Environmental pH Changes Research Articles

Growth and health responses to a long-term pH stress in Pacific white shrimp Litopenaeus vannamei

Environmental pH change is a challenge for growth and health of aquatic animals. In this study, the juveniles of Pacific white shrimp Litopenaeus vannamei were exposed to a higher pH 9.5 or a lower pH 6.5 versus a normal pH (pH 8.0) as the control for four weeks. Growth performance, antioxidant capacity, immune functions, and intestinal histological damage and microbiota were evaluated. The weight gain rate was significantly decreased in shrimp at pH 9.5 compared with the control shrimp at pH 8.0. The antioxidant capacities of shrimp as activities of SOD, GSH-Px, GST and MDA contents shown at pH 9.5 or pH 6.5 were significantly reduced relative to the control shrimp. The expressions of immune-related genes, including anti-lipopolysaccharide factor, lysozyme, protein toll, immune deficiency, and hemocyte protein-glutamine gamma-glutamyltransferase, were down-regulated and the intestine tissues were distorted. Histology analysis showed that shrimp gut epithelial cells at pH 6.5 separated from the basement membrane, while epithelial cell necrosis were found in shrimp at pH 9.5. The diversity of intestine microbiota was disturbed with lower Shannon and higher Simpson indices in shrimp at pH 6.5 than the control shrimp, but no differences were found in shrimp at pH 9.5. The intestine barrier functions were weakened as the KEGG analysis shown, where functions for protein or carbohydrate digestion and absorption at pH 6.5, and for antibiotics biosynthesis, including streptomycin, penicillin, cephalosporin, novobiocin, ansamycins and vancomycin, at pH 9.5 in shrimp were significantly decreased. All finding in this study suggest that long-term lower or higher pH stress could adversely affect gut microbiota functions, thus leading to shrimp reduced growth, immunity and antioxidant capacity, and even intestine histology damage.

Dynamic pH responsivity of triazole-based self-immolative linkers

Gating the release of chemical payloads in response to transient signals is an important feature of ‘smart’ delivery systems. Herein, we report a triazole-based self-immolative linker that can be reversibly paused or slowed and restarted throughout its elimination cascade in response to pH changes in both organic and organic-aqueous solvents. The linker is conveniently prepared using the alkyne–azide cycloaddition reaction, which introduces a 1,4-triazole ring that expresses a pH-sensitive intermediate during its elimination sequence. Using a series of model compounds, we demonstrate that this intermediate can be switched between active and dormant states depending on the presence of acid or base, cleanly gating the release of payload in response to a fluctuating external stimulus.

Preparation of photo-crosslinkable cinnamate modified hyaluronic acid for immobilization of horseradish peroxidase

Abstract In this work, a photo-responsive hydrogel membrane based on cinnamate-modified hyaluronic acid (HA-CM) was developed and safely cross-linked under UV light curing. The obtained material was effectively utilized for immobilization of horseradish peroxidase (HRP) enzyme via encapsulation and entrapment strategy with efficiency above 95%. The prepared HA-CM biopolymer was investigated before the UV curing using instrumental and spectral techniques including Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR). During the UV irradiation, the progress of the cross-linking reaction was monitored by the UV–vis light spectroscopy. In addition, when the photo-induced cross-linking had accomplished, the morphological appearance of the hydrogel membrane was recorded using a scanning electron microscope (SEM). The HRP immobilized in HA-CM membranes displayed remarkable stability against the environmental pH changes especially under alkaline media and shift the optimum pH to 8 compared to the free HRP, which exhibited the highest activity at pH 7. Also, the entrapped enzyme was able to preserve above 85% of its catalytic activity at higher temperature values where the free enzyme had deactivated by approximately 50%. Moreover, HA-CM-HRP maintained 87% of its activity after 10 sequential reuse cycles, which indicate the economic value of the employed immobilization strategy.

Effects of Environmental pH on Macrophage Polarization and Osteoimmunomodulation.

