N-acetyl-L-leucine for Niemann-Pick type C: a multinational double-blind randomized placebo-controlled crossover study.

BACKGROUND: Niemann-Pick disease type C (NPC) is a rare autosomal recessive neurodegenerative lysosomal disease characterized by multiple symptoms such as progressive cerebellar ataxia and cognitive decline. The modified amino acid N-acetyl-leucine has been associated with positive symptomatic and neuroprotective, disease-modifying effects in various studies, including animal models of NPC, observational clinical case studies, and a multinational, rater-blinded phase IIb clinical trial. Here, we describe the development of a study protocol (Sponsor Code "IB1001-301") for the chronic treatment of symptoms in adult and pediatric patients with NPC. METHODS: This multinational double-blind randomized placebo-controlled crossover phase III study will enroll patients with a genetically confirmed diagnosis of NPC patients aged 4 years and older across 16 trial sites. Patients are assessed during a baseline period and then randomized (1:1) to one of two treatment sequences: IB1001 followed by placebo or vice versa. Each sequence consists of a 12-week treatment period. The primary efficacy endpoint is based on the Scale for the Assessment and Rating of Ataxia, and secondary outcomes include cerebellar functional rating scales, clinical global impression, and quality of life assessments. DISCUSSION: Pre-clinical as well as observational and phase IIb clinical trials have previously demonstrated that IB1001 rapidly improved symptoms, functioning, and quality of life for pediatric and adult NPC patients and is safe and well tolerated. In this placebo-controlled cross-over trial, the risk/benefit profile of IB1001 for NPC will be evaluated. It will also give information about the applicability of IB1001 as a therapeutic paradigm for other rare and common neurological disorders. TRIAL REGISTRATIONS: The trial (IB1001-301) has been registered at www. CLINICALTRIALS: gov (NCT05163288) and www.clinicaltrialsregister.eu (EudraCT: 2021-005356-10). Registered on 20 December 2021.

Comparison of carcinogenic potency across life stages: implications for the assessment of transplacental cancer risk.

Cancer development from in utero exposure has been documented for a variety of agents with the most commonly studied compounds exhibiting mutagenic and genotoxic carcinogen properties. Age-dependent adjustment factors (ADAFs) are applied by the US Environmental Protection Agency to many such carcinogens when assessing cancer risk from early postnatal (PN) exposures; however, this approach has not been widely considered for transplacental (TP) exposure. To explore this question and further evaluate prenatal susceptibility a database of early life animal studies developed by California Office of Environmental Health Hazard Assessment (OEHHA) enhanced with additional literature searching was evaluated. Nine genotoxic carcinogens and one mixture (cigarette smoke) have data available via TP only, PN only and adult-only protocols. Potency comparisons across these lifestages displayed similarly greater potency in male liver and brain compared to adult-only exposure. Both TP and PN exposures were not markedly different than adult-only for other targets such as female liver and blood-borne tumors. Similarity in TP and PN targets and potency for carcinogen action suggests that a 10-fold ADAF may be applied for TP exposure as is currently applied to PN exposure. A similar conclusion was reached by OEHHA. The implications of this heightened TP vulnerability are greatest for less-than-lifetime exposure and this approach might be used to assess the level of cancer risk from exposures during pregnancy. A case example employing a mutagenic flame retardant was used to exemplify application of a TP ADAF for evaluating risks during pregnancy.

Whole genome comparative analysis of four Georgian grape cultivars.

