Effects of ultra-high pressure, thermal pasteurization, and ultra-high temperature sterilization on color and nutritional components of freshly-squeezed lettuce juice.

The aim of this study was to evaluate the effects of ultra-high pressure (UHP, 600 MPa/2 min), thermal pasteurization (TP, 95 °C/1 min) and ultra-high temperature (UHT, 115 °C/5 s) sterilization on the color, sensory evaluation, microorganisms, physicochemical characteristics and nutritional components of freshly-squeezed lettuce juice (FLJ). Results showed that three sterilization methods demonstrated desirable inactivation effects on total aerobic bacteria, yeast and mold, and there were no significant changes in the main nutritional components, including ash, protein, carbohydrate and total dietary fiber. However, UHT and TP significantly affected the color of FLJ from bright green to light brown and made chlorophyll, ß-carotene and vitamins (VE, VC, VK1, VB6, VB12, and folic acid) contents markedly decreased. By contrast, UHP maintained the original color, fresh-like sensory qualities, vitamins, and carotene of FLJ to the greatest extent. Our results provide a promising application of UHP in the large-scale processing of FLJ.

Ethanol fumigation combined with modified atmosphere packaging delays potato greening under light.

Potato greening under light causes post-harvest loss and compromises progress being made in the potato industry. In this study, we investigated the inhibiting effects of ethanol fumigation treatment on potato greening and water loss under light. This paper also examined the different treatments on the relationship between starch granule size and potato greening. Our results suggested that ethanol fumigation combined with modified atmosphere packaging have an effective in improving the overall visual quality and maintained a higher a* value and lower chlorophyll concentration. Ethanol fumigation combined with modified atmosphere packaging treatment deterred greening and water loss compared to the alone treatment. This was due to the larger starch granule size and fewer grana lamellae around the amyloplasts. Our results provide an effectively strategy that treating potatoes with 600 µL L-1 ethanol combined with modified atmosphere packaging to delay potato greening and explain the underlying mechanism of ethanol inhibition of potato greening.

Low temperature reduces potato wound formation by inhibiting phenylpropanoid metabolism and fatty acid biosynthesis.

Introduction: Potato tubers have the healing capacity to prevent surface water transpiration and pathogen invasion after mechanical damage. Previous research has shown the inability to form healing periderm in potatoes under low temperatures, but the potential mechanism is still unclear. Methods: To explore the effects and mechanisms of low-temperature potato healing, wounded potatoes were stored at low temperature (4°C) and room temperature (22°C), respectively. Results: In this study, compared with 22°C healing, low temperature reduced the content of hydrogen peroxide, and the down-regulation of StAMY23 inhibited the conversion of starch to sugar, alleviated the degradation of starch, and reduced the content of soluble sugars and sucrose. Meanwhile, inhibition of phenylalanine metabolism by suppression of StPAL1 and St4CL expression reduced lignin accumulation. Low temperature also down-regulated the expression of StKCS6, StFAOH, StGPAT5, and StPrx, causing the lower deposition amount of suberin in wounds of potato tubers. Discussion: The above results suggested that low temperature led to less wound tissue deposition at the wound surfaces via suppressing phenylpropanoid metabolism and fatty acid biosynthesis in potato tubers.

Stringent Starvation Protein Regulates Prodiginine Biosynthesis via Affecting Siderophore Production in Pseudoalteromonas sp. Strain R3.

