New records of a rare gibberfish, Gibberichthyslatifrons (Stephanoberyciformes, Gibberichthyidae), from the South China Sea, with comments on morphological characters.

The gibberfish Gibberichthyslatifrons is recorded for the first time from southwestern Taiwan, northern South China Sea, based on the collection of seven adults and subadults from the Dong-gang fish market, Pingtung. These specimens represent the northernmost extent of the adult range of the species, and they fill a distributional gap in the western Pacific Ocean. Our findings suggest that a population of the species has become recently established in the region. Detailed descriptions, otolith morphology, and fresh coloration are provided with comments on the morphological characteristics of the genus.

Adult morphology and redescription of Lestidiopsindopacificus (Ege, 1953), with comments on the features of related species (Teleostei, Aulopiformes, Paralepididae).

Two specimens representing the first known adults of Lestidiopsindopacificus (Ege, 1953) are reported and described from Taiwan, and the validity and generic assignment of this species are confirmed. The origin of the pelvic fin directly below the dorsal-fin base shows that L.indopacificus belongs to the L.mirabilis species complex. It can be separated from its congeners by the position of the nostrils above the posterior end of the maxilla, the light body color with unevenly distributed melanophores in adults, and a distinct combination of meristic values and other morphological characteristics. New geographic records are reported for the two other current members of this species complex, L.mirabilis (Ege, 1933) and L.extremus (Ege, 1953). The diagnostic features that separate these three very similar species are discussed.

Exploration of anti-leukemic effect of soft coral-derived 13-acetoxysarcocrassolide: Induction of apoptosis via oxidative stress as a potent inhibitor of heat shock protein 90 and topoisomerase II.

13-Acetoxysarcocrassolide (13-AC) is a marine cembranoid derived from the aquaculture soft coral of Lobophytum crassum. The cytotoxic effect of 13-AC against leukemia cells was previously reported but its mechanism of action is still unexplored. In the current study, we showed that 13-AC induced apoptosis of human acute lymphoblastic leukemia Molt4 cells, as evidenced by the cleavage of PARP and caspases, phosphatidylserine externalization, as well as the disruption of mitochondrial membrane potential. The use of N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, attenuated the cytotoxic effect induced by 13-AC. Molecular docking and thermal shift assay indicated that the cytotoxic mechanism of action of 13-AC involved the inhibition of heat shock protein 90 (Hsp 90) activity by eliciting the level of Hsp 70 and topoisomerase IIα in Molt4 cells. 13-AC also exhibited potent antitumor activity by reducing the tumor volume (48.3%) and weight (72.5%) in the in vivo Molt4 xenograft mice model. Our findings suggested that the marine cembranoid, 13-AC, acted as a dual inhibitor of Hsp 90 and topoisomerase IIα, exerting more potent apoptotic activity via the enhancement of ROS generation.

Sarcodia suae modulates the immunity and disease resistance of white shrimp Litopenaeus vannamei against Vibrio alginolyticus via the purine metabolism and phenylalanine metabolism.

Red seaweeds have several biofunctional properties, including immunomodulatory, antitumor, antioxidant, and antibacterial activities. In this study, we examined the effects of diets containing Sarcodia suae on the immune response, immune-related gene expressions, and disease resistance against Vibrio alginolyticus in white shrimp Litopenaeus vannamei. In addition, 1H NMR metabolomics was applied to analyze the metabolites extracted from shrimp fed with S. suae and their functions in regulating immunity. A diet containing only fish meal was used as the control diet (S0), and three diets containing different concentrations of S. suae powder, 2.5% (S2.5), 5% (S5), and 7.5% (S7.5) were used as experimental diets. Shrimp were fed diets for 20 days. Compared to the control group (S0), results showed that (1) shrimp fed diets supplemented with 5-7.5% of S. suae powder significantly increased anti-V. alginolyticus activity; (2) phagocytic activity (PA) increased in all shrimp fed with S. suae, but total haemocyte count (THC) only increased in S7.5 group; and (3) the expression of glutathione peroxidase (GPx) in haemocyte were significantly higher in S7.5 groups. Results from the 1H NMR analysis revealed that 19 heapatopancreatic metabolites were matched and identified among groups. Based on the KEGG enrichment analysis, the up-regulated metabolites in the shrimp fed S5 and S7.5 diets were primarily due to the metabolism of purine and phenylalanine and their respective pathways. Results from these trials reveal that diets containing S. suae can increase immune response, thereby increasing shrimp resistance to V. alginolyticus. The purine and phenylalanine metabolic pathways may be considered as the relevant pathways for optimizing immunomodulatory responses.

