Transinfection of Wolbachia wAlbB into Culex quinquefasciatus mosquitoes does not alter vector competence for Hawaiian avian malaria (Plasmodium relictum GRW4).

Avian malaria is expanding upslope with warmer temperatures and driving multiple species of Hawaiian birds towards extinction. Methods to reduce malaria transmission are urgently needed to prevent further declines. Releasing Wolbachia-infected incompatible male mosquitoes could suppress mosquito populations and releasing Wolbachia-infected female mosquitoes (or both sexes) could reduce pathogen transmission if the Wolbachia strain reduced vector competence. We cleared Culex quinquefasciatus of their natural Wolbachia pipientis wPip infection and transinfected them with Wolbachia wAlbB isolated from Aedes albopictus. We show that wAlbB infection was transmitted transovarially, and demonstrate cytoplasmic incompatibility with wild-type mosquitoes infected with wPip from Oahu and Maui, Hawaii. We measured vector competence for avian malaria, Plasmodium relictum, lineage GRW4, of seven mosquito lines (two with wAlbB; three with natural wPip infection, and two cleared of Wolbachia infection) by allowing them to feed on canaries infected with recently collected field isolates of Hawaiian P. relictum. We tested 73 groups (Ntotal = 1176) of mosquitoes for P. relictum infection in abdomens and thoraxes 6-14 days after feeding on a range of parasitemias from 0.028% to 2.49%, as well as a smaller subset of salivary glands. We found no measurable effect of Wolbachia on any endpoint, but strong effects of parasitemia, days post feeding, and mosquito strain on both abdomen and thorax infection prevalence. These results suggest that releasing male wAlbB-infected C. quinquefasciatus mosquitoes could suppress wPip-infected mosquito populations, but would have little positive or negative impact on mosquito vector competence for P. relictum if wAlbB became established in local mosquito populations. More broadly, the lack of Wolbachia effects on vector competence we observed highlights the variable impacts of both native and transinfected Wolbachia infections in mosquitoes.

HIV status affects PTSD symptom severity, psychophysiology, and heart rate variability in women with low but not high exposure to childhood maltreatment.

Objective: People living with HIV (PLWH) experience high rates of childhood trauma exposure, which is a significant risk factor for the development of posttraumatic stress disorder (PTSD). Because Black Americans living in urban environments are exposed to high levels of trauma, suffer from chronic PTSD, and are at increased risk for HIV infection, it is important to understand how HIV status interacts with childhood maltreatment to influence PTSD symptom severity and underlying psychophysiology. Methods: The current cross-sectional study assessed whether HIV status interacts with childhood maltreatment to influence PTSD symptom severity and heart rate variability during a dark-enhanced startle (DES) task in 88 Black women with (n=30) and without HIV (n=58). Results: HIV was associated with greater PTSD symptom severity only in women with low levels of childhood maltreatment (p=.024). Startle potentiation during DES was highest in women living without HIV and with high childhood maltreatment (p=.018). In women who had experienced low levels of childhood maltreatment, respiratory sinus arrhythmia (RSA) was lower during the dark phase of DES in women living without HIV than women living with HIV (WLWH), (p=.046). RSA during the light phase of DES was lower in WLWH than in women living without HIV (p=.042). Conclusion: In the current sample of Black women, HIV status was associated with PTSD symptom severity in a manner dependent on level of childhood maltreatment, suggesting that HIV status may be an important factor to consider for behavioral and pharmacological treatment strategies for PTSD. Additionally, HIV status is associated with lower percent potentiation to darkness and lower RSA during the light phase of DES, suggesting physiological mechanisms by which HIV may contribute to PTSD symptoms in individuals exposed to low levels of childhood maltreatment.

Evolutionary superscaffolding and chromosome anchoring to improve Anopheles genome assemblies.

BACKGROUND: New sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from 'finished'. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies. RESULTS: We evaluated and employed 3 gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies, we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: 6 with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and 3 with new assemblies based on re-scaffolding or long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: 7 for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further 7 with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi. CONCLUSIONS: Experimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our evaluations show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.

Chronic adolescent stress sex-specifically alters central and peripheral neuro-immune reactivity in rats.

