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Purpose: To investigate the anti-cancer potential of astaxanthin from Litopenaeus vannamei encapsulated in liposomes (ASX) to treat lung cancer A549 cells.Methods: Lung adenocarcinoma A549 cells were cultured and treated with ASX, following which cell viability and nuclear staining were performed. Generation of ROS was identified by the DCFH-DA assay while tetramethylrhodamine ethyl ester was used to determine the mitochondrial membrane potential. Flow cytometry was applied to investigate caspase-3/7 activity and cell cycle distribution.Results: ASX inhibited growth of A549 in a concentration- and time- dependent manner. The IC50 values at 24, 48 and 72 h were 53.73, 22.85, 17.46 μg/mL, respectively (p < 0.05). After incubation with ASX, the morphological changes were observed in A549 cells following Hoechst 33342/PI fluorescent staining. ASX increased ROS generation and was associated with the collapse of mitochondrial membrane potential, which subsequently triggered the activation of caspase-3/7 activity leading to apoptosis (p < 0.05). In addition, A549 cells accumulated in the G0/G1 phase.Conclusion: The results suggest that ASX is a valuable nutraceutical agent to target A549 lung cancer cells via ROS-dependent pathway as well as blockage of cell cycle progression. Keywords: Astaxanthin, Litopenaeus vannamei, Lung cancer, A549, Apoptosis

Objective: To evaluate changes of feline (Felis catus) oocytes proteins during in vitro maturation by using the proteomic approach. Methods: Immature oocytes (germinal vesicle) isolated from female cats were cultured and collected at 0 h and 24 h. After collection, oocytes were investigated into immature (germinal vesicle) and mature (metaphase II) stages. The qualitative profiles of the proteins at the immature and mature stages were determined by one-dimensional electrophoresis and liquid chromatography-mass spectrometry. Results: Our data revealed that following 24 h in vitro maturation the maturation rate (metaphase II stage) was 58.7%. Eighty-one of the 260 proteins analyzed were differentially expressed between the germinal vesicle stage and the metaphase II -arrest stage. Proteomic analysis of germinal vesicle and metaphase II oocytes showed abundant expression of proteins involved in transportation (10%), indicating that this was a major characteristic of germinal vesicle oocytes. Similarly, analysis of the proteome of metaphase II oocytes indicated that cell cycle proteins were overexpressed. Interestingly, proteins involved in DNA repair and apoptosis were only expressed in germinal vesicle oocytes and proteins involved in fertilization were only expressed in metaphase II oocytes. Conclusions: The overexpression of certain proteins in germinal vesicle and metaphase II is necessary for oocyte development and maturation. Our findings provide a valuable resource for further investigations into protein expression in oocytes at different developmental stages.

Background Dysregulation of immune response is associated with development of endometriosis. The study aim was to evaluate effect of combined oral contraceptive pills (COCs) consisting of ethinyl estradiol (EE) and desogestrel on the expression of macrophage, natural killer cells, and regulatory T cells of ovarian endometriotic cysts. Methods Endometriotic cyst wall tissues were collected from women with endometriosis who were treated (n = 22) with COCs (one table per day of EE 0.03 mg and desogestrel 0.15 mg administered for 28 to 35 days before surgery) or untreated (n = 22). The tissues were collected from endometriotic cyst wall during laparoscopic or laparotomy ovarian cystectomy. Immunohistochemistry for anti-CD68, anti-CD56, and anti-forkhead–winged helix transcription factor (FoxP3), a marker for macrophages, natural killer cells, and regulatory T cells, respectively, were investigated. Results The median (interquartile range [IQR]) number of anti-CD68 positive cells in the COC group was significantly lower than in the untreated group (12.7; 4.9–19.3) versus 45.7 (26.0–70.7), p Conclusions The effects of COC, containing EE 0.30 mg with desogestrel 0.15 mg, on the immune system was demonstrated by a significant decrease in the number of macrophages and an increase in natural killer and regulatory T cells.