Biodegradable bone materials can degrade gradually in response to physiological environment and simultaneously promote bone regeneration, thus avoiding the demand of secondary surgery. With the continuous degradation, the environmental pH around the materials could be significantly changed. In the current work, the effects of environmental pH on macrophage polarization and osteoimmunomodulation were investigated. The results showed that the viability of macrophages was inhibited when pH decreased from 7.4 to 6.2 while remained unchanged when pH increased to 8.6. The morphology of macrophages was sensitive to environmental pH changes. The roundish macrophages (pH 7.4) transformed fusiform and polygon-shaped cells at pH 6.6 and 8.2, respectively. The polarization state of macrophages was also found to be pH-dependent. The acidic environment (pH 6.6) tended to polarize macrophages to M2 phenotype, evidenced by the enhanced secretion of arginase-1 (Arg-1), downregulated expressions of M1-related genes such as CD86, tumor necrosis factor-α (TNF-α), interleukin-27 (IL-27), and IL-1β as well as upregulated gene expression of M2 surface marker CD206. The alkaline environment (pH 8.2) led to M1 polarization of macrophages, evidenced by the enhanced production of inducible nitric oxide synthase and upregulated expressions of M1-related genes such as CD11c, TNF-α, IL-1β, and IL-6. Moreover, the gene expressions of growth factors including bone morphogenetic protein-2 (BMP-2), BMP-6, and oncostatin-M (OSM) as well as toll-like receptor (TLR) pathways (TLR-3, TLR-4, and MyD88) were also upregulated at pH 8.2. In addition, the alkaline environment (pH 8.2) was found to exhibit a positive osteoimmunomodulatory effect because the microenvironment created by culturing macrophages in the culture medium with a pH value of 8.2 promoted the alkaline phosphatase (ALP) activity, collagen (Col) production, mineralization, as well as the gene expressions of ALP, Col-I, and osteocalcin (OCN) in osteoblasts. It is considered to be attributed to the enhanced productions of BMP-6 and OSM by macrophages in response to the alkaline environment (pH 8.2). The osteoimmunomodulatory effect of acidic environment (pH 6.6) was found to be weaker. Together, the results indicate that the behaviors of macrophages such as viability, morphology, and polarization state could be modulated by extracellular pH change. The development of novel biomaterials that can create a proper alkaline microenvironment at the materials/bone interface could be beneficial for enhanced bone regeneration.

PH-Responsive i-motif Conjugated Hyaluronic Acid/Polyethylenimine Complexes for Drug Delivery Systems.

i-motif is cytosine (C)-rich oligonucleotide (ODN) which shows pH-responsive structure change in acidic condition. Therefore, it has been utilized for the trigger of intercalated drug release, responding to environmental pH change. In this study, 2.76 molecules of i-motif binding ODNs (IBOs) were conjugated to each hyaluronic acid (HA) via amide bond linkages. Synthesis of HA-IBO conjugate (HB) was confirmed by FT-IR and agarose gel electrophoresis with Stains-All staining. After hybridization of HB with i-motif ODN (IMO), it was confirmed that doxorubicin (DOX) could be loaded in HB-IMO hybrid structure (HBIM) with 65.6% of drug loading efficiency (DLE) and 25.0% of drug loading content (DLC). At pH 5.5, prompt and significant DOX release from HBIM was observed due to the disruption of HBIM hybrid structure via i-motif formation of IMO, contrary to pH 7.4 condition. Then, HBIM was complexed with low molecular weight polyethylenimine (PEI1.8k), forming positively charged nanostructures (Z-average size: 126.0 ± 0.4 nm, zeta-potential: 16.1 ± 0.3 mV). DOX-loaded HBIM/PEI complexes displayed higher anticancer efficacy than free DOX in A549 cells, showing the potential for pH-responsive anticancer drug delivery systems.

PH-induced morphological changes of proteinaceous viral shells

Changes in environmental pH can induce morphological changes in empty proteinaceous shells of bacteriophages in vitro that are very similar to changes occurring in viral capsids in vivo after encapsidation of DNA. These changes in capsid shape and size cannot be explained with a simple elastic model alone. We propose a new theoretical framework that combines the elasticity of thin icosahedral shells with the pH dependence of capsid charge distribution. Minimization of the sum of elastic and electrostatic free energies leads to equilibrium shapes of viral shells that depend on a single elastic parameter and the detailed configuration of the imbedded protein charges. Based on the in vitro shell reconstructions of bacteriophage HK97 we elucidate the details of how the reversible transition between Prohead II and Expansion Intermediate II states of the HK97 procapsid is induced by pH changes, as well as some other features of the bacteriophage maturation.

