ArHsp90 is important in stress tolerance and embryo development of the brine shrimp, Artemia franciscana.

Females of the extremophile crustacean, Artemia franciscana, either release motile nauplii via the ovoviviparous pathway or encysted embryos (cysts) via the oviparous pathway. Cysts contain an abundant amount of the ATP-independent small heat shock protein that contributes to stress tolerance and embryo development, however, little is known of the role of ATP-dependent molecular chaperone, heat shock protein 90 (Hsp90) in the two processes. In this study, a hsp90 was cloned from A. franciscana. Characteristic domains of ArHsp90 were simulated from the deduced amino acid sequence, and 3D structures of ArHsp90 and Hsp90s of organisms from different groups were aligned. RNA interference was then employed to characterize ArHsp90 in A. franciscana nauplii and cysts. The partial knockdown of ArHsp90 slowed the development of nauplius-destined, but not cyst-destined embryos. ArHsp90 knockdown also reduced the survival and stress tolerance of nauplii newly released from A. franciscana females. Although the reduction of ArHsp90 had no effect on the development of diapause-destined embryos, the resulting cysts displayed reduced tolerance to desiccation and low temperature, two stresses normally encountered by A. franciscana in its natural environment. The results reveal that Hsp90 contributes to the development, growth, and stress tolerance of A. franciscana, an organism of practical importance as a feed source in aquaculture.

A combined toxicological impact on Artemia salina caused by the presence of dust particles, microplastics from cosmetics, and paracetamol.

Environmental pollution poses a significant and pressing threat to the overall well-being of aquatic ecosystems in modern society. This study showed that pollutants like dusts from AC filter, fan wings and Traffic dust PM 2.5 were exposed to Artemia salina in pristine form and in combination. The findings indicated that exposure to multi-pollutants had a detrimental effect on the hatching rates of A. salina cysts. Compared to untreated A. salina, the morphology of adult (7th day old) A. salina changed noticeably after each incubation period (24-120 h). Oxidative stress increased considerably as the exposure duration increased from 24 to 120 h compared to the control group. There was a time-dependent decline in antioxidant enzyme activity and total protein concentration. When all particles were used all together, the total protein content in A. salina decreased significantly. All particles showed a considerable decline in survival rate. Those exposed to traffic dust particles showed significantly higher levels of oxidative stress and antioxidant activity than those exposed to other particles.

Chromosome-Level Assembly of Artemia franciscana Sheds Light on Sex Chromosome Differentiation.

Since the commercialization of brine shrimp (genus Artemia) in the 1950s, this lineage, and in particular the model species Artemia franciscana, has been the subject of extensive research. However, our understanding of the genetic mechanisms underlying various aspects of their reproductive biology, including sex determination, is still lacking. This is partly due to the scarcity of genomic resources for Artemia species and crustaceans in general. Here, we present a chromosome-level genome assembly of A. franciscana (Kellogg 1906), from the Great Salt Lake, United States. The genome is 1 GB, and the majority of the genome (81%) is scaffolded into 21 linkage groups using a previously published high-density linkage map. We performed coverage and FST analyses using male and female genomic and transcriptomic reads to quantify the extent of differentiation between the Z and W chromosomes. Additionally, we quantified the expression levels in male and female heads and gonads and found further evidence for dosage compensation in this species.

Prolonged ecological changes can affect morphometrics and gene expression profile? Focusing on Hsp-70 and NLHS-induced Hsp-70 of Artemia urmiana.

