Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients

Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.     

Mitochondrial Carbonic Anhydrase VA Deficiency Resulting from CA5A Alterations Presents with Hyperammonemia in Early Childhood

Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child.     

Quantitative analysis of processive RNA degradation by the archaeal RNA exosome

RNA exosomes are large multisubunit assemblies involved in controlled RNA processing. The archaeal exosome possesses a heterohexameric processing chamber with three RNase-PH-like active sites, capped by Rrp4- or Csl4-type subunits containing RNA-binding domains. RNA degradation by RNA exosomes has not been studied in a quantitative manner because of the complex kinetics involved, and exosome features contributing to efficient RNA degradation remain unclear. Here we derive a quantitative kinetic model for degradation of a model substrate by the archaeal exosome. Markov Chain Monte Carlo methods for parameter estimation allow for the comparison of reaction kinetics between different exosome variants and substrates. We show that long substrates are degraded in a processive and short RNA in a more distributive manner and that the cap proteins influence degradation speed. Our results, supported by small angle X-ray scattering, suggest that the Rrp4-type cap efficiently recruits RNA but prevents fast RNA degradation of longer RNAs by molecular friction, likely by RNA contacts to its unique KH-domain. We also show that formation of the RNase-PH like ring with entrapped RNA is not required for high catalytic efficiency, suggesting that the exosome chamber evolved for controlled processivity, rather than for catalytic chemistry in RNA decay.     

Immunohistochemical comparison of whisker pad cutaneous innervation in Swiss Webster and hairless mice

To establish the mouse mutant, hairless (Hr), as a useful model for future analyses of target-ending interactions, we assessed the cutaneous innervation in the whisker pad after loss of primary hair targets. Postnatal (P) development of fur in Hr begins similarly to that of "normal" Swiss Webster (SW) mice. Around P10, hairs are shed and the follicles rendered permanently incompetent. Hair loss progresses rostrocaudally until the entire skin is denuded. Substantial alterations in the distribution and density of sensory and autonomic endings in the mystacial pad vibrissal and intervibrissal fur innervation were discovered. Pilo-neural complexes innervating fur hairs were dismantled in Hr. Epidermal innervation in SW was rich; only a few endings expressed growth-associated protein-43?kdal (GAP), suggesting limited changes in axonal elongation. Innervation in Hr formed a dense layer passing upward through the thickened epidermis, with substantial increases among all types of endings. Vibrissal follicle-sinus complexes were also hyperinnervated. Endings in Hr vibrissae and fur were strongly GAP-positive, suggesting reorganization of innervation. Dermal and vascular autonomic innervation in both strains co-localized tyrosine hydroxylase and neuropeptide Y, but only in Hr did neuropeptide Y co-localize calcitonin gene-related peptide (CGRP) and express GAP immunolabeling. Stereological quantitation of trigeminal ganglia revealed no differences in neuron number between Hr and SW, although there were small increases in cell volume in Hr trigeminal ganglion cells. These results suggested that a form of collateral sprouting was active in Hr mystacial pads, not in response to local injury, but as a result of loss of primary target tissues.     

Synthesis of 7-benzyl-5-(piperidin-1-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c][2,7]naphthyridin-1-ylamine and its analogs as bombesin receptor subtype-3 agonists

The original structure of a high-throughput screening hit obtained from an external vendor was revised based on multiple NMR studies. The active compound was re-synthesized via a novel route and its structure and biological activity as a BRS-3 agonist were unambiguously confirmed. Multi-gram quantities of the hit were prepared for pharmacokinetic and efficacy studies. The synthetic strategy allowed for the preparation of multiple analogs for SAR exploration.     

Epsilon substituted lysinol derivatives as HIV-1 protease inhibitors

A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-l-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.     

Effects of Source Water Quality on Chlorine Inactivation of Adenovirus,Coxsackievirus, Echovirus,and Murine Norovirus

