Understanding amphidromy in Hawai'i: 'O'opu nakea (Awaous stamineus).

Hawai'i is home to 'o'opu nakea (Awaous stamineus), a culturally significant, endemic, goby that exhibits an amphidromous life cycle characterized by a marine larval stage followed by post-larval recruitment to streams, where they live to become reproductive adults. However, it was recently suggested that their migration to the ocean might not be obligatory, as originally thought. Despite their importance in Hawaiian traditions and the ecology of Hawaiian freshwater ecosystems, we still lack a full understanding of their migratory patterns and life history due to the difficulties in determining the environmental migratory cues that set the timing and location of their migratory paths. This study examined environmental factors, such as mean annual rainfall, streamflow, and water chemistry, to determine if they play a role in whether A. stamineus spend their larval period in the ocean or their entire life cycle in freshwater streams. We sampled A. stamineus (n = 90) from three streams (Kahana, Kahalu'u, and Waimanalo) on the island of O'ahu, Hawai'i that represented the range of hydroclimatic gradient in wet-habitat conditions on the windward side of the island and characterized their migratory pattern using elemental analysis of sagittae, the largest pair of otoliths (calcareous ear structures). Based on otolith strontium:calcium and barium:calcium ratios, we determined if individuals spent their larval period in the ocean or the stream. We found that 100% of individuals displayed clear evidence of marine residence during their larval phase, regardless of the environmental factors the fish experienced. This study highlights the necessity of stream-ocean connectivity for the survival of A. stamineus and emphasizes the importance of stream-mouth conservation and management as it is a critical transition zone in stream-ocean-stream migratory pathways.

Reimagining drug manufacturing paradigm in today's pharmacy landscape.

The coronavirus disease 2019 pandemic has escalated the ongoing problem of critical medication shortages, which has serious implications for the health of our patients. Currently, active pharmaceutical ingredients (APIs) are synthesized in large-scale batch operations and shipped to drug product manufacturers, where they are produced on a large scale at centralized facilities. In the centralized drug manufacturing process, the formulation components, operations, and packaging are structured to favor long-term storage and shipment of resultant medicines to the point of care, making this process vulnerable to supply chain disruptions. We propose a rethinking of the drug manufacturing paradigm with an upgraded pharmaceutical compounding-based manufacturing paradigm. This paradigm will be based on integration of continuous manufacturing of APIs and manufacturing of medicines at the point of care with application of machine learning, artificial intelligence, and 3-dimensional printing. This paradigm will support implementation of precision medicine and customization according to patients' needs. The new model of drug manufacturing will be less dependent on the supply chain while ensuring availability of medicines in a cost-effective manner.

RNF168-Mediated Ubiquitin Signaling Inhibits the Viability of BRCA1-Null Cancers.

BRCA1 gene mutations impair homologous recombination (HR) DNA repair, resulting in cellular senescence and embryonic lethality in mice. Therefore, BRCA1-deficient cancers require adaptations that prevent excessive genomic alterations from triggering cell death. RNF168-mediated ubiquitination of γH2AX at K13/15 (ub-H2AX) serves as a recruitment module for the localization of 53BP1 to DNA break sites. Here, we found multiple BRCA1-mutant cancer cell lines and primary tumors with low levels of RNF168 protein expression. Overexpression of ectopic RNF168 or a ub-H2AX fusion protein induced cell death and delayed BRCA1-mutant tumor formation. Cell death resulted from the recruitment of 53BP1 to DNA break sites and inhibition of DNA end resection. Strikingly, reintroduction of BRCA1 or 53BP1 depletion restored HR and rescued the ability of cells to maintain RNF168 and ub-H2AX overexpression. Thus, downregulation of RNF168 protein expression is a mechanism for providing BRCA1-null cancer cell lines with a residual level of HR that is essential for viability. Overall, our work identifies loss of RNF168 ubiquitin signaling as a proteomic alteration that supports BRCA1-mutant carcinogenesis. We propose that restoring RNF168-ub-H2AX signaling, potentially through inhibition of deubiquitinases, could represent a new therapeutic approach. SIGNIFICANCE: This study explores the concept that homologous recombination DNA repair is not an all-or-nothing concept, but a spectrum, and that where a tumor stands on this spectrum may have therapeutic relevance.See related commentary by Wang and Wulf, p. 2720.