BACKGROUND: In recent years, many aquatic ecosystems, including Urmia Lake, have undergone severe ecological tensions. This lake, the largest natural habitat of the brine shrimp Artemia urmiana, has progressively desiccated and its salinity has dramatically increased over the last three decades. In the face of the long period environmental stresses, understanding the adaptation and ecological plasticity mechanisms is the most interesting challenges in genetic and applied ecology. These mechanisms may probably be driven by inducing expression of some genes involved in adaptation such as Hsp-70 and also adjusting morphological parameters. But they are yet to be understood. Hence, the present work aimed to study the mechanisms, along with testing the hypothesis that non-lethal heat shocked nauplii originating from drought period can evoke Hsp-70 expression more than those from rainy period. METHODS: This study measured and analyzed morphometrical characters of adult male and female Artemia urmiana over three decades. Then, the influence of three-decade ecological crisis on Hsp-70 and non-lethal heat shock (NLHS)-induced Hsp-70 expression levels of nauplii of Artemia urmiana habiting Urmia Lake using Real-time PCR technique, based on cyst collections in 1994 (rainy period) to 2020 (drought period), was evaluated. RESULTS: The morphometrics results showed that the morphological characters were significantly shrunk in 2020 compared to 1994 (CI 95%, p < 0.05). Furthermore, our results depicted that, Hsp-70 expression level was significantly upregulated in response to the prolonged ecological crisis, (CI 95%, P < 0.0001), and also interestingly, the nauplii exposed to longe-term ecological crisis (belong to 2020) were able to increase Hsp-70 expression more than other ones in response to environmental stressors including heat. CONCLUSIONS: The present results showed the involvement of Hsp-70 in the adaptation of Artemia urmiana to long term ecological alteration at the cost of shrinking morphometric parameters.

A cytological revisit on parthenogenetic Artemia and the deficiency of a meiosis-specific recombinase DMC1 in the possible transition from bisexuality to parthenogenesis.

Although parthenogenesis is widespread in nature and known to have close relationships with bisexuality, the transitional mechanism is poorly understood. Artemia is an ideal model to address this issue because bisexuality and "contagious" obligate parthenogenesis independently exist in its congeneric members. In the present study, we first performed chromosome spreading and immunofluorescence to compare meiotic processes of Artemia adopting two distinct reproductive ways. The results showed that, unlike conventional meiosis in bisexual Artemia, meiosis II in parthenogenic Artemia is entirely absent and anaphase I is followed by a single mitosis-like equational division. Interspecific comparative transcriptomics showed that two central molecules in homologous recombination (HR), Dmc1 and Rad51, exhibited significantly higher expression in bisexual versus parthenogenetic Artemia. qRT-PCR indicated that the expression of both genes peaked at the early oogenesis and gradually decreased afterward. Knocking-down by RNAi of Dmc1 in unfertilized females of bisexual Artemia resulted in a severe deficiency of homologous chromosome pairing and produced univalents at the middle oogenesis stage, which was similar to that of parthenogenic Artemia, while in contrast, silencing Rad51 led to no significant chromosome morphological change. Our results indicated that Dmc1 is vital for HR in bisexual Artemia, and the deficiency of Dmc1 may be correlated with or even possibly one of core factors in the transition from bisexuality to parthenogenesis.

From ecology to oncology: To understand cancer stem cell dormancy, ask a Brine shrimp (Artemia).

The brine shrimp (Artemia), releases embryos that can remain dormant for up to a decade. Molecular and cellular level controlling factors of dormancy in Artemia are now being recognized or applied as active controllers of dormancy (quiescence) in cancers. Most notably, the epigenetic regulation by SET domain-containing protein 4 (SETD4), is revealed as highly conserved and the primary control factor governing the maintenance of cellular dormancy from Artemia embryonic cells to cancer stem cells (CSCs). Conversely, DEK, has recently emerged as the primary factor in the control of dormancy exit/reactivation, in both cases. The latter has been now successfully applied to the reactivation of quiescent CSCs, negating their resistance to therapy and leading to their subsequent destruction in mouse models of breast cancer, without recurrence or metastasis potential. In this review, we introduce the many mechanisms of dormancy from Artemia ecology that have been translated into cancer biology, and herald Artemia's arrival on the model organism stage. We show how Artemia studies have unlocked the mechanisms of the maintenance and termination of cellular dormancy. We then discuss how the antagonistic balance of SETD4 and DEK fundamentally controls chromatin structure and consequently governs CSCs function, chemo/radiotherapy resistance, and dormancy in cancers. Many key stages from transcription factors to small RNAs, tRNA trafficking, molecular chaperones, ion channels, and links with various pathways and aspects of signaling are also noted, all of which link studies in Artemia to those of cancer on a molecular and/or cellular level. We particularly emphasize that the application of such emerging factors as SETD4 and DEK may open new and clear avenues for the treatment for various human cancers.