Smart gelatin films prepared using red cabbage (Brassica oleracea L.) extracts as solvent

This work studied the preparation of smart edible films based on gelatin and anthocyanins. Films were prepared by casting using aqueous or alcoholic extracts of red cabbage (Brassica oleracea L.) as solvent. The addition of these extracts to formulation provided films with important antioxidant properties (evaluated by ABTS.+and FRAP assays) but not antimicrobial activity and the capacity to sense environmental pH changes through the modification of its coloring. The response of these materials against pH changes was evaluated with buffers of different pH (1–14) and simulating their contact with liquid and semisolid foods, and with a container headspace at acid and alkaline pH. Alcoholic extracts were more effective than aqueous ones due to the higher anthocyanins concentration and variety, which exerted a plasticizing effect on the protein matrix. This effect was observed in an increase in film's elongation and solubility and a decrease in its Young's modulus. On the other hand, aqueous extracts seemed to favor the protein cross-linking, as they improved films' mechanical behavior. These materials could be used as active and intelligent packaging of food products, especially those susceptible to oxidation or whose deterioration is accompanied by pH changes.

Anticaries Activity of Curcumin on Decay Process in Human Tooth Enamel Samples (In Vitro Study)

BackgroundDental caries is a big public health problem which is prevalent in the highest degree worldwide, especially in lower socioeconomic levels. This study aims to investigate the anti-caries effects of Curcumin in Turmeric on the caries model of enamel under laboratory conditions. MethodsIn order to make a reliable model of caries, a Streptococcus mutans (ATCC 3568) bacterium was used. The biofilm was created on enamel slabs in 5 days. There were 5 groups of slabs. In order to create Cariogenic conditions, the slabs were exposed to Sucrose 10% three times a day and then, it was exposed to different concentrations of Curcumin (5, 10, 20 mg/ml). The slabs of one group were exposed to the normal saline after the exposure to Sucrose and called positive caries control group; and the other group was exposed to normal saline in each time of exposure which was called negative caries control groups. Environment PH was measured by PH meter two times in a day. After the laboratory phase, the slabs were washed and dried and Vickers hardness test was used to determine the superficial hardness and then the decrease rate of superficial hardness (SHL%) was measured by the approved formula. SPSS version 20 was used to analyse the data. Then, to analyse the data, One-way ANOVA with 95% confidence, Kolmogorov – Smirnov, and Tukey test was used. ResultsThe present study showed that Curcumin can have anti caries effect at the most level even in the least level of concentration used in this study (5 mg/ml). With regard to changes in PH of environment, we can declare that Curcumin results in reducing the dental caries by restraining the bacterial activity. ConclusionsRegarding that these results were received in laboratory conditions, further studies are needed.

Plasticity of the Pyruvate Node Modulates Hydrogen Peroxide Production and Acid Tolerance in Multiple Oral Streptococci

Commensal Streptococcus sanguinis and Streptococcus gordonii are pioneer oral biofilm colonizers. Characteristic for both is the SpxB-dependent production of H2O2, which is crucial for inhibiting competing biofilm members, especially the cariogenic species Streptococcus mutans H2O2 production is strongly affected by environmental conditions, but few mechanisms are known. Dental plaque pH is one of the key parameters dictating dental plaque ecology and ultimately oral health status. Therefore, the objective of the current study was to characterize the effects of environmental pH on H2O2 production by S. sanguinis and S. gordoniiS. sanguinis H2O2 production was not found to be affected by moderate changes in environmental pH, whereas S. gordonii H2O2 production declined markedly in response to lower pH. Further investigation into the pyruvate node, the central metabolic switch modulating H2O2 or lactic acid production, revealed increased lactic acid levels for S. gordonii at pH 6. The bias for lactic acid production at pH 6 resulted in concomitant improvement in the survival of S. gordonii at low pH and seems to constitute part of the acid tolerance response of S. gordonii Differential responses to pH similarly affect other oral streptococcal species, suggesting that the observed results are part of a larger phenomenon linking environmental pH, central metabolism, and the capacity to produce antagonistic amounts of H2O2IMPORTANCE Oral biofilms are subject to frequent and dramatic changes in pH. S. sanguinis and S. gordonii can compete with caries- and periodontitis-associated pathogens by generating H2O2 Therefore, it is crucial to understand how S. sanguinis and S. gordonii adapt to low pH and maintain their competitiveness under acid stress. The present study provides evidence that certain oral bacteria respond to environmental pH changes by tuning their metabolic output in favor of lactic acid production, to increase their acid survival, while others maintain their H2O2 production at a constant level. The differential control of H2O2 production provides important insights into the role of environmental conditions for growth competition of the oral flora.