Adversity during development is a reliable predictor of psychiatric disorders such as depression and anxiety which are increasingly recognized to have an immune component. We have previously demonstrated that chronic adolescent stress (CAS) in rats leads to depressive-like behavior in adulthood along with long-lasting changes to the hypothalamic-pituitary-adrenal axis and pro-inflammatory cytokine induction in the hippocampus. However, the mechanisms by which CAS promotes hippocampal inflammation are not yet defined. Here we tested the hypothesis that a history of CAS exaggerates induction of the pro-inflammatory NFκB pathway in the adult rat hippocampus without compromising the peripheral immune response. We also assessed potential sex differences because it is unclear whether females, who are twice as likely to suffer from mood disorders as males, are disproportionally affected by stress-primed inflammation. Male and female adolescent rats underwent a CAS paradigm or received no stress. Six weeks following the last stressor, all rats received a single systemic injection of either lipopolysaccharide or vehicle to unmask possible immune-priming effects of CAS. An NFκB signaling PCR array demonstrated that CAS exaggerated the expression of NFκB-related genes in the hippocampus of both males and females. Interestingly, targeted qPCR demonstrated that CAS potentiated the induction of hippocampal IL1B and REL mRNA in female rats only, suggesting that some immune effects of CAS are indeed sex-specific. In contrast to the hippocampal findings, indices of peripheral inflammation such as NFκB activity in the spleen, plasma IL-1ß, IL-6, TNF-α, and corticosterone were not impacted by CAS in female rats. Despite showing no pro-inflammatory changes to hippocampal mRNA, male CAS rats displayed lower plasma corticosterone response to LPS at 2 h after injection followed by an exaggerated plasma IL-1ß response at 4 h. This potentially blunted corticosterone response coupled with excessive innate immune signaling in the periphery is consistent with possible glucocorticoid resistance in males. In contrast, the effects of CAS manifested as excessive hippocampal immune reactivity in females. We conclude that while a history of exposure to chronic adolescent stress enhances adult immune reactivity in both males and females, the mechanism and manifestation of such alterations are sex-specific.

A cohort study of the effectiveness of insecticide-treated bed nets to prevent malaria in an area of moderate pyrethroid resistance, Malawi.

BACKGROUND: Insecticide-treated bed nets (ITNs) are the cornerstone of malaria control in sub-Saharan Africa but their effectiveness may be compromised by the spread of pyrethroid resistance among malaria vectors. The objective of this investigation was to assess the effectiveness of ITNs to prevent malaria in an area of Malawi with moderate pyrethroid resistance. METHODS: One deltamethrin ITN was distributed in the study area for every two individuals in each household plus one extra ITN for households with an odd number of residents. A fixed cohort of 1,199 children aged six to 59 months was seen monthly for one year and at sick visits to measure malaria infection and use of ITNs. Insecticide resistance among malaria vectors was measured. The effect of ITN use on malaria incidence was assessed, adjusting for potential confounders using generalized estimating equations accounting for repeated measures. RESULTS: There were 1,909 infections with Plasmodium falciparum over 905 person-years at risk (PYAR), resulting in an observed incidence of 2.1 infections per person-year (iPPY). ITNs were used during 97% of the PYAR. The main vector was Anopheles funestus: mortality in WHO tube assays after exposure to 0.05% deltamethrin was 38% (95% confidence interval (CI) 29-47), and resistance was due to elevated oxidase enzymes. After adjusting for potential confounders, the incidence of malaria infection among ITN users was 1.7 iPPY (95% CI 1.5-2.1) and among non-bed net users was 2.6 iPPY (95% CI 2.0-3.3). Use of ITNs reduced the incidence of malaria infection by 30% (rate ratio 0.7; 95% CI, 0.5-0.8) compared to no bed nets. CONCLUSION: ITNs significantly reduced the incidence of malaria infection in children in an area with moderate levels of pyrethroid resistance and considerable malaria transmission. This is the first study to show that ITNs provide protection in areas where pyrethroid-resistant An. funestus is the major malaria vector. Malaria control programmes should continue to distribute and promote ITNs in areas with low to moderate pyrethroid resistance; however, insecticide resistance may intensify further and it is not known whether ITNs will remain effective at higher levels of resistance. There is an urgent need to identify or develop new insecticides and technologies to limit the vulnerability of ITNs to insecticide resistance.

Rehabilitation of Combat-Related Injuries in the Veterans Administration: A Web of Support.

The Department of Veterans Affairs provides acute, subacute, and continuing rehabilitation for veterans using a hub-and-spoke system of hospitals and outpatient facilities. Using traumatic brain injury as an example, this commentary illustrates how this system provides interdisciplinary rehabilitative care to veterans throughout North Carolina.

Up-regulation of miR-182 expression after epigenetic modulation of human melanoma cells.