Probiotic, Bacillus subtilis E20 alters the immunity of white shrimp, Litopenaeus vannamei via glutamine metabolism and hexosamine biosynthetic pathway.

The purpose of this study was to profile the mechanisms of action of probiotic, Bacillus subtilis E20 in activating the immunity of white shrimp, Litopenaeus vannamei. Two groups of shrimp were studied. One group was fed a control diet without probiotic supplementation and the other was fed a probiotic-containing diet at a level of 109 cfu kg diet-1. After the 8-week feeding regimen, the metabolite composition in the hepatopancreas of shrimp were investigated using 1H nuclear magnetic resonance (1H NMR) based metabolomic analysis. Results from the 1H NMR analysis revealed that 16 hepatopancreatic metabolites were matched and identified among groups, of which 2 metabolites, creatinine and glutamine were significantly higher in probiotic group than in the control group. This result was confirmed by the reverse-phase high-performance liquid chromatography (RP-HPLC) and spectrophotometric analysis. Transcriptome analysis indicated the expressions of 10 genes associated with antioxidant enzymes, pattern recognition proteins and antimicrobial molecules, more active expression in the shrimp fed a diet supplemented with probiotic as compared to that of shrimp in control. In addition, the expressions of 4 genes involved with hexosamine biosynthesis pathway (HBP) and UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase for protein O-glycosylation were also higher in hepatopancreas of probiotic-treated shrimp than in shrimp fed a control diet. Western blot and enzyme-linked immunosorbent assay showed that heat shock factor 1, heat shock protein 70, and protein O-glycosylation in hepatopancreas were higher in probiotic group than the control group. These findings suggest that probiotic, B. subtilis E20 promotes the digestibility of glutamine in the diet, and that the increased glutamine in shrimp can be used as fuel for immune cells or may be used to regulate immune molecule expressions and protein O-glycosylation via the HBP to increase protein O-glycosylation, thereby improving the health of shrimp.

21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer Greatly Expands Mass Spectrometry Toolbox.

We provide the initial performance evaluation of a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer operating at the Environmental Molecular Sciences Laboratory at the Pacific Northwest National Laboratory. The spectrometer constructed for the 21T system employs a commercial dual linear ion trap mass spectrometer coupled to a FTICR spectrometer designed and built in-house. Performance gains from moving to higher magnetic field strength are exemplified by the measurement of peptide isotopic fine structure, complex natural organic matter mixtures, and large proteins. Accurate determination of isotopic fine structure was demonstrated for doubly charged Substance P with minimal spectral averaging, and 8158 molecular formulas assigned to Suwannee River Fulvic Acid standard with root-mean-square (RMS) error of 10 ppb. We also demonstrated superior performance for intact proteins; namely, broadband isotopic resolution of the entire charge state distribution of apo-transferrin (78 kDa) and facile isotopic resolution of monoclonal antibody under a variety of acquisition parameters (e.g., 6 s time-domains with absorption mode processing yielded resolution of approximately 1 M at m/z = 2700). Graphical Abstract ᅟ.

A high-efficiency induction of dopaminergic cells from human umbilical mesenchymal stem cells for the treatment of hemiparkinsonian rats.

The success rate in previous attempts at transforming human umbilical mesenchymal stem cells (HUMSCs) isolated from Wharton's jelly of the umbilical cord into dopaminergic cells was a mere 12.7%. The present study was therefore initiated to establish a more effective procedure for better yield of dopaminergic cells in such transformation for more effective HUMSC-based therapy for parkinsonism. To examine, in vitro, the effects of enhanced Nurr1 expression in HUMSCs on their differentiation, cells were processed through the three-stage differentiation protocol. The capacity of such cells to synthesize and release dopamine was measured by HPLC. The therapeutic effects of Nurr1-overexppressed HUMSCs were examined in 6-hydroxydopamine-lesioned rats by quantification of rotations in response to amphetamine. Enhanced Nurr1 expression in HUMSCs promoted the transformation into dopaminergic cells in vitro through stepwise culturing in sonic hedgehog, fibroblast growth factor-8, and neuron-conditioned medium. The success rate was about 71%, as determined by immunostaining for tyrosine hydroxylase and around 94 nM dopamine synthesis (intracellular and released into the culture medium), as measured by HPLC. Additionally, transplantation of such cells into the striatum of hemiparkinsonian rats resulted in improvement of their behavioral deficits, as indicated by amphetamine-evoked rotation scores. Viability of the transplanted cells lasted for at least 3 months as verified by positive staining for tyrosine hydroxylase. Nurr1, FGF8, Shh, and NCM can synergistically enhance the differentiation of HUMSCs into dopaminergic cells and may pave the way for HUMSC-based treatments for Parkinson's disease.

