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A desmoplastic stroma, enriched in cancer-associated fibroblasts (CAF), has been associated with resistance to therapy in patients with pancreatic ductal adenocarcinoma (PDAC). Here, after showing that chemotherapy promotes tumor fibrosis by increasing CAF frequency and activity, the authors develop a multi-paratopic VEGF decoy receptor for PD-L1 directed PlGF/VEGF blockade, promoting anti-fibrotic and anti-tumor effects in PDAC models.

The root traits and response strategies of plants play crucial roles in mediating interactions between plant root systems. Current research on the role of root exudates as underground chemical signals mediating these interactions has focused mainly on crops, with less attention given to desert plants in arid regions. In this study, we focused on the typical desert plant Haloxylon ammodendron and conducted a pot experiment using three root isolation methods (plastic film separation, nylon mesh separation, and no separation). We found that (1) as the degree of isolation increased, plant biomass significantly increased (p < 0.05), while root organic carbon content exhibited the opposite trend; (2) soil electrical conductivity (EC), soil total nitrogen (STN), soil total phosphorus (STP), and soil organic carbon (SOC) were significantly greater in the plastic film and nylon mesh separation treatments than in the no separation treatment (p < 0.05), and the abundance of Proteobacteria and Actinobacteriota was significantly greater in the plastic film separation treatment than in the no separation treatment (p < 0.05); (3) both plastic film and nylon mesh separations increased the secretion of alkaloids derived from tryptophan and phenylalanine in the plant root system compared with that in the no separation treatment; and (4) Pseudomonas, Proteobacteria, sesquiterpenes, triterpenes, and coumarins showed positive correlations, while both pseudomonas and proteobacteria were significantly positively correlated with soil EC, STN, STP, and SOC (p < 0.05). Aurachin D was negatively correlated with Gemmatimonadota and Proteobacteria, and both were significantly correlated with soil pH, EC, STN, STP, and SOC. The present study revealed strong negative interactions between the root systems of H. ammodendron seedlings, in which sesquiterpenoids, triterpenoids, coumarins, and alkaloids released by the roots played an important role in the subterranean competitive relationship. This study provides a deeper understanding of intraspecific interactions in the desert plant H. ammodendron and offers some guidance for future cultivation of this species in the northwestern region of China.

Foliar water uptake (FWU) has increasingly been regarded as a common approach for plants to obtain water under water-limited conditions. At present, the research on FWU has mostly focused on short-term experiments; the long-term FWU plant response remains unclear; Methods: Through a field in-situ humidification control experiment, the leaves of Calligonum ebinuricum N. A. Ivanova ex Soskov were humidified, and the changes of leaf water potential, gas exchange parameters and fluorescence physiological parameters of plants after long-term and short-term FWU were discussed; The main results were as follows: (1) After short-term humidification, the water potential of Calligonum ebinuricum decreased, the non-photochemical quenching (NPQ) increased, and the plant produced photoinhibition phenomenon, indicating that short-term FWU could not alleviate drought stress. (2) After long-term humidification, the leaf water potential, chlorophyll fluorescence parameter and net photosynthetic rate (Pn) increased significantly. That is to say, after long-term FWU, the improvement of plant water status promoted the occurrence of light reaction and carbon reaction, and then increased the net photosynthetic rate (Pn); Therefore, long-term FWU is of great significance to alleviate drought stress and promote Calligonum ebinuricum growth. This study will be helpful to deepen our understanding of the drought-tolerant survival mechanism of plants in arid areas.

ABSTRACT Antimicrobial resistance presents one of the most significant threats to human health, with the emergence of totally drug-resistant organisms. We have combined bioengineering, genetically modified bacteria, longitudinal readouts, and fluidics to develop a transformative platform to address the drug development bottleneck, utilizing Mycobacterium tuberculosis as the model organism. We generated microspheres incorporating virulent reporter bacilli, primary human cells, and an extracellular matrix by using bioelectrospray methodology. Granulomas form within the three-dimensional matrix, and mycobacterial stress genes are upregulated. Pyrazinamide, a vital first-line antibiotic for treating human tuberculosis, kills M. tuberculosis in a three-dimensional culture but not in a standard two-dimensional culture or Middlebrook 7H9 broth, demonstrating that antibiotic sensitivity within microspheres reflects conditions in patients. We then performed pharmacokinetic modeling by combining the microsphere system with a microfluidic plate and demonstrated that we can model the effect of dynamic antibiotic concentrations on mycobacterial killing. The microsphere system is highly tractable, permitting variation of cell content, the extracellular matrix, sphere size, the infectious dose, and the surrounding medium with the potential to address a wide array of human infections and the threat of antimicrobial resistance. IMPORTANCE Antimicrobial resistance is a major global threat, and an emerging concept is that infection should be studied in the context of host immune cells. Tuberculosis is a chronic infection that kills over a million people every year and is becoming progressively more resistant to antibiotics. Recent major studies of shorter treatment or new vaccination approaches have not been successful, demonstrating that transformative technologies are required to control tuberculosis. We have developed an entirely new system to study the infection of host cells in a three-dimensional matrix by using bioengineering. We showed that antibiotics that work in patients are effective in this microsphere system but not in standard infection systems. We then combined microspheres with microfluidics to model drug concentration changes in patients and demonstrate the effect of increasing antibiotic concentrations on bacterial survival. This system can be widely applied to address the threat of antimicrobial resistance and develop new treatments.