PURPOSE: We sought to investigate the epigenetic regulation of microRNAs (miRNAs) in melanoma. METHODS: We treated two highly metastatic human melanoma cell lines, C8161.9 and WM266-4, with the demethylating agents DAC (5-aza-2'-deoxycytidine) and trichostatin A. Locked nucleic acid-based miRNA expression profiling was utilized to examine the differential expression of miRNAs before and after treatment. RESULTS: We found that miR-182, a miRNA with oncogenic properties, was significantly up-regulated in human melanoma cells after epigenetic modulation. Genome sequence analysis revealed the presence of a prominent CpG island 8-10 kb upstream of mature miR-182. Methylation analysis showed that this genomic region was exclusively methylated in melanoma cells but not in human melanocytes, skin, or peripheral blood mononuclear cells. DISCUSSION: These results indicate that an epigenetic mechanism is likely involved in modulating the expression level of miR-182 in melanoma, and increased expression of oncogenic-like miR-182 could be a concern for melanoma patients after epigenetic therapy.

A personalized microRNA microarray normalization method using a logistic regression model.

MOTIVATION: MicroRNA (miRNA) is a set of newly discovered non-coding small RNA molecules. Its significant effects have contributed to a number of critical biological events including cell proliferation, apoptosis development, as well as tumorigenesis. High-dimensional genomic discovery platforms (e.g. microarray) have been employed to evaluate the important roles of miRNAs by analyzing their expression profiling. However, because of the small total number of miRNAs and the absence of well-known endogenous controls, the traditional normalization methods for messenger RNA (mRNA) profiling analysis could not offer a suitable solution for miRNA analysis. The need for the establishment of new adaptive methods has come to the forefront. RESULTS: Locked nucleic acid (LNA)-based miRNA array was employed to profile miRNAs using colorectal cancer cell lines under different treatments. The expression pattern of overall miRNA profiling was pre-evaluated by a panel of miRNAs using Taqman-based quantitative real-time polymerase chain reaction (qRT-PCR) miRNA assays. A logistic regression model was built based on qRT-PCR results and then applied to the normalization of miRNA array data. The expression levels of 20 additional miRNAs selected from the normalized list were post-validated. Compared with other popularly used normalization methods, the logistic regression model efficiently calibrates the variance across arrays and improves miRNA microarray discovery accuracy. AVAILABILITY: Datasets and R package are available at http://gauss.usouthal.edu/publ/logit/.

Genetic and environmental influences on cortisol reactivity to a psychosocial stressor in adolescents and young adults.

Individuals vary in their response to psychological and physiological stressors, and this reactivity can be captured using measures of cortisol. Previous research suggests cortisol reactivity is under some degree of genetic control; however, the measures used have varied widely. This study (N = 524) examined potential differences in heritability across varying cortisol metrics of stress reactivity following the Trier Social Stress Test (TSST) and whether these measures are genetically or environmentally interrelated. Participants included twins aged 15-20 years (56% female). Cortisol reactivity to the TSST was assessed via serial salivary cortisol samples collected pre- and post-TSST. Modest to moderate heritability estimates (12% [95CI: 1-36%] - 45% [95CI: 16-69%]) were observed across measures purported to capture stress reactivity (peak, area under the curve [AUC], baseline-to-peak change). Findings also demonstrate both shared and unique genetic and environmental influences between baseline cortisol and cortisol reactivity. Minimal to no additional genetic innovations above and beyond the contributions of peak cortisol were found for other measures of cortisol reactivity such as AUC. This study is one of the largest twin-based samples to examine the heritability of cortisol reactivity, and results suggest that simpler measures of cortisol reactivity demonstrate higher heritability compared to more complex measurements.

Designing Aedes (Diptera: Culicidae) Mosquito Traps: The Evolution of the Male Aedes Sound Trap by Iterative Evaluation.

Effective surveillance of Aedes aegypti (Linnaeus, Diptera: Culicidae) is critical to monitoring the impact of vector control measures when mitigating disease transmission by this species. There are benefits to deploying male-specific traps, particularly when a high level of catch-specificity is desired. Here, the rationale behind the developmental process of an entirely new trap which uses a sound lure to capture male Ae. aegypti, the male Aedes sound trap (MAST), is presented as a target product profile with findings from developmental trials of key trap components and performance. Trial results suggest that the presence of a black base associated with the trap influenced male catches as did variations in size of this base, to a degree. Trap entrance shape didn't influence catch rates, but entrance size did. No significant differences in catch rates were found when sound lures were set to intermittent or continuous playbacks, at volumes between 63-74 dB or frequencies of 450 Hz compared to 500 Hz. Additionally, adult males aged 3 days post-eclosion, were less responsive to sound lures set to 500 Hz than those 4 or 6 days old. Lastly, almost no males were caught when the MAST directly faced continual winds of 1.5 ms-1, but males were captured at low rates during intermittent winds, or if the trap faced away from the wind. The developmental process to optimising this trap is applicable to the development of alternate mosquito traps beyond Aedes sound traps and provides useful information towards the improved surveillance of these disease vectors.