A low noise single-transistor transimpedance preamplifier for Fourier-transform mass spectrometry using a T feedback network.

A novel single-transistor transimpedance preamplifier has been introduced for improving performance in Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry. A low noise junction field-effect transistor (JFET), BF862, is used as the main amplification stage of this trans-impedance preamplifier, and a T-shaped feedback network is introduced as both the feedback and the gate biasing solutions. The T feedback network has been studied using an operational amplifier (Op Amp), AD8099. Such a feedback system allows ~100-fold less feedback resistance at a given transimpedance, hence preserving bandwidth, which is beneficial to applications demanding high gain. The single-transistor preamplifier yields a tested transimpedance of ~10(4) Ω (80 dBΩ) in the frequency range between 1 kHz and 1 MHz (mass-to-charge ratio, m/z, of around 180-180k for a 12-T FT-ICR system), with a low power consumption of ~6 mW, which implies that this preamplifier is well suited to a 12-T FT-ICR mass spectrometer. In trading noise performance for higher trans-impedance, an alternative preamplifier design, an AD8099 preamplifier with the T feedback network, has also been studied with a capability of ~10(6) Ω (120 dBΩ) transimpedance in the same frequency range. The resistive components in the T feedback network reported here can be replaced by complex impedances, which allows adaptation of this feedback system to other frequency, transimpedance, and noise characteristics for applications not only in other mass spectrometers, such as Orbitrap, time-of-flight (TOF), and ion trap systems, but also in other charge/current detecting systems such as spectroscopy systems, microscopy systems, optical communication systems, or charge-coupled devices (CCDs).

Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation.

The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.

Use of top-down and bottom-up Fourier transform ion cyclotron resonance mass spectrometry for mapping calmodulin sites modified by platinum anticancer drugs.

Calmodulin (CaM) is a highly conserved, ubiquitous, calcium-binding protein; it binds to and regulates many different protein targets, thereby functioning as a calcium sensor and signal transducer. CaM contains 9 methionine (Met), 1 histidine (His), 17 aspartic acid (Asp), and 23 glutamine acid (Glu) residues, all of which can potentially react with platinum compounds; thus, one-third of the CaM sequence is a possible binding target of platinum anticancer drugs, which represents a major challenge for identification of specific platinum modification sites. Here, top-down electron capture dissociation (ECD) was used to elucidate the transition metal-platinum(II) modification sites. By using a combination of top-down and bottom-up mass spectrometric (MS) approaches, 10 specific binding sites for mononuclear complexes, cisplatin and [Pt(dien)Cl]Cl, and dinuclear complex [{cis-PtCl(2)(NH(3))}(2)(µ-NH(2)(CH(2))(4)NH(2))] on CaM were identified. High resolution MS of cisplatin-modified CaM revealed that cisplatin mainly targets Met residues in solution at low molar ratios of cisplatin-CaM (2:1), by cross-linking Met residues. At a high molar ratio of cisplatin:CaM (8:1), up to 10 platinum(II) bind to Met, Asp, and Glu residues. [{cis-PtCl(2)(NH(3))}(2)(µ-NH(2)(CH(2))(4)NH(2))] forms mononuclear adducts with CaM. The alkanediamine linker between the two platinum centers dissociates due to a trans-labilization effect. [Pt(dien)Cl]Cl forms {Pt(dien)}(2+) adducts with CaM, and the preferential binding sites were identified as Met51, Met71, Met72, His107, Met109, Met124, Met144, Met145, Glu45 or Glu47, and Asp122 or Glu123. The binding of these complexes to CaM, particularly when binding involves loss of all four original ligands, is largely irreversible which could result in their failure to reach the target DNA or be responsible for unwanted side-effects during chemotherapy. Additionally, the cross-linking of cisplatin to CaM might lead to the loss of the biological function of CaM or CaM-Ca(2+) due to limiting the flexibility of the CaM or CaM-Ca(2+) complex to recognize target proteins or blocking the binding region of target proteins to CaM.