Grapevine is the one of the most important fruit species in the world. Comparative genome sequencing of grape cultivars is very important for the interpretation of the grape genome and understanding its evolution. The genomes of four Georgian grape cultivars-Chkhaveri, Saperavi, Meskhetian green, and Rkatsiteli, belonging to different haplogroups, were resequenced. The shotgun genomic libraries of grape cultivars were sequenced on an Illumina HiSeq. Pinot Noir nuclear, mitochondrial, and chloroplast DNA were used as reference. Mitochondrial DNA of Chkhaveri closely matches that of the reference Pinot noir mitochondrial DNA, with the exception of 16 SNPs found in the Chkhaveri mitochondrial DNA. The number of SNPs in mitochondrial DNA from Saperavi, Meskhetian green, and Rkatsiteli was 764, 702, and 822, respectively. Nuclear DNA differs from the reference by 1,800,675 nt in Chkhaveri, 1,063,063 nt in Meskhetian green, 2,174,995 in Saperavi, and 5,011,513 in Rkatsiteli. Unlike mtDNA Pinot noir, chromosomal DNA is closer to the Meskhetian green than to other cultivars. Substantial differences in the number of SNPs in mitochondrial and nuclear DNA of Chkhaveri and Pinot noir cultivars are explained by backcrossing or introgression of their wild predecessors before or during the process of domestication. Annotation of chromosomal DNA of Georgian grape cultivars by MEGANTE, a web-based annotation system, shows 66,745 predicted genes (Chkhaveri-17,409; Saperavi-17,021; Meskhetian green-18,355; and Rkatsiteli-13,960). Among them, 106 predicted genes and 43 pseudogenes of terpene synthase genes were found in chromosomes 12, 18 random (18R), and 19. Four novel TPS genes not present in reference Pinot noir DNA were detected. Two of them-germacrene A synthase (Chromosome 18R) and (-) germacrene D synthase (Chromosome 19) can be identified as putatively full-length proteins. This work performs the first attempt of the comparative whole genome analysis of different haplogroups of Vitis vinifera cultivars. Based on complete nuclear and mitochondrial DNA sequence analysis, hypothetical phylogeny scheme of formation of grape cultivars is presented.

Is diacetyl a respiratory sensitizer? A reconsideration using QSAR, QMM, and competition experiments.

Concerns have been raised that diacetyl (DA) might be a respiratory sensitizer based on its LUMO energy similar to that of the respiratory allergen toluene-2,4-diisocyanate (TDI) and results of a local lymph node assay (LLNA) that reported an EC3 of 1.9%. To better understand the concerns, we performed a systematic literature review and experimental competition reactions between DA and TDI. The experimental evidence demonstrates that DA is at least 400-fold less reactive than TDI. The literature review finds evidence that the EC3 for DA is actually >11%. We conclude that DA is unlikely to have significant respiratory sensitization potential.

A novel DNMT3B splice variant expressed in tumor and pluripotent cells modulates genomic DNA methylation patterns and displays altered DNA binding.

DNA methylation is an epigenetic mark essential for mammalian development, genomic stability, and imprinting. DNA methylation patterns are established and maintained by three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Interestingly, all three DNMTs make use of alternative splicing. DNMT3B has nearly 40 known splice variants expressed in a tissue- and disease-specific manner, but very little is known about the role of these splice variants in modulating DNMT3B function. We describe here the identification and characterization of a novel alternatively spliced form of DNMT3B lacking exon 5 within the NH(2)-terminal regulatory domain. This variant, which we term DNMT3B3Delta5 because it is closely related in structure to the ubiquitously expressed DNMT3B3 isoform, is highly expressed in pluripotent cells and brain tissue, is downregulated during differentiation, and is conserved in the mouse. Creation of pluripotent iPS cells from fibroblasts results in marked induction of DNMT3B3Delta5. DNMT3B3Delta5 expression is also altered in human disease, with tumor cell lines displaying elevated or reduced expression depending on their tissue of origin. We then compared the DNA binding and subcellular localization of DNMT3B3Delta5 versus DNMT3B3, revealing that DNMT3B3Delta5 possessed significantly enhanced DNA binding affinity and displayed an altered nuclear distribution. Finally, ectopic overexpression of DNMT3B3Delta5 resulted in repetitive element hypomethylation and enhanced cell growth in a colony formation assay. Taken together, these results show that DNMT3B3Delta5 may play an important role in stem cell maintenance or differentiation and suggest that sequences encoded by exon 5 influence the functional properties of DNMT3B.

