Optimized protocol for the identification of lipid droplet proteomes using proximity labeling proteomics in cultured human cells.

Lipid droplets are endoplasmic reticulum-derived neutral lipid storage organelles that play critical roles in cellular lipid and energy homeostasis. Here, we present a protocol for the identification of high-confidence lipid droplet proteomes in a cell culture model. This approach overcomes limitations associated with standard biochemical fractionation techniques, employing an engineered ascorbate peroxidase (APEX2) to biotinylate endogenous lipid droplet proteins in living cells for subsequent purification and identification by proteomics. For complete details on the use and execution of this protocol, please refer to Bersuker et al. (2018).

A Proximity Labeling Strategy Provides Insights into the Composition and Dynamics of Lipid Droplet Proteomes.

Lipid droplet (LD) functions are regulated by a complement of integral and peripheral proteins that associate with the bounding LD phospholipid monolayer. Defining the composition of the LD proteome has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions. To overcome this limitation, we developed a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. Application of this approach to two different cell types identified the vast majority of previously validated LD proteins, excluded common contaminating proteins, and revealed new LD proteins. Moreover, quantitative analysis of LD proteome dynamics uncovered a role for endoplasmic reticulum-associated degradation in controlling the composition of the LD proteome. These data provide an important resource for future LD studies and demonstrate the utility of proximity labeling to study the regulation of LD proteomes.

Lipid disequilibrium disrupts ER proteostasis by impairing ERAD substrate glycan trimming and dislocation.

The endoplasmic reticulum (ER) mediates the folding, maturation, and deployment of the secretory proteome. Proteins that fail to achieve their native conformation are retained in the ER and targeted for clearance by ER-associated degradation (ERAD), a sophisticated process that mediates the ubiquitin-dependent delivery of substrates to the 26S proteasome for proteolysis. Recent findings indicate that inhibition of long-chain acyl-CoA synthetases with triacsin C, a fatty acid analogue, impairs lipid droplet (LD) biogenesis and ERAD, suggesting a role for LDs in ERAD. However, whether LDs are involved in the ERAD process remains an outstanding question. Using chemical and genetic approaches to disrupt diacylglycerol acyltransferase (DGAT)-dependent LD biogenesis, we provide evidence that LDs are dispensable for ERAD in mammalian cells. Instead, our results suggest that triacsin C causes global alterations in the cellular lipid landscape that disrupt ER proteostasis by interfering with the glycan trimming and dislocation steps of ERAD. Prolonged triacsin C treatment activates both the IRE1 and PERK branches of the unfolded protein response and ultimately leads to IRE1-dependent cell death. These findings identify an intimate relationship between fatty acid metabolism and ER proteostasis that influences cell viability.

Home use of two doses of misoprostol after mifepristone for medical abortion: a pilot study in Sweden and France.

OBJECTIVE: To test the feasibility, safety, and efficacy of home use of two doses of misoprostol for medical abortion (MA) in European settings. METHODS: One hundred thirty women (100 in Sweden, 30 in France) presenting for first-trimester MA were administered oral mifepristone in the clinic and sent home with two 400 microg doses of misoprostol, along with instructions to take the misoprostol at 24 h intervals. Women were also asked to complete a daily symptom diary. Outcomes of interest included effectiveness, side-effects, and adherence to and acceptability of the home-use regimen. RESULTS: Three women (all in France) were lost to follow-up. Of the remaining 127 women, 124 (98%) had a successful MA. All women adhered successfully to the home-use regimen, and satisfaction with home use was high (98%). Most women experienced noticeable, if transitory, side effects after both the first and second doses of misoprostol (97% and 94%, respectively). CONCLUSIONS: Misoprostol may successfully and satisfactorily be used at home as part of a MA regimen in European settings as it has been for years in the US. Further research to determine if two doses of misoprostol are more effective than a single dose would be useful.

Premarital sex, schoolgirl pregnancy, and school quality in rural Kenya.

