The dynamics of B-cell reconstitution post allogeneic hematopoietic stem cell transplantation: A real-world study.

BACKGROUND: The immune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is crucial for preventing infections and relapse and enhancing graft-versus-tumor effects. B cells play an important role in humoral immunity and immune regulation, but their reconstitution after allo-HSCT has not been well studied. METHODS: In this study, we analyzed the dynamics of B cells in 252 patients who underwent allo-HSCT for 2 years and assessed the impact of factors on B-cell reconstitution and their correlations with survival outcomes, as well as the development stages of B cells in the bone marrow and the subsets in the peripheral blood. RESULTS: We found that the B-cell reconstitution in the bone marrow was consistent with the peripheral blood (p = 0.232). B-cell reconstitution was delayed by the male gender, age >50, older donor age, the occurrence of chronic and acute graft-versus-host disease, and the infections of fungi and cytomegalovirus. The survival analysis revealed that patients with lower B cells had higher risks of death and relapse. More importantly, we used propensity score matching to obtain the conclusion that post-1-year B-cell reconstitution is better in females. Meanwhile, using mediation analysis, we proposed the age-B cells-survival axis and found that B-cell reconstitution at month 12 posttransplant mediated the effect of age on patient survival (p = 0.013). We also found that younger patients showed more immature B cells in the bone marrow after transplantation (p = 0.037). CONCLUSION: Our findings provide valuable insights for optimizing the management of B-cell reconstitution and improving the efficacy and safety of allo-HSCT.

Zhilong Huoxue Tongyu capsule alleviates myocardial fibrosis by improving endothelial cell dysfunction.

Background and aim: Zhilong Huoxue Tongyu (ZL) capsule is a classical traditional Chinese medicine (TCM) with satisfactory curative effects. Endothelial cell (EC) dysfunction plays an important role during myocardial fibrosis (MF). But the therapeutic effect of ZL capsule on EC dysfunction remains unknown in the development of MF. This study aims to investigate the effect of ZL capsule on EC dysfunction during MF in vivo. Experimental procedure: The model of MF is established in vivo by injecting isoproterenol for 14 days, simultaneously, we examined the therapeutic effect of ZL capsule on MF in vivo. An integrative approach combining biomarker examination, echocardiography and myocardial fibrosis condition using Hematoxylin-eosin staining, Masson staining, and Sirius red staining were performed to assess the efficacy of ZL capsule against MF. Subsequently, comprehensive immunofluorescence staining was performed to evaluate the therapeutic effect of ZL capsule on EC dysfunction. Results and conclusion: Prior to experiments, analysis of the published single-cell sequencing data was performed and it was discovered that EC dysfunction plays an important role. Further pharmacological results showed that ZL capsule could alleviate fibrosis injury and collagen fiber deposition. The mechanism investigation results showed that the endothelial-to-mesenchymal transition (EndMT) and MHC class-II (MHC-II) expression in EC were improved. In addition, ZL capsule can attenuate the inflammatory response during MF by intervening the activation of CD4+T cell mediated by EC. For the first time, we provided evidence that ZL capsule could improve MF by alleviating EC dysfunction via the regulation of EndMT and expression of MHC-II. Taxonomy classification by evise: Myocardial fibrosis, Chinese Herbal Medicine, Traditional Medicine, Endothelium, dysfunction, Endothelial-to-mesenchymal transition.

Xie Zhuo Tiao Zhi formula modulates intestinal microbiota and liver purine metabolism to suppress hepatic steatosis and pyroptosis in NAFLD therapy.

