Hijacking the self-replicating machine of bacteriophage for PCR-based cascade signal amplification in detecting SARS-CoV-2 viral marker protein in serum.

In this work, the nucleic acid detection of SARS-Cov-2 is extended to protein markers of the virus, utilizing bacteriophage. Specifically, the phage display technique enables the main protease of SARS-Cov-2 to control the self-replication of m13 phage, so that the presence of the viral protease can be amplified by phage replication as the first round of signal amplification. Then, the genome of replicated phage can be detected using polymer chain reaction (PCR), as the second round of signal amplification. Based on these two types of well-established biotechnology, the proposed method shows satisfactory sensitivity and robustness in the direct serum detection of the viral protease. These results may point to clinical application in the near future.

A recyclable self-supported nanoporous PdCu heterogeneous catalyst for aqueous Suzuki-Miyaura cross-coupling.

Nanoporous PdCu (NP-PdCu) was prepared by the dealloying strategy from a PdCuAl ternary alloy precursor and characterized systematically using SEM, TEM, XRD, and XPS. NP-PdCu was demonstrated to be a competent self-supported heterogenous catalyst for Suzuki-Miyaura cross-coupling, affording a series of synthetically valuable biaryl compounds in good to excellent yields. This catalyst could be easily separated from the product via centrifugation and reused several times without obvious loss of catalytic performance.

The effects of paeoniflorin injection on soluble triggering receptor expressed on myeloid-1 (sTREM-1) levels in severe septic rats.

Paeoniflorin (PAE) is the most abundant compound in Xuebijing injection widely used to treat sepsis. We aimed to investigate effect of PAE on expression of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in a rat model of sepsis. Wistar rats were divided into Normal, Model, and PAE groups (n=20 each). Endotoxin was administrated at 5 mg/ml/kg in Model and PAE rats to establish rat sepsis model. 1 h after endotoxin administration, PAE was administrated at 4 ml/kg in PAE group once per day for 3 days. Routine blood tests and biochemical indexes were assessed, including aspartate aminotransferase (AST) and creatine kinase-MB (CK-MB). The plasma sTREM-1 level was measured using quantitative ELISA. At the end of experiment, the small intestine, liver, kidney and lung were subjected to pathological examinations. A rat model of sepsis-induced multiple organ dysfunction syndrome (MODS) was established successfully with endotoxin administration (5 mg/ml/kg), evidenced by histo-pathological examinations, routine blood tests and biochemical indexes: platelet count decreased and white blood cell count increased (p<0.05), CK-MB and AST increased (p<0.05). PAE treatment significantly reduced the plasma levels of AST, CK-MB, and sTREM-1, compared to Model group (p<0.05). Meanwhile, sepsis-induced damages in the liver, lung, stomach and intestinal mucosa were also markedly ameliorated by PAE treatment. PAE demonstrated a significantly protective effect in a rat model of sepsis by decreasing plasma sTREM-1 level, reducing inflammation, preventing MODS and protecting organ functions.

Modification of starch metabolism in transgenic Arabidopsis thaliana increases plant biomass and triples oilseed production.

We have identified a novel means to achieve substantially increased vegetative biomass and oilseed production in the model plant Arabidopsis thaliana. Endogenous isoforms of starch branching enzyme (SBE) were substituted by either one of the endosperm-expressed maize (Zea mays L.) branching isozymes, ZmSBEI or ZmSBEIIb. Transformants were compared with the starch-free background and with the wild-type plants. Each of the maize-derived SBEs restored starch biosynthesis but both morphology and structure of starch particles were altered. Altered starch metabolism in the transformants is associated with enhanced biomass formation and more-than-trebled oilseed production while maintaining seed oil quality. Enhanced oilseed production is primarily due to an increased number of siliques per plant whereas oil content and seed number per silique are essentially unchanged or even modestly decreased. Introduction of cereal starch branching isozymes into oilseed plants represents a potentially useful strategy to increase biomass and oilseed production in related crops and manipulate the structure and properties of leaf starch.

Multimeric states of starch phosphorylase determine protein-protein interactions with starch biosynthetic enzymes in amyloplasts.

