Novel Lignin-Functionalized Waterborne Epoxy Composite Coatings with Excellent Anti-Aging, UV Resistance, and Interfacial Anti-Corrosion Performance.

Developing high-performance lignin anti-corrosive waterborne epoxy (WEP) coatings is conducive to the advancement of environmentally friendly coatings and the value-added utilization of lignin. In this work, a functionalized biomass waterborne epoxy composite coating is prepared using quaternized sodium lignosulfonate (QLS) as a functional nanofiller for mild carbon steel protection. The results showed that QLS has excellent dispersion and interface compatibility within WEP, and its abundant phenolic hydroxyl, sulfonate, quaternary ammonium groups, and nanoparticle structure endowed the coating with excellent corrosion inhibition and superior barrier properties. The corrosion inhibition efficiency of 100 mg L-1 QLS in carbon steel immersed in a 3.5 wt% NaCl solution reached 95.76%. Furthermore, the coating maintained an impedance modulus of 2.29 × 106  Ω cm2 (|Z|0.01 Hz ) after being immersed for 51 days in the high-salt system. In addition, QLS imparted UV-blocking properties and thermal-oxygen aging resistance to the coating, as evidenced by a |Z|0.01 Hz of 1.04 × 107  Ω cm2 after seven days of UV aging while still maintaining a similar magnitude as before aging. The green lignin/WEP functional coatings effectively withstand the challenging outdoor environment characterized by high salt concentration and intense UV radiation, thereby demonstrating promising prospects for application in metal protection.

Tpy-Mn(II)-Tpy (Tpy = 2,2':6',2″-terpyridine)-Based Pentagonal Prism for Green Photodriven Oxidation.

In coordination-driven metal-organic cages, the transition metal ions are generally utilized as linkages. Employment of its unique properties with the aim of achieving specific applications still presents great challenges. Herein, we report a decametric metal-organic cage named pentagonal prism (Mn20LC10) based on Tpy-Mn(II)-Tpy connectivity (Tpy = 2,2':6',2″-terpyridine) in which Mn(II) serves as a linker and endows the resulting metal-organic cage with good photosensitivity. In the photooxidation of benzaldehyde, pentagonal prism Mn20LC10 showed a significantly increased level of 1O2 production, the fastest conversion time, good recyclability, and substrate versatility due to its greatly improved intersystem crossing ability. Notably, the abundant active sites of metal pentagonal prism Mn20LC10 enable its photooxidation under solvent-free and daylight conditions. This work provides approaches for the development of inexpensive, green, and low-cost photosensitizer systems.

Construction of Single-Atom Catalysts for N, O Synergistic Coordination and Application to Electrocatalytic O2 Reduction.

Replacing expensive platinum oxygen reduction reaction (ORR) catalysts with atomically dispersed single-atom catalysts is an effective way to improve the energy conversion efficiency of fuel cells. Herein, a series of single-atom catalysts, TM-N2O2Cx (TM=Sc-Zn) with TM-N2O2 active units, were designed, and their catalytic performance for electrocatalytic O2 reduction was investigated based on density functional theory. The results show that TM-N2O2Cx exhibits excellent catalytic activity and stability in acidic media. The eight catalysts (TM=Sc, Ti, V, Cr, Mn, Fe, Co, and Ni) are all 4e- reaction paths, among which Sc-N2O2Cx, Ti-N2O2Cx, and V-N2O2Cx follow dissociative mechanisms and the rest are consistent with associative mechanisms. In particular, Co-N2O2Cx and Ni-N2O2Cx enable a smooth reduction in O2 at small overpotentials (0.44 V and 0.49 V, respectively). Furthermore, a linear relationship between the adsorption free energies of the ORR oxygen-containing intermediates was evident, leading to the development of a volcano plot for the purpose of screening exceptional catalysts for ORR. This research will offer a novel strategy for the design and fabrication of exceptionally efficient non-precious metal catalysts on an atomic scale.

Zebrafish Klf4 maintains the ionocyte progenitor population by regulating epidermal stem cell proliferation and lateral inhibition.