Assessment of the biological effect of metal ions and their complexes using Allium cepa and Artemia salina assays: a possible environmental implementation of biological inorganic chemistry.

The pollution of aquatic ecosystems due to the elevated concentration of a variety of contaminants, such as metal ions, poses a threat to humankind, as these ecosystems are in high relevance with human activities and survivability. The exposure in heavy metal ions is responsible for many severe chronic and pathogenic diseases and some types of cancer as well. Metal ions of the groups 11 (Cu, Ag, Au), 12 (Zn, Cd, Hg), 14 (Sn, Pb) and 15 (Sb, Bi) highly interfere with proteins leading to DNA damage and oxidative stress. While, the detection of these contaminants is mainly based on physicochemical analysis, the chemical determination, however, is deemed ineffective in some cases because of their complex nature. The development of biological models for the evaluation of the presence of metal ions is an attractive solution, which provides more insights regarding their effects. The present work critically reviews the reports published regarding the toxicity assessment of heavy metal ions through Allium cepa and Artemia salina assays. The in vivo toxicity of the agents is not only dose depended, but it is also strongly affected by their ligand type. However, there is no comprehensive study which compares the biological effect of chemical agents against Allium cepa and Artemia salina. Reports that include metal ions and complexes interaction with either Allium cepa or Artemia salina bio-indicators are included in the review.

Low-toxic, fluorescent labeled and size-controlled graphene oxide quantum dots@polystyrene nanospheres as reference material for quantitative determination and in vivo tracing.

Polystyrene (PS) is selected as a representative nanoplastic and persistent pollutant for its difficult degradation and wide application. The environmental risk assessment of PS is obstructed by the toxic dye-based fluorescent PS, which false positives could be induced by the leakage of dye. For high biocompatibility, low toxicity, hydrophilicity, good water dispersibility, strong fluorescent stability, graphene oxide quantum dots (o-CQDs) are selected and embedded into PS microspheres, i.e., o-CQDs@PS, by microemulsion polymerization and denoted as CPS. Meanwhile, the sizes of CPS, e.g., 100, 150, and 200 nm, could be controlled by optimizing the type and number of water-soluble initiators. The anti-interference, low toxicity, and in vivo fluorescent tracing of CPS are proven by the coexistence of metals (including Fe2+, Fe3+, K+, Ba2+, Al3+, Zn2+, Mg2+, Ca2+, and Na+) on the fluorescence intensity of CPS, the growth of Chlorella pyrenoidosa and Artemia cysts as aquatic phytoplankton and zooplankton cultured with CPS, and the transfer of CPS from water into brine shrimp. In the concentration range of 0.1-100 mg/L, CPS can be quantitatively determined, which is suitable for coastal water and wastewater treatment plants. Therefore, CPS with standard size is suitable as reference material of PS.

Expression of Abdominal-B in the brine shrimp, Artemia franciscana, expands our evolutionary understanding of the crustacean abdomen.

The brine shrimp, Artemia franciscana, has a body plan composed of 11 thoracic segments, followed by 2 genital segments, and then 6 additional abdominal segments. Previous studies of Artemia reported that expression of the posterior-most Hox gene, Abdominal-B (Abd-B), is restricted to the genital segments and is not observed posteriorly in the abdomen at any developmental stage. This report was remarkable because it suggested that the Artemia abdomen posterior to the genital segments was a novel body region of 6 segments that bore no homology to any region in other crustaceans and was unique amongst arthropods in being a Hox-free segmented domain outside of the head. In this study, we used RT-PCR, antibody staining, and in situ hybridization on various stages of Artemia nauplii to show that Abd-B mRNA and protein are in fact expressed throughout the abdominal segments during Artemia development, but this expression later retracts to the two genital segments (G1, G2) and the T11 appendages. This suggests that Abd-B does play a role in specifying abdominal segment identity in all crustaceans that have been examined and suggests a common evolutionary origin for the crustacean abdomen.