BACKGROUND: Current evidence indicates a rising global prevalence of Non-Alcoholic Fatty Liver Disease (NAFLD), which is closely associated to conditions such as obesity, dyslipidemia, insulin resistance, and metabolic syndrome. The relationship between the gut microbiome and metabolites in NAFLD is gaining attention understanding the pathogenesis and progression of dysregulated lipid metabolism and inflammation. The Xie Zhuo Tiao Zhi (XZTZ) decoction has been employed in clinical practice for alleviating hyperlipidemia and symptoms related to metabolic disorders. However, the pharmacological mechanisms underlying the effects of XZTZ remain to be elucidated. PURPOSE: The objective of this study was to examine the pharmacological mechanisms underlying the hypolipidemic and anti-inflammatory effects of XZTZ decoction in a mouse model of NAFLD, as well as the effects of supplementing exogenous metabolites on PO induced cell damage and lipid accumulation in cultured hepatocytes. METHODS: A high-fat diet (HFD) mouse model was established to examine the effects of XZTZ through oral gavage. The general condition of mice and the protective effect of XZTZ on liver injury were evaluated using histological and biochemical methods. Hematoxylin and eosin staining (H&E) staining and oil red O staining were performed to assess inflammatory and lipid accumulation detection, and cytokine levels were quantitatively analyzed. Additionally, the study included full-length 16S rRNA sequencing, liver transcriptome analysis, and non-targeted metabolomics analysis to investigate the relationship among intestinal microbiome, liver metabolic function, and XZTZ decoction. RESULTS: XZTZ had a significant impact on the microbial community structure in NAFLD mice. Notably, the abundance of Ileibacterium valens, which was significantly enriched by XZTZ, exhibited a negative correlation with liver injury biomarkers such as, alanine transaminase (ALT) and aspartate transaminase (AST) activity. Moreover, treatment with XZTZ led to a significant enrichment of the purine metabolism pathway in liver tissue metabolites, with inosine, a purine metabolite, showing a significant positive correlation with the abundance of I. valens. XZTZ and inosine also significantly enhanced fatty acid ß-oxidation, which led to a reduction in the expression of pro-inflammatory cytokines and the inhibition of liver pyroptosis. These effects contributed to the mitigation of liver injury and hepatocyte damage, both in vivo and vitro. Furthermore, the utilization of HPLC fingerprints and UPLC-Q-TOF-MS elucidated the principal constituents within the XZTZ decoction, including naringin, neohesperidin, atractylenolide III, 23-o-Acetylalisol B, pachymic acid, and ursolic acid which are likely responsible for its therapeutic efficacy. Further investigations are imperative to fully uncover and validate the pharmacodynamic mechanisms underlying these observations. CONCLUSION: The administration of XZTZ decoction demonstrates a protective effect on the livers of NAFLD mice by inhibiting lipid accumulation and reducing hepatocyte inflammatory damage. This protective effect is mediated by the upregulation of I.valens abundance in the intestine, highlighting the importance of the gut-liver axis. Furthermore, the presesnce of inosine, adenosine, and their derivatives are important in promoting the protective effects of XZTZ. Furthermore, the in vitro approaching, we provide hitherto undocumented evidence indicating that the inosine significantly improves lipid accumulation, inflammatory damage, and pyroptosis in AML12 cells incubated with free fatty acids.

Zhilong Huoxue Tongyu Capsules' Effects on ischemic stroke: An assessment using fecal 16S rRNA gene sequencing and untargeted serum metabolomics.

Zhilong Huoxue Tongyu capsule (ZHTC) is an effective traditional Chinese medicine compound for the treatment of ischemic stroke, which is widely used in clinical ischemic stroke patients. However, it is uncertain whether ZHTC affects ischemic stroke through gut microbiota and serum metabolites. In this study, a rat model of middle cerebral artery occlusion (MCAO) was prepared. By evaluating motor nerve function score, cerebral infarct size, brain tissue damage and intestinal barrier damage, it was found that ZHTC improved stroke-related symptoms in MCAO rats. Using 16S rRNA gene sequencing, fecal microbial transplantation (FMT), untargeted metabolomics, and spearman correlation analysis of gut microbiota and serum metabolites, we found that ZHTC can regulate the abundance of p_Firmicutes, p_Bacteroidota,p_Proteobacteria, g_Prevotella, and g_Lactobacillus, and regulated 23 differential metabolites. Spearman correlation analysis found that Arginine was positively correlated with p_Firmicutes, o_Clostridiales, c_Clostridia, and negatively correlated with p_Bacteroidetes, c_Bacteroidia,o_Bacteroidales; L-Lysine was negatively correlated with f_Christensenellaceae; L-methionine was positively correlated with o_Lactobacillales, f_Lactobacillaceae, and g_Lactobacillus. Altogether, this study shows for the first time that ZHTC can ameliorate ischemic stroke by modulating gut microbiota and metabolic disturbances. This lays the foundation for further revealing the causal relationship between ZHTC, gut dysbiosis, plasma metabolite levels and ischemic stroke, and provides a scientific explanation for the ameliorating effect of ZHTC on ischemic stroke.