Trait-based characterization of species transported on Japanese tsunami marine debris: Effect of prior invasion history on trait distribution.

Nearly 300 coastal marine species collected from >630 debris items from the 2011 Great East Japan earthquake and tsunami have landed alive along the North American Pacific coast and the Hawaiian Archipelago. We synthesized life history, environmental, and distributional traits for 103 of these species and compared species with (n=30) and without (n=62) known invasion histories. The species represent 12 phyla, and Mollusca, Crustacea, and Bryozoa accounted for 71 of the 103 species. The majority are native to the Northwest Pacific and the Central Indo-Pacific. Species with known invasion history were more common on artificial and hardpan substrates, in temperate reef, fouling, and flotsam habitats, at subtropical and tropical temperatures, and exhibited greater salinity tolerance than species with no prior invasion history. Thirty-five Japanese tsunami marine species without prior invasion history overlapped in ordination trait space with known invaders, indicating a subset of species in this novel assemblage that possess traits similar to species with known invasion history.

Transoceanic dispersal of the mussel Mytilus galloprovincialis on Japanese tsunami marine debris: An approach for evaluating rafting of a coastal species at sea.

Biofouled debris from the 2011 Great East Japan earthquake and tsunami has landed in the Northeast Pacific and along the Hawaiian Islands since 2012. As of 2017, >630 biofouled debris items with >320 living species of algae, invertebrates, and fish have been examined. The invasive mussel Mytilus galloprovincialis was present on >50% of those items. Size, reproduction, and growth of this filter-feeding species were examined to better understand long-distance rafting of a coastal species. The majority of mussels (79%) had developing or mature gametes, and growth rates averaged 0.075±0.018 SE mm/day. Structural and elemental (barium/calcium) analysis of mussel shells generated estimates of growth in coastal waters (mean=1.3 to 25mm total length), which provides an indication of residence times in waters along North America and the Hawaiian Islands prior to landing. Detailed studies of individual species contribute to our understanding of debris as a transport vector and aid efforts to evaluate potential risks associated with marine debris.

Tsunami-driven rafting: Transoceanic species dispersal and implications for marine biogeography.

The 2011 East Japan earthquake generated a massive tsunami that launched an extraordinary transoceanic biological rafting event with no known historical precedent. We document 289 living Japanese coastal marine species from 16 phyla transported over 6 years on objects that traveled thousands of kilometers across the Pacific Ocean to the shores of North America and Hawai'i. Most of this dispersal occurred on nonbiodegradable objects, resulting in the longest documented transoceanic survival and dispersal of coastal species by rafting. Expanding shoreline infrastructure has increased global sources of plastic materials available for biotic colonization and also interacts with climate change-induced storms of increasing severity to eject debris into the oceans. In turn, increased ocean rafting may intensify species invasions.

Beyond dichotomous life histories in partially migrating populations: cessation of anadromy in a long-lived fish.

Across animal taxa, migration allows individuals to exploit habitats and resources that predictably vary seasonally in suitability. Theory predicts that the "decision" to migrate or not is shaped by the relative fitness costs and benefits of exhibiting a given life history. Adoption of a migratory strategy is widely thought to reflect a dichotomous outcome; individuals are either resident or migratory, and continue to exhibit this life history until death. In fishes, anadromy and freshwater residency represents a well-studied life history dichotomy. Resident individuals may adopt a migratory life history later in life, but migratory individuals are not known to abandon this pattern. Here, we investigated the fitness benefits, as measured by body size, of residency and anadromy in a salmonid fish, Dolly Varden, Salvelinus malma, in Alaska, and reveal a novel life history: cessation of migration by older, larger individuals. Otolith microchemical analysis of Dolly Varden showed that while most fish migrated to sea at least once in their lives, lifelong resident fish exist in streams with close proximity to the ocean. Moreover, the probability of seaward migration in any year of life decreased annually after an individual's fourth year, and no fish migrated after their eighth year, while the oldest fish were captured in their 11th year. Migration conferred a size advantage in young fish, but the size benefits of marine foraging declined in older fish, at which time fish increasingly "retired from anadromy." Additionally, measurement of both natal otolith chemistry and the gonadosomatic index indicated a continued contribution to lifetime fitness, rather than senescence, in retired individuals. We suggest that the novel life history of reversion to residency by older fish is viable because foraging opportunities are subsidized by the predictable annual supply of energy-rich eggs and carcasses of spawning Pacific salmon.