The Group 3 LEA proteins of Artemia franciscana for cryopreservation.

Late embryogenesis-abundant (LEA) proteins are protective proteins that are enriched in the late stage of seed embryo development. LEA proteins play an important role in resisting abiotic stresses such as low temperature and drought. Artemia franciscana is the only animal known to express three different groups of LEA proteins in its life cycle, and the discovery has some applications for the cryopreservation of human cells. In this review, A. franciscana LEA proteins from Group 3 are systematically introduced, and the structure, location, function and application in cryopreservation are highlighted. As a nontoxic and effective cryoprotectant, A. franciscana LEA proteins are expected to provide a new method for cryopreservation of cells.

No short-term effect of sinking microplastics on heterotrophy or sediment clearing in the tropical coral Stylophora pistillata.

Investigations of encounters between corals and microplastics have, to date, used particle concentrations that are several orders of magnitude above environmentally relevant levels. Here we investigate whether concentrations closer to values reported in tropical coral reefs affect sediment shedding and heterotrophy in reef-building corals. We show that single-pulse microplastic deposition elicits significantly more coral polyp retraction than comparable amounts of calcareous sediments. When deposited separately from sediments, microplastics remain longer on corals than sediments, through stronger adhesion and longer periods of examination by the coral polyps. Contamination of sediments with microplastics does not retard corals' sediment clearing rates. Rather, sediments speed-up microplastic shedding, possibly affecting its electrostatic behaviour. Heterotrophy rates are three times higher than microplastic ingestion rates when corals encounter microzooplankton (Artemia salina cysts) and microplastics separately. Exposed to cysts-microplastic combinations, corals feed preferentially on cysts regardless of microplastic concentration. Chronic-exposure experiments should test whether our conclusions hold true under environmental conditions typical of inshore marginal coral reefs.

Purification and characterization of a highly thermostable GlcNAc-binding lectin from Collaea speciosa seeds.

Lectins from plants of the Diocleinae subtribe often exhibit specificity towards mannose/glucose and derived sugars, with some plants also displaying a second lectin specific to lactose/GalNAc. Here, we present a novel lectin from Collaea speciosa, named CsL, that displays specificity for GlcNAc/glucose. The lectin was extracted from Collaea speciosa seeds and purified by a single chromatographic step on a Sephadex G-50 matrix. In solution, the lectin appears as a dimeric protein composed of 25 kDa monomers. The protein is stable at pH 7-8 and dependent on divalent cations. CsL maintained its agglutination activity after heating to 90 °C for 1 h. Glycan array studies revealed that CsL binds to N-glycans with terminal GlcNAc residues, chitobiose and chitotriose moieties. The partial amino acid sequence of the lectin is similar to that of some lactose-specific lectins from the same subtribe. In contrast to other ConA-like lectins, CsL is not toxic to Artemia. Because of its remarkably different properties and specificity, this lectin could be the first member of a new group inside the Diocleinae lectins.

Histological study of gonadal tissues of adult Artemia salina (Linnaeus 1758) and immunohistochemistry by Caspase 3 and HSP70 to detect specific apoptosis markers on gonadal tissues after exposure to TBTCl.