Protein-protein interactions between starch phosphorylase (SP) and other starch biosynthetic enzymes were investigated using isolated maize endosperm amyloplasts and a recombinant maize enzyme. Plastidial SP is a stromal enzyme existing as a multimeric protein in amyloplasts. Biochemical analysis of the recombinant maize SP indicated that the tetrameric form was catalytically active in both glucan-synthetic and phosphorolytic directions. Protein-protein interaction experiments employing the recombinant SP as an affinity ligand with amyloplast extracts showed that the multimeric state of SP determined interactions with other enzymes of the starch biosynthetic pathway. The monomeric form of SP interacts with starch branching enzyme I (SBEI) and SBEIIb, whereas only SBEI interacts with the tetrameric form of SP. In all cases, protein-protein interactions were broken when amyloplast lysates were dephosphorylated in vitro, and enhanced following pre-treatment with ATP, suggesting a mechanism of protein complex formation regulated by protein phosphorylation. In vitro protein phosphorylation experiments with [γ-(32)P]-ATP show that SP is phosphorylated by a plastidial protein kinase. Evidence is presented which suggests SBEIIb modulates the catalytic activity of SP through the formation of a heteromeric protein complex.

Identification of multiple phosphorylation sites on maize endosperm starch branching enzyme IIb, a key enzyme in amylopectin biosynthesis.

Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser(649), Ser(286), and Ser(297). Two Ca(2+)-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser(649) and Ser(286) phosphorylation, and K2, responsible for Ser(649) and Ser(297) phosphorylation. The Ser(286) and Ser(297) phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel ß-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser(297) forms a stable salt bridge with Arg(665), part of a conserved Cys-containing domain in plant branching enzymes. Ser(649) conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application.

Glucan affinity of starch synthase IIa determines binding of starch synthase I and starch-branching enzyme IIb to starch granules.

The sugary-2 mutation in maize (Zea mays L.) is a result of the loss of catalytic activity of the endosperm-specific SS (starch synthase) IIa isoform causing major alterations to amylopectin architecture. The present study reports a biochemical and molecular analysis of an allelic variant of the sugary-2 mutation expressing a catalytically inactive form of SSIIa and sheds new light on its central role in protein-protein interactions and determination of the starch granule proteome. The mutant SSIIa revealed two amino acid substitutions, one being a highly conserved residue (Gly522→Arg) responsible for the loss of catalytic activity and the inability of the mutant SSIIa to bind to starch. Analysis of protein-protein interactions in sugary-2 amyloplasts revealed the same trimeric assembly of soluble SSI, SSIIa and SBE (starch-branching enzyme) IIb found in wild-type amyloplasts, but with greatly reduced activities of SSI and SBEIIb. Chemical cross-linking studies demonstrated that SSIIa is at the core of the complex, interacting with SSI and SBEIIb, which do not interact directly with each other. The sugary-2 mutant starch granules were devoid of amylopectin-synthesizing enzymes, despite the fact that the respective affinities of SSI and SBEIIb from sugary-2 for amylopectin were the same as observed in wild-type. The data support a model whereby granule-bound proteins involved in amylopectin synthesis are partitioned into the starch granule as a result of their association within protein complexes, and that SSIIa plays a crucial role in trafficking SSI and SBEIIb into the granule matrix.

Allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein-protein interactions.

Amylose extender (ae(-)) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae(-) maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein-protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae(-) mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272-Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16-20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-(32)P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn(2+)-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule.

The amylose extender mutant of maize conditions novel protein-protein interactions between starch biosynthetic enzymes in amyloplasts.

