-depleted plasma of rodents and humans. Here, this activity was shown to interact with complement in a calcium-dependent fashion, and antibody neutralization studies indicated preimmune IgM has a contributing role. Short-term exposure to cyclophosphamide (CPA) partially suppressed this activity in rodents and humans. At longer time points, cyclophosphamide also abrogated neutralizing antibody responses. Cyclophosphamide treatment of rats with large single or multiple intracerebral tumors substantially increased viral survival and propagation, leading to neoplastic regression.

Tumor relevant protein functional interactions identified using bipartite graph analyses.

An increased surge of -omics data for the diseases such as cancer allows for deriving insights into the affiliated protein interactions. We used bipartite network principles to build protein functional associations of the differentially regulated genes in 18 cancer types. This approach allowed us to combine expression data to functional associations in many cancers simultaneously. Further, graph centrality measures suggested the importance of upregulated genes such as BIRC5, UBE2C, BUB1B, KIF20A and PTH1R in cancer. Pathway analysis of the high centrality network nodes suggested the importance of the upregulation of cell cycle and replication associated proteins in cancer. Some of the downregulated high centrality proteins include actins, myosins and ATPase subunits. Among the transcription factors, mini-chromosome maintenance proteins (MCMs) and E2F family proteins appeared prominently in regulating many differentially regulated genes. The projected unipartite networks of the up and downregulated genes were comprised of 37,411 and 41,756 interactions, respectively. The conclusions obtained by collating these interactions revealed pan-cancer as well as subtype specific protein complexes and clusters. Therefore, we demonstrate that incorporating expression data from multiple cancers into bipartite graphs validates existing cancer associated mechanisms as well as directs to novel interactions and pathways.

Retrosellar intracranial extracerebral glioneuronal heterotopion: case report.

We report the case of an 18-year-old woman with an intradural retroclival retrosellar glioneuronal heterotopion. At the time of surgery, a well circumscribed pale-tan mass was identified posterior to and distinct from the posterior pituitary. Pathologic examination showed disorganized, non-neoplastic glial tissue characteristic of glioneuronal heterotopia. To our knowledge, this is the first report of such a lesion in this location.

Two G protein oncogenes in human endocrine tumors.

Somatic mutations in a subset of growth hormone (GH)-secreting pituitary tumors convert the gene for the alpha polypeptide chain (alpha s) of Gs into a putative oncogene, termed gsp. These mutations, which activate alpha s by inhibiting its guanosine triphosphatase (GTPase) activity, are found in codons for either of two amino acids, each of which is completely conserved in all known G protein alpha chains. The likelihood that similar mutations would activate other G proteins prompted a survey of human tumors for mutations that replace either of these two amino acids in other G protein alpha chain genes. The first gene so far tested, which encodes the alpha chain of Gi2, showed mutations that replaced arginine-179 with either cysteine or histidine in 3 of 11 tumors of the adrenal cortex and 3 of 10 endocrine tumors of the ovary. The mutant alpha i2 gene is a putative oncogene, referred to as gip2. In addition, gsp mutations were found in 18 of 42 GH-secreting pituitary tumors and in an autonomously functioning thyroid adenoma. These findings suggest that human tumors may harbor oncogenic mutations in various G protein alpha chain genes.

KLF6 depletion promotes NF-κB signaling in glioblastoma.

Dysregulation of the NF-κB transcription factor occurs in many cancer types. Krüppel-like family of transcription factors (KLFs) regulate the expression of genes involved in cell proliferation, differentiation and survival. Here, we report a new mechanism of NF-κB activation in glioblastoma through depletion of the KLF6 tumor suppressor. We show that KLF6 transactivates multiple genes negatively controlling the NF-κB pathway and consequently reduces NF-κB nuclear localization and downregulates NF-κB targets. Reconstitution of KLF6 attenuates their malignant phenotype and induces neural-like differentiation and senescence, consistent with NF-κB pathway inhibition. KLF6 is heterozygously deleted in 74.5% of the analyzed glioblastomas and predicts unfavorable patient prognosis suggesting that haploinsufficiency is a clinically relevant means of evading KLF6-dependent regulation of NF-κB. Together, our study identifies a new mechanism by which KLF6 regulates NF-κB signaling, and how this mechanism is circumvented in glioblastoma through KLF6 loss.