Plasma metabolomic profiles of breast cancer patients after short-term limonene intervention.

Limonene is a lipophilic monoterpene found in high levels in citrus peel. Limonene demonstrates anticancer properties in preclinical models with effects on multiple cellular targets at varying potency. While of interest as a cancer chemopreventive, the biologic activity of limonene in humans is poorly understood. We conducted metabolite profiling in 39 paired (pre/postintervention) plasma samples from early-stage breast cancer patients receiving limonene treatment (2 g QD) before surgical resection of their tumor. Metabolite profiling was conducted using ultra-performance liquid chromatography coupled to a linear trap quadrupole system and gas chromatography-mass spectrometry. Metabolites were identified by comparison of ion features in samples to a standard reference library. Pathway-based interpretation was conducted using the human metabolome database and the MetaCyc database. Of the 397 named metabolites identified, 72 changed significantly with limonene intervention. Class-based changes included significant decreases in adrenal steroids (P < 0.01), and significant increases in bile acids (P ≤ 0.05) and multiple collagen breakdown products (P < 0.001). The pattern of changes also suggested alterations in glucose metabolism. There were 47 metabolites whose change with intervention was significantly correlated to a decrease in cyclin D1, a cell-cycle regulatory protein, in patient tumor tissues (P ≤ 0.05). Here, oral administration of limonene resulted in significant changes in several metabolic pathways. Furthermore, pathway-based changes were related to the change in tissue level cyclin D1 expression. Future controlled clinical trials with limonene are necessary to determine the potential role and mechanisms of limonene in the breast cancer prevention setting.

Assessing the relative importance of local and regional processes on the survival of a threatened salmon population.

Research on regulatory mechanisms in biological populations often focuses on environmental covariates. An integrated approach that combines environmental indices with organismal-level information can provide additional insight on regulatory mechanisms. Survival of spring/summer Snake River Chinook salmon (Oncorhynchus tshawytscha) is consistently low whereas some adjacent populations with similar life histories experience greater survival. It is not known if populations with differential survival respond similarly during early marine residence, a critical period in the life history. Ocean collections, genetic stock identification, and otolith analyses were combined to evaluate the growth-mortality and match-mismatch hypotheses during early marine residence of spring/summer Snake River Chinook salmon. Interannual variation in juvenile attributes, including size at marine entry and marine growth rate, was compared with estimates of survival and physical and biological metrics. Multiple linear regression and multi-model inference were used to evaluate the relative importance of biological and physical metrics in explaining interannual variation in survival. There was relatively weak support for the match-mismatch hypothesis and stronger evidence for the growth-mortality hypothesis. Marine growth and size at capture were strongly, positively related to survival, a finding similar to spring Chinook salmon from the Mid-Upper Columbia River. In hindcast models, basin-scale indices (Pacific Decadal Oscillation (PDO) and the North Pacific Gyre Oscillation (NPGO)) and biological indices (juvenile salmon catch-per-unit-effort (CPUE) and a copepod community index (CCI)) accounted for substantial and similar portions of variation in survival for juvenile emigration years 1998-2008 (R2>0.70). However, in forecast models for emigration years 2009-2011, there was an increasing discrepancy between predictions based on the PDO (50-448% of observed value) compared with those based on the NPGO (68-212%) or biological indices (CPUE and CCI: 83-172%). Overall, the PDO index was remarkably informative in earlier years but other basin-scale and biological indices provided more accurate indications of survival in recent years.