Antiplatelet effect by p-cresol, a uremic and environmental toxicant, is related to inhibition of reactive oxygen species, ERK/p38 signaling and thromboxane A2 production.

P-cresol is a well-known uremic toxin and environmental toxicant that may affect platelet functions. In this study, p-cresol (1-5 µM) inhibited the arachidonic acid (AA)-induced platelet aggregation, with 47% and 82% of inhibition at concentrations of 2 and 5 µM, respectively. Under similar experimental condition, p-cresol showed little effect on the U46619-induced platelet aggregation. p-cresol (<500 µM) revealed no discernable cytotoxicity to platelets as analyzed by quantification of lactate dehydrogenase release. Antiplatelet effect of p-cresol was related to inhibition of thromboxane A(2) (TXA(2)) and prostaglandin D(2) (PGD(2)) formation. P-cresol (2-100 µM) partly inhibited the AA-induced reactive oxygen species (ROS) production as well as the extracellular signal-regulated kinase (ERK1/2) and p38 phosphorylation in platelets. P-cresol further inhibited the AA-induced aggregation of rabbit platelet-rich plasma (PRP) with an IC50 of 2 µM and aggregation of human PRP (IC50 = 13.6 µM). Intravenous administration of p-cresol (250-1000 nmole) into mice effectively suppressed the ex vivo platelet aggregation, whereas showed little effect on the value of RBC, hemoglobin (HGB), hematocrit, MCV, MCH, MCHC, platelets and lymphocyte counts. These results indicate that in acute p-cresol-poisoning and long-term exposure to cresol as in severe uremic patients, p-cresol may potentially inhibit blood clot formation and lead to hemorrhagic disorders via inhibition of platelet aggregation, ROS production, ERK/p38 activation and TXA(2) production.

Vacuum compatible sample positioning device for matrix assisted laser desorption∕ionization Fourier transform ion cyclotron resonance mass spectrometry imaging.

The high mass accuracy and resolving power of Fourier transform ion cyclotron resonance mass spectrometers (FT-ICR MS) make them ideal mass detectors for mass spectrometry imaging (MSI), promising to provide unmatched molecular resolution capabilities. The intrinsic low tolerance of FT-ICR MS to RF interference, however, along with typically vertical positioning of the sample, and MSI acquisition speed requirements present numerous engineering challenges in creating robotics capable of achieving the spatial resolution to match. This work discusses a two-dimensional positioning stage designed to address these issues. The stage is capable of operating in ∼1 × 10(-8) mbar vacuum. The range of motion is set to 100 mm × 100 mm to accommodate large samples, while the positioning accuracy is demonstrated to be less than 0.4 micron in both directions under vertical load over the entire range. This device was integrated into three different matrix assisted laser desorption∕ionization (MALDI) FT-ICR instruments and showed no detectable RF noise. The "oversampling" MALDI-MSI experiments, under which the sample is completely ablated at each position, followed by the target movement of the distance smaller than the laser beam, conducted on the custom-built 7T FT-ICR MS demonstrate the stability and positional accuracy of the stage robotics which delivers high spatial resolution mass spectral images at a fraction of the laser spot diameter.

Mass spectrometry evidence for cisplatin as a protein cross-linking reagent.

Cisplatin is a potent anticancer drug, which functions by cross-linking adjacent DNA guanine residues. However within 1 day of injection, 65-98% of the platinum in the blood plasma is protein-bound. It is generally accepted that cisplatin binds to methionine and histidine residues, but what is often underappreciated is that platinum from cisplatin has a 2+ charge and can form up to four bonds. Thus, it has the potential to function as a cross-linker. In this report, the cross-linking ability of cisplatin is demonstrated by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with the use of standard peptides, the 16.8 kDa protein calmodulin (CaM), but was unsuccessful for the 64 kDa protein hemoglobin. The high resolution and mass accuracy of FTICR MS along with the high degree of fragmentation of large peptides afforded by collisionally activated dissociation (CAD) and electron capture dissociation (ECD) are shown to be a valuable means of characterizing cross-linking sites. Cisplatin is different from current cross-linking reagents by targeting new functional groups, thioethers, and imidazoles groups, which provides complementarity with existing cross-linkers. In addition, platinum(II) inherently has two positive charges which enhance the detection of cross-linked products. Higher charge states not only promote the detection of cross-linking products with less purification but result in more comprehensive MS/MS fragmentation and can assist in the assignment of modification sites. Moreover, the unique isotopic pattern of platinum flags cross-linking products and modification sites by mass spectrometry.