A Low-Powered and Highly Selective Trap for Male Aedes (Diptera: Culicidae) Surveillance: The Male Aedes Sound Trap.

As Aedes aegypti (Linnaeus, Diptera: Culicidae) expands its global distribution and vectors a range of debilitating arboviruses there is an increased need for enhanced mosquito surveillance. Consequently, we developed a Male Aedes Sound Trap (MAST) that requires minimal power and is highly species-specific. Two different versions of the MAST were developed, one that uses synthetic pyrethroid to kill captured mosquitoes (MAST Spray) and another which has an internal divider to create a killing chamber in which a sticky panel can be placed to capture mosquitoes (MAST Sticky). We compared weekly capture rates of male Ae. aegypti and bycatch from the two MAST versions to those from BG-Sentinel (BGS) traps and Sound-producing BG-Gravid Aedes Traps (SGATs) throughout Cairns, northern Australia. Weekly mean male Ae. aegypti catches did not significantly differ between trap types. However, the rate of positive weekly detections of male Ae. aegypti was lower for the MAST Sticky than the other three trap types. The MASTs sampled significantly fewer mosquitoes other than male Ae. aegypti, than either the BGS trap or the SGAT. Also, the MASTs and SGATs all caught significantly less non-Culicidae bycatch than the BGS traps. Consequently, we have developed a versatile male Ae. aegypti trap which is potentially of great benefit to Ae. aegypti surveillance programs.

Outcomes from international field trials with Male Aedes Sound Traps: Frequency-dependent effectiveness in capturing target species in relation to bycatch abundance.

Aedes aegypti and Aedes albopictus vector dengue, chikungunya and Zika viruses. With both species expanding their global distributions at alarming rates, developing effective surveillance equipment is a continuing priority for public health researchers. Sound traps have been shown, in limited testing, to be highly species-specific when emitting a frequency corresponding to a female mosquito wingbeat. Determining male mosquito capture rates in sound traps based on lure frequencies in endemic settings is the next step for informed deployment of these surveillance tools. We field-evaluated Male Aedes Sound Traps (MASTs) set to either 450 Hz, 500 Hz, 550 Hz or 600 Hz for sampling Aedes aegypti and/or Aedes albopictus and compared catch rates to BG-Sentinel traps within Pacific (Madang, Papua New Guinea) and Latin American (Molas, Mexico and Orange Walk Town, Belize) locations. MASTs set to 450-550 Hz consistently caught male Ae. aegypti at rates comparable to BG-Sentinel traps in all locations. A peak in male Ae. albopictus captures in MASTs set at 550 Hz was observed, with the lowest mean abundance recorded in MASTs set to 450 Hz. While significantly higher abundances of male Culex were sampled in MASTs emitting lower relative frequencies in Molas, overall male Culex were captured in significantly lower abundances in the MASTs, relative to BG-Sentinel traps within all locations. Finally, significant differences in rates at which male Aedes and Culex were positively detected in trap-types per weekly collections were broadly consistent with trends in abundance data per trap-type. MASTs at 550 Hz effectively captured both male Ae. aegypti and Ae. albopictus while greatly reducing bycatch, especially male Culex, in locations where dengue transmission has occurred. This high species-specificity of the MAST not only reduces staff-time required to sort samples, but can also be exploited to develop an accurate smart-trap system-both outcomes potentially reducing public health program expenses.

Functional characterization of the progestagen-associated endometrial protein gene in human melanoma.

