Thirteen Cases of Valeryl Fentanyl in Michigan: A Call for Expanding Opioid Testing.

ABSTRACT: In this report, we describe 13 cases of drug overdose in Michigan in which valeryl fentanyl was found in postmortem blood. Valeryl fentanyl is a schedule I opioid that is rarely found in drug overdoses in the United States. Although little data exist on the mortality and morbidity associated with valeryl fentanyl, its molecular structure indicates that it would be less potent than fentanyl.When analyzing blood samples for valeryl fentanyl, samples from peripheral sites were sometimes negative for quantitative levels; however, samples from central sites in the same decedent were positive. This could indicate unique pharmacokinetics for valeryl fentanyl, which could have implications for other fentanyl analogs. Given the paucity of pharmacodynamic information, the prohibition of its use, the potential to buttress law enforcement efforts in monitoring drug trafficking trends, and to determine the efficacy of current regulations, laboratories should test for valeryl fentanyl. When testing for valeryl fentanyl, and likely other fentanyl analogs, the site of sample collection is important: central sources of blood are preferred to peripheral sources.

Girardia dorotocephala transcriptome sequence, assembly, and validation through characterization of piwi homologs and stem cell progeny markers.

Planarian flatworms are popular models for the study of regeneration and stem cell biology in vivo. Technical advances and increased availability of genetic information have fueled the discovery of molecules responsible for stem cell pluripotency and regeneration in flatworms. Unfortunately, most of the planarian research performed worldwide utilizes species that are not natural habitants of North America, which limits their availability to newcomer laboratories and impedes their distribution for educational activities. In order to circumvent these limitations and increase the genetic information available for comparative studies, we sequenced the transcriptome of Girardia dorotocephala, a planarian species pandemic and commercially available in North America. A total of 254,802,670 paired sequence reads were obtained from RNA extracted from intact individuals, regenerating fragments, as well as freshly excised auricles of a clonal line of G. dorotocephala (MA-C2), and used for de novo assembly of its transcriptome. The resulting transcriptome draft was validated through functional analysis of genetic markers of stem cells and their progeny in G. dorotocephala. Akin to orthologs in other planarian species, G. dorotocephala Piwi1 (GdPiwi1) was found to be a robust marker of the planarian stem cell population and GdPiwi2 an essential component for stem cell-driven regeneration. Identification of G. dorotocephala homologs of the early stem cell descendent marker PROG-1 revealed a family of lysine-rich proteins expressed during epithelial cell differentiation. Sequences from the MA-C2 transcriptome were found to be 98-99% identical to nucleotide sequences from G. dorotocephala populations with different chromosomal number, demonstrating strong conservation regardless of karyotype evolution. Altogether, this work establishes G. dorotocephala as a viable and accessible option for analysis of gene function in North America.

The atypical MAPK ERK3 potently suppresses melanoma cell growth and invasiveness.

Mitogen-activated protein kinase 6 (MAPK6) represents an atypical MAPK also known as extracellular signal-regulated kinase 3 (ERK3), which has been shown to play roles in cell motility and metastasis. ERK3 promotes migration and invasion of lung cancer cells and head and neck cancer cells by regulating the expression and/or activity of proteins involved in cancer progression. For instance, ERK3 upregulates matrix metallopeptidases and thereby promotes cancer cell invasiveness, and it phosphorylates tyrosyl-DNA phosphodiesterase 2, thereby enhancing chemoresistance in lung cancer. Here we discovered that ERK3 plays a converse role in melanoma. We observed that BRAF, an oncogenic Ser/Thr kinase, upregulates ERK3 expression levels by increasing both ERK3 messenger RNA levels and protein stability. Interestingly, although BRAF's kinase activity was required for upregulating ERK3 gene transcription, BRAF stabilized ERK3 protein in a kinase-independent fashion. We further demonstrate that ERK3 inhibits the migration, proliferation and colony formation of melanoma cells. In line with this, high level of ERK3 predicted increased survival among patients with melanomas. Taken together, these results indicate that ERK3 acts as a potent suppressor of melanoma cell growth and invasiveness.

Vulvodynia: What We Know and Where We Should Be Going.