DNMT1 and DNMT3B modulate distinct polycomb-mediated histone modifications in colon cancer.

DNA methylation patterns are established and maintained by three DNA methyltransferases (DNMT): DNMT1, DNMT3A, and DNMT3B. Although essential for development, methylation patterns are frequently disrupted in cancer and contribute directly to carcinogenesis. Recent studies linking polycomb group repression complexes (PRC1 and PRC2) to the DNMTs have begun to shed light on how methylation is targeted. We identified previously a panel of genes regulated by DNMT3B. Here, we compare these with known polycomb group targets to show that approximately 47% of DNMT3B regulated genes are also bound by PRC1 or PRC2. We chose 44 genes coregulated by DNMT3B and PRC1/PRC2 to test whether these criteria would accurately identify novel targets of epigenetic silencing in colon cancer. Using reverse transcription-PCR, bisulfite genomic sequencing, and pyrosequencing, we show that the majority of these genes are frequently silenced in colorectal cancer cell lines and primary tumors. Some of these, including HAND1, HMX2, and SIX3, repressed cell growth. Finally, we analyzed the histone code, DNMT1, DNMT3B, and PRC2 binding by chromatin immunoprecipitation at epigenetically silenced genes to reveal a novel link between DNMT3B and the mark mediated by PRC1. Taken together, these studies suggest that patterns of epigenetic modifiers and the histone code influence the propensity of a gene to become hypermethylated in cancer and that DNMT3B plays an important role in regulating PRC1 function.

Invasion suppressor cystatin E/M (CST6): high-level cell type-specific expression in normal brain and epigenetic silencing in gliomas.

DNA hypermethylation-mediated gene silencing is a frequent and early contributor to aberrant cell growth and invasion in cancer. Malignant gliomas are the most common primary brain tumors in adults and the second most common tumor in children. Morbidity and mortality are high in glioma patients because tumors are resistant to treatment and are highly invasive into surrounding brain tissue rendering complete surgical resection impossible. Invasiveness is regulated by the interplay between secreted proteases (eg, cathepsins) and their endogenous inhibitors (cystatins). In our previous studies we identified cystatin E/M (CST6) as a frequent target of epigenetic silencing in glioma. Cystatin E/M is a potent inhibitor of cathepsin B, which is frequently overexpressed in glioma. Here, we study the expression of cystatin E/M in normal brain and show that it is highly and moderately expressed in oligodendrocytes and astrocytes, respectively, but not in neurons. Consistent with this, the CST6 promoter is hypomethylated in all normal samples using methylation-specific PCR, bisulfite genomic sequencing, and pyrosequencing. In contrast, 78% of 28 primary brain tumors demonstrated reduced/absent cystatin E/M expression using a tissue microarray and this reduced expression correlated with CST6 promoter hypermethylation. Interestingly, CST6 was expressed in neural stem cells (NSC) and markedly induced upon differentiation, whereas a glioma tumor initiating cell (TIC) line was completely blocked for CST6 expression by promoter methylation. Analysis of primary pediatric brain tumor-derived lines also showed CST6 downregulation and methylation in nearly 100% of 12 cases. Finally, ectopic expression of cystatin E/M in glioma lines reduced cell motility and invasion. These results demonstrate that epigenetic silencing of CST6 is frequent in adult and pediatric brain tumors and occurs in TICs, which are thought to give rise to the tumor. CST6 methylation may therefore represent a novel prognostic marker and therapeutic target specifically altered in TICs.

DNA methyltransferase 3B (DNMT3B) mutations in ICF syndrome lead to altered epigenetic modifications and aberrant expression of genes regulating development, neurogenesis and immune function.