Dysregulation of the immune response contribute to a significant role in endometriosis. This research examined macrophages and natural killer (NK) cells numbers in endometriotic lesions and their association with the different lesion colors: red, black, and white. To investigate the amount of the CD68 and CD56 in eutopic endometrium and different type of the endometriotic lesions.A cross-sectional analytic study was conducted. Women suspected endometriosis requiring laparoscopic surgery between July 2016 and January 2017 were recruited. Their lesions were classified as red, black, or white and these lesions were excised by standard laparoscopic surgery. Twenty-four endometriotic lesions from each color group were obtained from 45 women who met the inclusion criteria. One type of lesion was collected from 25 women. Two different lesion types and three-color lesion types were collected from the same women in 13 and 7 subjects, respectively. Immunohistochemistry staining with anti-human mouse cluster of differentiation (CD) 68 monoclonal antibody for macrophages and mouse anti-human CD56 monoclonal antibody for NK cells were performed.The number of CD68 macrophages in red lesions was higher than in black and white lesions [median (25The dynamic changes in the immune cells in ectopic endometrium were specific to the macrophages but not to the NK cells, as demonstrated by the highest number of CD68 macrophages in the red lesion, the earliest established ectopic endometrium. NK cells in endometriosis may have a role in the uterus.

Background: The differential diagnosis between uterine fibroid and adenomyosis is sometimes difficult; a precise diagnosis is required in women with infertility because of the different choice of treatments. Ultrasound elastography (UE) is a novel technique to evaluate the elasticity or the stiffness of the tissue of interest. The present study aims to compare UE shear wave velocity (SWV) among normal uterine myometrium, uterine fibroid, and adenomyosis, and assess the accuracy of shear wave elastography in the diagnosis of adenomyosis.Materials and Methods: This cross-sectional study recruited 25 subjects for each group (control, adenomyosis, and fibroid) from April 2019 to April 2020. Transvaginal UE using an Aplio 500 (Toshiba Medical Systems, Japan) with ultrasound mapping for point of tissue biopsy was performed for all subjects. The diagnosis was confirmed by histology. Masson’s trichrome staining for collagen was performed and quantified.Results: The mean ± standard deviation (SD) for SWV was 3.44 ± 0.95 m/seconds (control group), 4.63 ± 1.45 m/seconds (adenomyosis group), and 4.53 ± 1.07 m/seconds (fibroid group). The mean SWV differed when comparing normal myometrium and adenomyosis after adjustments for age and endometrial pathology (P=0.019). The cut-off point of SWV at 3.465 m/seconds could differentiate adenomyosis from the normal uterus with an 80% sensitivity, 80% specificity, and an area under the curve (AUC) of 0.80 (95% confidence interval [CI]: 0.68-0.93) (P

We previously analyzed the central nervous system (CNS) transcriptome and found three isotypes of long neuropeptide F (MrNPF-I, -II, -III) and four isoforms of short NPF (sMrNPF) in the giant freshwater prawn, Macrobrachium rosenbergii. We now validate the complete sequences of the MrNPF-I and -II precursor proteins, which show high similarity (91-95 %) to NPFs of the penaeus shrimp (PsNPF). MrNPF-I and -II precursors share 71 % amino acid identity, whereas the mature 32-amino-acid MrNPF-I and 69-amino-acid MrNPF-II are identical, except for a 37-amino-acid insert within the middle part of the latter. Both mature MrNPFs are almost identical to PsNPF-I and -II except for four amino acids at the mid-region of the peptides. Reverse transcription plus the polymerase chain reaction revealed that transripts of MrNPF-I and -II were expressed in various parts of CNS including the eyestalk, brain and thoracic and abdominal ganglia, with the highest expression occurring in the brain and thoracic ganglia and with MrNPF-I showing five- to seven-fold higher expression than MrNPF-II. These peptides were also expressed in the midgut hindgut, and hepatopancreas, with MrNPF-I expression in the former two organs being at the same level as that in the brain and thoracic ganglia and about 4-fold higher than NPF-II. The expression of NPFs was also detected in the testes and spermatic duct but appeared much weaker in the latter. Other tissues that also expressed a considerable amount of NPF-I included the hematopoeitic tissue, heart and muscle. By immunohistochemistry, we detected MrNPFs in neurons of clusters 2, 3 and 4 and neuropils ME, MT and SG of the optic ganglia, neurons in cluster 6 and neuropils AMPN, PMPN, PT, PB and CB of the medial protocerebrum, neurons in clusters 9 and 11 and neurophils ON and OGTN of the deutocerebrum and neurons in clusters 14, 15 and 16 and neuropils TN and AnN of the tritocerebrum. Because of their high degree of conservation and strong and wide-spread expression in tissues other than CNS, we believe that, in addition to being a neuromodulator in controlling feeding, MrNPFs also play critical roles in tissue homeostasis. This should be further explored.