Study of the volatilization rules of volatile oil and the sustained-release effect of volatile oil solidified by porous starch.

Volatile oil from traditional Chinese medicine has various biological activities and has pharmacological activities in the central nervous system, digestive system, cardiovascular system, respiratory system, etc. These oils are widely used in clinical practice. However, the development of their clinical applications is restricted due to the disadvantages of volatile oils, such as high stimulation, high volatility and poor stability. To improve the stability of a volatile oil in the preparation process, its volatilization and stable release must be controlled. In this paper, porous starch was used as a solid carrier material, and liquid volatile oil was solidified by physical adsorption. GC-MS was used to determine the chemical constituents of the volatile oil, solidified powder and tablets, and the volatilization rules of 34 chemical constituents were analysed statistically. The solidified volatile oil/porous starch powder was characterized by XRD, TGA and DSC, and the VOCs of the volatile oil before and after solidification were analysed by portable GC-MS. Finally, the stable release of the volatile oil could be optimized by changing the porous starch ratio in the formulation. Volatilization was shown to be closely related to the peak retention time and chemical composition, which was consistent with the theory of flavour. The physical properties and chemical composition of the volatile oil did not change after curing, indicating that the adsorption of the volatile oil by porous starch was physical adsorption. In this paper, the porous starch-solidified volatile oil had a slow-release effect, and the production process is simple, easy to operate, and has high application value.

Sedative and hypnotic effects of Perilla frutescens essential oil through GABAergic system pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine believes that depression syndrome has become one of the core pathogenesis of insomnia. The pharmacology of traditional Chinese medicine points out that Perilla frutescens has the effect of regulating Qi and relieving depression, promoting Qi circulation to relieve pain, so Perilla frutescens may have the potential therapeutic effect on insomnia. Related studies have reported the sedative and hypnotic effects of Perilla frutescens, but these studies have not yet explored the mechanism of sedative and hypnotic effects of Perilla frutescens essential oil (PFEO) through inhalation administration. AIM OF THE STUDY: The purpose of this study is to explore the underlying sedative and hypnotic mechanisms of PFEO through the GABAergic system pathways. MATERIALS AND METHODS: Established the PCPA insomnia model of mice, The open field test, pentobarbital-induced falling asleep rate, latency of sleeping time, and duration of sleeping time experiments were used to evaluate the behavior of mice, the enzyme-linked immunosorbent assay was used to analyze the content of 5-HT and GABA in hypothalamus and cerebral cortex. Immunohistochemical experiment, Western blot experiment and RT-PCR experiment were used to study the mechanism of PFEO through GABAergic pathway to regulate insomnia. The main volatile constituents of PFEO were analyzed by gas chromatography-mass spectrometry (GC-MS). RESULTS: The inhalation of PFEO has sedative and hypnotic effects, which reduce significantly the autonomic activity of PCPA insomnia mice, increase falling asleep rate, shorten latency of sleeping time, and prolong duration of sleeping time; the results of enzyme-linked immunosorbent assay show that PFEO increase the content of 5-HT and GABA in hypothalamus and cerebral cortex. The results showed that inhalation of PFEO increase the expression of GABAAα1 and GABAAα2 positive cells, increase the level of GABAAα1 and GABAAα2 protein and also increase the level of GABAAα1 mRNA and GABAAα2 mRNA in the hypothalamus and cerebral cortex. The highest content of PFEO is Perillaldehyde (54.37%), followed by 1,4-Cineole (7.42%), Acetaldehyde diethyl acetal (6.61%), D-Limonene (5.09%), Eucalyptol (4.94%), etc. CONCLUSION: The inhalation of PFEO has sedative and hypnotic effects, it is speculated that the mechanism of which may be the sedative and hypnotic effects through the GABAergic pathway.

[Network pharmacology research on high frequency use of Pericarpium Citri Reticulatae and Aucklandiae Radix herb pair in treatment of constipation with aromatic traditional Chinese medicine based on data mining].