Genome-wide DNA methylation patterns are established and maintained by the coordinated action of three DNA methyltransferases (DNMTs), DNMT1, DNMT3A and DNMT3B. DNMT3B hypomorphic germline mutations are responsible for two-thirds of immunodeficiency, centromere instability, facial anomalies (ICF) syndrome cases, a rare recessive disease characterized by immune defects, instability of pericentromeric satellite 2-containing heterochromatin, facial abnormalities and mental retardation. The molecular defects in transcription, DNA methylation and chromatin structure in ICF cells remain relatively uncharacterized. In the present study, we used global expression profiling to elucidate the role of DNMT3B in these processes using cell lines derived from ICF syndrome and normal individuals. We show that there are significant changes in the expression of genes critical for immune function, development and neurogenesis that are highly relevant to the ICF phenotype. Approximately half the upregulated genes we analyzed were marked with low-level DNA methylation in normal cells that was lost in ICF cells, concomitant with loss of repressive histone modifications, particularly H3K27 trimethylation, and gains in transcriptionally active H3K9 acetylation and H3K4 trimethylation marks. In addition, we consistently observed loss of binding of the SUZ12 component of the PRC2 polycomb repression complex and DNMT3B to derepressed genes, including a number of homeobox genes critical for immune system, brain and craniofacial development. We also observed altered global levels of certain histone modifications in ICF cells, particularly ubiquitinated H2AK119. Therefore, this study provides important new insights into the role of DNMT3B in modulating gene expression and chromatin structure and reveals new connections between DNMT3B and polycomb-mediated repression.

Pharmacologic inhibition of epigenetic modifications, coupled with gene expression profiling, reveals novel targets of aberrant DNA methylation and histone deacetylation in lung cancer.

Lung cancer is the leading cause of cancer-related deaths in the United States due, in large part, to the lack of early detection methods. Lung cancer arises from a complex series of genetic and epigenetic changes leading to uncontrolled cell growth and metastasis. Unlike genetic changes, epigenetic changes, such as DNA methylation and histone acetylation, are reversible with currently available pharmaceuticals and are early events in lung tumorigenesis detectable by non-invasive methods. In order to better understand how epigenetic changes contribute to lung cancer, and to identify new disease biomarkers, we combined pharmacologic inhibition of DNA methylation and histone deacetylation in non-small cell lung cancer (NSCLC) cell lines, with genome-wide expression profiling. Of the more than 200 genes upregulated by these treatments, three of these, neuronatin, metallothionein 3 and cystatin E/M, were frequently hypermethylated and transcriptionally downregulated in NSCLC cell lines and tumors. Interestingly, four other genes, cylindromatosis, CD9, activating transcription factor 3 and oxytocin receptor, were dominantly regulated by histone deacetylation and were also frequently downregulated in lung tumors. The majority of these genes also suppressed NSCLC growth in culture when ectopically expressed. This study therefore reveals new putative NSCLC growth regulatory genes and epigenetic disease biomarkers that may enhance early detection strategies and serve as therapeutic targets.

Epigenomic profiling reveals novel and frequent targets of aberrant DNA methylation-mediated silencing in malignant glioma.

Malignant glioma is the most common central nervous system tumor of adults and is associated with a significant degree of morbidity and mortality. Gliomas are highly invasive and respond poorly to conventional treatments. Gliomas, like other tumor types, arise from a complex and poorly understood sequence of genetic and epigenetic alterations. Epigenetic alterations leading to gene silencing, in the form of aberrant CpG island promoter hypermethylation and histone deacetylation, have not been thoroughly investigated in brain tumors, and elucidating such changes is likely to enhance our understanding of their etiology and provide new treatment options. We used a combined approach of pharmacologic inhibition of DNA methylation and histone deacetylation, coupled with expression microarrays, to identify novel targets of epigenetic silencing in glioma cell lines. From this analysis, we identified >160 genes up-regulated by 5-aza-2'-deoxycytidine and trichostatin A treatment. Further characterization of 10 of these genes, including the putative metastasis suppressor CST6, the apoptosis-inducer BIK, and TSPYL5, whose function is unknown, revealed that they are frequent targets of epigenetic silencing in glioma cell lines and primary tumors and suppress glioma cell growth in culture. Furthermore, we show that other members of the TSPYL gene family are epigenetically silenced in gliomas and dissect the contribution of individual DNA methyltransferases to the aberrant promoter hypermethylation events. These studies, therefore, lay the foundation for a comprehensive understanding of the full extent of epigenetic changes in gliomas and how they may be exploited for therapeutic purposes.