A gain and bandwidth enhanced transimpedance preamplifier for Fourier-transform ion cyclotron resonance mass spectrometry.

The nature of the ion signal from a 12-T Fourier-transform ion cyclotron resonance mass spectrometer and the electronic noise were studied to further understand the electronic detection limit. At minimal cost, a new transimpedance preamplifier was designed, computer simulated, built, and tested. The preamplifier design pushes the electronic signal-to-noise performance at room temperature to the limit, because of its enhanced tolerance of the capacitance of the detection device, lower intrinsic noise, and larger flat mid-band gain (input current noise spectral density of around 1 pA/√Hz when the transimpedance is about 85 dBΩ). The designed preamplifier has a bandwidth of ~3 kHz to 10 MHz, which corresponds to the mass-to-charge ratio, m/z, of approximately 18 to 61 k at 12 T. The transimpedance and the bandwidth can be easily adjusted by changing the value of passive components. The feedback limitation of the circuit is discussed. With the maximum possible transimpedance of 5.3 MΩ when using an 0402 surface mount resistor, the preamplifier was estimated to be able to detect ~110 charges in a single scan.

Detection of myofibrillar proteins using a step gradient minigel with an ambiguous interface.

Myosin heavy chain (MHC), actin, titin, and nebulin are four major myofibrillar proteins that interact with each other. However, it is difficult to analyze the four proteins simultaneously on the same minigel due to their broad range of molecular weights. Numerous gradient gels are normally used to detect these myofibrillar proteins. The conventional step gradient gel provides better separation of the four major proteins, but several proteins accumulate at the interfaces between different gradient layers. To eliminate the obvious interfaces, we employed a plastic syringe filled with 12 and 4% acrylamide solutions simultaneously and then established an improved step gradient minigel with an ambiguous interface. It was determined by blue dextran in-gel visualization and scanning densitometry that the acrylamide concentration at the ambiguous interface gradually changed. Coomassie blue staining and immunoblotting revealed that the four proteins were successfully separated and transferred for analysis. This gel system is simple to prepare and easy to use, and it is a reliable method for analyzing myofibrillar proteins or other protein mixtures with broad molecular masses.

Age- and region-dependent alterations in the GABAergic innervation in the brain of rats treated with amphetamine.

Mechanisms underlying the pathogenesis of psychotic disorders were explored by monitoring the expression of GABAergic neurons in an animal model. Male rats of postnatal days 21 and 60 were intraperitoneally injected with amphetamine (Amph), 5 mgkg, or saline three times daily for 6 d. After 1-d or 14-d withdrawal from Amph, they were challenged on day 8 (W1d) or on day 21 (W14d) with a single same dosage and then perfused. Immunostaining on the brain sections using an anti-glutamic acid decarboxylase (GAD67) antiserum revealed that the Amph treatment increased the densities of the GAD67-immunoreactive boutons by approx. 36 to 79% above controls in the layers of motor and somatosensory cortices of the W1d juvenile, whereas for those of W14d, the densities resembled controls. For the Amph-treated adults of both W1d and W14d, the GAD67 immunoreactivity increased 56-133% in these layers. In the striatum, the GAD67 densities responded to Amph in a similar manner to the neocortices. However, for the nucleus accumbens, the GAD67 terminals were up-regulated by 22-64% in all Amph-injected rats of both ages. In the hippocampal CA1CA3 region of the Amph-administered juvenile, increases of 24-27% of GAD67 terminals occurred for W1d and W14d animals. By contrast, however, in the W1d Amph-injected adult, there were increases of 42-48% in CA1-CA3, at W14d the GAD67 boutons resembled controls or were reduced. An age-dependent correlation was implicated between behavioural and immunostaining observations. The data support the view that inhibitory regulation is involved in neuronal responses to chronic psychostimulant administration and reflect differential neuronal plasticity among the developing and adult brain regions.