Background: Several types of research have been recently carried out on the biological effects of TBTs, including investigations of genitals in invertebrates in response to exposure to TBTs in marine water. Aim: The objective of this research was to investigate the acute effects of tributyltin chloride (TBTCl) on gonads in the adult stage of Artemia salina by use normal histology and immunohistochemistry (IHC) (Caspase 3 and HSP70) to see specific apoptosis markers. Methods: After exposure of A. salina to different concentrations of TBTCl (25, 50, 100, 200, and 300 ng.l-1), 50 adult A. salina (25 male and 25 female) were selected randomly from each concentration to histologically study the gonads. The gonad tissue was sectioned (5 µm) and some slides were stained with hematoxylin and eosin and others were stained with IHC avidin-biotin complex, and were examined under a light microscope. Results: The results showed significant differences (p < 0.05) in histological lesions between different concentrations of TBTCl. The histological lesions in the testis and ovary section were undifferentiated cells, degenerating yolk globules, and follicle cells enveloping the oocyte which was then compared with control tissue, and these effects were found to be increased in females more than in males with the highest concentration of TBTCl. Immunohistochemistry (IHC) showed that positive immunostaining was observed in the testis and ovary as brownish deposits to Caspase 3 and HSP70 antibody after exposure to TBTCl, while the testis and ovary section in control tissue had no immunoreactivity to Caspase 3 and HSP70 antibody; these effects were profoundly increased with the highest concentration of TBTCl in females more than in males. Finally, the histological lesions and IHC (Caspase 3 and HSP70) revealed that the apoptosis and immune system stress of A. salina gonad tissue damage in females were more sensitive to TBTCl toxicity as compared to white males. Conclusion: In general, the present study aimed to observe the effects TBTCl on A. salina gonads by using histological sections and IHC (Caspase 3 and HSP70), which were evaluated for the first time and have been proven to possess an important function in apoptosis marker and immune system stress in Artemia. Finally, the specific mechanisms through which TBTCl affects A. salina Caspase 3 and HSP70 expression need further investigation.

Evaluation of Indonesian mangrove Xylocarpus granatum leaves ethyl acetate extract as potential anticancer drug.

Local Xylocarpus granatum leaves were extracted by ethyl acetate solvent and characterized by TLC fingerprinting and 2D 1H NMR spectroscopy to contain phenolic compounds as well as several organic and amino acids as metabolic byproducts, such as succinic acid and acetic acid. Traces of flavonoids and other non-categorized phenolic compounds exhibited intermediate antioxidant activity (antioxidant IC50 84.93 ppm) as well as anticancer activity against HeLa, T47D, and HT-29 cell lines; which the latter being most effective against HT-29 with Fraction 5 contained the strongest activity (anticancer IC50 23.12 ppm). Extracts also behaved as a natural growth factor and nonlethal towards brine shrimps as well as human adipose-derived stem cell hADSC due to antioxidative properties. A stability test was performed to examine how storage conditions factored in bioactivity and phytochemical structure. Extracts were compared with several studies about X. granatum leaves extracts to evaluate how ethnogeography and ecosystem factored on biologically active compounds. Further research on anticancer or antioxidant mechanism on cancer cells is needed to determine whether the extract is suitable as a candidate for an anticancer drug.

Target enzymes are stabilized by AfrLEA6 and a gain of α-helix coincides with protection by a group 3 LEA protein during incremental drying.

Anhydrobiotic organisms accumulate late embryogenesis abundant (LEA) proteins, a family of intrinsically disordered proteins (IDPs) reported to improve cellular tolerance to water stress. Here we show that AfrLEA6, a Group 6 LEA protein only recently discovered in animals, protects lactate dehydrogenase (LDH), citrate synthase (CS) and phosphofructokinase (PFK) against damage during desiccation. In some cases, protection is enhanced by trehalose, a naturally-occurring protective solute. An open question is whether gain of secondary structure by LEA proteins during drying is a prerequisite for this stabilizing function. We used incremental drying (equilibration to a series of relative humidities, RH) to test the ability of AfrLEA2, a Group 3 LEA protein, to protect desiccation-sensitive PFK. AfrLEA2 was chosen due to its exceptional ability to protect PFK. In parallel, circular dichroism (CD) spectra were obtained for AfrLEA2 across the identical range of relative water contents. Protection of PFK by AfrLEA2, above that observed with trehalose and BSA, coincides with simultaneous gain of α-helix in AfrLEA2. At 100% RH, the CD spectrum for AfrLEA2 is typical of random coil, while at decreasing RH, the spectrum shows higher ellipticity at 191 nm and minima at 208 and 220 nm, diagnostic of α-helix. This study provides experimental evidence linking the gain of α-helix with stabilization of a target protein across a graded series of hydration states. Mechanistically, it is intriguing that certain other functions of these IDPs, like preventing aggregation of target proteins, can occur in fully hydrated cells and apparently do not require gain of α-helix.