Purpose: To investigate the capacity of aqueous Pseuderanthemum paltiferum leaf extracts (PPA) to induce apoptosis in A549 human lung cancer cells and the possible mechanisms of action. Methods: Human lung cancer A549 cells were cultured in the presence of PPA (0 - 1000 μg/mL). Cell viability was assessed by MTT assay while morphological alterations in the cells were observed by Hoechst 33342/PI double staining. Intracellular reactive oxygen species (ROS) levels and subsequent changes of mitochondrial membrane potential were also investigated. Involvement of caspase-3 activation in the apoptotic pathway was determined. Results: PPA inhibited the growth of A549 cells in a concentration- and time-dependent manner. Major phenotypic apoptotic cell death was evidenced in microscopic images. Furthermore, treatment of A549 cells with PPA resulted in a significant increase in the production of ROS accompanied by attenuation of mitochondrial membrane potential, thus inducing the activation of caspase-3 activity ( p < 0.05). Conclusion: PPA exerts anti-cancer activity by suppression of cell viability and induction of ROSmediated mitochondrial dependent apoptosis in A549 cells, and may be a potential candidate for the development of a therapeutic agent for lung cancer. Keywords: Pseuderanthemum palatiferum , Apoptosis, Reactive oxygen species, Mitochondria, Lung cancer

Background: The domestic cat serves as an animal model for assisted reproductive studies of endangered felid species. To date, there are no data on the protein alterations following cryopreservation of oocytes in felid family. Methods: Immature (germinal vesicle) domestic cat oocytes were vitrified in the vitrification solution containing 35% ethylene glycol, 20% DMSO and 0.5 mM sucrose. The vitrified-warmed oocytes were matured (metaphase II) in vitro and subjected to proteomic analysis using 1DE SDS-PAGE prefractionation combined with LC-MS/MS. Results: A total of 1712 proteins were identified in in vitro matured oocytes. Of the 1712 proteins, 1454 proteins were found in both groups, whereas, 258 proteins were differentially expressed between control and vitrified-warmed groups. In vitrified-warmed oocytes, the missing proteins were membrane and nuclear proteins; whereas, apoptosis and DNA repair proteins were overrepresented. Conclusions: The identified missing and overexpressed proteins in vitrified-warmed oocytes represent potential markers of cryoinjuries and the developmental pathways of oocytes. The findings of differential expressed proteins may contribute to effective ways of proteome analysis of oocyte/embryo quality in order to assess safety of cryopreservation in felid species.

Although Depo-medroxyprogesterone acetate (DMPA), an injectable contraceptive progestin, is very effective for pain relief and prevention of recurrence in women with endometriosis, there is no report on the mechanism of this medication about cell proliferation and apoptosis.To investigate the effects of DMPA on cell proliferation and apoptosis in the eutopic endometrium of women with endometriosis.A randomized controlled study was conducted in 28 women with endometriosis. The DMPA-treated group included 14 women who were scheduled to undergo laparoscopic surgery after 150 mg of DMPA injections. The control group included 14 women who were scheduled to undergo the surgery without DMPA injection. The endometrial tissue was obtained from each woman by endometrial aspiration before surgery. The ELISA formats of PCNA and the quantitative colorimetric analysis of TUNEL were used for estimating cell proliferation and apoptosis of the eutopic endometrium.There were no differences in the women characteristics between the two groups. The relative level of cell proliferation was significantly less in the DMPA than the control groups (1.08±0.57 vs. 1.73±0.50, p = 0.014). Whereas the relative level of cell apoptosis was greater in the DMPA group than that in the control group (1.12±0.36 vs. 0.82±0.39, p = 0.034).Three months of 150 mg DMPA treatment could suppress cell proliferation and enhance cell apoptosis of the eutopic endometrium of women with endometriosis.