OBJECTIVE: The aim of the study was to review the current nomenclature and literature examining microbiome cytokine, genomic, proteomic, and glycomic molecular biomarkers in identifying markers related to the understanding of the pathophysiology and diagnosis of vulvodynia (VVD). MATERIALS AND METHODS: Computerized searches of MEDLINE and PubMed were conducted focused on terminology, classification, and "omics" variations of VVD. Specific MESH terms used were VVD, vestibulodynia, metagenomics, vaginal fungi, cytokines, gene, protein, inflammation, glycomic, proteomic, secretomic, and genomic from 2001 to 2016. Using combined VVD and vestibulodynia MESH terms, 7 references were identified related to vaginal fungi, 15 to cytokines, 18 to gene, 43 to protein, 38 to inflammation, and 2 to genomic. References from identified publications were manually searched and cross-referenced to identify additional relevant articles. A narrative synthesis of the articles was conducted; however, meta-analysis was not conducted because of substantial heterogeneity in the studies and limited numbers of control-matched studies. RESULTS: Varying definitions of VVD complicate a meta-analysis, and standard definitions will better allow for comparisons of studies and enhance the applicability of evidence to patient populations. Although data are still limited, genomic and molecular diagnostic testings continue to be investigated as potential tools for the diagnosis of VVD. CONCLUSIONS: Standardized nomenclature will allow for comparability of studies and progress in research related to the pathophysiology of VVD and to facilitate clinical decision making and treatment choices. Although the current understanding of the pathogenesis of VVD is limited, there are new opportunities to explore potential diagnostic markers differences in women with VVD, which may lead to targeted therapy.

Alignment of Mitotic Chromosomes in Human Cells Involves SR-Like Splicing Factors Btf and TRAP150.

Serine-arginine-rich (SR) or SR-like splicing factors interact with exon junction complex proteins during pre-mRNA processing to promote mRNA packaging into mature messenger ribonucleoproteins (mRNPs) and to dictate mRNA stability, nuclear export, and translation. The SR protein family is complex, and while many classical SR proteins have well-defined mRNA processing functions, those of other SR-like proteins is unclear. Here, we show that depletion of the homologous non-classical serine-arginine-rich (SR) splicing factors Bcl2-associated transcription factor (Btf or BCLAF) and thyroid hormone receptor-associated protein of 150 kDa (TRAP150) causes mitotic defects. We hypothesized that the depletion of these SR-like factors affects mitosis indirectly through an altered expression of mitotic checkpoint regulator transcripts. We observed an altered abundance of transcripts that encode mitotic regulators and mitotic chromosome misalignment defects following Btf and/or TRAP150 depletion. We propose that, in addition to their previously reported roles in maintaining mRNA distribution, Btf and TRAP150 control the abundance of transcripts encoding mitotic regulators, thereby affecting mitotic progression in human cells.

Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue.

As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and "amplifiability" for aCGH and other downstream applications. Phenol-chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues.

Son maintains accurate splicing for a subset of human pre-mRNAs.

Serine-arginine-rich (SR) proteins play a key role in alternative pre-mRNA splicing in eukaryotes. We recently showed that a large SR protein called Son has unique repeat motifs that are essential for maintaining the subnuclear organization of pre-mRNA processing factors in nuclear speckles. Motif analysis of Son highlights putative RNA interaction domains that suggest a direct role for Son in pre-mRNA splicing. Here, we used in situ approaches to show that Son localizes to a reporter minigene transcription site, and that RNAi-mediated Son depletion causes exon skipping on reporter transcripts at this transcription site. A genome-wide exon microarray analysis was performed to identify human transcription and splicing targets of Son. Our data show that Son-regulated splicing encompasses all known types of alternative splicing, the most common being alternative splicing of cassette exons. We confirmed that knockdown of Son leads to exon skipping in pre-mRNAs for chromatin-modifying enzymes, including ADA, HDAC6 and SetD8. This study reports a comprehensive view of human transcription and splicing targets for Son in fundamental cellular pathways such as integrin-mediated cell adhesion, cell cycle regulation, cholesterol biosynthesis, apoptosis and epigenetic regulation of gene expression.

Disorientation effects, circulating small ribonucleic acid, and genetic susceptibility on static postural stability.

Background: Motion Sickness increases risk of performance deficits and safety of flight concerns. The etiology of motion sickness is poorly understood. Here, we attempted to quantify the physiological effects of motion sickness on static balance and determine the genetic predictors associated with these effects. Methods: 16 subjects underwent a disorientation stimulus to induce motion sickness. Motion sickness susceptibility was identified using the Motion Sickness Susceptibility Questionnaire. Postural balance outcomes were measured using two tasks, and small ribonucleic acid profiles were assessed with blood draws before motion sickness stimulus. Differences in postural sway before and after the stimulus as well as effect modification of susceptibility were assessed. A random forest followed by regression tree analysis was constructed for each postural sway variable to determine top genetic and covariate predictors. Findings: Significant differences existed in mean postural balance responses between before and after stimulus. Individuals with longer stimulus survival experienced a greater (but insignificant) perception of sway, even if not displaying increased sway for all conditions. Circulation small ribonucleic acids were differentially expressed between individuals with long and short stimulus survival, many of these microRNA have purported targets in genes related to vestibular disorders. Interpretation: We found motion sickness produces transient motor dysfunction in a healthy military population. Small ribonucleic acids were differentially expressed between subjects with long and short stimulus survival times.