Antagonistic Activity Against Pathogenic Vibrio Isolates of Bioflocculant-Producing Bacteria Isolated from Shrimp Ponds.

<b>Background and Objectives:</b> Biofloc culture system has been used in aquaculture as an effective technology for water treatment due to many advantages of being biodegradable and environmentally friendly. This study aims to isolate bioflocculant-producing bacteria antagonistic to pathogenic <i>Vibrio</i> species from Pacific white shrimp ponds in Thua Thien Hue, Vietnam. <b>Materials and Methods:</b> <i>Vibrio</i> isolates were isolated by screening on medium with and without antibiotics. The resistance of <i>Vibrio</i> to antimicrobial agents was assessed by Minimum Inhibitory Concentration (MIC). Bioflocs formed in shrimp cultures were used to screen bioflocculant-producing bacteria. The identification of bacteria was performed by 16S rRNA sequencing. The flocculating activity was measured by a test with kaolin clay suspension. To evaluate the antagonistic activity against <i>Vibrio</i> isolates, an agar well diffusion assay was used. <b>Results:</b> The screening results have found that <i>Vibrio</i> isolates such as <i>V. parahaemolyticus</i> KS02 and <i>V. alginolyticus</i> KS08 from shrimp ponds can be resistant to many antibiotics with the highest resistance rate up to 66.49%. Four bioflocculant-producing isolates were obtained and identified as <i>Bacillus</i> species. Among them, <i>Bacillus velezensis </i>B9 when grown in YPG medium supplemented with 3% sucrose and 0.7% peptone had the highest bioflocculation with an activity of 49.2%. Two isolates of <i>B. subtilis</i> B2 and <i>Bacillus</i> sp. B6 had quite strong antagonistic activities against vibriosis shown in the zones of inhibition on the assay plates with diameters of about 20 mm. <b>Conclusion:</b> The present study has found some <i>Bacillus</i> isolates had bioflocculant-producing efficiency and inhibited pathogenic <i>Vibrio</i> bacteria. These <i>Bacillus</i> isolates will potentially be used as inoculum for bioflocculation to improve shrimp production.

Stress-dependent conformational changes of artemin: Effects of heat and oxidant.

Artemin is an abundant thermostable protein in Artemia embryos and it is considered as a highly efficient molecular chaperone against extreme environmental stress conditions. The conformational dynamics of artemin have been suggested to play a critical role in its biological functions. In this study, we have investigated the conformational and functional changes of artemin under heat and oxidative stresses to identify the relationship between its structure and function. The tertiary and quaternary structures of artemin were evaluated by fluorescence measurements, protein cross-linking analysis, and dynamic light scattering. Based on the structural analysis, artemin showed irreversible substantial conformational lability in responses to heat and oxidant, which was mainly mediated through the hydrophobic interactions and dimerization of the chaperone. In addition, the chaperone-like activity of heated and oxidized artemin was examined using lysozyme refolding assay and the results showed that although both factors, i.e. heat and oxidant, at specific levels improved artemin potency, simultaneous incubation with both stressors significantly triggered the chaperone activation. Moreover, the heat-induced dimerization of artemin was found to be the most critical factor for its activation. It was suggested that oxidation presumably acts through stabilizing the dimer structures of artemin through formation of disulfide bridges between the subunits and strengthens its chaperoning efficacy. Accordingly, it is proposed that artemin probably exists in a monomer-oligomer equilibrium in Artemia cysts and environmental stresses and intracellular portion of protein substrates may shift the equilibrium towards the active dimer forms of the chaperone.

Activin A affects feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish (Danio rerio) larvae.