Saponins are secondary metabolites that provide medicinal benefits in controlling body homeostasis and metabolic functions. Sea cucumber has been consumed in many Asian countries due to their health benefits. Active chemicals found in sea cucumber include natural source of saponins which are enriched in their tissues, including the Cuvierian tubules and the body wall. Tissue origin of the saponin biosynthesis and accumulation is limitedly known. The present study is to indicate major compositions and distributions of saponins in the body wall of Holothuria leucospilota. Structurally, their body wall consisted of the pigmented layer of the epidermis, the dermal connective tissues, and inner muscular layers. Interestingly, release of the pigmented granules from the epidermis was related to detection of epidermal saponins. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) revealed identical mass spectra in the saponin extracts and compared to the known compounds of holothurians. To investigate the release of epidermal saponins, the epidermis dissolved in either butanol or distilled water were analyzed and presented the saponin masses with two prominent masses of m/z 1,243.3 (holothurin A and scabraside B) and 1,259.3 (holothurin A3). MALDI-IMS also demonstrated strong signals of the known saponins which were only localized in the epidermis of the body wall. Taken together, this study shows that granule release from epidermal pigmented cells is somehow related to the amount of epidermal saponins released to surrounding seawater. Hence, the future research in the sea cucumber better focuses on epidermal cells that are the enriched site of saponins, although several active compounds require further investigation.

Background Apium graveolens L. is a traditional Chinese medicine prescribed as a treatment for hypertension, gout, and diabetes. This study aimed to determine the neuroprotective effects of A. graveolens extract against a Parkinson’s disease (PD) model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57BL/6 mice. Methods Male C57BL/6 mice treated with MPTP were orally dosed with A. graveolens extract daily for 21 days. Behavioral tests, including a rotarod apparatus, a narrow beam test, a drag test, a grid walk test, a swimming test, and a resting tremor evaluation, were performed. Thereafter, the mice were sacrificed, and monoamine oxidase A and B activity, lipid peroxidation activity, and superoxide anion levels were measured. Immunohistochemical staining of tyrosine hydroxylase was performed to identify dopaminergic neurons. Results We found that treatment with A. graveolens at dose of 375 mg/kg demonstrated the highest effect and led to significant improvements in behavioral performance, oxidative stress parameters, and monoamine oxidase A and B activity compared with the untreated group (p

Holothuria scabra is the potential source of terpene with high antioxidant capacity and one of the most valuable species in the trade. The results indicated that antioxidant activity of crude methanol extracts and three pure compounds, Friedelin, 3-Hydroxybenzaldehyde and 4-Hydroxybenzaldehyde, from sea cucumber was determined using DPPH, Folin–Cioccalteau reagent. The results indicated the total phenolic contents at 30.52.28 ± 0.21 GAE/g dry weight equivalent and the effective concentration (EC50) value were found to be 33.77 ± 0.24, 14.63 ± 0.01, 14.62 ± 0.01 and 14.78 ± 0.11 mg/ml whole body of Holothuria scabra, Friedelin, 3-Hydroxybenzaldehyde and 4-Hydroxybenzaldehyde, respectively. In conclusion, the methanol extract and pure compounds of whole body parts of sea cucumber showed the highest antioxidant activity with EC50 value of 33.77 ± 0.24, 14.63 ± 0.01, 14.62 ± 0.01 and 14.68 ± 0.11 mg/ml. It also showed the highest total phenolic content at 30.52 ± 0.21 GAE/g dry weight.

Proteomic analysis of oocytes can help identify proteins that are involved in female meiotic maturation and early embryonic development. Many proteins with well-defined functions have been identified during oocyte maturation. High levels of MPF, MAPK, Mos and low levels of cAMP play an essential role in the resumption of meiosis I. Following germinal vesicle breakdown, chromosome condensation and spindle formation occurred at metaphase I by assembly of the meiotic apparatus, which includes the proteins NuMA, γ-tubulin and Polo-like kinase 1. The metaphase II arrest is a result of high levels of MPF and MAPK. Proteins involved in the stress response and redox regulation, including peroxiredoxin, GST and HSF1, are also necessary for protection against oxidative stress. During fertilization, the sperm-egg interaction requires egg surface proteins, oocyte zona pellucida, molecular chaperones, GPI-anchored proteins and CD9 to recognize sperm proteins and prevent polyspermy. Following gamete fusion, resumption and complete of meiosis II is induced by GTP and CaM kinase II activation, which inactivates MPF and activation of the anaphase promoting complex/cyclosome results in sister chromatid separation. Decondensation of the sperm head begins after zona penetration and GSH and NPM2 are necessary for male pronuclear formation. MAPK inactivation is required for pronuclear formation. At the cleavage stage, the maternal effect proteins PADI6, FLOPED and FILIA are essential for embryonic progression past the two-cell stage. After cell adhesion, cell junctions and the cytoskeleton play an important role in compaction of the morula. Par6, Par3 and protein kinase C are components of the apical polarity complex and are important for formation of the blastocoel cavity. During the blastocyst stage, TEAD4 and CDX2 are required for trophoectoderm formation. This proteomic analysis of oocytes has improved our understanding of the molecular processes that regulate oocyte maturation, fertilization and pre-implantation in mammals.