Epigenetic silencing of the tumor suppressor cystatin M occurs during breast cancer progression.

Cystatin M is a secreted inhibitor of lysosomal cysteine proteases. Several lines of evidence indicate that cystatin M is a tumor suppressor important in breast malignancy; however, the mechanism(s) that leads to inactivation of cystatin M during cancer progression is unknown. Inspection of the human cystatin M locus uncovered a large and dense CpG island within the 5' region of this gene (termed CST6). Analysis of cultured human breast tumor lines indicated that cystatin M expression is either undetectable or in low abundance in several lines; however, enhanced gene expression was measured in cells cultured on the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC). Increased cystatin M expression does not correlate with a cytotoxic response to 5-aza-dC; rather, various molecular approaches indicated that the CST6 gene was aberrantly methylated in these tumor lines as well as in primary breast tumors. Moreover, 60% (12 of 20) of primary tumors analyzed displayed CST6 hypermethylation, indicating that this aberrant characteristic is common in breast malignancies. Finally, preinvasive and invasive breast tumor cells were microdissected from nine archival breast cancer specimens. Of the five tumors displaying CST6 gene methylation, four tumors displayed methylation in both ductal carcinoma in situ and invasive breast carcinoma lesions and reduced expression of cystatin M in these tumors was confirmed by immunohistochemistry. In summary, this study establishes that the tumor suppressor cystatin M is a novel target for epigenetic silencing during mammary tumorigenesis and that this aberrant event can occur before development of invasive breast cancer.

Pharmacokinetics of a novel closantel/ivermectin injection in cattle.

Two studies are described on the pharmacokinetics of a combination anthelmintic consisting of ivermectin and closantel for use in cattle. In the first, the pharmacokinetics of both active drugs in the combination were compared with the formulation with either ivermectin or closantel removed. No differences in the pharmacokinetics were observed, indicating that neither the absorption nor distribution of ivermectin or closantel in the combination were influenced by the presence of the other. In the second study the pharmacokinetics of ivermectin and closantel in the combination product were compared with control formulations of each. No difference was found between the closantel formulations. With ivermectin it was noted that absorption and excretion were more rapid and Cmax higher in the combination, although the AUC of both formulations were not significantly different.

Inactivation of Wnt inhibitory factor-1 (WIF1) expression by epigenetic silencing is a common event in breast cancer.

The Wnt signaling pathway is a powerful and prominent oncogenic mechanism dysregulated in numerous cancer types. While evidence from transgenic mouse models and studies of human tumors clearly indicate that this pathway is of likely importance in human breast cancer, few clues as to the exact molecular nature of Wnt dysregulation have been uncovered in this tumor type. Here, we show that the Wnt inhibitory factor-1 (WIF1) gene, which encodes a secreted protein antagonistic to Wnt-dependent signaling, is targeted for epigenetic silencing in human breast cancer. We show that cultured human breast tumor cell lines display absent or low levels of WIF1 expression that are increased when cells are cultured with the DNA demethylating agent 5-aza-2'-deoxycytidine. Furthermore, the WIF1 promoter is aberrantly hypermethylated in these cells as judged by both methylation-specific PCR and bisulfite genomic sequencing. Using a panel of patient-matched breast tumors and normal breast tissue, we show that WIF1 expression is commonly diminished in breast tumors when compared with normal tissue and that this correlates with WIF1 promoter hypermethylation. Analysis of a panel of 24 primary breast tumors determined that the WIF1 promoter is aberrantly methylated in 67% of these tumors, indicating that epigenetic silencing of this gene is a frequent event in human breast cancer. Using an isogenic panel of cell lines proficient or deficient in the DNA methyltransferases (DNMTs) DNMT1 and/or DNMT3B, we show that hypermethylation of the WIF1 promoter is attributable to the cooperative activity of both DNMT1 and DNMT3B. Our findings establish the WIF1 gene as a target for epigenetic silencing in breast cancer and provide a mechanistic link between the dysregulation of Wnt signaling and breast tumorigenesis.

