Water temperature effects on embryonic development and GnRH-like expression in kisslip cuttlefish (Sepia lycidas) hinting the potential role of GnRH-like peptide.

Gonadotropin-releasing hormone (GnRH)-like peptides are multifunctional neuropeptides involved in cardiac control, early ontogenesis, and reproduction in cephalopods. However, the precise role of GnRH-like peptides in embryonic development and juvenile growth in cephalopods remains unknown. In this study, we showed that GnRH-like peptides are involved in the embryonic development of kisslip cuttlefish (Sepia lycidas). We confirmed that higher water temperatures induced early hatching. Simultaneously, we found that brain GnRH-like peptide gene expression gradually increased with increasing hatching speed. However, the rise in water temperature within a suitable range had no effect on the juvenile sex ratio or early gonadal development. Our results indicate that GnRH-like peptides may play an accelerating role in embryonic development; however, they are not involved in sex determination or early gonadal development in kisslip cuttlefish.

Gene network analyses of Sepia esculenta larvae exposed to copper and cadmium: A comprehensive investigation of oxidative stress, immune response, and toxicological mechanisms.

Copper (Cu) and Cadmium (Cd), prevalent heavy metals in marine environments, have known implications in oxidative stress, immune response, and toxicity in marine organisms. Sepia esculenta, a cephalopod of significant economic value along China's eastern coastline, experiences alterations in growth, mobility, and reproduction when subjected to these heavy metals. However, the specific mechanisms resulting from heavy metal exposure in S. esculenta remain largely uncharted. In this study, we utilized transcriptome and four oxidative, immunity, and toxicity indicators to assess the toxicological mechanism in S. esculenta larvae exposed to Cu and Cd. The measurements of Superoxide Dismutase (SOD), Malondialdehyde (MDA), Glutathione S-Transferase (GST), and Metallothioneins (MTs) revealed that Cu and Cd trigger substantial oxidative stress, immune response, and metal toxicity. Further, we performed an analysis on the transcriptome data through Weighted Gene Co-expression Network Analysis (WGCNA) and Protein-Protein Interaction (PPI) network analysis. Our findings indicate that exposure methods and duration influence the type and the extent of toxicity and oxidative stress within the S. esculenta larvae. We took an innovative approach in this research by integrating WGCNA and PPI network analysis with four significant physiological indicators to closely examine the toxicity and oxidative stress profiles of S. esculenta upon exposure to Cu and Cd. This investigation is vital in decoding the toxicological, immunological, and oxidative stress mechanisms within S. esculenta when subjected to heavy metals. It provides foundational insights capable of advancing invertebrate environmental toxicology and informs S. esculenta artificial breeding practices.

Effect of polycyclic aromatic hydrocarbons on homeobox gene expression during embryonic development of cuttlefish, Sepia officinalis.

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POPs) commonly found in marine environments. Their bioaccumulation can cause harm to aquatic organisms, including invertebrates, particularly during the early stages of embryonic development. In this study, we evaluated, for the first time, the patterns of PAH accumulation in both capsule and embryo of common cuttlefish (Sepia officinalis). In addition, we explored the effects of PAHs by analysing the expression profiles of seven homeobox genes [i.e., gastrulation brain homeobox (GBX), paralogy group labial/Hox1 (HOX1), paralogy group Hox3 (HOX3), dorsal root ganglia homeobox (DRGX), visual system homeobox (VSX), aristaless-like homeobox (ARX) and LIM-homeodomain transcription factor (LHX3/4)]. We found that PAH levels in egg capsules were higher than those observed in chorion membranes (35.1 ± 13.3 ng/g vs 16.4 ± 5.9 ng/g). Furthermore, PAHs were also found in perivitellin fluid (11.5 ± 5.0 ng/ml). Naphthalene and acenaphthene were the congeners present at highest concentrations in each analysed egg component suggesting higher bioaccumulation rates. Embryos with high concentrations of PAHs also showed a significant increase in mRNA expression for each of the analysed homeobox genes. In particular, we observed a 15-fold increase in the ARX expression levels. Additionally, the statistically significant variation in homeobox gene expression patterns was accompanied by a concomitant increase in mRNA levels of both aryl hydrocarbon receptor (AhR) and estrogen receptor (ER). These findings suggest that bioaccumulation of PAHs may modulate developmental processes of cuttlefish embryos by targeting homeobox gene-mediated transcriptional outcomes. Mechanisms underlying the upregulation of homeobox genes could be related to the ability of PAHs to directly activate AhR- or ER-related signaling pathways.