Prodiginines are a family of red-pigmented secondary metabolites with multiple biological activities. The biosynthesis of prodiginines is affected by various physiological and environmental factors. Thus, prodiginine biosynthesis regulation is highly complex and multifaceted. Although the regulatory mechanism for prodiginine biosynthesis has been extensively studied in Serratia and Streptomyces species, little is known about that in the marine betaproteobacterium Pseudoalteromonas In this study, we report that stringent starvation protein A (SspA), an RNA polymerase-associated regulatory protein, is required for the biosynthesis of prodiginine in Pseudoalteromonas sp. strain R3. The strain lacking sspA (ΔsspA) fails to produce prodiginine, which resulted from the downregulation of the prodiginine biosynthetic gene (pig) cluster. The effect of SspA on prodiginine biosynthesis is independent of histone-like nucleoid structuring protein (H-NS) and RpoS (σS). Further analysis demonstrates that the ΔsspA strain has a significant decrease in the transcription of the siderophore biosynthesis gene (pvd) cluster, leading to the inhibition of siderophore production and iron uptake. The ΔsspA strain regains the ability to synthesize prodiginine by cocultivation with siderophore producers or the addition of iron. Therefore, we conclude that SspA-regulated prodiginine biosynthesis is due to decreased siderophore levels and iron deficiency. We further show that the iron homeostasis master regulator Fur is also essential for pig transcription and prodiginine biosynthesis. Overall, our results suggest that SspA indirectly regulates the biosynthesis of prodiginine, which is mediated by the siderophore-dependent iron uptake pathway.IMPORTANCE The red-pigmented prodiginines are attracting increasing interest due to their broad biological activities. As with many secondary metabolites, the biosynthesis of prodiginines is regulated by both environmental and physiological factors. At present, studies on the regulation of prodiginine biosynthesis are mainly restricted to Serratia and Streptomyces species. This work focused on the regulatory mechanism of prodiginine biosynthesis in Pseudoalteromonas sp. R3. We found that stringent starvation protein A (SspA) positively regulates prodiginine biosynthesis via affecting the siderophore-dependent iron uptake pathway. The connections among SspA, iron homeostasis, and prodiginine biosynthesis were investigated. These findings uncover a novel regulatory mechanism for prodigiosin biosynthesis.

Stringent starvation protein A and LuxI/LuxR-type quorum sensing system constitute a mutual positive regulation loop in Pseudoalteromonas.

Bacteria commonly exhibit social activities through acyl-homoserine lactones (AHLs)-based quorum sensing (QS) systems to form their unique social network. The sigma factor RpoS is an important regulator that controls QS system in different bacteria. However, the upstream of RpoS involving regulation on QS system remains unclear. In Escherichia coli RpoS is regulated by stringent starvation protein A (SspA), which is dependent of histone-like nucleoid structuring protein (H-NS). To date, the connection between SspA and QS system is essentially unknown. Here, we characterized a typical LuxI/LuxR-type QS system in marine bacterium Pseudoalteromonas sp. T1lg65 which can produce four types of AHLs. The luxI encoding AHLs synthase and luxR encoding AHLs-responsive receptor are co-transcribed, providing advantages in rapidly amplifying QS signaling. Notably, SspA positively regulated luxI/luxR transcription by activating RpoS expression, which is mediated by H-NS. Interestingly, LuxR in turn positively regulated SspA expression. Therefore, SspA and QS system constitute a mutual positive regulation loop in T1lg65. In view of the crucial roles of SspA and QS system in environmental adaption, we believe that the improvement of bacterial tolerance to marine environments could be related to rapidly tuning SspA-involved QS programming.

SspA positively controls exopolysaccharides production and biofilm formation by up-regulating the algU expression in Pseudoalteromonas sp. R3.

Biofilm formation enhances the survival and persistence of microorganisms in response to environmental stresses. It has been revealed that stringent starvation protein A (SspA) can function as an important regulator dealing with environmental stresses for bacterial survival. However, the connection between SspA and biofilm formation is essentially unclear yet. In this study, we presented evidence showing SspA positively controls biofilm formation by up-regulating exopolysaccharides (EPS) production in marine bacterium Pseudoalteromonas sp. R3. Both qPCR and lacZ reporter system congruously revealed that SspA positively controls the expression of EPS biosynthesis gene cluster. Unlike generally accepted thought that SspA regulates bacterial physiology by inhibiting the expression of histone-like nucleotide structuring protein (H-NS) gene, the function of SspA on EPS production and biofilm formation in Pseudoalteromonas sp. R3 is H-NS-independent. Instead, SspA positively regulates the expression of sigma factor AlgU-encoding gene, thus affecting EPS biosynthesis and biofilm formation. In view of the important role of SspA in biofilm formation, we believe that the improvement of tolerance to marine environmental stresses could be related to tuning of SspA-involved biofilm formation.

Aryl Fluoride Activation Through Palladium-Magnesium Bimetallic Cooperation: A Mechanistic and Computational Study.

Herein is described a mechanistic study of a palladium-catalyzed cross-coupling of aryl Grignard reagents to fluoroarenes that proceeds via a low-energy heterobimetallic oxidative addition pathway. Traditional oxidative additions of aryl chlorides to Pd complexes are known to be orders of magnitude faster than with aryl fluorides, and many palladium catalysts do not activate aryl fluorides at all. The experimental and computational studies outlined herein, however, support the view that at elevated Grignard : ArX ratios (i.e. 2.5 : 1) a Pd-Mg heterobimetallic mechanism predominates, leading to a remarkable decrease in the energy required for Ar-F bond activation. The heterobimetallic transition state for C-X bond cleavage is proposed to involve simultaneous Pd backbonding to the arene and Lewis acid activation of the halide by Mg to create a low-energy transition state for oxidative addition. The insights gained from this computational study led to the development of a phosphine ligand that was shown to be similarly competent for Ar-F bond activation.