This study aimed to investigate the effect of astaxanthin (ASX) extracted and ASX powder from shrimp (Litopenaeus vannamei) shells on Wistar rats with Alzheimer&rsquo, s disease, induced by amyloid-&beta, (1-42) peptides. In this task, the rats were divided into eight groups: (1) Control, (2) sham operate, (3) negative control (vehicle) + A&beta, 1-42, (4) ASX extract+A&beta, 1-42, (5) commercial ASX + A&beta, 1-42, (6) ASX powder + A&beta, 1-42, (7) blank powder + A&beta, 1-42, and (8) vitamin E + A&beta, 1-42. All treatments were orally administrated for 30 days. At 14- and 29-days post injection, animals were observed in behavioral tests. On the 31st day, animals were sacrificed, the hippocampus and cortex were collected. Those two brain areas were then homogenized and stored for biochemical and histological analysis. The results showed that the A&beta, 1-42 infused group significantly reduced cognitive ability and increased memory loss, as assessed by the Morris water maze test, novel object recognition test, and novel object location test. Moreover, the A&beta, 1-42 infused group exhibited a deterioration of oxidative markers, including glutathione peroxidase enzymes (GPx), lipid peroxidation (MDA), products of protein oxidation, and superoxide anion in the cortex and the hippocampus. Meanwhile, ASX powder (10 mg/kg body weight) showed a significant reduction in cognitive and memory impairments and oxidative stress which is greater than ASX extract in the same dose of compound or vitamin E (100 mg/kg body weight). Our study indicates the beneficial properties of ASX in alleviation of cognitive functions and reducing neurodegeneration in Wistar rats induced by amyloid-&beta, (1-42) peptides.

The organic matrix proteins of molluscan nacre are known to engage biocompatible and osteoinductive properties. In this study, we aimed to extract Haliotis diversicolor nacreous proteins and assayed their effect on pre-osteoblastic cell differentiation. We found that acid extracted nacreous proteins (AEP) consisted primarily of a major protein band of 25 kDa and three other minor proteins. AEP enhanced alkaline phosphatase (ALP) activity of MC3T3-E1 cells both in time- and concentration-dependent manners. Transcriptional up-regulation of osteogenic markers, including collagen type I (COL-I), osteopontin (OPN) and osteocalcin (OCN), was also apparent in days 7 and 14 upon AEP treatments. At the translational level, higher protein expression of COL-I was evident in AEP treated cells, and the protein was presumably laid down as extracellular matrix. Further de novo sequencing of an AEP major protein revealed a match with the abalone mantle protein sometsuke. Conclusively, we demonstrated that H. diversicolor AEP contains a factor, potentially the mantle protein sometsuke, which may impart in the osteoblastic cell differentiation.

The crustacean X-organ–sinus gland (XO–SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus ( Ppel -CHH), and compared it with crab CHH-type I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel -CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel -CHH and Ppel -MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9 kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XO–SG, raises new questions concerning the established mode of MIH action.

Phosphorylated sperm proteins are crucial for sperm maturation and capacitation as a priori to their fertilization with eggs. In the freshwater prawn, Macrobrachium rosenbergii, a male reproduction-related protein (Mar-Mrr) was known to be expressed only in the spermatic ducts as a protein with putative phosphorylation and may be involved in sperm capacitation in this species. We investigated further the temporal and spatial expression of the Mar-Mrr gene using RT-PCR and in situ hybridization and the characteristics and fate of the protein using immunblotting and immunocytochemistry. The Mar-Mrr gene was first expressed in 4-week-old post larvae and the protein was produced in epithelial cells lining the spermatic ducts, at the highest level in the proximal region and decreased in the middle and distal parts. The native protein had a MW of 17 kDa and a high degree of serine/threonine phosphorylation. It was transferred from the epithelial cells to become a major protein at the anterior region of the sperm. We suggest that it is involved in sperm capacitation and fertilization in this open thelycal species and this is being investigated.