In the skin and gill epidermis of fish, ionocytes develop alongside keratinocytes and maintain body fluid ionic homeostasis that is essential for adaptation to environmental fluctuations. It is known that ionocyte progenitors in zebrafish embryos are specified from p63+ epidermal stem cells through a patterning process involving DeltaC (Dlc)-Notch-mediated lateral inhibition, which selects scattered dlc+ cells into the ionocyte progenitor fate. However, mechanisms by which the ionocyte progenitor population is modulated remain unclear. Krüppel-like factor 4 (Klf4) transcription factor was previously implicated in the terminal differentiation of mammalian skin epidermis and is known for its bifunctional regulation of cell proliferation in a tissue context-dependent manner. Here, we report novel roles for zebrafish Klf4 in the ventral ectoderm during embryonic skin development. We found that Klf4 was expressed in p63+ epidermal stem cells of the ventral ectoderm from 90% epiboly onward. Knockdown or knockout of klf4 expression reduced the proliferation rate of p63+ stem cells, resulting in decreased numbers of p63+ stem cells, dlc-p63+ keratinocyte progenitors and dlc+ p63+ ionocyte progenitor cells. These reductions subsequently led to diminished keratinocyte and ionocyte densities and resulted from upregulation of the well-known cell cycle regulators, p53 and cdkn1a/p21. Moreover, mutation analyses of the KLF motif in the dlc promoter, combined with VP16-klf4 or engrailed-klf4 mRNA overexpression analyses, showed that Klf4 can bind the dlc promoter and modulate lateral inhibition by directly repressing dlc expression. This idea was further supported by observing the lateral inhibition outcomes in klf4-overexpressing or knockdown embryos. Overall, our experiments delineate novel roles for zebrafish Klf4 in regulating the ionocyte progenitor population throughout early stem cell stage to initiation of terminal differentiation, which is dependent on Dlc-Notch-mediated lateral inhibition.

Recombinant platelet-derived growth factor-BB alleviates osteoarthritis in a rat model by decreasing chondrocyte apoptosis in vitro and in vivo.

Osteoarthritis (OA) is a common joint disease that mainly affects the diarthrodial joints. Treatments for OA include non-pharmacological interventions, topical and oral therapies, intra-articular therapies and joint surgery. However, all the treatments mentioned above mainly aim to control the symptoms instead of improving or reversing the joint condition. In this research, we observed the effect of recombinant platelet-derived growth factor (PDGF)-BB on OA in a monosodium iodoacetate (MIA)-induced rat model and revealed the possible mechanisms. In vitro, the level of inflammation in the chondrocytes was gradually alleviated, and the apoptosis rate was gradually decreased by PDGF-BB at increasing concentrations. The levels of p-p38, Bax and caspase-3 decreased, and the level of p-Erk increased with increasing PDGF-BB concentration. In vivo, PDGF-BB could significantly reverse chondrocyte and matrix loss. Furthermore, high concentrations of PDGF-BB could alleviate cartilage hyperplasia to remodel the tissue. The level of collagen II was up-regulated, and the levels of collagen X and apoptosis were down-regulated by increasing concentrations of PDGF-BB. In conclusion, recombinant PDGF-BB alleviated OA by down-regulating caspase-3-dependent apoptosis. The effects of PDGF-BB on OA mainly include inhibiting chondrocyte loss, reducing cartilage hyperplasia and osteophyte formation, and regulating collagen anabolism.

Ti3AlC2 MXene nanosheets as a novel corrosion inhibitor for carbon steel in 0.5 M sulfuric acid solution.

Herein we report that Ti3AlC2 MXene nanosheets were identified as a highly effective cathodic protection corrosion inhibitor for carbon steel in hydrochloric acid solution. Ti3AlC2 Mxene nanosheets form a stable inhibition layer on metal surfaces due to their high adsorption capacity and act as a barrier or protective film to prevent attacks from corrosive substances and thus lead to an extended metal service life.

The Effects of Heat Treatment on the Microstructure and Mechanical Properties of a Selective Laser Melted AlCoFeNi Medium-Entropy Alloy.

A single body-centered cubic (BCC)-structured AlCoFeNi medium-entropy alloy (MEA) was prepared by the selective laser melting (SLM) technique. The hardness of the as-built sample was around 32.5 HRC. The ultimate tensile strength (UTS) was around 1211 MPa, the yield strength (YS) was around 1023 MPa, and the elongation (El) was around 10.8%. A novel BCC + B2 + face-centered cubic (FCC) structure was formed after aging. With an increase in aging temperature and duration, the number of fine grains increased, and more precipitates were observed. After aging at 450 °C for 4 h, the formed complex polyphase structure significantly improved the mechanical properties. Its hardness, UTS, YS, and El were around 45.7 HRC, 1535 MPa, 1489 MPa, and 8.5%, respectively. The improvement in mechanical properties was mainly due to Hall-Petch strengthening, which was caused by fine grains, and precipitation strengthening, which was caused by an increase in precipitates after aging. Meanwhile, the FCC precipitates made the alloy have good toughness. The complex interaction of multiple strengthening mechanisms leads to a good combination of strength, hardness, and toughness.