Urban youths' perspectives on violence and the necessity of fighting.

OBJECTIVES: To assess youth perceptions of the causes and consequences of violence generally, the causes and consequences of fighting specifically, and to determine how best to approach fighting in the context of violence prevention activities. METHODS: Thirteen structured focus group interviews with youths from three high violence urban settings: a large, urban high school, a training center for disadvantaged youths, and a school for adjudicated youths. Participants were 120 urban, predominately African-American youths and young adults ages 14-22 years (mean: 17.2 years). Seven focus groups were conducted with females, and six with males. RESULTS: Adolescents identified the causes of violence on multiple levels including: individual, family, interpersonal, and community level factors. Most youths (89%) had been in a physical fight. Participants felt that fighting was not "right", but identified situations in which it was necessary. Specifically, fighting was used as a problem solving tool, and could prevent escalation of violence. Youths felt that the adults in their lives, including physicians, were generally ill equipped to give advice about violence, as adults' experiences were so removed from their own. Participants looked to experienced role models to offer problem solving and harm reduction strategies. Youths were open to receiving anticipatory guidance about violence and fighting from primary care physicians they felt comfortable with, and who showed respect for their experiences. CONCLUSIONS: Interventions that include blanket admonitions against fighting should be reassessed in light of youth perceptions that fighting plays a complex role in both inciting and preventing more serious violence.

Simvastatin-diltiazem drug interaction resulting in rhabdomyolysis and hepatitis.

Simvastatin, a hydroxymethyl glutarate coenzyme A (HMG-CoA) reductase inhibitor, is a commonly used cholesterol lowering agent. The long-term safety profile of simvastatin, established over ten-years of clinical use, is excellent. Both rhabdomyolysis and hepatitis, however, are recognized toxic effects of this medication, and generally occur when the patients are taking more than 40 mg of simvastatin a day. Potent inhibitors of the cytochrome P450 3A4 (CYP3A4) enzyme increase the incidence of simvastatin toxicity. Calcium channel blockers are weak inhibitors of the CYP3A4 enzyme. Diltiazem is known to increase the serum concentration of simvastatin. Many patients who take both simvastatin and diltiazem require lower doses of simvastatin to achieve the recommended reduction in cholesterol. Since diltiazem is known to increase plasma levels of lovastatin, a similar phenomenon may occur with simvastatin. Our patient had been stable for three years on simvastatin therapy. His rhabdomyolysis and hepatitis coincided with the addition of diltiazem. This is the first report of the combined toxicities of rhabdomyolysis and hepatitis being induced by the addition of diltiazem to simvastatin therapy. This patient serves as a reminder to the clinician of the potential interaction of these two commonly used drugs.

Cat cuddler's cough.

Pasteurella multocida typically causes cutaneous infections in humans following animal bites or scratches. Primary pulmonary disease, however, can occur in humans after inhalation of airborne particles or by aspiration of colonized or infected nasopharyngeal secretions containing this organism. Symptoms of P. multocida pulmonary infection in humans are variable, ranging from cough with or without hemoptysis to severe prostration. P. multocida infection of the lower respiratory tree has a predilection for elderly patients with underlying lung pathology, especially chronic obstructive pulmonary disease and bronchiectasis. This report reminds the clinician that P. multocida can cause pulmonary infection in patients without underlying lung disease, and stresses the importance of careful history when presented with an indolent infection.

A rare cause of dyspnea and fibrocystic pulmonary disease.

Tracheobronchomegaly (TBM) is a disorder of the major airways characterized by an enlargement of the trachea and main stem bronchi. It is thought to result from a congenital connective tissue defect that results in the trachea and main bronchi becoming flaccid. Although TBM is considered rare, it may be more common than is generally recognized, and may be a seldom-diagnosed cause of chronic lung disease.