Human gliomas with wild-type p53 express bcl-2.

Human astrocytomas frequently overexpress wild-type p53, which suggests that gliomas have evolved a mechanism to subvert p53-mediated apoptosis. bcl-2 inhibits apoptosis mediated by p53, and it is expressed in several human cancers. We therefore examined a series of human gliomas to determine whether bcl-2 is expressed and whether this expression is associated with tumors which have wild-type p53. Twenty-eight paraffin-embedded gliomas (3 WHO grade II, 13 grade III, 12 grade IV) were immunohistochemically stained for bcl-2 and p53. p53 mutations were identified with single strand conformation polymorphism and DNA sequencing. Sixteen of 28 (57%) tumors expressed bcl-2, and bcl-2 expression was associated with wild-type p53 (P < 0.01). Among gliomas which overexpressed p53, bcl-2 was positive in 7 of 7 tumors with wild-type p53 but in only 1 of 7 with mutant p53 (P < 0.01). We conclude that bcl-2 is frequently expressed in human gliomas and that expression is more common in tumors with wild-type p53.

Computational Identification of Tumor Anatomic Location Associated with Survival in 2 Large Cohorts of Human Primary Glioblastomas.

BACKGROUND AND PURPOSE: Tumor location has been shown to be a significant prognostic factor in patients with glioblastoma. The purpose of this study was to characterize glioblastoma lesions by identifying MR imaging voxel-based tumor location features that are associated with tumor molecular profiles, patient characteristics, and clinical outcomes. MATERIALS AND METHODS: Preoperative T1 anatomic MR images of 384 patients with glioblastomas were obtained from 2 independent cohorts (n = 253 from the Stanford University Medical Center for training and n = 131 from The Cancer Genome Atlas for validation). An automated computational image-analysis pipeline was developed to determine the anatomic locations of tumor in each patient. Voxel-based differences in tumor location between good (overall survival of >17 months) and poor (overall survival of <11 months) survival groups identified in the training cohort were used to classify patients in The Cancer Genome Atlas cohort into 2 brain-location groups, for which clinical features, messenger RNA expression, and copy number changes were compared to elucidate the biologic basis of tumors located in different brain regions. RESULTS: Tumors in the right occipitotemporal periventricular white matter were significantly associated with poor survival in both training and test cohorts (both, log-rank P < .05) and had larger tumor volume compared with tumors in other locations. Tumors in the right periatrial location were associated with hypoxia pathway enrichment and PDGFRA amplification, making them potential targets for subgroup-specific therapies. CONCLUSIONS: Voxel-based location in glioblastoma is associated with patient outcome and may have a potential role for guiding personalized treatment.

Regression of experimental brain tumors with 6-thioxanthine and Escherichia coli gpt gene therapy.

The identification of transgenes with antitumor activity is critical to the development of gene therapy of cancer. Retrovirus-mediated transfer of the Escherichia coli gpt gene into rat C6 glioma cells without subsequent selection still inhibited the proliferation of this mixed polyclonal population upon addition of the prodrug, 6-thioxanthine, with an ID50 of 4.1 microM, whereas parental C6 cells were not affected at a concentration of 500 microM. In a time-course assay, effects of the prodrug on the mixed polyclonal cell proliferation required at least 10 days of exposure. In mixed co-cultures, a bystander effect was not present over the first 4 days of prodrug exposure, but required trypsinization of the co-cultures and replating at lower densities. This "modified" bystander assay thus revealed a 50% decrease in C6 cell proliferation, even when the initial ratio of gpt-expressing to parental C6 cells was as low as 1:19. In a nude mouse model of subcutaneous tumors, co-grafts of C6 glioma and gpt-retrovirus producer cells displayed retarded growth upon exposure to 6-thioxanthine (6-TX). In a nude mouse model of intracerebral tumors, grafting of the gpt-retrovirus producer cells leads to an 80% reduction in intracerebral tumor volumes after 6-TX treatment. This reduction results in a 28% increase in the mean time of survival of animals that harbor intracerebral tumors (p < 0.0005). These antitumor effects indicate that the gpt/6-TX enzyme/prodrug pair is a promising alternative to the thymidine kinase gene and ganciclovir combination in the gene therapy of cancer.