Differential gene expression in tomato fruit and Colletotrichum gloeosporioides during colonization of the RNAi\u2013SlPH tomato line with reduced fruit acidity and higher pH

BackgroundThe destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi–SlPH tomato lines was sequenced and gene-expression patterns were compared.ResultsC. gloeosporioides inoculation of the RNAi–SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi–SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi–SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi–SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi–SlPH tomato, it mainly upregulates the nitrate metabolic process.ConclusionsModulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi–SlPH fruit, and limited colonization on the WT acidic fruit. The change in environmental pH resulted in different defense responses for the two tomato lines. Interestingly, the WT line showed upregulation of jasmonate pathways and glutamate accumulation, supporting the reduced symptom development and increased ammonia accumulation, as the fungus might utilize glutamate to accumulate ammonia and increase environmental pH for better expression of pathogenicity factors. This was not found in the RNAi–SlPH line which downregulated sugar metabolism and upregulated the phenylpropanoid pathway, leading to host susceptibility.

Smart Microparticles with a pH-responsive Macropore for Targeted Oral Drug Delivery

The development of a smart microencapsulation system programmed to actively respond to environmental pH change has long been recognized a key technology in pharmaceutical and food sciences. To this end, we developed hollow microparticles (MPs) with self-controlled macropores that respond to environmental pH change, using an Oil-in-Water emulsion technique, for oral drug delivery. We observed that freeze-drying of MPs induced closure of macropores. The closing/opening behavior of macropores was confirmed by exposing MPs encapsulating different ingredients (sulforhodamine b, fluorescent nanoparticles, and lactase) to simulated gastrointestinal (GI) fluids. MPs maintained their intact, closed pore structure in gastric pH, and subsequent exposure to intestinal pH resulted in pore opening and ingredients release. Further, MPs displayed higher protection (>15 times) than commercial lactase formulation, indicating the protective ability of the system against harsh GI conditions. This study showed development of a hybrid MP system combining the advantages of solid particles and hollow capsules, exhibiting easy solvent-free loading mechanism and smart protection/release of encapsulates through controllable macropores. Ultimately, our MPs system strives to usher a new research area in smart drug delivery systems and advance the current oral drug delivery technology by solving major challenges in targeted delivery of pH-sensitive therapeutics.

Preparation and controlled release behavior of halloysite/2-mercaptobenzothiazole nanocomposite with calcined halloysite as nanocontainer

2-Mercaptobenzothiazole was loaded in calcined halloysite to prepare nanocomposite through vacuum loading and polyelectrolyte layers coating. The microstructure and morphology of treated nanotubes and synthetic composite were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy and automatic gas adsorption analyzer. The total loading and release behavior of 2-mercaptobenzothiazole in tubes with and without polyelectrolyte shell were investigated with the help of UV–Vis spectrophotometer.Results show that calcination keeps the basic tube structure, expands the inner diameter and shortens the tube length. Halloysite treated at 550°C has the maximum loading of 8.2wt%. The release time of inhibitor in uncoated halloysite is 3–10days, which is closely related to the calcination temperature, and the release rules conform to the first order kinetic model. Polyelectrolyte shell makes it controlled and sustained for composite to release 2-mercaptobenzothiazole under the same condition. When the environmental pH changes from neutral to acidic or alkaline to dissociate the polyelectrolyte layers, 2-mercaptobenzothiazole will be released at different rates immediately. It is concluded that 550°C treated halloysite is a worthy candidate to prepare halloysite/2-mercaptobenzothiazole composite.