Using data from nearly 600 adolescents aged 12-19 in combination with data collected from 33 primary schools that the adolescents attended, this report explores whether certain aspects of the school environment affect the initiation of premarital sex among girls and boys in three districts of Kenya. The results suggest that, although neither the school nor the home appears to influence whether boys engage in sex prior to marriage, for girls, a school characterized by a gender-neutral atmosphere appears to reduce the risk of their engaging in premarital sex. Furthermore, although policymakers in Kenya are clearly concerned with the problem of "schoolgirl pregnancy," the data indicate that in this sample, pregnancy is not the primary reason that girls leave school.

Dermal squamo-melanocytic tumor: a unique biphenotypic neoplasm of uncertain biological potential.

We report four cases of an unusual cutaneous squamo-melanocytic neoplasm with histological features of malignancy and uncertain biological potential. These tumors developed on the face of middle-aged and older adults. Clinically, a purple-black nodule ranged in size from 3 to 10 mm in maximum diameter. After complete excision, neither recurrence nor metastasis has been observed (mean follow-up time, 3.25 years). Histologically, a discrete dermal nodule surrounded by a fibroblastic stroma was composed of large islands of mitotically active atypical epithelioid cells. The nodule was not connected to the epidermis in three of four cases. Two types of cells were either diffusely admixed or clustered in small groups within the nodule. Small, atypical, epithelioid cells containing finely granular brown pigment, proven to be melanin, constituted the first cell type. The second type consisted of atypical squamoid cells, some with abundant pink cytoplasm, giving rise to squamous pearls. A lentigo maligna was present in one case. The remaining three cases had neither significant intraepidermal melanocytic nor keratinocytic atypia. Immunohistochemical studies indicated that the melanin-containing epithelioid cells expressed S-100 antigens, and the squamoid cells expressed cytokeratins. A small population of tumor cells did not label with either of the antibodies. These four tumors (along with a previously reported, apparently identical tumor arising in the setting of lentigo maligna) represent a unique biphasic dermal neoplasm with histological features of malignancy but, at this time, uncertain biological behavior. Although none have recurred or metastasized, the follow-up time is too short in our estimation to guarantee a benign course. These neoplasms are easily recognized by their characteristic features. Further follow-up evaluations should allow determination of their biologic potential.

The changing nature of adolescence in the Kassena-Nankana District of northern Ghana.

This study reports the results of a primarily qualitative investigation of adolescent reproductive behavior in the Kassena-Nankana District, an isolated rural area in northern Ghana, where traditional patterns of marriage, family formation, and social organization persist. The study is based on in-depth interviews and focus-group discussions with adolescents, parents, chiefs, traditional leaders, youth leaders, and health workers, supplemented by quantitative data from the 1996 wave of a panel survey of women of reproductive age conducted by the Navrongo Health Research Centre. The social environment that adolescent boys and girls in the Kassena-Nankana District encounter and its links to reproductive behavior are described. The principal question is whether even in this remote rural area, the social environment has been altered in ways that have undermined traditional sexual and reproductive patterns. The survey data indicate a considerable increase in girls' education and the beginning of a decline in the incidence of early marriage. The qualitative data suggest that social institutions, systems, and practices such as female circumcision that previously structured the lives of adolescent boys and girls have eroded, leading to an apparent increase in premarital sexual activity.

Atypical melanocytic nevi of the genital type with a discussion of reciprocal parenchymal-stromal interactions in the biology of neoplasia.