Antioxidant Peptides from Sepia esculenta Hydrolyzate Attenuate Oxidative Stress and Fat Accumulation in Caenorhabditis elegans.

The hydrolysate of golden cuttlefish (Sepia esculenta) was prepared by using papain, and then, it was further separated by ultrafiltration, gel filtration chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The peptide components of the active fraction were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and then two novel peptides, SeP2 (DVEDLEAGLAK, 1159.27 Da) and SeP5 (EITSLAPSTM, 1049.22 Da), were obtained and displayed significant alleviation effects on oxidative stress in Caenorhabditis elegans. Studies indicated that S. esculenta antioxidant peptides (SePs) increase superoxide dismutase (SOD) activity but reduce reactive oxygen species (ROS) and malondialdehyde (MDA) levelsin oxidation-damaged nematodes. Using transgenic CF1553 nematodes, the sod-3p::GFP expression in the worms treated with SePs was significantly higher than that of the control nematodes. Real-time PCR also demonstrated that the expression of stress-related genes such as sod-3 is up-regulated by SePs. Furthermore, studies showed that SePs could obviously decrease fat accumulation as well as reduce the elevated ROS and MDA levels in high-fat nematodes. Taken together, these results indicated that SePs are capable of the activation of antioxidant defense and the inhibition of free radicals and lipid peroxidation, play important roles in attenuating oxidative stress and fat accumulation in C. elegans, and might have the potential to be used in nutraceutical and functional foods.

An FMRFamide Neuropeptide in Cuttlefish Sepia pharaonis: Identification, Characterization, and Potential Function.

Neuropeptides are released by neurons that are involved in a wide range of brain functions, such as food intake, metabolism, reproduction, and learning and memory. A full-length cDNA sequence of an FMRFamide gene isolated from the cuttlefish Sepia pharaonis (designated as SpFMRFamide) was cloned. The predicted precursor protein contains one putative signal peptide and four FMRFamide-related peptides. Multiple amino acid and nucleotide sequence alignments showed that it shares 97% similarity with the precursor FMRFamides of Sepiella japonica and Sepia officinalis and shares 93% and 92% similarity with the SpFMRFamide gene of the two cuttlefish species, respectively. Moreover, the phylogenetic analysis also suggested that SpFMRFamide and FMRFamides from S. japonica and S. officinalis belong to the same sub-branch. Tissue expression analysis confirmed that SpFMRFamide was widely distributed among tissues and predominantly expressed in the brain at the three development stages. The combined effects of SpFMRFamide+SpGnRH and SpFLRFamide+SpGnRH showed a marked decrease in the level of the total proteins released in the CHO-K1 cells. This is the first report of SpFMRFamide in S. pharaonis and the results may contribute to future studies of neuropeptide evolution or may prove useful for the development of aquaculture methods for this cuttlefish species.

Sepia Ink Oligopeptide Induces Apoptosis of Lung Cancer Cells via Mitochondrial Pathway.