Identification and characterization of a LuxI/R-type quorum sensing system in Pseudoalteromonas.

Bacteria usually produce, release and detect quorum sensing (QS)-based signal molecules, and successively orchestrate gene expression in respond to environmental changes. Pseudoalteromonas are typical marine bacteria, but knowledge on their QS systems is extremely fragmentary. In this study, genome sequencing of Pseudoalteromonas sp. R3 was performed. Accordingly, a QS working model including three sets of hierarchically organized QS systems was proposed in strain R3. Among them, the typical LuxI/R-type QS system using acyl-homoserine lactones (AHLs) as signal molecules was characterized. Sequence similarity analysis indicated luxI encoding AHLs synthase is novel. The luxR encoding AHLs receptor is directly adjacent to luxI downstream. Notably, mutagenesis demonstrated LuxI and LuxR affect each other at transcriptional level, and both control the AHLs formation. Interestingly, it was found that LuxI/R-type QS system positively involves resistance to streptomycin. Thin-layer chromatography analysis showed strain R3 can produce 3-OH-C6-HSL and C8-HSL, which was supported by heterologous expression of LuxI in Escherichia coli. Sequence alignment analysis indicated that the N-terminal region of LuxI is more conservative than the C-terminal region, revealing the importance of N-terminal region in AHLs synthesis. The obtained findings enrich our knowledge on LuxI/R-type QS system in Pseudoalteromonas and its regulation on adaptation to marine environments.

Dissemination of Genetic Acquisition/Loss Provides a Variety of Quorum Sensing Regulatory Properties in Pseudoalteromonas.

A bstract: Quorum sensing (QS) enables single-celled bacteria to communicate with chemical signals in order to synchronize group-level bacterial behavior. Pseudoalteromonas are marine bacteria found in versatile environments, of which QS regulation for their habitat adaptation is extremely fragmentary. To distinguish genes required for QS regulation in Pseudoalteromonas, comparative genomics was deployed to define the pan-genomics for twelve isolates and previously-sequenced genomes, of which acyl-homoserine lactone (AHL)-based QS traits were characterized. Additionally, transposon mutagenesis was used to identify the essential QS regulatory genes in the selected Pseudoalteromonas isolate. A remarkable feature showed that AHL-based colorization intensity of biosensors induced by Pseudoalteromonas most likely correlates with QS regulators genetic heterogeneity within the genus. This is supported by the relative expression levels of two of the main QS regulatory genes (luxO and rpoN) analyzed in representative Pseudoalteromonas isolates. Notably, comprehensive QS regulatory schema and the working model proposed in Pseudoalteromonas seem to phylogenetically include the network architectures derived from Escherichia coli, Pseudomonas, and Vibrio. Several associated genes were mapped by transposon mutagenesis. Among them, a right origin-binding protein-encoding gene (robp) was functionally identified as a positive QS regulatory gene. This gene lies on a genomic instable region and exists in the aforementioned bioinformatically recruited QS regulatory schema. The obtained data emphasize that the distinctly- and hierarchically-organized mechanisms probably target QS association in Pseudoalteromonas dynamic genomes, thus leading to bacterial ability to accommodate their adaption fitness and survival advantages.

NIXANTPHOS: a highly active ligand for palladium catalyzed Buchwald-Hartwig amination of unactivated aryl chlorides.

Xantphos is one of the two most common ligands used in palladium catalyzed Buchwald-Hartwig amination reactions, because of its broad scope and high probability of success. It does not, however, work well with unactivated aryl chlorides. Herein NIXANTPHOS is compared to Xantphos and an array of mono- and bidentate phosphines. NIXANTPHOS outperforms Xantphos and all other bidentate ligands examined. Under the optimal reaction conditions, unactivated aryl chlorides afford the expected products in good to excellent yield with as low as 0.05 mol% (500 ppm) palladium loading.

Effects of the extract of Ginkgo biloba on the differentiation of bone marrow mesenchymal stem cells in vitro.