Utilizing gene microarray profiling of melanoma samples, we have recently identified a novel gene overexpressed in both thick primary and metastatic melanomas. This gene, progestagen-associated endometrial protein (PAEP), has never before been implicated in the oncogenic processes of melanoma, with its true function in oncogenesis and tumour progression relatively unknown. Overexpression of the PAEP gene in freshly procured thick primary and metastatic melanoma samples (58%) and daughter cell lines (77%) is confirmed by quantitative RT-PCR, immunohistochemistry, Western blotting and mass spectrometric analysis. We suggest that PAEP gene overexpression is involved with melanoma tumour progression as well as an aggressive phenotype. Transfection of melanoma cells with PAEP small interfering RNA (siRNA) reveals a significant decrease in soft agar colony formation and a marked inhibition of both cell migration and cell invasion. Furthermore, we establish stable melanoma transfectants via PAEP lentiviral small hairpin RNA (shRNA), examine their growth characteristics in a murine xenograft model and reveal that tumour growth is significantly inhibited in two separate melanoma cell lines. Our data strongly implicate the PAEP gene as a tumour growth promoter with oncogenic properties and a potential therapeutic target for patients with advanced melanoma.

Osteoporosis treatment following hip fracture: how rates vary by service.

OBJECTIVES: Osteoporosis is a prevalent condition among older people. It is often undiagnosed until patients suffer fragility fractures. Previous studies have shown low rates of initiating osteoporosis treatment during the acute hip fracture hospitalization. It is not clear if this varies by the treating service. We compared the rates of instituting osteoporosis treatment during the acute hospitalization for fragility hip fractures. METHODS: Rates of initiating treatment among previously untreated patients were compared between the orthopedic, medicine, and rehabilitation services using retrospective cross-sectional chart review at an academic medical center. Between January 2005 and August 2008, 191 patients admitted with a fragility hip fracture survived to be discharged from the hospital. RESULTS: There were 67 (35%) patients who were started on some form of osteoporosis treatment during their acute hospital stay. Factors statistically associated with starting treatment included having a discharge diagnosis of osteoporosis (P < 0.0001) and treating service (P < 0.0001). Orthopedics was the least likely of the 3 treating services to initiate treatment, while medicine was the most likely. CONCLUSIONS: Overall rates of osteoporosis treatment initiation were low at 35% of the 191 patients' records surveyed. Efforts to increase adherence during the acute hospital stay should be explored. A promising intervention includes instituting an osteoporosis consultative service to improve the likelihood of starting osteoporosis treatment post fragility fracture.

Investigating Male Aedes aegypti (Diptera: Culicidae) Attraction to Different Oviposition Containers Using Various Configurations of the Sound Gravid Aedes Trap.

Aedes aegypti (Linnaeus), the primary vectors of the arboviruses dengue virus and Zika virus, continue to expand their global distributions. In efforts to better control such species, several mosquito control programs are investigating the efficacy of rearing and releasing millions of altered male Aedes throughout landscapes to reduce populations and disease transmission risk. Unfortunately, little is known about Ae. aegypti, especially male, dispersal behaviors within urban habitats. We deployed Sound-producing Gravid Aedes Traps (SGATs) in Cairns, northern Australia, to investigate male Ae. aegypti attraction to various oviposition container configurations. The traps were arranged to include: 1) water only, 2) organically infused water, 3) infused water and L3 larvae, 4) infused water and a human-scented lure, and lastly 5) no water or olfactory attractant (dry). Our data suggest that males were more attracted to SGATs representing active larval sites than potential larval sites, but were equally attracted to dry SGATs relative to those containing water and/or infusion. Additionally, we found that female Ae. aegypti were equally attracted to wet SGATs, with or without infusion, but not dry ones. These results suggest that male Ae. aegypti within northern Australia are more attracted to active larval sites and equally attracted to dry containers as wet or infused ones. Additionally, female Ae. aegypti are unlikely to enter dry containers. Such findings contribute to our understanding of potentially attractive features for local and released Ae. aegypti throughout the northern Australian urban landscape.

Efficient production of male Wolbachia-infected Aedes aegypti mosquitoes enables large-scale suppression of wild populations.

The range of the mosquito Aedes aegypti continues to expand, putting more than two billion people at risk of arboviral infection. The sterile insect technique (SIT) has been used to successfully combat agricultural pests at large scale, but not mosquitoes, mainly because of challenges with consistent production and distribution of high-quality male mosquitoes. We describe automated processes to rear and release millions of competitive, sterile male Wolbachia-infected mosquitoes, and use of these males in a large-scale suppression trial in Fresno County, California. In 2018, we released 14.4 million males across three replicate neighborhoods encompassing 293 hectares. At peak mosquito season, the number of female mosquitoes was 95.5% lower (95% CI, 93.6-96.9) in release areas compared to non-release areas, with the most geographically isolated neighborhood reaching a 99% reduction. This work demonstrates the high efficacy of mosquito SIT in an area ninefold larger than in previous similar trials, supporting the potential of this approach in public health and nuisance-mosquito eradication programs.