Motion sickness decreases low back function and changes gene expression in military aircrew.

BACKGROUND: Motion sickness and low back disorders are prevalent and debilitating conditions that affect the health, performance, and operational effectiveness of military aircrews. This study explored the effects of a motion sickness stimulus on biomechanical and genetic factors that could potentially be involved in the causal pathways for both disorders. METHODS: Subjects recruited from a military population were exposed to either a mild (n = 12) or aggressive (n = 16) motion sickness stimulus in a Neuro-Otologic Test Center. The independent variable of interest was the motion sickness stimulus exposure (before vs. after), though differences between mild and aggressive stimuli were also assessed. Dependent measures for the study included motion sickness exposure duration, biomechanical variables (postural stability, gait function, low back function, lumbar spine loading), and gene expression. FINDINGS: Seven of twelve subjects experiencing the mild motion sickness stimulus endured the full 30 min in the NOTC, whereas subjects lasted an average of 13.2 (SD 5.0) minutes in the NOTC with the aggressive motion sickness stimulus. Mild motion sickness exposure led to a significant decrease in the postural stability measure of sway area, though the aggressive motion sickness exposure led to a statistically significant increase in sway area. Both stimuli led to decreases in low back function, though the decrease was only statistically significant for the mild protocol. Both stimuli also led to significant changes in gene expression. INTERPRETATION: Motion sickness may alter standing balance, decrease low back function, and lead to changes in the expression of genes with roles in osteogenesis, myogenesis, development of brain lymphatics, inflammation, neuropathic pain, and more. These results may provide preliminary evidence for a link between motion sickness and low back disorders.

MDM4 Isoform Expression in Melanoma Supports an Oncogenic Role for MDM4-A.

The p53 tumor suppressor integrates upstream signals such as DNA damage and active oncogenes to initiate cell cycle arrest or apoptosis. This response is critical to halting inappropriate growth signals. As such, p53 activity is lost in cancer. In melanoma, however, the p53 gene is intact in a reported 94% of human cases. Rather than direct mutation, p53 is held inactive through interaction with inhibitory proteins. Here, we examine the expression of the two primary inhibitors of p53, MDM2 and MDM4, in genomic databases and biopsy specimens. We find that MDM4 is frequently overexpressed. Moreover, changes in splicing of MDM4 occur frequently and early in melanomagenesis. These changes in splicing must be considered in the design of therapeutic inhibitors of the MDM2/4 proteins for melanoma.

Randomized controlled trial of fractionated laser resurfacing on aged skin as prophylaxis against actinic neoplasia.

BACKGROUNDThe loss of insulin-like growth factor 1 (IGF-1) expression in senescent dermal fibroblasts during aging is associated with an increased risk of nonmelanoma skin cancer (NMSC). We tested how IGF-1 signaling can influence photocarcinogenesis during chronic UVB exposure to determine if fractionated laser resurfacing (FLR) of aged skin, which upregulates dermal IGF-1 levels, can prevent the occurrence of actinic keratosis (AK) and NMSC.METHODSA human skin/immunodeficient mouse xenografting model was used to test the effects of a small molecule inhibitor of the IGF-1 receptor on chronic UVB radiation. Subsequently, the durability of FLR treatment was tested on a cohort of human participants aged 65 years and older. Finally, 48 individuals aged 60 years and older with considerable actinic damage were enrolled in a prospective randomized clinical trial in which they underwent a single unilateral FLR treatment of one lower arm. Numbers of AKs/NMSCs were recorded on both extremities for up to 36 months in blinded fashion.RESULTSXenografting studies revealed that chronic UVB treatment with a topical IGF-1R inhibitor resulted in a procarcinogenic response. A single FLR treatment was durable in restoring appropriate UVB response in geriatric skin for at least 2 years. FLR resulted in sustained reduction in numbers of AKs and decreased numbers of NMSCs in the treated arm (2 NMSCs) versus the untreated arm (24 NMSCs).CONCLUSIONThe elimination of senescent fibroblasts via FLR reduced the procarcinogenic UVB response of aged skin. Thus, wounding therapies are a potentially effective prophylaxis for managing high-risk populations.TRIAL REGISTRATIONClinicalTrials.gov (NCT03906253).FUNDINGNational Institutes of Health, Veterans Administration.