The amylose extender (ae(-)) mutant of maize lacks starch branching enzyme IIb (SBEIIb) activity, resulting in amylopectin with reduced branch point frequency, and longer glucan chains. Recent studies indicate isozymes of soluble starch synthases form high molecular weight complexes with SBEII isoforms. This study investigated the effect of the loss of SBEIIb activity on interactions between starch biosynthetic enzymes in maize endosperm amyloplasts. Results show distinct patterns of protein-protein interactions in amyloplasts of ae(-) mutants compared with the wild type, suggesting functional complementation for loss of SBEIIb by SBEI, SBEIIa, and SP. Coimmunoprecipitation experiments and affinity chromatography using recombinant proteins showed that, in amyloplasts from normal endosperm, protein-protein interactions involving starch synthase I (SSI), SSIIa, and SBEIIb could be detected. By contrast, in ae(-) amyloplasts, SSI and SSIIa interacted with SBEI, SBEIIa, and SP. All interactions in the wild-type were strongly enhanced by ATP, and broken by alkaline phosphatase, indicating a role for protein phosphorylation in their assembly. Whilst ATP and alkaline phosphatase had no effect on the stability of the protein complexes from ae(-) endosperm, radiolabelling experiments showed SP and SBEI were both phosphorylated within the mutant protein complex. It is proposed that, during amylopectin biosynthesis, SSI and SSIIa form the core of a phosphorylation-dependent glucan-synthesizing protein complex which, in normal endosperm, recruits SBEIIb, but when SBEIIb is absent (ae(-)), recruits SBEI, SBEIIa, and SP. Differences in stromal protein complexes are mirrored in the complement of the starch synthesizing enzymes detected in the starch granules of each genotype, reinforcing the hypothesis that the complexes play a functional role in starch biosynthesis.

Starch biosynthetic enzymes from developing maize endosperm associate in multisubunit complexes.

Mutations affecting specific starch biosynthetic enzymes commonly have pleiotropic effects on other enzymes in the same metabolic pathway. Such genetic evidence indicates functional relationships between components of the starch biosynthetic system, including starch synthases (SSs), starch branching enzymes (BEs), and starch debranching enzymes; however, the molecular explanation for these functional interactions is not known. One possibility is that specific SSs, BEs, and/or starch debranching enzymes associate physically with each other in multisubunit complexes. To test this hypothesis, this study sought to identify stable associations between three separate SS polypeptides (SSI, SSIIa, and SSIII) and three separate BE polypeptides (BEI, BEIIa, and BEIIb) from maize (Zea mays) amyloplasts. Detection methods included in vivo protein-protein interaction tests in yeast (Saccharomyces cerevisiae) nuclei, immunoprecipitation, and affinity purification using recombinant proteins as the solid phase ligand. Eight different instances were detected of specific pairs of proteins associating either directly or indirectly in the same multisubunit complex, and direct, pairwise interactions were indicated by the in vivo test in yeast. In addition, SSIIa, SSIII, BEIIa, and BEIIb all comigrated in gel permeation chromatography in a high molecular mass form of approximately 600 kD, and SSIIa, BEIIa, and BEIIb also migrated in a second high molecular form, lacking SSIII, of approximately 300 kD. Monomer forms of all four proteins were also detected by gel permeation chromatography. The 600- and 300-kD complexes were stable at high salt concentration, suggesting that hydrophobic effects are involved in the association between subunits.

Analysis of protein complexes in wheat amyloplasts reveals functional interactions among starch biosynthetic enzymes.

Protein-protein interactions among enzymes of amylopectin biosynthesis were investigated in developing wheat (Triticum aestivum) endosperm. Physical interactions between starch branching enzymes (SBEs) and starch synthases (SSs) were identified from endosperm amyloplasts during the active phase of starch deposition in the developing grain using immunoprecipitation and cross-linking strategies. Coimmunoprecipitation experiments using peptide-specific antibodies indicate that at least two distinct complexes exist containing SSI, SSIIa, and either of SBEIIa or SBEIIb. Chemical cross linking was used to identify protein complexes containing SBEs and SSs from amyloplast extracts. Separation of extracts by gel filtration chromatography demonstrated the presence of SBE and SS forms in protein complexes of around 260 kD and that SBEII forms may also exist as homodimers. Analysis of cross-linked 260-kD aggregation products from amyloplast lysates by mass spectrometry confirmed SSI, SSIIa, and SBEII forms as components of one or more protein complexes in amyloplasts. In vitro phosphorylation experiments with gamma-(32)P-ATP indicated that SSII and both forms of SBEII are phosphorylated. Treatment of the partially purified 260-kD SS-SBE complexes with alkaline phosphatase caused dissociation of the assembly into the respective monomeric proteins, indicating that formation of SS-SBE complexes is phosphorylation dependent. The 260-kD SS-SBEII protein complexes are formed around 10 to 15 d after pollination and were shown to be catalytically active with respect to both SS and SBE activities. Prior to this developmental stage, SSI, SSII, and SBEII forms were detectable only in monomeric form. High molecular weight forms of SBEII demonstrated a higher affinity for in vitro glucan substrates than monomers. These results provide direct evidence for the existence of protein complexes involved in amylopectin biosynthesis.