The distributions of neuropeptides in paraffin-embedded tissue sections (PETS) of the eyestalk, brain, and thoracic ganglia of the shrimp Penaeus monodon were visualized by imaging mass spectrometry (IMS). Peptide signals were obtained from PETS without affecting morphological features. Twenty-nine neuropeptides comprising members of FMRFamide, SIFamides, crustacean hyperglycaemic hormone, orcokinin-related peptides, tachykinin-related peptides, and allatostatin A were detected and visualized. Among these findings we first identified tachykinin-related peptide as a novel neuropeptide in this shrimp species. We found that these neuropeptides were distributed at specific areas in the three neural organs. In addition, 28 peptide sequences derived from 4 types of constitutive proteins, including actin, histones, arginine kinase, and cyclophilin A were also detected. All peptide sequences were verified by liquid chromatography–tandem mass spectrometry. The use of IMS on acetic acid-treated PETS enabled us to identify peptides and obtain their specific localizations in correlation with the undisturbed histological structure of the tissue samples.

The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5-7 μm in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13-15 μm in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease.

We have developed an imaging mass spectrometer ― the atmospheric-pressure mass microscope ― with high spatial resolution, which is composed of an atmospheric pressure ion-source chamber for matrix-assisted laser desorption/ionization (AP-MALDI) and a quadrupole ion trap time-of-flight (QIT-TOF) mass spectrometer. The production of 2-dimensional mass spectrum intensity by the atmospheric-pressure mass microscope with a very fine scan pitch enables us to visualize the distribution of lipids and their metabolites in frozen sections without any specific probes or labels. In this study, we applied AP-MALDI-QIT-TOF imaging mass spectrometry to investigate the distribution of lipids in the compound eye-associated eyestalk of the blue swimming crab, Portunus pelagicus. The eyestalk of crustaceans responds to many signaling substances, and most of these substances are involved in lipid metabolism. The merged ion image of five significant biomolecules clearly showed seven separate zones in the eye and eyestalk. The results indicate that the lipid components of each cell layer of the compound eye are different. This study provides information regarding lipid compositions and locations on real tissue that correlate with function. Imaging mass spectrometry (IMS) with high spatial resolution could thus be a powerful tool for imaging and measurement of various samples for a first molecular screening.

Testis maturation, germ cell development and function of sperm, are related to lipid composition. Phosphatidylcholines (PCs) play a key role in the structure and function of testes. As well, increases of polyunsaturated fatty acids (PUFA) and highly unsaturated fatty acids (HUFA), especially arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are essential for male fertility. This study is the first report to show the composition and distribution of PCs and total fatty acids (FAs) in three groups of seminiferous tubules (STs) classified by cellular associations [i.e., A (STs with mostly early germ cells), B (STs with mostly spermatids), and C (STs with spermatozoa)], in three morphotypes of Macrobrachium rosenbergii, [i.e., small male (SM), orange claw male (OC), and blue claw male (BC)]. Thin layer chromatography exhibited levels of PCs reaching maxima in STs of group B. Imaging mass spectrometry showed remarkably high signals corresponding to PC (16:0/18:1), PC (18:0/18:2), PC (18:2/20:5), and PC (16:0/22:6) in STs of groups A and B. Moreover, most signals were detected in the early developing cells and the intertubular area, but not at the area containing spermatozoa. Finally, gas chromatography-mass spectrometry indicated that the major FAs present in the testes were composed of 14:0, 16:0, 17:0, 18:0, 16:1, 18:1, 18:2, 20:1, 20:2, 20:4, 20:5, and 22:6. The testes of OC contained the greatest amounts of these FAs while the testes of BC contained the least amounts of these FAs, and there was more EPA (20:5) in the testes of SM and OC than those in the BC. The increasing amounts of FAs in the SM and OC indicate that they are important for spermatogenesis and spermiogenesis. This knowledge will be useful in formulating diets containing PUFA and HUFA for prawn broodstocks in order to improve testis development, and lead to increased male fecundity.

Background: Holothuria scabra is one of the most commercially important species found in the Pacific region. The sea cucumber extracts have been widely reported to have beneficial health effects. The aim of this study was to determine the nutritional compositions of H. scabra , and compare its important nutritional contents with that of other species. Methods: The sea cucumbers were dissected, sliced into small pieces, and then freeze-dried. The nutritional compositions, including proximate composition, amino acids, fatty acids, collagen, GABA, Vitamin A, C, and E of the whole body and body wall of H. scabra , were analyzed. Results: H. scabra contained a high quantity of protein (22.50% in whole body and 55.18% in body wall) and very low lipids (1.55% in whole body and 1.02% in body wall). The three most abundant amino acids found in both the whole body and body wall were glycine, glutamic acid, and proline. The main fatty acids found in the whole body were stearic acid and nervonic acid, and in the body wall were arachidonic acid and stearic acid. The whole body and body wall also contained high levels of essential amino acids, essential fatty acids, and collagen, in addition to moderate amounts of vitamin E and low amounts of GABA and vitamin C. Conclusions: The sea cucumber, H. scabra , contained high quantity of protein and very low lipid. It contained high essential amino acids, essential fatty acids, nervonic and arachidonic acids, and collagen, which also contained GABA, vitamin C, and vitamin E. Keywords : sea cucumber; Holothuria scabra ; nutrition components; functional food