Based on data mining and through the method of network pharmacology, we analyzed the mechanism of high-frequency use of herb pair in the treatment of constipation with aromatic traditional Chinese medicine in this study. Through data mining, aromatic traditional Chinese medicine was obtained for the treatment of constipation and Pericarpium Citri Reticulatae and Aucklandiae Radix herb pair was used as the research object. The volatile oil from Pericarpium Citri Reticulatae and Aucklandiae Radix was extracted by steam distillation, and the chemical compositions of the volatile oil were detected by gas chromatography-mass spectrometry(GC-MS). The targets of volatile oil from Pericarpium Citri Reticulatae and Aucklandiae Radix were searched by PubChem, TCMSP, STITCH and Swiss Target Prediction databases. The targets of constipation were predicted and screened in OMIM, Genecards-Search Resuits and TTD databases. The obtained targets were introduced into Cytoscape 3.7.1 to construct protein-protein interaction(PPI) network diagram for GO and KEGG pathway enrichment analysis by using R language. The network diagram of "component-target-pathway" was constructed according to the results of KEGG enrichment. Discovery Studio 2.5 software was used to verify the molecular docking between the components and the targets. Among them, the most frequently used pair of aromatic traditional Chinese medicine in the treatment of constipation was Pericarpium Citri Reticulatae and Aucklandiae Radix. A total of 33 compounds were detected by GC-MS, and a total of 180 common action targets of Pericarpium Citri Reticulatae and Aucklandiae Radix on volatile oil in the treatment of constipation were predicted. The key targets included CYP19 A1, PPARA, PGR, ACHE, SLC6 A2 and so on. GO enrichment analysis showed that the activities of Pericarpium Citri Reticulatae and Aucklandiae Radix on volatile oil were mainly involved in the biological processes such as circulatory system, blood circulation, and steroid hormone binding. In KEGG enrichment pathway, neuroactive ligand-receptor interaction, endocrine resistance, Ca~(2+) signal pathway and IL-17 signaling pathway showed significant effect on constipation. The results of molecular docking showed that PGR, the target protein related to the treatment of constipation, had a good binding with gamma-linolenic acid, dihydro-alpha-ionone, alpha-eudesmol, caryophyllene oxide and beta-ionone. The results show that by using data mining technology and network pharmacology, it is revealed that the active components of Pericarpium Citri Reticulatae and Aucklandiae volatile oil in high frequency use of aromatic traditional Chinese medicine can be used totreat constipation mainly through CYP19 A1, PPARA, PGR, ACHE, SLC6 A2 and other targets, providing a new idea and method for the further study of aromatic traditional Chinese medicine in the treatment of constipation.

The Combination of Aquilaria sinensis (Lour.) Gilg and Aucklandia costus Falc. Volatile Oils Exerts Antidepressant Effects in a CUMS-Induced Rat Model by Regulating the HPA Axis and Levels of Neurotransmitters.

The Aquilaria sinensis (Lour.) Gilg (CX)-Aucklandia costus Falc. (MX) herbal pair is frequently used in traditional Chinese medicine prescriptions for treating depression. The volatile oil from CX and MX has been shown to have good pharmacological activities on the central nervous system, but its curative effect and mechanism in the treatment of depression are unclear. Therefore, the antidepressant effect of the volatile oil from CX-MX (CMVO) was studied in chronic unpredictable mild stress (CUMS) rats. The suppressive effects of CMVO (25, 50, 100 µL/kg) against CUMS-induced depression-like behavior were evaluated using the forced swimming test (FST), open field test (OFT) and sucrose preference test (SPT). The results showed that CMVO exhibited an antidepressant effect, reversed the decreased sugar preference in the SPT and prolongation of immobility time in the FST induced by CUMS, increased the average speed, time to enter the central area, total moving distance, and enhanced the willingness of rats to explore the environment in the OFT. Inhalational administration of CMVO decreased levels of adrenocorticotropic hormone and corticosterone in serum and the expression of corticotropin-releasing hormone mRNA in the hypothalamus, which indicated regulation of over-activation of the hypothalamic-pituitary-adrenal (HPA) axis. In addition, CMVO restored levels of 5-hydroxytryptamine (5-HT), dopamine, norepinephrine and acetylcholine in the hippocampus. The RT-PCR and immunohistochemistry results showed that CMVO up-regulated the expression of 5-HT1A mRNA. This study demonstrated the antidepressant effect of CMVO in CUMS rats, which was possibly mediated via modulation of monoamine and cholinergic neurotransmitters and regulation of the HPA axis.