[Impact of patient compliance on the outcomes in hypertensive patients receiving hydrochlorothiazide based combination therapy with spironolactone or captopril].

OBJECTIVE: To explore the impact of patient compliance on the long-term outcomes in hypertensive patients receiving hydrochlorothiazide (HCTZ) based combination therapy with spironolactone or captopril. METHODS: A total of 853 patients with mild to moderate hypertension were recruited and randomly divided into HCTZ group (HCTZ 12.5 mg q.d), spironolactone group (HCTZ 12.5 mg q.d and spironolactone 20 mg q.d), and captopril group (HCTZ 12.5 mg q.d and captopril 25 mg bid) after 2-week placebo washout period and 6-week loading period for HCTZ. Since the efficacy of combination therapy was proven to be better than monotherapy 1 year after therapy beginning, patients in HCTZ group were randomly assigned to spironolactone group or captopril group. The patients were followed up for 4 years. Patients were divided to compliance (n = 424) or non-compliance group (n = 429) according test drug taking questionnaire. During the follow-up time, the blood pressure and the outcomes were recorded monthly, and blood biochemical parameters were determined once a year. RESULTS: At the end of follow up, incidence of cardio-cerebral vascular events was significantly lower in compliance group (2 fatal, 8 non-fatal) than that in noncompliance group (7 fatal, 21 non-fatal, P < 0.05). Systolic blood pressure [-(19.4 +/- 20.6) mm Hg, 1 mm Hg = 0.133 kPa] and diastolic blood pressure [-(10.7 +/- 13.5) mm Hg] were significantly reduced compared values at baseline and noncompliance group (all P < 0.001) while the reduction did not reach statistically significance in noncompliance group [-(7.3 +/- 18.2) mm Hg and -(3.5 +/- 10.2) mm Hg, all P > 0.05 vs. baseline]. The serum BUN, Cr and UA levels in the compliance group were significantly higher and the serum K(+), CHO, LDL-C level were significantly lower than baseline values. The serum BUN, UA levels in the compliance group were significantly higher while the serum K(+), cholesterol levels were significantly lower than those in the noncompliance group (all P < 0.05). CONCLUSIONS: This study indicates that patient compliance could affect the long-term outcome and antihypertensive efficacy in hypertensive patients receiving HCTZ based combination therapy with spironolactone or captopril.

[Chronic effects of low-dose hydrochlorothiazide in patients with mild to moderate essential hypertension].

OBJECTIVE: To investigate the chronic efficacy of low-dose hydrochlorothiazide (HCTZ) in patients with mild-to-moderate hypertension. METHODS: After a 2-weeks placebo run-in period, 232 patients with mild or moderate hypertension were recruited and received HCTZ (12.5 mg once daily) therapy for one year. Patient compliance and blood pressure were monitored and serum BUN, Cr, glucose, electrolytes, and lipids were measured before, 6 weeks and 1 year after treatment. RESULTS: (1) Reduction of SBP, DBP and MAP were more significantly at 1 year [(10.45 +/- 17.28) mm Hg, (8.45 +/- 11.06) mm Hg, (9.12 +/- 10.88) mm Hg] than that at 6 weeks post therapy [(6.01 +/- 16.05) mm Hg, (2.90 +/- 10.33) mm Hg, (3.94 +/- 10.68) mm Hg, all P < 0.05]. Blood pressure were reduced to normal in 35.1% patients at 1 year and in 20.3% patients at 6 weeks (P < 0.05). (2) No patient developed diabetes mellitus or hypokalemia during therapy while the serum uric acid at 1 year post therapy was significantly higher than that at before therapy (P < 0.05). CONCLUSION: The study indicates that low dose HCTZ is an effective and safe antihypertensive agent for patients with mild-to-moderate hypertension and uric acid changes during therapy need to be monitored.