Anti-inflammatory and Chondroprotective Effects of Platelet-derived Growth Factor-BB on Osteoarthritis Rat Models.

Osteoarthritis (OA) is a common and challenging joint disease that mainly affects the diarthrodial joints. Traditionally, except for surgery for severe cases, treatments for OA mainly focus on relieving pain and improving joint function. However, these treatments are not effective for cartilage repair and induce only symptomatic relief. Platelet-derived growth factor (PDGF)-BB, a member of the PDGF cytokine family, has been proved to have effects on protecting the chondrocytes via multiple mechanisms. In this study, we further focused on the effects of PDGF-BB on OA and found that PDGF-BB could attenuate OA development by inhibiting inflammation and enhancing cell proliferation via JAK2/STAT3, PI3K/AKT, and p38 signaling pathways and PKA-mediated regulation of SOX-9/RunX-2. This article demonstrates the feasibility of PDGF-BB application as a treatment for OA. This is the first article that reports that PDGF-BB attenuates OA development via PKA-mediated regulation of SOX-9 and RunX-2.

Retention of ion channel genes expression increases Japanese medaka survival during seawater reacclimation.

Euryhaline teleosts exhibit varying acclimability to survive in environments that alternate between being hypotonic and hypertonic. Such ability is conferred by ion channels expressed by ionocytes, the ion-regulating cells in the gills or skin. However, switching between environments is physiologically challenging, because most channels can only perform unidirectional ion transportation. Coordination between acute responses, such as gene expression, and long-term responses, such as cell differentiation, is believed to strongly facilitate adaptability. Moreover, the pre-acclimation to half seawater salinity can improve the survivability of Japanese medaka (Oryzias latipes) during direct transfer to seawater; here, the ionocytes preserve hypertonic acclimability while performing hypotonic functions. Whether acclimability can be similarly induced in a closed species and their corresponding responses in terms of ion channel expression remain unclear. In the present study, Japanese medaka pre-acclimated in brackish water were noted to have higher survival rates while retaining higher expression of the three ion channel genes ATP1a1a.1, ATP1b1b, and SLC12a2a. This retention was maintained up to 2 weeks after the fish were transferred back into freshwater. Notably, this induced acclimability was not found in its close kin, Indian medaka (Oryzias dancena), the natural habitat of which is brackish water. In conclusion, Japanese medaka surpassed Indian medaka in seawater acclimability after experiencing exposure to brackish water, and this ability coincided with seawater-retention gene expression.

Effects and action mechanisms of individual cytokines contained in PRP on osteoarthritis.

Osteoarthritis (OA) is defined as a degenerative joint disease that can affect all tissues of the joint, including the articular cartilage, subchondral bone, ligaments capsule, and synovial membrane. The conventional nonoperative treatments are ineffective for cartilage repair and induce only symptomatic relief. Platelet-rich plasma (PRP) is a platelet concentrate derived from autologous whole blood with a high concentration of platelets, which can exert anti-inflammatory and regenerative effects by releasing multiple growth factors and cytokines. Recent studies have shown that PRP exhibits clinical benefits in patients with OA. However, high operational and equipment requirements greatly limit the application of PRP to OA treatment. Past studies have indicated that high-concentration PRP growth factors and cytokines may be applied as a commercial replacement for PRP. We reviewed the relevant articles to summarize the feasibility and mechanisms of PRP-based growth factors in OA. The available evidence suggests that transforming growth factor-α and ß, platelet-derived growth factors, epidermal growth factor, insulin-like growth factor-1, and connective tissue growth factors might benefit OA, while vascular endothelial growth factor, tumor necrosis factor-α, angiopoietin-1, and stromal cell derived factor-1α might induce negative effects on OA. The effects of fibroblast growth factor, hepatocyte growth factor, platelet factor 4, and keratinocyte growth factor on OA remain uncertain. Thus, it can be concluded that not all cytokines released by PRP are beneficial, although the therapeutic action of PRP has a valuable potential to improve.

Fructus cannabis protein extract powder as a green and high effective corrosion inhibitor for Q235 carbon steel in 1 M HCl solution.