More information is needed on the disinfection efficacy of chlorine for viruses in source water. In this study, chlorine disinfection efficacy was investigated for USEPA Contaminant Candidate List viruses coxsackievirus B5 (CVB5), echovirus 1 (E1), murine norovirus (MNV), and human adenovirus 2 (HAdV2) in one untreated groundwater source and two partially treated surface waters. Disinfection experiments using pH 7 and 8 source water were carried out in duplicate, using 0.2 and 1 mg/liter free chlorine at 5 and 15°C. The efficiency factor Hom (EFH) model was used to calculate disinfectant concentration × contact time (CT) values (mg·min/liter) required to achieve 2-, 3-, and 4-log₁₀ reductions in viral titers. In all water types, chlorine disinfection was most effective for MNV, with 3-log₁₀ CT values at 5°C ranging from ≤0.020 to 0.034. Chlorine disinfection was least effective for CVB5 in all water types, with 3-log₁₀ CT values at 5°C ranging from 2.3 to 7.9. Overall, disinfection proceeded faster at 15°C and pH 7 for all water types. Inactivation of the study viruses was significantly different between water types, but no single source water had consistently different inactivation rates than another. CT values for CVB5 in one type of source water exceeded the recommended CT values set forth by USEPA''s Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems using Surface Water Sources. The results of this study demonstrate that water quality plays a substantial role in the inactivation of viruses and should be considered when developing chlorination plans.Disinfection processes are critical for the reduction of infectious virus concentrations in source water, because viruses are less efficiently removed by primary treatment of drinking water (e.g., coagulation and filtration) than are other pathogen types of concern (e.g., bacteria and protozoa). Over the years, many disinfection studies have focused on the inactivation of viruses in purified and buffered, demand-free, reagent-grade water (RGW). However, relatively few investigators have examined the impact of water quality during the disinfection process, even though water quality has been found to be a significant factor for inactivation of viruses.Several researchers found that the inactivation rate of poliovirus by free chlorine increased as the ionic concentration of water increased. In one study, poliovirus 1 was inactivated three times faster in boric acid buffer than in purified water (3). In addition, several investigators found that when the ionic content of buffered water was raised by the addition of NaCl or KCl, poliovirus 1 was inactivated two to four times faster than in the buffered water alone (2, 16, 17). In another study, poliovirus 1 was inactivated 10 times more rapidly in drinking water than in purified water (4).Studies conducted with natural waters have demonstrated both increased and decreased disinfection efficacy of chlorine in these waters compared to purified or buffered waters. In a study comparing chlorine disinfection in purified water and Potomac estuarine water, coxsackievirus A9 was inactivated more rapidly in the source water. The remaining study viruses (coxsackievirus B1, echovirus 7, adenovirus 3, poliovirus 1, and reovirus 3) were all inactivated more slowly in the source water (13). Bacteriophage MS2 was inactivated more slowly by free chlorine in two types of surface water than in buffered, demand-free water. However, there was no difference between the inactivation rates of this virus in the buffered water and groundwater (10). In another study, both feline calicivirus and adenovirus 40 were inactivated more slowly in treated groundwater than in buffered, demand-free water (21).The United States Environmental Protection Agency''s (USEPA) Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems using Surface Water Sources (Guidance Manual) recommends disinfectant concentration × contact time (CT) values of 4, 6, and 8 to achieve 2-, 3-, and 4-log₁₀ inactivation, respectively, with chlorine at 5°C and pH 6 to 9 (23). These CT values, which incorporate a safety factor of 3, were obtained from inactivation experiments conducted with monodispersed hepatitis A virus (HAV) in buffered, demand-free water. As water quality can significantly affect the disinfection efficacy of chlorine, it is unclear whether these CT value recommendations are sufficient for inactivation of viruses in source water. More information is needed to systematically examine the role of water quality in chlorine disinfection of viruses.The objective of the present study was to examine the disinfection efficacy of free chlorine on selected viruses from USEPA''s Contaminant Candidate List (CCL) (22) in one untreated and two partially treated source waters from distinct geographical regions. By comparing the efficacy of chlorine disinfection in the source water types to disinfection in buffered, chlorine-demand-free RGW (7), the impact of water quality could be examined. The four representative CCL viruses selected for this study included human adenovirus 2 (HAdV2), echovirus 1 (E1), coxsackievirus B5 (CVB5), and murine norovirus (MNV), a surrogate for human norovirus (22). The viruses were selected because they were previously found to be the least effectively inactivated viruses of their type in RGW (6). Disinfection experiments were carried out in duplicate in pH 7 and 8 source water at 5 and 15°C using 0.2 and 1 mg/liter free chlorine. Inactivation curves were plotted using Microsoft Excel, and CT values were calculated using the efficiency factor Hom (EFH) model (9).     

Mechanical force affects expression of an in vitro metastasis-like phenotype in HCT-8 cells

Cancer deaths are primarily caused by metastases, not by the parent tumor. During metastasis, malignant cells detach from the parent tumor, and spread through the circulatory system to invade new tissues and organs. The physical-chemical mechanisms and parameters within the cellular microenvironment that initiate the onset of metastasis, however, are not understood. Here we show that human colon carcinoma (HCT-8) cells can exhibit a dissociative, metastasis-like phenotype (MLP) in vitro when cultured on substrates with appropriate mechanical stiffness. This rather remarkable phenotype is observed when HCT-8 cells are cultured on gels with intermediate-stiffness (physiologically relevant 21-47 kPa), but not on very soft (1 kPa) and very stiff (3.6 GPa) substrates. The cell-cell adhesion molecule E-Cadherin, a metastasis hallmark, decreases 4.73 ± 1.43 times on cell membranes in concert with disassociation. Both specific and nonspecific cell adhesion decrease once the cells have disassociated. After reculturing the disassociated cells on fresh substrates, they retain the disassociated phenotype regardless of substrate stiffness. Inducing E-Cadherin overexpression in MLP cells only partially reverses the MLP phenotype in a minority population of the dissociated cells. This important experiment reveals that E-Cadherin does not play a significant role in the upstream regulation of the mechanosensing cascade. Our results indicate, during culture on the appropriate mechanical microenvironment, HCT-8 cells undergo a stable cell-state transition with increased in vitro metastasis-like characteristics as compared to parent cells grown on standard, very stiff tissue culture dishes. Nuclear staining reveals that a large nuclear deformation (major/minor axis ratio, 2:5) occurs in HCT-8 cells when cells are cultured on polystyrene substrates, but it is markedly reduced (ratio, 1:3) in cells grown on 21 kPa substrates, suggesting the cells are experiencing different intracellular forces when grown on stiff as compared to soft substrates. Furthermore, MLP can be inhibited by blebbistatin, which inactivates myosin II activity and relaxes intracellular forces. This novel finding suggests that the onset of metastasis may, in part, be linked to the intracellular forces and the mechanical microenvironment of the tumor.