UNLABELLED: Melanocytic lesions of the genital area, especially those on the vulva, may present great difficulty in histological interpretation. A histological diagnosis of malignant melanoma was made in more than one third of 56 genital area melanocytic lesions submitted in consultation to the authors. The median age of the patients with these lesions was 25 years. This article is a clinicopathological study of these lesions and distinguishes them from malignant melanoma. The stroma of the lesions of the genital area was different from the stroma seen in the dysplastic nevi and melanoma. The differences in the stromal form in the diverse lesions is useful in diagnosis and is of profound biological significance. The stroma in the reported lesions and in some lesions of melanocytic neoplasia is described in detail, and its biological significance is discussed. Three sets of cases are used in this comparative study to delineate the clinicopathological nature and the biology of the genital nevi. The 56 cases submitted in consultation constitute the primary series of our work (The Clark Cutaneous Pathology Laboratory Series). These are compared with a series of cases from the Pigmented Lesion Group of the University of Pennsylvania and Pathology Services, Inc, and another series of cases from the Genetic Epidemiology Branch of the National Cancer Institute. The two series used for comparative study contain approximately the same number of cases of dysplastic nevi and malignant melanoma as there are atypical melanocytic nevi of the genital type in the primary series. The total number of cases was studied by comparison of their attributes in a relational database. The clinical data of the primary series was acquired through the use of a questionnaire completed by the contributors. The 56 cases presented two distinctive pathological pictures. One of these is termed atypical melanocytic nevi of the genital type (AMNGT), whereas dysplastic nevi (DN) formed the second of the two pathological pictures. There were 36 AMNGT and 14 DN. The remaining six cases were common nevi without atypia or ill-defined melanocytic hyperplasias. The lesions of AMNGT are usually located on the vulva, but they are seen on the perineum and, rarely, on the mons pubis and in the axilla. Lesions similar to AMNGT have been seen uncommonly on the male genitalia. The stromal patterns were distinctive and related to specific melanocytic lesions. An unclassified (unclass) or nonspecific stromal pattern was associated with AMNGT; a pattern of regression with differentiation (diff-regress) dominated the stroma of common dermal nevi; concentric eosinophilic fibroplasia (cef) and lamellar fibroplasia (lf) were present in dysplastic nevi; fibroplasia with a plaquelike lymphocytic infiltrate (fl) and diffuse eosinophilic fibroplasia (def) were noted in radial growth phase melanoma; and fibroplasia with angiogenesis (fa) or an absence of evidence for parenchymal stromal reciprocal interactions (nopsi) marked thick or deeply invasive vertical growth phase melanomas. Recommendations for management of the lesions are suggested. CONCLUSIONS: One kind of atypical melanocytic proliferation in the genital area forms a distinctive clinicopathological entity that can be distinguished from melanoma and dysplastic nevi, the AMNGT. Such lesions are more common on the labia minora or the mucosa of the clitoral region than they are on the labia majora. The other common atypical melanocytic proliferation of this area is a dysplastic nevus, which is much more common on the labia majora than on the labia minora. The reciprocal interactions between parenchyma and stroma are discussed as homeostatic processes, a continually functioning template maintaining tissue, organ, and organismal form and function. The progressive disorganization of this template in neoplasia is illustrated and is considered to be a cardinal element in the biology of neoplasia.

Immunological characterisation of the acrosomal filament in the marine shrimp Sicyonia ingentis.

During sperm/egg interaction, the sperm of Sicyonia ingentis undergoes a unique biphasic acrosome reaction. After acrosomal vesicle exocytosis, the sperm generates an acrosomal filament, over a period of 4-6 min, that is approximately 10 microns in length. Neither actin filaments nor normal microtubules have been demonstrated at the ultrastructural level of this unusual filament. Using a battery of cytoskeleton-directed antibodies the biochemical nature of this filament has been investigated. Antibodies to actin and tubulin do not label the subacrosome or acrosomal filament, but do recognise actin and tubulin in other shrimp tissues. Antibodies to tau, MAP2, and neurofilament medium and heavy subunits were all localised to the subacrosomal region of the sperm. It is interesting, however, that only the two clones of neurofilament monoclonal antibodies recognised the acrosomal filament. Electrophoretic and Western blot analysis in conjunction with amino acid sequencing revealed that the proteins localised to the acrosomal filament are of a unique sequence and may represent a new type of protein of centrosomal origin.