Synthesis of cribrostatin 6.

The synthesis of cribrostatin 6 (1) is described. A regioselective bromination, a biaryl coupling, and an intramolecular cyclization are the key steps in the synthesis.

Synthesis of santiagonamine.

The first total synthesis of santiagonamine (1) is achieved in 12 steps from isovanillin. A palladium-catalyzed Ullmann cross-coupling reaction and a photocyclization are the key steps in the synthesis.

RB reversibly inhibits DNA replication via two temporally distinct mechanisms.

The retinoblastoma (RB) tumor suppressor is a critical negative regulator of cellular proliferation. Repression of E2F-dependent transcription has been implicated as the mechanism through which RB inhibits cell cycle progression. However, recent data have suggested that the direct interaction of RB with replication factors or sites of DNA synthesis may contribute to its ability to inhibit S phase. Here we show that RB does not exert a cis-acting effect on DNA replication. Furthermore, the localization of RB was distinct from replication foci in proliferating cells. While RB activation strongly attenuated the RNA levels of multiple replication factors, their protein expression was not diminished coincident with cell cycle arrest. During the first 24 h of RB activation, components of the prereplication complex, initiation factors, and the clamp loader complex (replication factor C) remained tethered to chromatin. In contrast, the association of PCNA and downstream components of the processive replication machinery was specifically disrupted. This signaling from RB occurred in a manner dependent on E2F-mediated transcriptional repression. Following long-term activation of RB, we observed the attenuation of multiple replication factors, the complete cessation of DNA synthesis, and impaired replicative capacity in vitro. Therefore, functional distinctions exist between the "chronic" RB-mediated arrest state and the "acute" arrest state. Strikingly, attenuation of RB activity reversed both acute and chronic replication blocks. Thus, continued RB action is required for the maintenance of two kinetically and functionally distinct modes of replication inhibition.

Int7G24A variant of transforming growth factor-beta receptor type I is associated with invasive breast cancer.

PURPOSE: The transforming growth factor-beta (TGF-beta) signaling pathway has been frequently implicated in breast cancer. An intronic variant (Int7G24A) of TGF-beta receptor type I (TGFBR1) is associated with kidney and bladder cancers in our recent study. We hypothesize that this germline variant may be involved in development and progression of breast cancer. EXPERIMENTAL DESIGN: Case-control studies were designed from archived paraffin-embedded tissue specimens from the same geographic area with a homogenous ethnic population. We analyzed 223 patients (25 with preinvasive tumors and 198 with invasive and metastatic breast cancers) and 153 noncancer controls. The Int7G24A was identified by PCR-RFLP. Another germline deletion (TGFBR1*6A) and somatic mutations in the TGFBR1 were also analyzed by PCR and single-strand conformational polymorphism. RESULTS: The Int7G24A allele was evident in 32% of patients with preinvasive neoplasms and 48% of patients with invasive breast cancers compared with 26% controls (P = 0.00008). In addition, 11 (5.6%) homozygous Int7G24A carriers were found in patients with invasive breast cancers, whereas only 3 (2%) homozygous carriers were found in the control group. The TGFBR1*6A allele was not significantly associated with breast cancer patients and only one somatic mutation was found in 71 breast cancers. CONCLUSION: These data suggest that the germline Int7G24A variant may represent a risk factor for invasive breast cancer and a marker for breast cancer progression. A separate study with a larger sample size is warranted to validate the association of the Int7G24A with human breast cancer.

Application of the Stephan et al. Chest Radiograph Comparison Method to Decomposed Human Remains.

This manuscript describes the use of comparative radiography of the chest to facilitate positive identification of human remains in advanced stages of decomposition. The method reported by Stephan et al. for positive identification of dry, disarticulated skeletal elements was used on semifleshed, decomposing remains. Positive identification was established through multiple points of concordance observed in radiographs of the left and right clavicles and the C5-T1 vertebrae. This case study demonstrates the applicability of the Stephan et al.'s method in cases involving decomposing remains.

Unbiased analysis of RB-mediated transcriptional repression identifies novel targets and distinctions from E2F action.