BACKGROUND/AIMS: Our previous study suggested the anti-tumor activity of sepia ink oligopeptide (SIO). Here we sought to investigate the underlying molecular mechanism. METHODS: Cell proliferation was evaluated by cell counting kit-8 (CCK-8) assay. Cell apoptosis was determined by Annexin V/Propidium Iodide (PI) staining. The mitochondria pathway was characterized by quantification of Bcl-2, Bax, Caspase-9 and Cyto-C. The death receptor pathway was analyzed by determinement of Fas, Caspase-8 and NIK. The endoplasmic reticulum (ER)-dependent pathway was determined by measurement the expression of CHOP, Caspase-12, GRP78 and Calpain. The associated gene expression was quantified by RT-PCR and protein level was determined by immunoblotting. RESULTS: We demonstrated treatment with structurally modified SIO (CSIO, 5 µM) significantly inhibited cell proliferation and induced apoptosis in lung cancer cell line A549. The mitochondrial pathway, death receptor pathway and ER stress induced apoptosis were stimulated upon CSIO treatment. The administration with respective inhibitors including midiv-1 (50 µM for 2 h), PDTC (20 µM PDTC for 30 min) and ALLN (20 mM ALLN for 5 h) readily reversed the apoptosis inducing effect of CSIO. CONCLUSION: Our data demonstrates that CSIO is capable of induction apoptosis in lung cancer cell line, which is mediated by all three classical apoptotic pathways. Our results warrant further in vivo investigations of the anti-tumor potential of CSIO.

Proteomics analysis of crude squid ink isolated from Sepia esculenta for their antimicrobial, antibiofilm and cytotoxic properties.

The present study deals with the proteomics analysis of crude squid ink isolated from Sepia esculenta for their antibacterial, antifungal, antibiofilm and cytotoxic properties. To achieve this, SDS-PAGE was used to separate proteins as bands, In-gel trypsin digested and analyzed by MALDI-TOF mass spectrometry. A total of 4 bands were identified by MASCOT search analysis namely astacin-like squid metalloprotease type I (ASMT-I), 70 kDa neurofilament protein (NP), uncharacterized protein LOC106181966 isoform X1 (UP-Iso-X1) and Ommochrome-binding protein (Oc-BP). Further, the obtained crude squid proteins were subjected to antimicrobial and antibiofilm activities against pathogenic bacterial and fungal strains respectively. Further, MTT assay was also carried out to deliberately explain the cytotoxic ability of crude squid ink protein against MCF-7 breast cancer cell lines. The results from the study revealed that, the proteins are shown to be toxic against pathogenic strains and breast cancer cell lines in a dose-dependent manner. More importantly, the proteins are well enough to eradicate biofilms substantiated by light and confocal laser scanning microscopic observations. Altogether, the crude squid ink proteins hampered the growth of breast cancer cells with an IC50 value of 65.3 ±â€¯0.46 µg mL-1. In conclusion, it is believed that the proteins from crude squid ink will provide new insights in hampering bacterial biofilms and cancer in near future.

Melanin quantification by in vitro and in vivo analysis of near-infrared fluorescence.

Objective measurements of melanin can provide important information for differentiating melanoma from benign pigmented lesions and in assessing pigmentary diseases. Herein, we evaluate near-infrared (NIR) fluorescence as a possible tool to quantify melanin. Various concentrations of in vitro Sepia melanin in tissue phantoms were measured with NIR fluorescence and diffuse reflectance spectroscopy. Similar optic measurements were conducted in vivo on 161 normal human skin sites. Diffuse reflectance spectroscopy was used to quantify the melanin content via Stamatas-Kollias algorithm. At physiologic concentrations, increasing in vitro melanin concentrations demonstrated higher fluorescence that was linearly correlated (R2  = 0.99, p < .001). At higher concentrations, the fluorescence signal plateaued. A linear relationship was also observed with melanin content in human skin (R2  = 0.59, p < .001). Comparing the fluorescence and reflectance signals with in vitro and in vivo samples, the estimated melanin concentration in human skin ranged between 0 and 1.25 mg/ml, consistent with previous quantitative studies involving invasive methods.

In vitro and in vivo anticancer activity of posterior salivary gland toxin from the cuttlefish Sepia pharaonis, Ehrenberg (1831).