The Fructus cannabis protein extract powder (FP), was firstly used as a green and high effective corrosion inhibitor through a simple water-extraction method. The composition and surface property of FP were characterized by FTIR, LC/MS, UV, XPS, water contact angle and AFM force-curve measurements. Results indicate that FP contains multiply functional groups, such as NH, CO, CN, CO, etc. The adsorption of FP on the carbon steel surface makes it higher hydrophobicity and adhesion force. The corrosion inhibition performance of FP was researched by electrochemical impedance, polarization curve and differential capacitance curve. Moreover, the inhibitive stability of FP, and the effects of temperature and chloride ion on its inhibition property were also investigated. The above results indicate that the FP exhibits excellent corrosion inhibition efficiency (~98 %), and possesses certain long-term inhibitive stability with inhibition efficiency higher than 90 % after 240 h immersion in 1 M HCl solution. The high temperature brings about the FP desorption on the carbon steel surface, while high concentration of chloride ion facilitates the FP adsorption. The adsorption mechanism of FP follows the Langmuir isotherm adsorption. This work will provide an insight for protein as a green corrosion inhibitor.

A New Imidazole Derivative for Corrosion Inhibition of Q235 Carbon Steel in an Acid Environment.

Q235 carbon steel is a commonly used engineering material, but its application in marine environments is limited by its susceptibility to corrosion, especially localized corrosion that can lead to material perforation. Effective inhibitors are crucial to addressing this issue, particularly in acidic environments where localized areas become increasingly acidic. This study reports the synthesis of a new imidazole derivative corrosion inhibitor and evaluates its effectiveness in corrosion inhibition performance using potentiodynamic polarization curve and electrochemical impedance spectroscopy techniques. High-resolution optical microscopy and scanning electron microscopy were employed for surface morphology analysis. Fourier-transform infrared spectroscopy was used to explore the protection mechanisms. The results demonstrate that the self-synthesized imidazole derivative corrosion inhibitor offers an excellent corrosion protection performance for Q235 carbon steel in a 3.5 wt. % NaCl acidic solution. This inhibitor can provide a new strategy for carbon steel corrosion protection.

Corrosion Behavior of the AlCoCrFeNi2.1 Eutectic High-Entropy Alloy in Chloride-Containing Sulfuric Acid Solutions at Different Temperatures.

In this work, the influence of temperature on the corrosion behavior of AlCoCrFeNi2.1 eutectic high-entropy alloy in a chloride-containing sulfuric acid solution was investigated using electrochemical measurement, X-ray photoelectron spectroscopy, and scanning electron microscopy. Results show that the passive film of AlCoCrFeNi2.1 is stable in chloride-containing sulfuric acid solutions at low temperatures, while an unstable film forms on the alloy at high temperatures. Furthermore, temperature changes the proportion of hydroxide and oxide in Fe and Cr, but it has no noticeable effect on Al and Ni, which is a significant factor on the passive behavior. L12 phase exhibits good corrosion resistance at different temperatures. Pitting occurred on B2 phase in the chloride-containing sulfuric acid solution at a low temperature of 5 °C, while pitting and dissolution take place on AlCoCrFeNi2.1 in the acid solution at room temperature and above.

Anti-Corrosion Mechanism of Parsley Extract and Synergistic Iodide as Novel Corrosion Inhibitors for Carbon Steel-Q235 in Acidic Medium by Electrochemical, XPS and DFT Methods.

The parsley extract (PLE) was prepared using absolute ethyl alcohol. The PLE and synergistic iodide were firstly utilized as efficacious corrosion inhibitors to slow down the corrosion rate of carbon steel-Q235 in 0.5 mol/L H2SO4 solution. The anti-corrosion performance was researched by weight loss method, electrochemical tests, surface analysis and quantum chemistry calculation. Results of electrochemical and weight loss tests show that the synergetic PLE and I- exhibit the optimal corrosion inhibition efficiency 99%. The combined inhibitor displays the favorable long-term corrosion inhibition effect, and the inhibition efficiency can maintain more than 90% after 144 h immersion. The introduction of I- makes carbon steel surface with higher negative charge amount, which could be beneficial to the interaction between corrosion inhibitor and Fe atoms. The adsorption behavior obeys the Langmuir isotherm adsorption, and involves chemical and physical adsorption. On the basis of electrochemical consequences and theoretical calculation, the adsorption process and anti-corrosion mechanisms are further explored.