The retinoblastoma tumor suppressor, RB, is thought to inhibit cell cycle progression through transcriptional repression. E2F-regulated genes have been viewed as presumptive targets of RB-mediated repression. However, we found that specific E2F targets were not regulated in a consistent manner by the action of a RB allele that is refractory to cyclin-dependent kinase/cyclin-mediated phosphorylation (PSM-RB) when compared with E2F2 overproduction. Therefore, we used Affymetrix GeneChips as an unbiased approach to identify RB targets. We found that expression of PSM-RB significantly attenuates >200 targets, the majority of which are involved in cell cycle control (DNA replication or G2-M), DNA repair, or transcription/chromatin structure. The observed repression was due to the action of RB and not merely a manifestation of altered cell cycle distribution. Additionally, the majority of RB repression targets were confirmed through the blockade of endogenous RB phosphorylation via p16ink4a overexpression. Thus, these results have utility in assigning RB pathway activation in more complex systems of cell cycle inhibition (e.g., mitogen withdrawal, senescence, or DNA damage checkpoint). As expected, a significant fraction of RB-repressed genes have promoters that are bound/regulated by E2F family members. However, targets were identified that are distinct from genes known to be stimulated by overexpression of specific E2F proteins. Moreover, the relative action of RB versus E2F2 overexpression on specific genes demonstrates that a simple opposition model does not explain the relative contribution of RB to gene regulation. Thus, this study provides the first unbiased description of RB-repressed genes, thereby delineating new aspects of RB-mediated transcriptional control and novel targets involved in diverse cellular processes.

Copy number variation in archival melanoma biopsies versus benign melanocytic lesions.

BACKGROUND: Skin melanocytes can give rise to benign and malignant neoplasms. Discrimination of an early melanoma from an unusual/atypical benign nevus can represent a significant challenge. However, previous studies have shown that in contrast to benign nevi, melanoma demonstrates pervasive chromosomal aberrations. OBJECTIVE: This substantial difference between melanoma and benign nevi can be exploited to discriminate between melanoma and benign nevi. METHODS: Array-comparative genomic hybridization (aCGH) is an approach that can be used on DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues to assess the entire genome for the presence of changes in DNA copy number. In this study, high resolution, genome-wide single-nucleotide polymorphism (SNP) arrays were utilized to perform comprehensive and detailed analyses of recurrent copy number aberrations in 41 melanoma samples in comparison with 21 benign nevi. RESULTS: We found statistically significant copy number gains and losses within melanoma samples. Some of the identified aberrations are previously implicated in melanoma. Moreover, novel regions of copy number alterations were identified, revealing new candidate genes potentially involved in melanoma pathogenesis. CONCLUSIONS: Taken together, these findings can help improve melanoma diagnosis and introduce novel melanoma therapeutic targets.

Model systems for flavoenzyme activity: interplay of hydrogen bonding and aromatic stacking in cofactor redox modulation.

[structure: see text] A model system has been developed to study the synergy between aromatic stacking and hydrogen bonding in the binding of a flavin derivative. The results show that the identity of both the hydrogen bonding and pi-stacking units strongly determine the overall receptor affinity for flavin in both the oxidized and radical anion forms.

Sarcoptes scabiei mites modulate gene expression in human skin equivalents.

The ectoparasitic mite, Sarcoptes scabiei that burrows in the epidermis of mammalian skin has a long co-evolution with its hosts. Phenotypic studies show that the mites have the ability to modulate cytokine secretion and expression of cell adhesion molecules in cells of the skin and other cells of the innate and adaptive immune systems that may assist the mites to survive in the skin. The purpose of this study was to identify genes in keratinocytes and fibroblasts in human skin equivalents (HSEs) that changed expression in response to the burrowing of live scabies mites. Overall, of the more than 25,800 genes measured, 189 genes were up-regulated >2-fold in response to scabies mite burrowing while 152 genes were down-regulated to the same degree. HSEs differentially expressed large numbers of genes that were related to host protective responses including those involved in immune response, defense response, cytokine activity, taxis, response to other organisms, and cell adhesion. Genes for the expression of interleukin-1α (IL-1α) precursor, IL-1ß, granulocyte/macrophage-colony stimulating factor (GM-CSF) precursor, and G-CSF precursor were up-regulated 2.8- to 7.4-fold, paralleling cytokine secretion profiles. A large number of genes involved in epithelium development and keratinization were also differentially expressed in response to live scabies mites. Thus, these skin cells are directly responding as expected in an inflammatory response to products of the mites and the disruption of the skin's protective barrier caused by burrowing. This suggests that in vivo the interplay among these skin cells and other cell types, including Langerhans cells, dendritic cells, lymphocytes and endothelial cells, is responsible for depressing the host's protective response allowing these mites to survive in the skin.