Posterior salivary gland (PSG) toxins are high molecular weight toxins secreted by cephalopods and gastropods which possess immense potentials in biomedical applications. In the present study, the biomedical potentials of the PSG toxin from the cuttlefish, S. pharaonis was determined in vitro and in vivo. The cytostatic potentials of the PSG toxin was determined by the lymphocyte migration inhibition assay. The PSG toxin (50 µg/ml) effectively inhibited the migration of lymphocytes across the agarose gel matrix under the presence of lipopolysaccharide mitogen. The cytotoxicity of the PSG toxin against cancer cell lines was determined using the MTT assay. The PSG toxin exhibited highest cytotoxicity against the MCF-7 breast cancer cells (IC50-10.64 µM) followed by KB, HeLa and A549 cells. The PSG toxin also exhibited proportional release of LDH leakage by mitochondrial damage with an IC50-13.85 µM against MCF-7 breast cancer cells. Flow cytometry analysis revealed that the PSG toxin induced apoptosis in MCF-7 cells by cell cycle arrest at G0/G1 phase. The PSG toxin (80 mg/kg b.w.) exhibited pronounced reduction (29%) in tumor growth in experimentally induced breast carcinoma in female Balb/C mice, in vivo. Hematological analysis illustrated the restoration of blood and biochemical parameters by the PSG toxin in mice induced with tumor. Histopathology studies also revealed the restitution of morphological features in the mammary tumor and vital organs in mice treated with the PSG toxin without any observed toxicity and adverse effects. The PSG toxin further exhibited commendable potentials in the prevention of tumor metastasis into immediate organs viz lungs, thus functioning as an anti-metastatic agent. The results of the present study showed that the PSG toxin exhibited immense promise as a potential peptide based anticancer agent, in future.

Clue to Understanding the Janus Behavior of Eumelanin: Investigating the Relationship between Hierarchical Assembly Structure of Eumelanin and Its Photophysical Properties.

The contradictory biological function of eumelanin (photoprotection vs photosensitization) has long been a topic of debate in a wide range of disciplines such as chemistry, physics and biology. For understanding full spectrum of eumelanin's photobiological aspect, revealing how eumelanin's complex structural organization dictates its photophysical properties is critical step. Here, we report a practical approach to controlling the hierarchically assembled structure of natural eumelanin, which leads to disassembly of its structure into subunits and oxidized subunits, respectively. Based on the well-characterized model system, it was possible to systematically determine how the photophysical properties of eumelanin are ruled by its hierarchical assembly organization. Particularly, our experiments reveal that the chemical oxidation of eumelanin's subunits, which leads to delamination of their stacked layer structure, is critical to significantly increase their photochemical reactivity to generate ROS under UV irradiation. This result provides clear experimental evidence that oxidative degradation of eumelanin, which might be induced by phagosomal enzymatic activity in the process of melanomagenesis, is responsible for triggering the negative photobiological role of eumelanin such as ROS source needed for development of malignant melanoma.

Nonenzymatic Transformation of Amorphous CaCO3 into Calcium Phosphate Mineral after Exposure to Sodium Phosphate in Vitro: Implications for in Vivo Hydroxyapatite Bone Formation.

Studies indicate that mammalian bone formation is initiated at calcium carbonate bioseeds, a process that is driven enzymatically by carbonic anhydrase (CA). We show that amorphous calcium carbonate (ACC) and bicarbonate (HCO3 (-) ) cause induction of expression of the CA in human osteogenic SaOS-2 cells. The mineral deposits formed on the surface of the cells are rich in C, Ca and P. FTIR analysis revealed that ACC, vaterite, and aragonite, after exposure to phosphate, undergo transformation into calcium phosphate. This exchange was not seen for calcite. The changes to ACC, vaterite, and aragonite depended on the concentration of phosphate. The rate of incorporation of phosphate into ACC, vaterite, and aragonite, is significantly accelerated in the presence of a peptide rich in aspartic acid and glutamic acid. We propose that the initial CaCO3 bioseed formation is driven by CA, and that the subsequent conversion to calcium phosphate/calcium hydroxyapatite (exchange of carbonate by phosphate) is a non-enzymatic exchange process.

Antifungal and antihepatotoxic effects of sepia ink extract against oxidative stress as a risk factor of invasive pulmonary aspergillosis in neutropenic mice.