Metabolomic characterization of nipple aspirate fluid by (1)H NMR spectroscopy and GC-MS.

Nipple aspirate fluid (NAF) is a noninvasively obtained biofluid from the duct openings of the breast. NAF components are constantly secreted, metabolized, and reabsorbed by the epithelial lining of the lactiferous ducts of the breast. NAF has been studied as a potential breast tissue surrogate for the discovery of novel breast cancer risk, early detection, and treatment response biomarkers. We report the first unsupervised metabolite characterization of nipple aspirate fluid using NMR and GC-MS using convenience samples previously collected from four premenopausal and four postmenopausal women. A total of 38 metabolites were identified using the two analytical techniques, including amino acids, organic acids, fatty acids, and carbohydrates. Analytical reproducibility of metabolites in NAF by GC-MS was high across different extraction and analysis days. Overall, 31 metabolites had a coefficient of variation below 20%. By GC-MS, there were eight metabolites unique to NAF, 19 unique to plasma, and 24 shared metabolites. Correlative analysis of shared metabolites between matched NAF and plasma samples from pre- and postmenopausal women shows almost no correlations, with the exception being lactic acid, which was significantly negatively correlated (R(2) = 0.57; P = 0.03). These results suggest that NAF is metabolically distinct from plasma and that the application of metabolomic strategies may be useful for future studies investigating breast cancer risk and intervention response biomarkers.

Expression of epidermal growth factor, transforming growth factor-ß1 and adiponectin in nipple aspirate fluid and plasma of pre and post-menopausal women.

BACKGROUND: Nipple aspirate fluid (NAF) contains large amounts of protein thought to reflect the microenvironment of the breast, and is of interest in breast cancer prevention research. The correlation between specific NAF proteins to plasma concentrations have not been well studied in healthy women. We collected matched NAF and plasma from 43 healthy pre and postmenopausal women participating in an early phase clinical study to compare the levels of putative cancer protein biomarkers. We compared baseline NAF and plasma levels of epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-ß1), and adiponectin and evaluated menopausal status and body mass index (BMI) as potential modifying factors. FINDINGS: NAF and plasma levels of EGF, TGF-ß1 and adiponectin were not correlated. EGF and TGF-ß1 levels in NAF of premenopausal women were significantly higher than postmenopausal women (P's < 0.01). These differences by menopausal status were not observed in plasma. Both NAF and plasma adiponectin levels were non-significantly higher in postmenopausal women. NAF biomarker levels were not associated with BMI whereas plasma EGF, TGF-ß1 and adiponectin levels in postmenopausal women were all inversely correlated with BMI (P's < 0.05). CONCLUSIONS: Protein biomarkers differ significantly between NAF and plasma and are affected differently by both BMI and menopausal status. This study demonstrates important differences in biological information gained by characterizing biomarkers in NAF compared to plasma and suggests each sample source may independently inform on breast cancer risk.

Elemental markers in elasmobranchs: effects of environmental history and growth on vertebral chemistry.

Differences in the chemical composition of calcified skeletal structures (e.g. shells, otoliths) have proven useful for reconstructing the environmental history of many marine species. However, the extent to which ambient environmental conditions can be inferred from the elemental signatures within the vertebrae of elasmobranchs (sharks, skates, rays) has not been evaluated. To assess the relationship between water and vertebral elemental composition, we conducted two laboratory studies using round stingrays, Urobatis halleri, as a model species. First, we examined the effects of temperature (16°, 18°, 24°C) on vertebral elemental incorporation (Li/Ca, Mg/Ca, Mn/Ca, Zn/Ca, Sr/Ca, Ba/Ca). Second, we tested the relationship between water and subsequent vertebral elemental composition by manipulating dissolved barium concentrations (1x, 3x, 6x). We also evaluated the influence of natural variation in growth rate on elemental incorporation for both experiments. Finally, we examined the accuracy of classifying individuals to known environmental histories (temperature and barium treatments) using vertebral elemental composition. Temperature had strong, negative effects on the uptake of magnesium (DMg) and barium (DBa) and positively influenced manganese (DMn) incorporation. Temperature-dependent responses were not observed for lithium and strontium. Vertebral Ba/Ca was positively correlated with ambient Ba/Ca. Partition coefficients (DBa) revealed increased discrimination of barium in response to increased dissolved barium concentrations. There were no significant relationships between elemental incorporation and somatic growth or vertebral precipitation rates for any elements except Zn. Relationships between somatic growth rate and DZn were, however, inconsistent and inconclusive. Variation in the vertebral elemental signatures of U. halleri reliably distinguished individual rays from each treatment based on temperature (85%) and Ba exposure (96%) history. These results support the assumption that vertebral elemental composition reflects the environmental conditions during deposition and validates the use of vertebral elemental signatures as natural markers in an elasmobranch. Vertebral elemental analysis is a promising tool for the study of elasmobranch population structure, movement, and habitat use.