Sedative and hypnotic effects of compound Anshen essential oil inhalation for insomnia.

BACKGROUNDS: The chemical composition of many essential oils indicates that they have sedative and hypnotic effects, but there is still a lack of systematic studies on the sedative and hypnotic effects of essential oils. In addition, aromatherapy does not seem to have the side effects of many traditional psychotropic substances, which is clearly worthwhile for further clinical and scientific research. The clinical application of essential oils in aromatherapy has received increasing attention, and detailed studies on the pharmacological activities of inhaled essential oils are increasingly needed. HYPOTHESIS/PURPOSE: As insomniacs are usually accompanied by symptoms of depression and anxiety of varying degrees, based on the theory of aromatherapy of Traditional Chinese Medicine, this experiment is to study a Compound Anshen essential oil that is compatible with Lavender essential oil, Sweet Orange essential oil, Sandalwood essential oil and other aromatic medicine essential oils with sedative and hypnotic effects, anti-anxiety and anti-depression effects. To study the sedative and hypnotic effects of Compound Anshen essential oil inhaled and the main chemical components of Compound Anshen essential oil, and to compare and analyze the pharmacodynamics of diazepam, a commonly used drug for insomnia. METHODS: The Open field test and Pentobarbital-induced sleep latency and sleep time experiments were used to analyze and compare the sedative and hypnotic effects of inhaling Compound Anshen essential oil and the administration of diazepam on mice. The changes of 5-HT and GABA in mouse brain were analyzed by Elisa. The main volatile constituents of Compound Anshen essential oil were analyzed by gas chromatography-mass spectrometry (GC-MS). RESULTS: Inhalation of Compound Anshen essential oil can significantly reduce the spontaneous activity of mice, reduce latency of sleeping time and prolong duration of sleeping time. The results of enzyme-linked immunosorbent assay showed that Compound Anshen essential oil can increase the content of 5-HT and GABA in mouse brain. The main volatile chemical constituents of the Compound Anshen essential oil are D-limonene (24.07%), Linalool (21.98%), Linalyl acetate (15.37%), α-Pinene (5.39%), and α-Santalol (4.8%). CONCLUSION: The study found that the inhalation of Compound Anshen essential oil has sedative and hypnotic effect. This study provides a theoretical basis for further research and development of the sedative and hypnotic effects of Compound Anshen essential oil based on the theory of aromatherapy.

The Mechanism of Compound Anshen Essential Oil in the Treatment of Insomnia Was Examined by Network Pharmacology.

The active component-target network and protein-protein interaction network of Compound Anshen essential oil were constructed. The target functions and related pathways were analyzed to explore the mechanism of Compound Anshen essential oil in the treatment of insomnia. GC-MS was used to detect the chemical composition of Compound Anshen essential oil, and the TCMSP, STITCH, TTD, and DrugBank databases were searched to predict and screen the targets of Compound Anshen essential oil in the treatment of insomnia. Cytoscape software was used to construct the network diagrams of the active component-action target and protein-protein interaction networks, ClueGO software was used to analyze the GO enrichment and KEGG pathway of the target, and the systemsDock website database was used for molecular docking. The analysis of the network results showed that the activity of Compound Anshen essential oil mainly involves biological processes such as the phospholipase C-activating G protein-coupled receptor signaling pathway, response to ammonium ions, calcium ion transport into the cytosol, and chloride transport. The results of molecular docking showed that linalool, caryophyllene, dibutyl phthalate, (-)-4-terpineol, and (-)-α-terpineol have good binding activity with ADRB2, DRD2, ESR1, KCNH2, NR1H4, NR1I2, NR1I3, and TRPV1 targets. This study demonstrates the multicomponent, multitarget, and multichannel characteristics of Compound Anshen essential oil and provides a new therapeutic idea and method for further research on the mechanism of Compound Anshen essential oil in the treatment of insomnia.