The objective of the present study was to assess the effect of alginate-encapsulated infectious pancreatic necrosis virus antigens in inducing the immune response of Atlantic salmon as booster vaccines. One year after intraperitoneal injection with an oil-adjuvanted vaccine, post-smolts were orally boosted either by 1) alginate-encapsulated IPNV antigens (ENCAP); 2) soluble antigens (UNENCAP) or 3) untreated feed (control). This was done twice, seven weeks apart. Sampling was done twice, firstly at 7 weeks post 1st oral boost and the 2nd, at 4 weeks after the 2nd oral boost. Samples included serum, head kidney, spleen and hindgut. Serum antibodies were analyzed by ELISA while tissues were used to assess the expression of IgM, IgT, CD4, GATA3, FOXP3, TGF-β and IL-10 genes by quantitative PCR. Compared to controls, fish fed with ENCAP had a significant increase (p

A key enzyme for the biosynthesis and bioengineering of heparin, 3-O-sulfotransferase-1 (3-OST-1), was expressed and purified in Gram-positive Bacillus subtilis and Bacillus megaterium. Western blotting, protein sequence analysis, and enzyme activity measurement confirmed the expression. The enzymatic activity of 3-OST-1 expressed in Bacillus species were found to be similar to those found when expressed in Escherichia coli. The endotoxin level in 3-OST-1 from B. subtilis and B. megaterium were 10(4)-10(5)-fold lower than that of the E. coli-expressed 3-OST-1, which makes the Bacillus expression system of particular interest for the generation of pharmaceutical grade raw heparin from nonanimal sources.

Heparin is a widely used clinical anticoagulant that is prepared from pig intestine. A contamination of heparin in 2008 has led to a reexamination of animal-derived pharmaceuticals. A bioengineered heparin prepared by bacterial fermentation and chemical and enzymatic processing is currently under development. This study examines the challenges of reducing or removing endotoxins associated with this process that are necessary to proceed with preclinical in vivo evaluation of bioengineered heparin. The current process is assessed for endotoxin levels, and strategies are examined for endotoxin removal from polysaccharides and enzymes involved in this process., (© 2012 International Union of Biochemistry and Molecular Biology, Inc.)

The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sulfotransferases are enzymes that catalyze the transfer of sulfo groups from a donor, for example 3'-phosphoadenosine 5'-phosphosulfate, to an acceptor, for example the amino or hydroxyl groups of a small molecule, xenobiotic, carbohydrate, or peptide. These enzymes are important targets in the design of novel therapeutics for treatment of a variety of diseases. This review examines assays used for this important class of enzyme, paying particular attention to sulfotransferases acting on carbohydrates and peptides and the major challenges associated with their analysis.

In 2008, heparin (active pharmaceutical ingredient, API) lots were associated with anaphylactoid-type reactions. Oversulfated chondroitin sulfate (OSCS), a semi-synthetic glycosaminoglycan (GAG), was identified as a contaminant and dermatan sulfate (DS) as an impurity. While DS has no known toxicity, OSCS was toxic leading to patient deaths. Heparins, prepared before these adverse reactions, needed to be screened for impurities and contaminants. Heparins were analyzed using high-field (1)H-NMR spectroscopy. Heparinoids were mixed with a pure heparin and analyzed by (1)H-NMR to assess the utility of (1)H-NMR for screening heparin adulterants. Sensitivity of heparinoids to deaminative cleavage, a method widely used to depolymerize heparin, was evaluated with polyacrylamide gel electrophoresis to detect impurities and contaminants, giving limits of detection (LOD) ranging from 0.1% to 5%. Most pharmaceutical heparins prepared between 1941 and 2008 showed no impurities or contaminants. Some contained DS, CS, and sodium acetate impurities. Heparin prepared in 2008 contained OSCS contaminant. Heparin adulterated with heparinoids showed additional peaks in their high-field (1)H-NMR spectra, clearly supporting NMR for monitoring of heparin API with an LOD of 0.5-10%. Most of these heparinoids were stable to nitrous acid treatment suggesting its utility for evaluating impurities and contaminants in heparin API., ((c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association)

An oversulfated chondroitin sulfate (OSCS) was identified as a contaminant to pharmaceutical heparin and severe anaphylactoid reactions were ascribed to this contaminant. An examination of the biochemistry underlying both the anticoagulant activity and the toxic effects of oversulfated chondroitin sulfate was undertaken. This study demonstrates that the anticoagulant activity of this oversulfated chondroitin sulfate is primarily dependent on heparin cofactor II mediated inhibition of thrombin. Heparin and oversulfated chondroitin sulfate binding to coagulation, kinin-kallikrein and complement proteins were studied by surface plasmon resonance. While oversulfated chondroitin sulfate binds tightly to antithrombin III, unlike heparin, OSCS does not induce antithrombin III to undergo the conformational change required for its inactivation of thrombin and factor Xa. In contrast to heparin, oversulfated chondroitin sulfate tightly binds factor XIIa suggesting a biochemical mechanism for the factor XIIa-based enhancement of vasoactive bradykinin production.

The novel low-molecular-weight chitosan polysulfate (MW 5120-26,200 Da) was prepared using the depolymerization of chitosan with papain (EC. 3.4.22.2). The sulfonation of depolymerized products was performed using chlorosulfonic acid in N,N-dimethylformamide under semi-heterogeneous conditions. The structures of the products were characterized by FTIR, (13)C NMR, and (1)H NMR (1D, 2D NMR) spectroscopy. The present study sheds light on the mechanism of anticoagulant activity of chitosan polysulfate. Anticoagulant activity was investigated by an activated partial thromboplastin assay, a thrombin time assay, a prothrombin time assay, and thrombelastography. Surface plasmon resonance also provided valuable data for understanding the relationship between the molecular binding of sulfated chitosan to two important blood clotting regulators, antithrombin III and heparin cofactor II. These results show that the principal mechanism by which this chitosan polysulfate exhibits anticoagulant activity is mediated through heparin cofactor II and is dependent on polysaccharide molecular weight.