Human breast tissue disposition and bioactivity of limonene in women with early-stage breast cancer.

Limonene is a bioactive food component found in citrus peel oil that has shown chemopreventive and chemotherapeutic activities in preclinical studies. We conducted an open-label pilot clinical study to determine the human breast tissue disposition of limonene and its associated bioactivity. We recruited 43 women with newly diagnosed operable breast cancer electing to undergo surgical excision to take 2 grams of limonene daily for two to six weeks before surgery. Blood and breast tissue were collected to determine drug/metabolite concentrations and limonene-induced changes in systemic and tissue biomarkers of breast cancer risk or carcinogenesis. Limonene was found to preferentially concentrate in the breast tissue, reaching high tissue concentration (mean = 41.3 µg/g tissue), whereas the major active circulating metabolite, perillic acid, did not concentrate in the breast tissue. Limonene intervention resulted in a 22% reduction in cyclin D1 expression (P = 0.002) in tumor tissue but minimal changes in tissue Ki67 and cleaved caspase-3 expression. No significant changes in serum leptin, adiponectin, TGF-ß1, insulin-like growth factor binding protein-3 (IGFBP-3), and interleukin-6 (IL-6) levels were observed following limonene intervention. There was a small but statistically significant postintervention increase in insulin-like growth factor I (IGF-I) levels. We conclude that limonene distributed extensively to human breast tissue and reduced breast tumor cyclin D1 expression that may lead to cell-cycle arrest and reduced cell proliferation. Furthermore, placebo-controlled clinical trials and translational research are warranted to establish limonene's role for breast cancer prevention or treatment.

Safety and Feasibility of Topical Application of Limonene as a Massage Oil to the Breast.

BACKGROUND: Limonene, a major component in citrus oil, has demonstrated anti-cancer effects in preclinical mammary cancer models. However, the effective oral dose translates to a human dose that may not be feasible for chronic dosing. We proposed to evaluate topical application of limonene to the breast as an alternative dosing strategy. MATERIALS AND METHODS: We conducted a mouse disposition study to determine whether limonene would be bio available in the mammary tissue after topical application. SKH-1 mice received topical or oral administration of limonene in the form of orange oil every day for 4 weeks. Plasma and mammary pads were collected 4 hrs after the final dosing. We also conducted an exploratory clinical study to evaluate the safety and feasibility of topically applied limonene in the form of orange oil to the breast. Healthy women were recruited to apply orange oil containing massage oil to their breasts daily for four weeks. Safety and feasibility were assessed by reported adverse events, clinical labs, and usage compliance. Pre and post-intervention nipple aspirate fluid (NAF) and plasma were collected for limonene concentration determination. RESULTS: The mouse disposition study showed that topical and oral orange oil administration resulted in similar mammary tissue disposition of limonene with no clinical signs of toxicity. In the clinical study, the topical application of limonene containing massage oil to the breast was found to be safe with high levels of usage compliance for daily application, although NAF and plasma limonene concentrations were not significantly changed after the massage oil application. CONCLUSIONS: Our studies showed that limonene is bio available in mammary tissue after topical orange oil application in mice and this novel route of administration to the breast is safe and feasible in healthy women.

Resveratrol modulates drug- and carcinogen-metabolizing enzymes in a healthy volunteer study.