From local resynchronization to global pattern recovery in the zebrafish segmentation clock.

Integrity of rhythmic spatial gene expression patterns in the vertebrate segmentation clock requires local synchronization between neighboring cells by Delta-Notch signaling and its inhibition causes defective segment boundaries. Whether deformation of the oscillating tissue complements local synchronization during patterning and segment formation is not understood. We combine theory and experiment to investigate this question in the zebrafish segmentation clock. We remove a Notch inhibitor, allowing resynchronization, and analyze embryonic segment recovery. We observe unexpected intermingling of normal and defective segments, and capture this with a new model combining coupled oscillators and tissue mechanics. Intermingled segments are explained in the theory by advection of persistent phase vortices of oscillators. Experimentally observed changes in recovery patterns are predicted in the theory by temporal changes in tissue length and cell advection pattern. Thus, segmental pattern recovery occurs at two length and time scales: rapid local synchronization between neighboring cells, and the slower transport of the resulting patterns across the tissue through morphogenesis.

Resistance band training after triamcinolone acetonide injection for subacromial bursitis: A randomized clinical trial.

OBJECTIVE: To investigate the effect of progressive resistance training using resistance (elastic) bands on subacromial bursitis following triamcinolone acetonide injection. DESIGN: Randomized clinical trial. PATIENTS: A total of 68 patients with subacromial bursitis were randomized to a triamcinolone acetonide group or a triamcinolone acetonide plus resistance band training group. METHODS: Visual analogue scale, Constant scores, range of motion, proprioception, and muscle strength were evaluated at pretreatment and at 3, 12 and 24 weeks' follow-up. Re-treatment ratio was calculated at one-year follow-up. RESULTS: At 3 and 12 weeks, both the triamcinolone acetonide group and triamcinolone acetonide plus resistance band training group showed a significant improvement in Visual analogue scale score, Constant score, range of motion, proprioception and muscle strength. Although the scores in the triamcinolone acetonide group had not increased at 24 weeks compared with baseline, the scores in the triamcinolone acetonide plus resistance band training group showed continued improvement at 24 weeks. A lower proportion of patients in the triamcinolone acetonide plus resistance band training bands group than in the triamcinolone acetonide group had received re-treatment at one-year follow-up (12.1% vs 82.9%). CONCLUSION: Progressive resistance training with resistance (elastic) bands has the advantages of extending the benefits of corticosteroid injection and maintaining long-term effects on shoulder function in patients with subacromial bursitis.

Exposure to silver impairs learning and social behaviors in adult zebrafish.

Silver and silver nanoparticles are used in several consumer products, particularly sterilizing agents. Ag+ released from the particles causes physiological damages of aquatic organisms. However, the effects of silver on neural and behavioral functions of fish remain unclear. Here, we used zebrafish as a model to investigate the impacts of silver on social, learning and memory behaviors in teleost. Adult zebrafish showed mortality rates of 12.875% and 100% on 72 h exposure to 30 and ≥ 50 ppb of silver nitrate, respectively. Silver accumulation in the brain increased on exposure to 10 and 30 ppb of AgNO3. The physical fitness of the zebrafish, measured by novel tank diving test and swimming performance, decreased after 72 h incubation in 30 ppb of AgNO3. Exposure to 10 ppb of AgNO3 impaired social preference, social recognition, learning, and memory, but did not affect anxiety level, aggressiveness, and shoaling behavior. In situ hybridization of c-fos mRNA showed that AgNO3 treatment decreased neural activity in the brain areas crucial for learning, memory, and social behaviors, including the medial and dorsal zones of the dorsal telencephalic area. In conclusion, 72 h exposure to AgNO3 in a sublethal level impaired learning and social behaviors, indicating neurotoxicity in adult zebrafish.

LPA1 is essential for lymphatic vessel development in zebrafish.