Investigation of the effect of laponite and crosslinkers on thermal, mechanical, swelling and conductive properties of the phtagel composite films

Phytagel (PHG)-laponite (L) composites were prepared in the form of films by using PHG known to be biocompatible with the body, L clay which has electrical conductivity, and weak and strong crosslinkers, i.e., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (E) and Cu2+, respectively. Composites were obtained by using laponite in two different ratios and only one concentration of Cu2+. Bare PHG and PHG films crosslinked with E were also prepared simultaneously and characterization of all the films are presented comparatively. Cu2+ ions are more effective crosslinkers than E molecules for PHG chains and increased the mechanical strength of composites as revealed in swelling studies and differential scanning calorimetry analysis. It was determined that laponite and Cu2+ ions increased the conductivity of PHG hydrogel and conductivity decreased when the amount of laponite is increased. Wet film samples had conductivity between 0.95 × 10−8 and 5.94 × 10−8 S cm−1 values, while dry films were not conductive. Wet PHG, PHG-E, PHG-E-L1, and PHG-E-L2 films showed bending deformation towards the anode under electric field of 25 V in magnitude, while wet PHG-Cu-L2 films tended to bend towards the cathode side. It was concluded that these composites are intelligent hydrogel composites which can respond to stimuli such as environmental pH and electric field changes and can be considered for use in studies involving low electrical conductivity.

Phospholipids, fatty acids and hemoglobin in rat erythrocytes under stress conditions (swimming at low temperature)

The phospholipid and fatty acid composition of rat erythrocytes was studied after stress exposure—swimming until drowning. This kind of stress was found to increase the content of phospholipids typical for the outer membrane layer (phosphatidylcholine by 13% and sphingomyelin by 23%). In contrast, the content of acid phospholipids, referring to the inner membrane layer, decreased (phosphatidylethanolamine by 16%, phosphatidylserine by 14% and monophosphoinositide by 23%). Our data indicate that under stress conditions the erythrocyte membrane undergoes certain structural changes, which appear to affect its functional properties. At the same time, the content of saturated and unsaturated fatty acids, as well as their “unsaturation index”, remain basically intact under the above stress conditions, probably, preserving functional properties of the erythrocyte membrane by compensating its impaired phospholipid structure. Based on the analysis of absorption spectra of lipid extracts, stress was established to induce a 2-fold spectrum enhancement in the heme-specific range of 390–410 nm. The appearance of heme in the extract indicates hemoglobin saponification induced by changes in pH of the erythrocyte internal environment. Indeed, during lipid extraction hemoglobin converts into a disordered state due to the effect not only of temperature and pH of the medium, but also of organic solvents, having a lower capacity to form hydrogen bonds than water. Probably, a small portion of phospholipids undergoes trans-esterification during their extraction from erythrocytes by the chloroform–methanol mixture.

Germination Requirements Differ between Fenoxaprop-P-ethyl Resistant and Susceptible Japanese Foxtail (Alopecurus japonicus) Biotypes

Herbicide-resistant (R biotype) and -sensitive (S biotype) individuals were identified from the same population, and seed was increased for each biotype for three generations. We conducted laboratory experiments to determine the effects of temperature, light, salt stress, osmotic stress, pH, and burial depth on the germination and emergence of resistant and sensitive biotypes of Japanese foxtail. The results revealed that there was no difference in the final germination rate between the two biotypes under different temperature conditions, but time to obtain 50% germination or emergence (tE50) and mean germination time of the R biotype were higher than that of the S biotype at 10 C and 15/10 C 12-h day/night regime. In dark conditions, the final germination rate of the S biotype was higher and lower than that of the R biotype at 10 and 25 C, respectively. The overall germination rate of the R biotype was lower than that in the S biotype, and extended germination time was required in extreme conditions, such as 250 mM NaCl and −0.4 MPa osmotic potential. The change in environmental pH had no effect on the germination of the two biotypes. Emergence of the R biotype was lower than the S biotype when seed was buried at least 8 cm deep in an organic matter substrate. This study demonstrated the pleiotropic effects of a resistance allele on seed germination and emergence under different environmental conditions. Deep tillage could be used to reduce the growth and spread of resistant Japanese foxtail individuals.