Resveratrol has been shown to exhibit cancer-preventive activities in preclinical studies. We conducted a clinical study to determine the effect of pharmacologic doses of resveratrol on drug- and carcinogen-metabolizing enzymes. Forty-two healthy volunteers underwent baseline assessment of cytochrome P450 (CYP) and phase II detoxification enzymes. CYP1A2, CYP2D6, CYP2C9, and CYP3A4 enzyme activities were measured by the metabolism of caffeine, dextromethorphan, losartan, and buspirone, respectively. Blood lymphocyte glutathione S-transferase (GST) activity and GST-pi level and serum total and direct bilirubin, a surrogate for UDP-glucuronosyl transferase (UGT) 1A1 activity, were measured to assess phase II enzymes. After the baseline evaluation, study participants took 1 g of resveratrol once daily for 4 weeks. Enzyme assessment was repeated upon intervention completion. Resveratrol intervention was found to inhibit the phenotypic indices of CYP3A4, CYP2D6, and CYP2C9 and to induce the phenotypic index of 1A2. Overall, GST and UGT1A1 activities were minimally affected by the intervention, although an induction of GST-pi level and UGT1A1 activity was observed in individuals with low baseline enzyme level/activity. We conclude that resveratrol can modulate enzyme systems involved in carcinogen activation and detoxification, which may be one mechanism by which resveratrol inhibits carcinogenesis. However, pharmacologic doses of resveratrol could potentially lead to increased adverse drug reactions or altered drug efficacy due to inhibition or induction of certain CYPs. Further clinical development of resveratrol for cancer prevention should consider evaluation of lower doses of resveratrol to minimize adverse metabolic drug interactions.

Adipose tissue accumulation of d-limonene with the consumption of a lemonade preparation rich in d-limonene content.

d-limonene is a bioactive food component found in high concentration in citrus peel oil with anticancer effects in preclinical studies of mammary carcinogenesis. Extrapolation of preclinical data to human cancer is limited, in part, by inadequate information on the oral bioavailability and tissue disposition of d-limonene in humans. As a fat-soluble compound, d-limonene is more likely to deposit in fatty tissues such as the breast. To assess disposition of d-limonene in humans, we conducted a pilot study of oral d-limonene-rich lemonade. Following a 1-wk washout period devoid of citrus, healthy adults consumed 40 oz. of freshly prepared lemonade containing 500 to 600 mg d-limonene daily for 4 wk. On the first and last consumption days, blood and buttock fat biopsy were collected. Matched preintervention and postintervention fat biopsies (n = 7), and matched preintervention and postintervention plasma samples (n = 6), were analyzed for d-limonene levels using gas chromatography and mass spectrometry. There was a significant increase in d-limonene levels in the fat biopsies after 4 wk (P = 0.009); initial levels ranged from nondetectable to 7.79 micromol/kg tissue, and postintervention levels ranged from 53.6 to 294 micromol/kg tissue. Plasma d-limonene levels increased from 0.35 to 0.72 micromol/l initially to postintervention levels of 0.54 to 1.65 micromol/l (P = 0.016). Postintervention adipose d-limonene levels were 51.0 to 195 times higher than plasma levels (P = 0.009). Our results demonstrate accumulation of d-limonene in adipose tissue after oral dosing and support additional studies of d-limonene for chemoprevention in tissues such as the breast that are comprised of a significant fat fraction.

Determination of d-limonene in adipose tissue by gas chromatography-mass spectrometry.

We developed a novel method for analyzing d-limonene levels in adipose tissue. Fat samples were subjected to saponification followed by solvent extraction. d-Limonene in the sample extract was analyzed using gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring. Linear calibration curves were established over the mass range of 79.0-2529 ng d-limonene per 0.1g of adipose tissue. Satisfactory within-day precision (R.S.D. 6.7-9.6%) and accuracy (%difference of -2.7 to 3.8%) and between-day precision (R.S.D. 6.0-10.7%) and accuracy (%difference of 1.8-2.6%) were achieved. The assay was successfully applied to human fat biopsy samples from a d-limonene feeding trial.