Molecular phylogeny and biogeography of the marine shrimp Penaeus.

The evolutionary relationships among 13 species representing all six subgenera of the shrimp genus Penaeus were examined using 558 bp of mitochondrial (mt) DNA from the cytochrome oxidase subunit I gene. Analyses of this sequence revealed high genetic divergence between species (d = 8-24%), a finding which contrasts with previous work, which indicated that genetic diversity, based on electrophoretic analysis of allozymes, was extremely low in Penaeus. Three tree-building methods (maximum parsimony, neighbor joining, and maximum likelihood) were concordant in indicating that current subgenera assignments do not reflect evolutionary partitions within the genus Penaeus. While the molecular phylogenies cast doubt on the validity of subgenera, the observed relationships are concordant with biogeographic boundaries across the tropical range of Penaeus. Both the western Atlantic and eastern Pacific contain monophyletic species pairs which cluster together in all analyses. The Indo-Pacific contains a putative basal taxa (P. indicus), the deepest mtDNA lineages, and the highest diversity, including representatives of all three primary lineages observed in Penaeus. These data are consistent with the suggestion by Dall et al. (1990) that Penaeus arose in the Indo-Pacific and radiated eastward and westward to account for the current circumtropical distribution of the genus. This phylogenetic framework for Penaeus will enhance the scientific foundations for wildlife resource management and breeding experiments (hybridization and related manipulations) designed to improve the commercial value of captive strains.

Clinically recognized dysplastic nevi. A central risk factor for cutaneous melanoma.

OBJECTIVE: To investigate the relationship of number and type of nevi to the development of melanoma. DESIGN: Case-control study. SETTING: Outpatient clinics in referral hospitals. PATIENTS: Cases were 716 consecutive patients with newly diagnosed melanoma identified at 2 melanoma centers between January 1, 1991, and December 31, 1992. Stratified random sampling of patients from outpatient clinics was used to identify 1014 participating controls of the same age, sex, race, and geographic distribution as the melanoma cases. All study subjects underwent an interview, a complete skin examination, photography of the most atypical nevi, and, if the patient was willing, a biopsy of the most atypical nevus. MAIN OUTCOME MEASURES: Number and type of nevi on the entire body were systematically reported. All diagnoses of clinically dysplastic nevi were confirmed by expert examiners. RESULTS: Risk for melanoma was strongly related to number of small nevi, large nondysplastic nevi, and clinically dysplastic nevi. In the absence of dysplastic nevi, increased numbers of small nevi were associated with an approximately 2-fold risk, and increased numbers of both small and large nondysplastic nevi were associated with a 4-fold risk. One clinically dysplastic nevus was associated with a 2-fold risk (95% confidence interval, 1.4-3.6), while 10 or more conferred a 12-fold increased risk (95% confidence interval, 4.4-31). Congenital nevi were not associated with increased risk of melanoma. CONCLUSIONS: Although nondysplastic nevi confer a small risk, clinically dysplastic nevi confer substantial risk for melanoma. On the basis of nevus number and type, clinicians can identify a population at high risk of this epidemic cancer for screening and intervention.

Cell-cell association directed mitotic spindle orientation in the early development of the marine shrimp Sicyonia ingentis.