CCHamide-2 Is an Orexigenic Brain-Gut Peptide in Drosophila.

The neuroendocrine peptides CCHamide-1 and -2, encoded by the genes ccha1 and -2, are produced by endocrine cells in the midgut and by neurons in the brain of Drosophila melanogaster. Here, we used the CRISPR/Cas9 technique to disrupt the ccha1 and -2 genes and identify mutant phenotypes with a focus on ccha-2 mutants. We found that both larval and adult ccha2 mutants showed a significantly reduced food intake as measured in adult flies by the Capillary Feeding (CAFE) assay (up to 72% reduced food intake compared to wild-type). Locomotion tests in adult flies showed that ccha2 mutants had a significantly reduced locomotor activity especially around 8 a.m. and 8 p.m., where adult Drosophila normally feeds (up to 70% reduced locomotor activity compared to wild-type). Reduced larval feeding is normally coupled to a delayed larval development, a process that is mediated by insulin. Accordingly, we found that the ccha2 mutants had a remarkably delayed development, showing pupariation 70 hours after the pupariation time point of the wild-type. In contrast, the ccha-1 mutants were not developmentally delayed. We also found that the ccha2 mutants had up to 80% reduced mRNA concentrations coding for the Drosophila insulin-like-peptides-2 and -3, while these concentrations were unchanged for the ccha1 mutants. From these experiments we conclude that CCHamide-2 is an orexigenic peptide and an important factor for controlling developmental timing in Drosophila.

The A- and B-type muscarinic acetylcholine receptors from Drosophila melanogaster couple to different second messenger pathways.

Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors (GPCRs) that are activated by the agonists acetylcholine and muscarine and blocked by several antagonists, among them atropine. In mammals five mAChRs (m1-m5) exist of which m1, m3, and m5 are coupled to members of the Gq/11 family and m2 and m4 to members of the Gi/0 family. We have recently shown that Drosophila melanogaster and other arthropods have two mAChRs, named A and B, where the A-type has the same pharmacology as the mammalian mAChRs, while the B-type has a very low affinity to muscarine and no affinity to classical antagonists such as atropine. Here, we find that the D. melanogaster A-type mAChR is coupled to Gq/11 and D. melanogaster B-type mAChR to Gi/0. Furthermore, by comparing the second and third intracellular loops of all animal mAChRs for which the G protein coupling has been established, we could identify several amino acid residues likely to be specific for either Gq/11 or Gi/0 coupling. Using these hallmarks for specific mAChR G protein interaction we found that all protostomes with a sequenced genome have one mAChR coupled to Gq/11 and one to four mAChRs coupled to Gi/0. Furthermore, in protostomes, probably all A-type mAChRs are coupled to Gq/11 and all B-type mAChRs to G0/i.

[Study on preparation of andrographolide composite particles based on mechanical crushing method and its dissolution].

OBJECTIVE: To prepare andrographolide composite particles, and evaluate their particle structure and dissolution. METHOD: The mechanical crushing method was adopted to prepare andrographolide and polyethylene glycol (PEG) 6000 composite particles. The structures were characterized by the scanning electron microscope (SEM) and the differential scanning calorimeter (DSC). The contact angles were determined by the contact angle analyzer. The in vitro dissolution curve was detected. RESULT: Andrographolide and PEG 6000 gave rise to coated composite particle structures, with the decrease in the crystallinity of andrographolide. The in vitro dissolution rate of composite particles was significantly obvious than that of its raw materials, ultrafine powder and their physical mixtures. CONCLUSION: Andrographolide composite particles based on the mechanical crushing method could notably enhance the in vitro dissolution of andrographolide.

Transient kinetics and rate-limiting steps for the processive cellobiohydrolase Cel7A: effects of substrate structure and carbohydrate binding domain.