The balance of osteogenesis and adipogenesis in bone marrow mesenchymal stem cells (BMSCs) is disrupted in osteoporosis. This study was designed to investigate the effects of extract of Ginkgo biloba (EGB) on proliferation, osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells in vitro. The effect of EGB on proliferation was evaluated by CCK-8 assay and flow cytometry. Osteogenic differentiation was evaluated by Alizarin Red S staining and Alkaline phosphatase assay. Adipogenic differentiation was evaluated by Oil Red O staining. Quantitative real-time polymerase chain reaction (Real-time PCR) was used to detect the expression of osteogenic specific genes (BMP-2, Runx2 and Colla1) and adipogenic specific genes (ap2, PPARγ). EGB did not significantly affect proliferation of BMSCs. However, it increased the calcium accumulation and significantly promoted the activity of alkaline phosphatase, especially when the concentration of EGB reached 150 µg/mL. EGB dose-dependently inhibited the adipogenic ability of BMSCs. The osteogenic-related genes (BMP-2, Runx2, Colla1) were overexpressed while the expression of genes involved in adipogenesis, such as PPAR-γ and ap2, was decreasing with the increase of EGB concentration. Our data proves that EGB inhibited adipocyte differentiation and enhanced osteogenic differentiation in BMSCs, but had no effect on the proliferation of BMSCs.

Pollutants removal in subsurface infiltration systems by shunt distributing wastewater with/without intermittent aeration under different shunt ratios.

Matrix dissolved oxygen (DO), removal of COD, TP and nitrogen in subsurface infiltration systems (SISs), named SIS A (without intermittent aeration and shunt distributing wastewater), SIS B (with shunt distributing wastewater) and SIS C (with intermittent aeration and shunt distributing wastewater) were investigated. Aerobic conditions were developed in 50cm depth and anoxic or anaerobic conditions were not changed in 80 and 110cm depth by intermittent aeration. Under appropriate shunt ratios, shunt distributing wastewater improved denitrification and had little influence on COD, TP and NH3-N removal. Under the optimal shunt ratio of 1:2 for SIS C, high average removal rates of COD (90.06%), TP (93.17%), NH3-N (88.20%) and TN (85.79%) were obtained, which were higher than those in SIS A (COD: 82.56%, TP: 92.76%, NH3-N: 71.08%, TN: 49.24%) and SIS B (COD: 81.12%, TP: 92.58%, NH3-N: 69.14%, TN: 58.73%) under the optimal shunt ratio of 1:3.

Positional Selectivity in C-H Functionalizations of 2-Benzylfurans with Bimetallic Catalysts.

Metal-catalyzed carbon-carbon bond-forming reactions are a mainstay in the synthesis of pharmaceutical agents. A long-standing problem plaguing the field of transition metal catalyzed C-H functionalization chemistry is control of selectivity among inequivalent C-H bonds in organic reactants. Herein we advance an approach to direct site selectivity in the arylation of 2-benzylfurans founded on the idea that modulation of cooperativity in bimetallic catalysts can enable navigation of selectivity. The bimetallic catalysts introduced herein exert a high degree of control, leading to divergent site-selective arylation reactions of both sp(2) and sp(3) C-H bonds of 2-benzylfurans. It is proposed that the selectivity is governed by cation-π interactions, which can be modulated by choice of base and accompanying additives [MN(SiMe3)2, M = K or Li·12-crown-4].

Palladium-Catalyzed Asymmetric Allylic Alkylations with Toluene Derivatives as Pronucleophiles.

The first two highly enantioselective palladium-catalyzed allylic alkylations with benzylic nucleophiles, activated with Cr(CO)3 , have been developed. These methods enable the enantioselective synthesis of α-2-propenyl benzyl motifs, which are important scaffolds in natural products and pharmaceuticals. A variety of cyclic and acyclic allylic carbonates are competent electrophilic partners furnishing the products in excellent enantioselectivity (up to 99 % ee and 92 % yield). This approach was employed to prepare a nonsteroidal anti-inflammatory drug analogue.

Synthesis of triarylmethanols via tandem arylation/oxidation of diarylmethanes.

A tandem arylation/oxidation of diarylmethanes for the convenient synthesis of unsymmetrical triarylmethanols bearing different aryl and heteroaryl groups is described. A Pd(OAc)2-NiXantphos catalyst system efficiently catalyzed arylation of weakly acidic sp3-hybridized C-H bonds of diarylmethanes with aryl bromides, and the arylation products were then oxidized in situ to carbinols by simply opening the reaction flasks to air. The triarylmethanol products were obtained in 35-98% yield.