During early cleavages of Sicyonia ingentis embryos, mitotic spindle orientations differ between blastomeres and change in a predictable manner with each successive mitosis. From 2nd through 7th cleavages, spindles orient at a 90 degrees angle with respect to the spindle of the parent blastomere. Thus, spindle orientation is parallel to the cleavage plane that formed the blastomere. To determine if specific spindle orientations were intrinsic properties of individual blastomeres, we altered blastomere associations and asked how mitotic spindle orientation was affected in successive cleavages using laser scanning confocal microscopy. Linear embryos were constructed by dissociating 4-cell embryos and recombining the blastomeres in a linear array. The ensuing cleavage (3rd embryonic cleavage) of these linear embryos was parallel to the long axis of the embryo, resulting in four parallel pairs of blastomeres which lay in a common plane that was parallel to the substratum. The 4th cleavage produced a linear embryo with the 16 blastomeres arranged in four parallel quartets. Then, in preparation for 5th cleavage, spindles oriented at a 45 degrees angle (not parallel as in normal development) with respect to the previous cleavage plane. When 8-cell linear embryos were separated into linear half-embryos, subsequent spindle orientations were not like those observed for intact 8-cell linear embryos, but rather regressed to the orientation seen in 4-cell linear embryos. We suggest that the reorientation of mitotic spindles during early cleavage of S. ingentis is neither an intrinsic property nor age dependent, but rather is cell contact related. Further, these results in conjunction with observations of non-manipulated embryos suggest that spindle poles (centrosomes) avoid cytoplasmic regions adjacent to where there is cell-cell contact during early development.

Apparent anticipation in familial melanoma.

Genetic anticipation refers to progressively earlier age at diagnosis (AAD) and/or increasing severity of a disorder in successive generations. Previous examination of 23 familial melanoma kindreds revealed evidence for a dramatic reduction in AAD in successive generations. To further evaluate evidence for anticipation, we examined the AAD, number of tumours, and tumour thickness in affected parent-offspring (P-O) pairs from these 23 kindreds. Number of tumours and tumour thickness were also evaluated by generation. There were statistically significant intergenerational differences for AAD and number of tumours in the 47 affected P-O pairs. Median AAD decreased from 48 years to 28 years. The median AAD in the offspring (AADo) of a parent with CMM (median 28, mean 29.3 +/- 10.1 years, n = 51) was significantly different from the median AADo of a parent without CMM (42, 42.3 +/- 13.9 years, n = 55) (P < 0.001). In addition, parents with AAD less than the median of 48 years had offspring with a median AAD (26, 25.8 +/- 8.5 years, n = 21) significantly different from those of parents with AAD > or = 48 years (32, 34.5 +/- 10.2 years, n = 26) (P = 0.005). Ninety-four percent (44/47) of the P-O pairs showed positive anticipation, with 62% showing anticipation of > 15 years. There was little difference based on the affected parent's sex. In the 106 CMM cases there were no significant differences in tumour number (P = 0.08) or tumour thickness (P = 0.85) by generation. Number of tumours was however significantly different in cases with AAD < 34 years (n = 52, median 1.5, mean 2.3 +/- 2.4) vs cases with AAD > or = 34 years (n = 54, 1.0, 1.6 +/- 1.7) (P < 0.001). Thus these 23 familial melanoma kindreds showed evidence for anticipation as defined by a decrease in AAD in successive generations. Although increased surveillance may partly explain the results, additional studies should evaluate melanoma risk factors, genetic and/or environmental, across generations to examine the reasons for the apparent anticipation.

A prognostic model for predicting 10-year survival in patients with primary melanoma. The Pigmented Lesion Group.

OBJECTIVE: To develop a prognostic model, based on clinical and pathologic data that are routinely available to the clinician, that would estimate the chance for survival of a patient with primary cutaneous melanoma after definitive surgical therapy. DESIGN: Cohort analytical study. SETTING: University medical center. PATIENTS: 488 patients with primary cutaneous melanoma who had no apparent metastatic disease. Patients were followed prospectively for at least 10 years. An independent validation sample of 142 patients was used to assess the stability of the model. MEASUREMENTS: Six clinical and pathologic variables that predict survival and are readily available to the clinician were used to develop a prediction model. The variables were tested for their association with death by using a univariate logistic regression model. Point estimates were generated for the probability of surviving melanoma at 10 years. Variables that were statistically significantly associated with survival were retained for testing in a logistic regression model. RESULTS: 488 patients were followed prospectively for a median of 13.5 years (minimum, 10.0 years; maximum, 20.5 years). The overall 10-year survival of the study group was 78%. Four variables were found to be independent predictors of survival. Presented as adjusted odds ratios, from strongest to weakest relative predictive strength, these variables were tumor thickness (odds ratio, 50.8), site of primary melanoma (odds ratio, 4.4), age of the patient (odds ratio, 3.0), and sex of the patient (odds ratio, 2.0). The four-variable model was significantly more accurate than tumor thickness alone, particularly for predicting death. Overall, use of the model reduced the error rate of the prediction of death by 50%. CONCLUSIONS: A prognostic model that uses four readily accessible variables more accurately predicts outcome in patients with primary melanoma than does tumor thickness alone. This four-variable model can identify patients at high risk for the recurrence of disease, an identification that becomes increasingly important as adjuvant therapies are developed for treatment of melanoma.