BACKGROUND: There is a great need for novel strategies to overcome the high mortality associated with invasive pulmonary aspergillosis (IPA) in immunocompromised patients. To evaluate the antifungal and antihepatotoxic potentials of Sepia ink extract, its effect on liver oxidative stress levels was analyzed against IPA in neutropenic mice using amphotercin B as a reference drug. MATERIALS AND METHODS: Eighty neutropenic infected mice were randomly assigned into four main groups. The 1(st) group was treated with saline, neutropenic infected (NI), the 2(nd) group was treated with ink extract (200 mg/kg) (IE) and the 3(rd) group was treated with amphotericin B (150 mg/kg) (AMB) and 4(th) group was treated with IE plus AMB. Treatment was started at 24 h after fungal inoculation (1×10(9) conidia/ml). RESULTS: The present study revealed good in vitro and in vivo antifungal activity of IE against A. fumigatus. IE significantly reduced hepatic fungal burden and returns liver function and histology to normal levels. Compared with the untreated infected group, mice in the IE, AMB, and IE+ AMB groups had increased glutathione reduced (GSH) and superoxide dismutase (SOD) and significantly reduced malondialdehyde (MDA) levels at 24 and 72 h after inoculation with A. fumigatus conidia. CONCLUSION: It is then concluded that in combination with antifungal therapy (AMB), IE treatment can reduce hepatic fungal burden, alleviate hepatic granulomatous lesions and oxidative stress associated with IPA in neutropenic mice.

Prophenoloxidase system, lysozyme and protease inhibitor distribution in the common cuttlefish Sepia officinalis.

The immune system of cephalopods remains poorly understood. The aim of this study was to determine the specific activity of immune enzymes in epithelial barriers, circulatory and digestive systems of the common cuttlefish Sepia officinalis. Three enzyme groups with putative functions in immunity were investigated: phenoloxidases (POs), lysozymes and protease inhibitors (PIs). Consistent with a role in immunity, highest PO activities were found in the integument as well as the respiratory and circulatory organs under zymogenic (proPO) and active form. Surprisingly, high PO activities were also found in the digestive gland and its appendages. Similarly, high lysozyme activities were detected in the integument and circulatory organs, but also in the posterior salivary glands, highlighting the implication of this antibacterial enzyme group in most tissues exposed to the environment but also within the circulatory system. Albeit highest in digestive organs, the ubiquitous detection of PI activity in assayed compartments suggests immune function(s) in a wide range of tissues. Our study reports proPO/PO, lysozyme and PI distributions in S. officinalis body compartments for the first time, and thus provides the fundamental basis for a better understanding of the humoral immune system in cephalopods as well as invertebrates.

Sepia ink oligopeptide induces apoptosis in prostate cancer cell lines via caspase-3 activation and elevation of Bax/Bcl-2 ratio.

Sepia ink oligopeptide (SIO) is a tripeptide extracted from Sepia ink. To test the hypothesis that SIO inhibits prostate cancer by inducing apoptosis, the effects of SIO on the proliferation of three human prostate cancer cell lines were examined using a CCK-8 assay. SIO significantly inhibited the proliferation of DU-145, PC-3 and LNCaP cells in a time- and dose-dependent manner. Flow cytometry studies showed that exposing DU-145, PC-3 and LNCaP cells to 5, 10, or 15 mg/mL SIO for 24 h increased the percentage of the early-stage apoptotic cells from 11.84% to 38.26% (DU-145), 22.76% to 39.96% (PC-3) and 5.05% to 16.11% (LNCaP), respectively. In addition, typical morphologic changes were observed in the cells with acridine orange/ethidium bromide staining. SIO treatment induced strong S and G2/M phase cell cycle arrest in a dose-dependent manner in DU-145 and LNCaP. In contrast, SIO treatment induced strong Sub G1 and G0/G1 phase cell cycle arrest in a dose-dependent manner in PC-3. SIO exposure for 24 h decreased the expression of the anti-apoptotic protein Bcl-2 and increased the expression of the apoptogenic protein Bax. Moreover, the Bax/Bcl-2expression ratio was increased. Concurrently, the expression of caspase-3 was upregulated. These data support our hypothesis that SIO has anticarcinogenic properties.