Palladium-catalyzed α-arylation of aryl acetic acid derivatives via dienolate intermediates with aryl chlorides and bromides.

To date, examples of α-arylation of carboxylic acids remain scarce. Using a deprotonative cross-coupling process (DCCP), a method for palladium-catalyzed γ-arylation of aryl acetic acids with aryl halides has been developed. This protocol is applicable to a wide range of aryl bromides and chlorides. A procedure for the palladium-catalyzed α-arylation of styryl acetic acids is also described.

NiXantphos: a deprotonatable ligand for room-temperature palladium-catalyzed cross-couplings of aryl chlorides.

Although the past 15 years have witnessed the development of sterically bulky and electron-rich alkylphosphine ligands for palladium-catalyzed cross-couplings with aryl chlorides, examples of palladium catalysts based on either triarylphosphine or bidentate phosphine ligands for efficient room temperature cross-coupling reactions with unactivated aryl chlorides are rare. Herein we report a palladium catalyst based on NiXantphos, a deprotonatable chelating aryldiphosphine ligand, to oxidatively add unactivated aryl chlorides at room temperature. Surprisingly, comparison of an extensive array of ligands revealed that under the basic reaction conditions the resultant heterobimetallic Pd-NiXantphos catalyst system outperformed all the other mono- and bidentate ligands in a deprotonative cross-coupling process (DCCP) with aryl chlorides. The DCCP with aryl chlorides affords a variety of triarylmethane products, a class of compounds with various applications and interesting biological activity. Additionally, the DCCP exhibits remarkable chemoselectivity in the presence of aryl chloride substrates bearing heteroaryl groups and sensitive functional groups that are known to undergo 1,2-addition, aldol reaction, and O-, N-, enolate-α-, and C(sp(2))-H arylations. The advantages and importance of the Pd-NiXantphos catalyst system outlined herein make it a valuable contribution for applications in Pd-catalyzed arylation reactions with aryl chlorides.

Chemo- and regioselective C(sp³)-H arylation of unactivated allylarenes by deprotonative cross-coupling.

The combination of aryl bromides, allylbenzene, base and a palladium catalyst usually results in a Heck reaction. Herein we combine these same reagents, but override the Heck pathway by employing a strong base. In the presence of LiN(SiMe3)2, allylbenzene derivatives undergo reversible deprotonation. Transmetalation of the resulting allyllithium intermediate to LPdAr(Br) and reductive elimination provide the 1,1-diarylprop-2-enes, which are not accessible by the Heck reaction. The regioselectivity in this deprotonative cross-coupling process is catalyst-controlled and very high.

Raising the pKa limit of "soft" nucleophiles in palladium-catalyzed allylic substitutions: application of diarylmethane pronucleophiles.

The Tsuji-Trost allylic substitution reaction provides a useful and efficient approach to construct C-C bonds between sp(3)-hybridized carbons. The widely accepted paradigm for classifying the mode of attack of nucleophiles on palladium π-allyl intermediates in the Tsuji-Trost reaction is based on the pKa of the pronucleophile: (1) stabilized or "soft" carbon nucleophiles and heteroatom nucleophiles (e.g., pronucleophiles with pKa's < 25), and (2) unstabilized or "hard" nucleophiles (those from pronucleophiles with pKa's > 25). One of the keys to the continuing development of allylic substitution processes remains broadening the scope of "soft" nucleophiles. Herein we report a general method for the room temperature Pd-catalyzed allylic substitution with diarylmethane derivatives (pKa's up to 32). The synthetic significance of the method is that it provides a rapid access to products containing allylated diarylmethyl motifs. The method is general for a wide range of nucleophiles derived from diarylmethanes and heterocyclic derivatives. A procedure for the Pd-catalyzed allylic substitutions to afford diallylation products with quaternary centers is also described. With triarylmethanes and alkylated diarylmethanes the corresponding allylated products are isolated. We anticipate that the described method will be a valuable complement to the existing arsenal of nucleophiles in Pd-catalyzed allylic substitutions. Mechanistic studies show that the nucleophile derived from diphenylmethane undergoes external attack on π-allyl palladium species under our reaction conditions. This unexpected observation indicates that diarylmethane derivatives behave as "soft" or stabilized nucleophiles. The results of this study indicate that the cutoff between "soft" and "hard" nucleophiles should be raised from a pronucleophile pKa of 25 to at least 32.