Author Correction: Efficient production of male Wolbachia-infected Aedes aegypti mosquitoes enables large-scale suppression of wild populations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Authentication scheme for routine verification of genetically similar laboratory colonies: a trial with Anopheles gambiae.

BACKGROUND: When rearing morphologically indistinguishable laboratory strains concurrently, the threat of unintentional genetic contamination is constant. Avoidance of accidental mixing of strains is difficult due to the use of common equipment, technician error, or the possibility of self relocation by adult mosquitoes ("free fliers"). In many cases, laboratory strains are difficult to distinguish because of morphological and genetic similarity, especially when laboratory colonies are isolates of certain traits from the same parental strain, such as eye color mutants, individuals with certain chromosomal arrangements or high levels of insecticide resistance. Thus, proving genetic integrity could seem incredibly time-consuming or impossible. On the other hand, lacking proof of genetically isolated laboratory strains could question the validity of research results. RESULTS: We present a method for establishing authentication matrices to routinely distinguish and confirm that laboratory strains have not become physically or genetically mixed through contamination events in the laboratory. We show a specific example with application to Anopheles gambiae sensu stricto strains at the Malaria Research and Reference Reagent Resource Center. This authentication matrix is essentially a series of tests yielding a strain-specific combination of results. CONCLUSION: These matrix-based methodologies are useful for several mosquito and insect populations but must be specifically tailored and altered for each laboratory based on the potential contaminants available at any given time. The desired resulting authentication plan would utilize the least amount of routine effort possible while ensuring the integrity of the strains.

The 14-3-3sigma gene promoter is methylated in both human melanocytes and melanoma.

BACKGROUND: Recent evidence demonstrates that 14-3-3sigma acts as a tumor suppressor gene inactivated by methylation of its 5' CpG islands in epithelial tumor cells, while remaining un-methylated in normal human epithelia. The methylation analysis of 14-3-3sigma has been largely overlooked in melanoma. METHODS: The methylation status of 14-3-3sigma CpG island in melanocytes and melanoma cells was analyzed by methylation-specific sequencing (MSS) and quantitative methylation-specific PCR (Q-MSP). 14-3-3sigma mRNA and protein expression in cell lines was detected by real-time RT-PCR and western blot. Melanoma cells were also treated by 5-aza-2'-deoxycytidine (DAC), a demethylating agent, and/or histone deacetylase inhibitor, Trichostatin A (TSA), to evaluate their effects on 14-3-3sigma gene expression. RESULTS: 14-3-3sigma is hypermethylated in both human melanocytes and most melanoma cells in a lineage-specific manner, resulting in the silencing of 14-3-3sigma gene expression and the active induction of 14-3-3sigma mRNA and protein expression following treatment with DAC. We also observed a synergistic effect upon gene expression when DAC was combined with TSA. The promoter methylation status of 14-3-3sigma was analyzed utilizing Q-MSP in 20 melanoma tissue samples and 10 cell lines derived from these samples, showing that the majority of melanoma samples maintain their hypermethylation status of the 14-3-3sigma gene. CONCLUSION: 14-3-3sigma is hypermethylated in human melanoma in a cell-linage specific manner. Spontaneous demethylation and re-expression of 14-3-3sigma is a rare event in melanoma, indicating 14-3-3sigma might have a tentative role in the pathogenesis of melanoma.

Epigenetics in human melanoma.

BACKGROUND: Recent technological advances have allowed us to examine the human genome in greater detail than ever before. This has opened the door to an improved understanding of the gene expression patterns involved with cancer. METHODS: A review of the literature was performed to determine the role of epigenetic modifications in human melanoma. We focused the search on histone deacetylation, methylation of gene promoter regions, demethylation of CpG islands, and the role of microRNA. We examined the relationship between human melanoma epigenetics and their importance in tumorigenesis, tumor progression, and inhibition of metastasis. The development and clinical application of select pharmacologic agents are also discussed. RESULTS: We identified several articles that have extensively studied the role of epigenetics in melanoma, further elucidating the complex processes involved in gene regulation and expression. Several new agents directly affect epigenetic mechanisms in melanoma, with divergent affects on the metastatic potential of melanoma. CONCLUSIONS: Epigenetic mechanisms have emerged as having a central role in gene regulation of human melanoma, including the identification of several putative tumor suppressor genes and oncogenes. Further research will focus on the development of novel therapeutics that will likely target and alter such epigenetic changes.