Two-locus linkage analysis of cutaneous malignant melanoma/dysplastic nevi.

Previous linkage analyses of 19 cutaneous malignant melanoma/dysplastic nevi (CMM/DN) kindreds showed significant evidence of linkage and heterogeneity to both chromosomes 1p and 9p. Five kindreds also showed evidence of linkage (Z>0.7) to both regions. To further examine these findings, we conducted two-trait-locus, two-marker-locus linkage analysis. We examined one homogeneity and one heterogeneity single-locus model (SL-Hom and SL-Het), and two-locus (2L) models: an epistatic model (Ep), in which CMM was treated as a genuine 2L disease, and a heterogeneity model (Het), in which CMM could result from disease alleles at either locus. Both loci were modeled as autosomal dominant. The LOD scores for CMM alone were highest using the SL-Het model (Z = 8.48, theta = .0). There was much stronger evidence of linkage to chromosome 9p than to 1p for CMM alone; the LOD scores were approximately two times greater on 9p than on 1p. The change in LOD scores from an evaluation of CMM alone to CMM/DN suggested that a chromosome 1p locus (or loci) contributed to both CMM and CMM/DN, whereas a 9p locus contributed more to CMM alone. For both 2L models, the LOD scores from 1p were greater for CMM/DN than for CMM alone (Ep: Z=4.63 vs. 3.83; Het: 4.94 vs. 3.80, respectively). In contrast, for 9p, the LOD scores were substantially lower with CMM/DN than with CMM alone (Ep: 4.64 vs. 7.06; Het: 5.38 vs. 7.99, respectively). After conditioning on linkage to the other locus, only the 9p locus consistently showed significant evidence for linkage to CMM alone. Thus, the application of 2L models may be useful to help unravel the complexities of familial melanoma.

Melanocytic nevi, dysplastic nevi, and malignant melanoma in children from melanoma-prone families.

BACKGROUND: Melanocytic nevi, particularly dysplastic nevi (DN), are important markers of increased risk of malignant melanoma in adults, but little is known about their prevalence and relation to melanoma in children. OBJECTIVE: Our purpose was to define the prevalence of DN, number of nevi, and their relation to the risk of melanoma in children younger than 20 years of age from melanoma-prone families. METHODS: One hundred twenty-five persons younger than 20 years of age, from 23 melanoma-prone families, underwent clinical evaluation with nevus counts, photography, and biopsy of suspected melanocytic lesions and were observed for development of DN and melanoma. RESULTS: In melanoma-prone families, 37% of children had DN. The patients were divided into four categories: those with melanoma, DN (without melanoma), indeterminant (largely because of age at examination), and unaffected. The risk of melanoma was assessed by nevus number and presence of DN. High nevus number was strongly correlated with the presence of DN. The risk of the development of melanoma in children from melanoma-prone families appeared most related to the presence of DN (relative risk, 45; 95% confidence intervals, 2.6-786.4) and started at an early age. Of note, all children in whom melanoma developed had DN. CONCLUSION: Family history of melanoma and the presence of DN defines children with a high risk for melanoma developing at an early age.