Neurotransmitters and neurohormones are agents that control gonad maturation in decapod crustaceans. Of these, serotonin (5-HT) and dopamine (DA) are neurotransmitters with known antagonist roles in female reproduction, whilst gonadotropin-releasing hormones (GnRHs) and corazonin (Crz) are neurohormones that exercise both positive and negative controls in some invertebrates. However, the effects of these agents on the androgenic gland (AG), which controls testicular maturation and male sex development in decapods, via insulin-like androgenic gland hormone (IAG), are unknown. Therefore, we set out to assay the effects of 5-HT, DA, l-GnRH-III, oct-GnRH and Crz, on the AG of small male Macrobrachium rosenbergii (Mr), using histological studies, a BrdU proliferative cell assay, immunofluorescence of Mr-IAG, and ELISA of Mr-IAG. The results showed stimulatory effects by 5-HT and l-GnRH-III through significant increases in AG size, proliferation of AG cells, and Mr-IAG production (P < 0.05). In contrast, DA and Crz caused inhibitory effects on the AG through significant decreases in AG size, proliferation of AG cells, and Mr-IAG production (P < 0.05). Moreover, the prawns treated with Crz died before day 16 of the experimental period. We propose that 5-HT and certain GnRHs can be now used to stimulate reproduction in male M. rosenbergii, as they induce increases in AG and testicular size, IAG production, and spermatogenesis. The mechanisms by which these occur are part of our on-going research.

Ovary maturation, oocyte differentiation, and embryonic development in shrimp are highly dependent on nutritional lipids taken up by female broodstocks. These lipids are important as energy sources as well as for cell signaling. In this study, we report on the compositions of major lipids, i.e. phosphatidylcholines (PCs), triacylglycerols (TAGs), and fatty acids (FAs), in the ovaries of the banana shrimp, Penaeus merguiensis, during ovarian maturation. Thin-layer chromatography analysis showed that the total PC and TAG signal intensities increased during ovarian maturation. Further, by using gas chromatography, we found that (1) FAs 14:0, 16:1, 18:1, 18:2, 20:1, and 22:6 proportionally increased as ovarian development progressed to more mature stages; (2) FAs 16:0, 18:0, 20:4, and 20:5 proportionally decreased; and (3) FAs 15:0, 17:0, and 20:2 remained unchanged. By using imaging mass spectrometry, we found that PC 16:0/16:1 and TAG 18:1/18:2/22:6 were detected in oocytes stages 1 and 2. PCs 16:1/20:4, 16:0/22:6, 18:3/22:6, 18:1/22:6, 20:5/22:6, and 22:6/22:6 and TAGs 16:0/16:1/18:3, 16:0/18:1/18:3, 16:0/18:1/18:1, and 16:0/18:2/22:6 were present in all stages of oocytes. In contrast, the PC- and TAG-associated FAs 20:4, 20:5, and 22:6 showed high signal intensities in stage 3 and 4 oocytes. These FAs may act as nutrition sources as well as signaling molecules for developing embryos and the hatching process. Knowledge of lipid compositions and localization could be helpful for formulating the diet for female broodstocks to promote fecundity and larval production.

We have developed a mass microscopy technique, i.e., a microscope combined with high-resolution matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), which is a powerful tool for investigating the spatial distribution of biomolecules without any time-consuming extraction, purification, and separation procedures for biological tissue sections. Mass microscopy provides clear images about the distribution of hundreds of biomolecules in a single measurement and also helps in understanding the cellular profile of the biological system. The sample preparation and the spatial resolution and speed of the technique are all important steps that affect the identification of biomolecules in mass microscopy. In this Award Lecture Review, we focus on some of the recent developments in clinical applications to show how mass microscopy can be employed to assess medical molecular morphology.