Chromosome 19q deletions in human gliomas overlap telomeric to D19S219 and may target a 425 kb region centromeric to D19S112.

Chromosome 19q harbors a tumor suppressor gene that is involved in astrocytoma, oligodendroglioma and mixed glioma tumorigenesis. We had previously mapped this gene to an approximately 5 megabase region of chromosome 19q13.2-13.3 between APOC2 and HRC. To narrow the location of this tumor suppressor further, we studied 138 gliomas for loss of allelic heterozygosity at six microsatellite polymorphisms between APOC2 and HRC, including a newly described polymorphism in the ERCC2 gene. Allelic loss occurred in 48 gliomas (35%), including 25 of 41 oligodendroglial tumors (61%). Four cases had proximal breakpoints within the APOC2-HRC region, two telomeric to ERCC2 and two telomeric to D19S219. In addition, one of the latter tumors had an interstitial deletion between D19S219 and D19S112, a distance of only 425 kilobases surrounding the DM (myotonic dystrophy) gene. These findings suggest that the glioma tumor suppressor on chromosome 19q maps to 19q13.3, telomeric to D19S219 and perhaps centromeric to D19S112. The data exclude a number of candidate genes from 19q13.2-13.3, including a putative phosphatase gene and the DNA repair/metabolism genes ERCC1, ERCC2 and probably LIG1.

Clinical characteristics of acromegalic patients whose pituitary tumors contain mutant Gs protein.

Activating mutations in the gene for the alpha-chain of Gs, the stimulatory regulator of adenylyl cyclase, have been identified in human GH-secreting pituitary tumors. Using the polymerase chain reaction and allele-specific oligonucleotide hybridization, we screened 25 GH-secreting tumors for the presence of the activating mutations. We also reviewed the clinical charts of the patients from whom the tumors were removed. Of 25 tumors, 10 (40%) contained activating mutations. Patients in the mutation-positive group came to surgery with smaller tumors and had lower GH levels. The activating mutations identify a subgroup of GH-secreting pituitary tumors that probably arise from a shared oncogenic mechanism.

Retrovirus-mediated gene therapy of experimental brain neoplasms using the herpes simplex virus-thymidine kinase/ganciclovir paradigm.

Recent results in experimental brain tumors indicate that transfer of sensitizing genes to tumor cells in vivo with subsequent drug treatment can reduce tumor masses and prolong the survival of rodents. In the present study, the 9L rat gliosarcoma model was used to evaluate the therapeutic effectiveness of the herpes simplex virus-thymidine kinase (HSV-tk) gene, delivered by a retrovirus vector, against tumor cells in the rat brain after systemic application of the nucleoside analogue ganciclovir (GCV). The HSV-tk gene was inserted into a retroviral vector (pMFG), which was produced using the amphotropic packaging cell line CRIP-MFG-S-HSV-TK. Packaging cells were implanted into established 9L tumors in the brains of syngeneic rats to effect gene delivery to tumor cells, followed by intraperitoneal GCV injections. Treated animals survived significantly longer (more than twice as long) than did the control groups. Brains from GCV-treated and nontreated animals were examined immunohistochemically at different time intervals after grafting of CRIP-MFG-S-HSV-TK cells and GCV treatment. Tumors in GCV-treated animals were significantly smaller as compared with nontreated animals at all time points. Sections stained immunohistochemically for HSV-TK confirmed gene transfer to tumor cells, which could be distinguished from packaging cells by different morphology and immunohistochemical staining for the retroviral envelope protein gp70. Approximately 45% of the cells in tumors implanted with CRIP-MFG-S-HSV-TK cells, but not treated with GCV, showed immunocytochemical staining for HSV-TK, demonstrating a high-efficiency of retrovirus-mediated gene transfer. Tumors in rats treated with packaging cells and GCV showed only 9% HSV-TK-positive cells after treatment, indicating that most cells expressing the HSV-tk gene were killed. The success of this therapeutic modality in experimental animals depends in large parts on the high efficiency of gene delivery and on the immune response against tumor cells.