Development of a P((MAA-co-NVP)-g-EG) Hydrogel Platform for Oral Protein Delivery: Effects of Hydrogel Composition on Environmental Response and Protein Partitioning.

Hydrogels based upon terpolymers of methacrylic acid, N-vinyl pyrrolidone, and poly(ethylene glycol) are developed and characterized for their ability to respond to changes in environmental pH and to partition protein therapeutics of varying molecular weights and isoelectric points. P((MAA-co-NVP)-g-EG) hydrogels are synthesized with PEG-based cross-linking agents of varying length and incorporation densities. The composition is confirmed using FT-IR spectroscopy and shows peak shifts indicating hydrogen bonding. Scanning electron microscopy reveals microparticles with an irregular, planar morphology. The pH-responsive behavior of the hydrogels is confirmed under equilibrium and dynamic conditions, with the hydrogel collapsed at acidic pH and swollen at neutral pH. The ability of the hydrogels to partition model protein therapeutics at varying pH and ionic strength is evaluated using three model proteins: insulin, porcine growth hormone, and ovalbumin. Finally, the microparticles are evaluated for adverse interactions with two model intestinal cell lines and show minimal cytotoxicity at concentrations below 5 mg mL-1 .

Environmental pH and the Requirement for the Extrinsic Proteins of Photosystem II in the Function of Cyanobacterial Photosynthesis.

In one of the final stages of cyanobacterial Photosystem II (PS II) assembly, binding of up to four extrinsic proteins to PS II stabilizes the oxygen-evolving complex (OEC). Growth of cyanobacterial mutants deficient in certain combinations of these thylakoid-lumen-associated polypeptides is sensitive to changes in environmental pH, despite the physical separation of the membrane-embedded PS II complex from the external environment. In this perspective we discuss the effect of environmental pH on OEC function and photoautotrophic growth in cyanobacteria with reference to pH-sensitive PS II mutants lacking extrinsic proteins. We consider the possibilities that, compared to pH 10.0, pH 7.5 increases susceptibility to PS II-generated reactive oxygen species (ROS) causing photoinhibition and reducing PS II assembly in some mutants, and that perturbations to channels in the lumenal regions of PS II might alter the accessibility of water to the active site as well as egress of oxygen and protons to the thylakoid lumen. Reduced levels of PS II in these mutants, and reduced OEC activity arising from the disruption of substrate/product channels, could reduce the trans-thylakoid pH gradient (ΔpH), leading to the impairment of photosynthesis. Growth of some PS II mutants at pH 7.5 can be rescued by elevating CO2 levels, suggesting that the pH-sensitive phenotype might primarily be an indirect result of back-pressure in the electron transport chain that results in heightened production of ROS by the impaired photosystem.

Identification and phylogeny of putative PEPC genes in three toxin‐producing Karenia (Dinophyta) species

Dense blooms of toxin-producing Karenia brevis increase local surface ocean pH through CO2 uptake. To identify genes that may contribute to bloom-related environmental pH and pCO2 changes, transcriptomes with RNA from K.brevis Wilson cultures that had been acclimated to low CO2 (250ppm) or recent CO2 (350ppm) pCO2 levels were assembled. Among the annotated transcripts were PEPC, PPDK, and PEPCK enzymes found in the model C4 carbon fixation pathway. Previous studies have demonstrated that the enzymatic activity of PEPC, PPDK, and/or PEPCK in some algae species, including marine diatoms, is influenced by variations in dissolved inorganic carbon. We found significantly similar PEPC, PPDK, and PEPCK enzymes in the transcriptomes of K.brevis and two sister species Karenia papilionacea, and Karenia mikimotoi. One or more isoforms of PEPC were also identified in the transcriptomes of thirty additional photosynthetic phytoplankton species from nine phyla. Phylogenetic trees were constructed with neighbor joining and maximum likelihood techniques to characterize the evolutionary relationship among phytoplankton, terrestrial plant C4, and terrestrial plant C3 PEPC sequences. Based on the nucleotide trees constructed during this study, the Karenia PEPC transcripts were more closely related to the terrestrial C4 genes than the terrestrial C3 genes. Furthermore, PEPC phylogeny among phytoplankton closely resembles phylogenetic trees constructed with ribosomal RNA. This study confirmed that the toxin-producing dinoflagellates K.brevis, K.mikimotoi, and K. papilionacea express putative PEPC, PEPCK, and PPDK transcripts.