Lysophosphatidic acid (LPA) has long been implicated in regulating vascular development via endothelial cell-expressed G protein-coupled receptors. However, because of a lack of notable vascular defects reported in LPA receptor knockout mouse studies, the regulation of vasculature by LPA receptors in vivo is still uncertain. Using zebrafish as a model, we studied the gene expression patterns and functions of an LPA receptor, LPA(1), during embryonic development, in particular, vascular formation. Whole-mount in situ hybridization experiments revealed that zebrafish lpa(1) (zlpa(1)) was ubiquitously expressed early in development, and its expression domains were later localized to the head region and the vicinity of the dorsal aorta. The expression of zlpa(1) surrounding the dorsal aorta suggests its role in vasculature development. Knocking down of zLPA(1) by injecting morpholino (MO) oligonucleotides at 0.625-1.25 ng per embryo resulted in the absence of thoracic duct and edema in pericardial sac and trunk in a dose-dependent manner. These zlpa(1)-MO-resulted defects could be specifically rescued by ectopic expression of zlpa(1). In addition, overexpression of vegf-c, a well-known lymphangiogenic factor, also partially ameliorated the inhibition of thoracic duct development. Taken together, these results demonstrate that LPA(1) is necessary for lymphatic vessel formation during embryonic development in zebrafish.

Sodium Hyaluronate and Platelet-Rich Plasma for Partial-Thickness Rotator Cuff Tears.

PURPOSE: The treatment of partial-thickness rotator cuff tears (PTRCT) remains controversial. Few studies have focused on the conservative and new measurements of small to medium PTRCT. The use of sodium hyaluronate (SH) or platelet-rich plasma (PRP) as a method for rotator cuff repair requires further investigation. The aim of this study was to evaluate the combined use of SH and PRP in the treatment of small to medium PTRCT. STUDY DESIGN: A double-blinded randomized trial was used in this study. METHODS: Individuals with PTRCT detected by clinical examination and magnetic resonance imaging (MRI) were included in this study. The patients were randomly assigned to receive subacromial injections of normal saline, SH, PRP, or SH + PRP once a week for 4 wk. The primary outcome measure was the Constant score, and the secondary outcomes included the American Shoulder and Elbow Surgeons (ASES) and the visual analog scale scores. All of the clinical outcomes were assessed at pretreatment and 1, 3, 6, and 12 months posttreatment. MRI was used to evaluate the evolution of the cuff defect after 1 yr. RESULTS: The PRP group and the SH + PRP group showed a significantly higher Constant score and ASES score after the treatments. There were significant differences between the SH + PRP group and the SH or PRP group at 12 months in the Constant, visual analog scale, and ASES scores. MRI results showed that the tear size significantly decreased in both the PRP and the SH + PRP groups, especially in the SH + PRP group. CONCLUSION: Our study provided evidence of the efficacy of PRP injection in the healing of small to medium PTRCT. Moreover, the combined injection of SH and PRP yielded a better clinical outcome than SH or PRP alone.

Expression and water calcium dependence of calcium transporter isoforms in zebrafish gill mitochondrion-rich cells.

BACKGROUND: Freshwater fish absorb Ca2+ predominantly from ambient water, and more than 97% of Ca2+ uptake is achieved by active transport through gill mitochondrion-rich (MR) cells. In the current model for Ca2+ uptake in gill MR cells, Ca2+ passively enters the cytosol via the epithelium Ca2+ channel (ECaC), and then is extruded into the plasma through the basolateral Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA). However, no convincing molecular or cellular evidence has been available to support the role of specific PMCA and/or NCX isoforms in this model. Zebrafish (Danio rerio) is a good model for analyzing isoforms of a gene because of the plentiful genomic databases and expression sequence tag (EST) data. RESULTS: Using a strategy of BLAST from the zebrafish genome database (Sanger Institute), 6 isoforms of PMCAs (PMCA1a, PMCA1b, PMCA2, PMCA3a, PMCA3b, and PMCA4) and 7 isoforms of NCXs (NCX1a, NCX1b, NCX2a, NCX2b, NCX3, NCX4a, and NCX4b) were identified. In the reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, 5 PMCAs and 2 NCXs were ubiquitously expressed in various tissues including gills. Triple fluorescence in situ hybridization and immunocytochemistry showed the colocalization of zecac, zpmca2, and zncx1b mRNAs in a portion of gill MR cells (using Na+-K+-ATPase as the marker), implying a subset of ionocytes specifically responsible for the transepithelial Ca2+ uptake in zebrafish gills. The gene expressions in gills of high- or low-Ca2+-acclimated zebrafish by quantitative real-time PCR analysis showed that zecac was the only gene regulated in response to environmental Ca2+ levels, while zpmcas and zncxs remained steady. CONCLUSION: The present study provides molecular evidence for the specific isoforms of Ca2+ transporters, zECaC, zPMCA2, and zNCX1b, supporting the current Ca2+ uptake model, in which ECaC may play a role as the major regulatory target for this mechanism during environmental challenge.