Mechanisms of protein degradation in mantle muscle and proposed gill remodeling in starved Sepia officinalis.

Cephalopods have relatively high rates of protein synthesis compared to rates of protein degradation, along with minimal carbohydrate and lipid reserves. During food deprivation on board protein is catabolized as a metabolic fuel. The aim of the current study was to assess whether biochemical indices of protein synthesis and proteolytic mechanisms were altered in cuttlefish, Sepia officinalis, starved for 7 days. In mantle muscle, food deprivation is associated with a decrease in protein synthesis, as indicated by a decrease in the total RNA level and dephosphorylation of key signaling molecules, such as the eukaryote binding protein, 4E-BP1 (regulator of translation) and Akt. The ubiquitination-proteasome system (UPS) is activated as shown by an increase in the levels of proteasome ß-subunit mRNA, polyubiquitinated protein, and polyubiquitin mRNA. As well, cathepsin activity levels are increased, suggesting increased proteolysis through the lysosomal pathway. Together, these mechanisms could supply amino acids as metabolic fuels. In gill, the situation is quite different. It appears that during the first stages of starvation, both protein synthesis and protein degradation are enhanced in gill. This is based upon increased phosphorylation of 4E-BP1 and enhanced levels of UPS indicators, especially 20S proteasome activity and polyubiquitin mRNA. It is proposed that an increased protein turnover is related to gill remodeling perhaps to retain essential hemolymph-borne compounds.

Steroidogenesis in the brain of Sepia officinalis and Octopus vulgaris.

The presence of vertebrate-like steroids, steroidogenic enzymes and steroid receptors has been reported exclusively in cephalopods gonads. The role played by these steroids has been also recently investigated. We here give evidence of steroidogenic activity in the brain of cephalopods. The activity of two key steroidogenic enzymes: 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD is present in the lobes of the nervous system of both Sepia and Octopus. Such enzymes convert pregnenolone to progesterone and androstenedione to testosterone respectively. Binding experiments seem to assign a functional role to the androgens in the brain of cephalopods. According to the present results, the absence of any progesterone binding moiety supports the hypothesis that progesterone may be a metabolite product along the steroidogenic chain leading to androgens. The presence of steroidogenic enzymes in specific lobes of the central nervous system is discussed in terms of the possible role that steroids can play in the sexual differentiation of the brain and in influencing some coded behaviours of cephalopods, such as learning processes.

Retention of the cyanobacterial neurotoxin beta-N-methylamino-l-alanine in melanin and neuromelanin-containing cells--a possible link between Parkinson-dementia complex and pigmentary retinopathy.

beta-N-methylamino-l-alanine (BMAA), a neurotoxic amino acid produced by cyanobacteria, has been suggested to be involved in the etiology of a neurodegenerative disease complex which includes Parkinson-dementia complex (PDC). In PDC, neuromelanin-containing neurons in substantia nigra are degenerated. Many PDC patients also have an uncommon pigmentary retinopathy. The aim of this study was to investigate the distribution of (3)H-BMAA in mice and frogs, with emphasis on pigment-containing tissues. Using autoradiography, a distinct retention of (3)H-BMAA was observed in melanin-containing tissues such as the eye and neuromelanin-containing neurons in frog brain. Analysis of the binding of (3)H-BMAA to Sepia melanin in vitro demonstrated two apparent binding sites. In vitro-studies with synthetic melanin revealed a stronger interaction of (3)H-BMAA with melanin during synthesis than the binding to preformed melanin. Long-term exposure to BMAA may lead to bioaccumulation in melanin- and neuromelanin-containing cells causing high intracellular levels, and potentially changed melanin characteristics via incorporation of BMAA into the melanin polymer. Interaction of BMAA with melanin may be a possible link between PDC and pigmentary retinopathy.