Gelatin based films capable of modifying its color against environmental pH changes

The aim of this work was to develop biodegradable protein-based films capable of sense pH changes. These protein films were prepared by casting from aqueous solutions of bovine gelatin, glycerol and three acid-base indicators: methyl orange (MO), neutral red (NR) and bromocresol green (BCG), at pH 2, 6 and 11. All resulting protein films were homogeneous, thin and had different colors depending on pH and the indicator used. The response of these materials was evaluated simulating their contact with liquid and semisolid media, and with a container headspace at acid and alkaline pH. In all tests, developed protein films could modify their color after being in contact with media of different pH. The physicochemical properties of films were also affected differently by the presence of each acid-base indicator. While the addition of BCG did not significantly modify the properties of control gelatin films, except its color; the incorporation of MO and NR into film-forming solutions significantly improved mechanical properties and decreased the water solubility and moisture content of the resulting protein films without affecting their water vapor permeability.

(Invited) Modulating the Activity of Protein Functionalized Gold Nanoparticles

It is of great potential to apply nanomaterials in biomedical detections. Gold nanoparticles (AuNPs) are widely used due to good biocompatibility, conveniently modification and specific properties. In order to provide AuNPs with more bioactivities, AuNPs are functionalized with different active proteins. The effectiveness of protein functionalized AuNPs in the applications depends mostly on the structure and activity of the immobilized proteins. However, it is observed that the bioactivities of protein functionalized AuNPs is difficult to remain at their original level in most cases. The reason is that the proteins always lose the optimal structures and activities after immobilized on AuNPs either by covalent or nonspecific conjugations. The ability to maintain and modulate protein activity in these functionalized AuNPs becomes the key issue in this field. To solve this problem, we systematically study the bioactivity of protein functionalized AuNPs by immobilizing the proteins on AuNPs by specific orientation of proteins, different surface densities of proteins, and the co-immobilization of proteins and stimuli-responsive polymers. Moreover, the activity of protein functionalized AuNPs is further modulated under different conditions in our work, including different environmental temperatures, pH change, and enzymatic digestion. Different orientations of proteins on AuNP are achieved by introducing the specific amino acid, cysteine, by mutation at fixed sites of the proteins for binding to gold with Au-S bond. The stimuli-responsive polymers with different molecular weights are also functionalized with thiol group to immobilize on AuNPs. Meanwhile, the surface density of proteins and polymer/protein ratio on AuNPs are investigated by the molar ratios of proteins, polymers and nanoparticles in the immobilization systems. The results show that site-directed orientation and surface density of the proteins immobilized on AuNPs can both modulate the activity of protein functionalized AuNPs, and changing the surface density is not as effective as changing the protein orientation on AuNPs for modulating their activities. Moreover, both the molecular weight of stimuli-responsive polymers and polymer/protein molar ratio on AuNPs significantly affect the activity of protein functionalized AuNPs under different conditions. For example, pyrophosphatase (PPase) and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) co-immobilized AuNPs can be almost used as an “on/off” switch for the modulation of bioactivity; furthermore, approximately 90% activity of the functionalized AuNPs is still remained after digested by trypin for 90 min. The fabrication of protein functionalized AuNPs with polymer co-modification provides a new way to produce the responsive nanohybrid with high sensitivity and tunable activity to the environmental stimuli. These results indicate that protein functionalized AuNPs have great potential in biomedical detection, targeted delivery, controllable biocatalysis, and molecular/cellular recognition. The work was supported by National Natural Science Foundation of China (21374070 and 21474072), and the National Natural Science Foundation Fund for Distinguished Young Scholars (21125418).