Selective delivery of herpes virus vectors to experimental brain tumors using RMP-7.

RMP-7, a bradykinin analog, has been shown to selectively open the blood-tumor barrier for the delivery of chemotherapeutic drugs to brain tumors. In contrast to bradykinin, RMP-7 has no hypotensive effects and has been approved for human use. This study was initiated to determine whether RMP-7 would open the blood-tumor barrier to virus vectors encoding tumor-killing genes in an experimental model. The herpes virus vector used, hrR3, which encodes virus thymidine kinase gene and the lacZ reporter gene, is defective in a gene encoding ribonucleotide reductase, replicates selectively in dividing tumor cells and not in postmitotic neural cells. It was determined that an optimum dose of RMP-7 (1.5-3.0 microg/kg over 10-15 minutes) enhanced viral delivery to brain tumors in rats bearing intracranial 9 L gliosarcomas when infused through the carotid artery immediately prior to virus vector application. Maximum expression of the lacZ reporter gene occurred at 3 days after intracarotid infusion. By 8 days, transgene expression was largely confined to tumor foci away from the main tumor mass. Viral delivery was essentially specific to tumor cells, with little transgene expression elsewhere in the brain. Minimal uptake and pathology was noted in the kidney, spleen, and liver. These findings indicate that intracarotid delivery of RMP-7 can augment the selective delivery of virus vectors to brain tumors in an experimental rat model, with the potential for application to human brain tumors.

Therapy of primary CNS lymphoma with methotrexate-based chemotherapy and deferred radiotherapy: preliminary results.

Disease-free survival in primary CNS lymphoma has improved with the advent of methotrexate-based pre-irradiation chemotherapy. Prolonged response durations have been noted in six of eight patients refusing radiation therapy in two of our prior series. We have treated an additional 11 patients with methotrexate-based chemotherapy without subsequent planned irradiation. Some received maintenance chemotherapy. Most have had durable responses with little or no toxicity. Prolonged responses can be maintained without radiation therapy, thus avoiding potential long-term radiation toxicity.

Von Hippel-Lindau disease and erythrocytosis: radioimmunoassay of erythropoietin in cyst fluid from a brainstem hemangioblastoma.

A 59-year-old woman with von Hippel-Lindau disease developed erythrocytosis and a recurrent intracranial hemangioblastoma. Radioimmunoassay showed an elevated level of erythropoietin in her serum. Cyst fluid from the tumor also contained erythropoietin, concentrated a thousandfold relative to the serum level. Production of erythropoietin by hemangioblastomas may explain the erythrocytosis present in some patients with von Hippel-Lindau disease.

Aneurysmal compression of the anterior visual pathways.

Ten patients with aneurysmal compression of the anterior visual pathways had visual loss, unilateral in 4 and bilateral in 6. There was no typical clinical presentation. Visual loss was acute or gradual, acuity sometimes fluctuated, and visual field testing was highly variable. The aneurysms were supraclinoid (four patients), carotid-ophthalmic (two), anterior communicating-anterior cerebral (three), and intracavernous carotid (one). Nine patients had successful clipping of their aneurysm, and in one, ipsilateral common carotid ligation was performed. Postoperatively, visual acuity was improved in six cases, unchanged in three, and worse in one.