Activin A belongs to the superfamily of transforming growth factor-ß and plays an important role in hormone regulation and tissue development. However, few research studies have been conducted on the effect of activin A on feeding organs in fish. In this study, the zebrafish (Danio rerio) larvae were treated with 1 ng ml-1 activin A for 8 days continuously. The haematoxylin and eosin (H&E) staining section results revealed that the transverse inner diameter of the pharynx and oesophagus significantly increased on the third and eighth days after treatment compared with the control group (P < 0.05). On the eighth day, the cross-sectional area of the pharyngeal muscle increased by 8638 µm2 compared to the control group (P < 0.05). The RNA in situ hybridization results also showed that the expression of skeletal muscle-specific genes (myog and myod) was significantly increased in pharyngeal muscle on the eighth day. Furthermore, the qRT-PCR results showed the expression of gh gene was significantly increased on the eighth day (P < 0.05). At the same time, more larvae in activin A group were able to feed larger brine shrimp (Artemia) than in the control group on the eighth day. In conclusion, activin A could affect feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish larvae. This study is the first to report that the development of the pharynx and oesophagus can directly affect food intake in fish larvae, which provides a theoretical basis for the study of food intake of fish at an early stage.

A novel group 6 LEA protein from diapause embryos of Artemia franciscana is cytoplasmically localized.

The expression of late embryogenesis abundant (LEA) proteins is one mechanism by which anhydrobiotic organisms survive periods of severe water loss. Artemia franciscana is an animal extremophile that uniquely expresses LEA proteins concurrently from Groups 1, 3, and 6. In this study we examine the subcellular localization of AfrLEA6, a Group 6 LEA protein from embryos of A. franciscana. Immunohistochemistry reveals that AfrLEA6 is located in the cytoplasm of diapause embryos and does not co-localize with nuclei or mitochondria. Due to a trace contaminant arising from chitin-based affinity chromatography during AfrLEA6 purification, the primary antiserum displayed affinities for both AfrLEA6 as well as Artemia chitin-binding proteins. This contaminant (fusion protein of intein plus chitin binding domain) co-migrates with AfrLEA6 during SDS-PAGE. Pre-adsorption of the antiserum with dechorionated embryos was required to remove the non-specific fluorescence in the embryonic cuticular membrane. Results of this study are consistent with the apparent importance of distributing multiple types of LEA proteins across many subcellular locations in anhydrobiotic organisms.

Ultra-Micro-Scale-Fractionation (UMSF) as a Powerful Tool for Bioactive Molecules Discovery.

Herein is detailed the development and validation of an ultra-micro-scale-fractionation (UMSF) technique for the discovery of plant-based, bioactive molecules, coupling the advantages of ultra-performance liquid chromatography mass spectrometry (UPLC-MS) separations with microtiter plate-based bioassay screens. This novel one-step approach simultaneously uses UPLC to collect chemical profile information, while performing high-resolution fractionation, greatly improving workflow compared to methods relying on high-performance liquid chromatography (HPLC), solid phase extraction or flash systems for chromatographic separations. Using the UMSF technique, researchers are able to utilize smaller quantities of starting materials, reduce solvent consumption during fractionation, reduce laborious solvent dry down times, replace costly single-use solid-phase-extraction cartridges with reusable analytical-sale UPLC columns, reduce fractionation times to less than 10 min, while simultaneously generating chemical profile data of active fractions and enjoying superior chromatographic resolution. Using this technique, individual bioactive components can be readily purified, identified, and bioassayed in one step from crude extracts, thereby eliminating ambiguous synergistic effects often reported in plant-based natural products research. A successful case-study is presented illustrating the versatility of this technique in identifying lupulone as the principal cytotoxic component from hops (Humulus lupulus L.), using a brine shrimp (Artemia franciscana) model. These results confirm and expand upon previous cell-based bioassay studies using a more complex, multicellular organism, and add to our understanding of structure-function activity relationships for secondary metabolites in hops and the Cannabaceae plant family.