Cellobiohydrolases are exoacting, processive enzymes that effectively hydrolyze crystalline cellulose. They have attracted considerable interest because of their role in both natural carbon cycling and industrial enzyme cocktails used for the deconstruction of cellulosic biomass, but many mechanistic and regulatory aspects of their heterogeneous catalysis remain poorly understood. Here, we address this by applying a deterministic model to real-time kinetic data with high temporal resolution. We used two variants of the cellobiohydrolase Cel7A from Hypocrea jecorina , and three types of cellulose as substrate. Analysis of the pre-steady-state regime allowed delineation rate constants for both fast and slow steps in the enzymatic cycle and assessment of how these constants influenced the rate of hydrolysis at quasi-steady state. Processive movement on the cellulose strand advanced with characteristic times of 0.15-0.7 s per step at 25 °C, and the rate was highest on amorphous substrate. The cellulose binding module was found to raise this rate on crystalline, but not on amorphous, substrate. The rapid processive movement signified high intrinsic reactivity, but this parameter had marginal influence on the steady-state rate. This was because dissociation and association were slower and, hence, rate limiting. Specifically, the dissociation from the strand was found to occur with characteristic times of 45-100 s. This meant that dissociation was the bottleneck, except at very low substrate loads (0.5-1 g/L), where association became slower.

Expression patterns of the Drosophila neuropeptide CCHamide-2 and its receptor may suggest hormonal signaling from the gut to the brain.

The insect neuropeptides CCHamide-1 and -2 are recently discovered peptides that probably occur in all arthropods. Here, we used immunocytochemistry, in situ hybridization, and quantitative PCR (qPCR), to localize the two peptides in the fruitfly Drosophila melanogaster. We found that CCHamide-1 and -2 were localized in endocrine cells of the midgut of larvae and adult flies. These endocrine cells had the appearance of sensory cells, projecting processes close to or into the gut lumen. In addition, CCHamide-2 was also localized in about forty neurons in the brain hemispheres and ventral nerve cord of larvae. Using qPCR we found high expression of the CCHamide-2 gene in the larval gut and very low expression of its receptor gene, while in the larval brain we found low expression of CCHamide-2 and very high expression of its receptor. These expression patterns suggest the following model: Endocrine CCHamide-2 cells in the gut sense the quality of food components in the gut lumen and transmit this information to the brain by releasing CCHamide-2 into the circulation; subsequently, after binding to its brain receptors, CCHamides-2 induces an altered feeding behavior in the animal and possibly other homeostatic adaptations.

Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods.

Muscarinic acetylcholine receptors (mAChRs) play a central role in the mammalian nervous system. These receptors are G protein-coupled receptors (GPCRs), which are activated by the agonists acetylcholine and muscarine, and blocked by a variety of antagonists. Mammals have five mAChRs (m1-m5). In this study, we cloned two structurally related GPCRs from the fruit fly Drosophila melanogaster, which, after expression in Chinese hamster ovary cells, proved to be muscarinic acetylcholine receptors. One mAChR (the A-type; encoded by gene CG4356) is activated by acetylcholine (EC50, 5 × 10(-8) M) and muscarine (EC50, 6 × 10(-8) M) and blocked by the classical mAChR antagonists atropine, scopolamine, and 3-quinuclidinyl-benzilate (QNB), while the other (the B-type; encoded by gene CG7918) is also activated by acetylcholine, but has a 1,000-fold lower sensitivity to muscarine, and is not blocked by the antagonists. A- and B-type mAChRs were also cloned and functionally characterized from the red flour beetle Tribolium castaneum. Recently, Haga et al. (Nature 2012, 482: 547-551) published the crystal structure of the human m2 mAChR, revealing 14 amino acid residues forming the binding pocket for QNB. These residues are identical between the human m2 and the D. melanogaster and T. castaneum A-type mAChRs, while many of them are different between the human m2 and the B-type receptors. Using bioinformatics, one orthologue of the A-type and one of the B-type mAChRs could also be found in all other arthropods with a sequenced genome. Protostomes, such as arthropods, and deuterostomes, such as mammals and other vertebrates, belong to two evolutionarily distinct lineages of animal evolution that split about 700 million years ago. We found that animals that originated before this split, such as cnidarians (Hydra), had two A-type mAChRs. From these data we propose a model for the evolution of mAChRs.

An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose.

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross-linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH-catalyzed reaction with cellobiose, was recorded under constant-potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 µA mM(-1) cm(-2)), low detection limit (25 nM), and fast response time (t(95%) ≈ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH-biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the ß-anomer of cello-oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real-time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose.