Reconstitution of the Early Stage of Chetomin Biosynthesis in Aspergillus fumigatus Leads to the Production of Epipolythiodioxopiperazines.

Using CRISPR-Cas9 technology and a microhomology-mediated end-joining repair system, we substituted genes of the gliotoxin pathway in Aspergillus fumigatus with genes responsible for chetomin biosynthesis from Chaetomium cochliodes, leading to the production of three new epipolythiodioxopiperazines (ETPs). This work represents the first successful endeavor to produce ETPs in a non-native host. Additionally, the simultaneous disruption of five genes in a single transformation marks the most extensive gene knockout event in filamentous fungi to date.

Urease of Aspergillus fumigatus Is Required for Survival in Macrophages and Virulence.

The number of patients suffering from fungal diseases has constantly increased during the last decade. Among the fungal pathogens, the airborne filamentous fungus Aspergillus fumigatus can cause chronic and fatal invasive mold infections. So far, only three major classes of drugs (polyenes, azoles, and echinocandins) are available for the treatment of life-threatening fungal infections, and all present pharmacological drawbacks (e.g., low solubility or toxicity). Meanwhile, clinical antifungal-resistant isolates are continuously emerging. Therefore, there is a high demand for novel antifungal drugs, preferentially those that act on new targets. We studied urease and the accessory proteins in A. fumigatus to determine their biochemical roles and their influence on virulence. Urease is crucial for the growth on urea as the sole nitrogen source, and the transcript and protein levels are elevated on urea media. The urease deficient mutant displays attenuated virulence, and its spores are more susceptible to macrophage-mediated killing. We demonstrated that this observation is associated with an inability to prevent the acidification of the phagosome. Furthermore, we could show that a nickel-chelator inhibits growth on urea. The nickel chelator is also able to reverse the effects of urease on macrophage killing and phagosome acidification, thereby reducing virulence in systemic and trachea infection models. IMPORTANCE The development of antifungal drugs is an urgent task, but it has proven to be difficult due to many similarities between fungal and animal cells. Here, we characterized the urease system in A. fumigatus, which depends on nickel for activity. Notably, nickel is not a crucial element for humans. Therefore, we went further to explore the role of nickel-dependent urease in host-pathogen interactions. We were able to show that urease is important in preventing the acidification of the phagosome and therefore reduces the killing of conidia by macrophages. Furthermore, the deletion of urease shows reduced virulence in murine infection models. Taken together, we identified urease as an essential virulence factor of A. fumigatus. We were able to show that the application of the nickel-chelator dimethylglyoxime is effective in both in vitro and in vivo infection models. This suggests that nickel chelators or urease inhibitors are potential candidates for the development of novel antifungal drugs.

Topographical study of scapular foramina and scapular nutrient foramina in dried skeletons.

PURPOSE: The aim of our study is to study the prevalence and anatomy of scapular foramina (SF) and scapular nutrient foramina (SNF) in dried skeletons from the Northeastern Thai population. METHODS: A total of 150 dried scapulae were investigated. Both SF and SNF were identified using a metal wire with a diameter of 0.36 mm. The number, locations, lengths, and diameters of SF were recorded. Subsequently, SNF were identified using the same metal wire. Their number and locations were recorded. Two observers performed the evaluations and measurements. RESULTS: SF were present in 78.0% of scapulae. They could have up to five openings. Eighteen types were found. On average they were longer in males (21.7 ± 5.0 mm) than females (19.45 ± 4.6 mm). The mean diameters of both the superior and inferior openings were significantly greater in females (p < 0.01). SNF, in contrast, were present in 100% of scapulae. They were located in the supraspinous fossa (36.7%), subscapular fossa (31.3%), infraspinous fossa (22.8%), and peri-glenoid area (10.0%). CONCLUSION: Unlike previous studies, the present study suggests that SF are normal anatomical findings, present in 78.0% of the scapulae investigated. Surgeons should be aware of both SNF and SF when operating or interpreting radiological findings.

Cross-Sectional Area of the Tibial Nerve in Diabetic Peripheral Neuropathy Patients: A Systematic Review and Meta-Analysis of Ultrasonography Studies.

Background: There is a link between diabetic peripheral neuropathy (DPN) progression and the increase in the cross-sectional area (CSA) of the tibial nerve at the ankle. Nevertheless, no prior meta-analysis has been conducted to evaluate its usefulness for the diagnosis of DPN. Methods: We searched Google Scholar, Scopus, and PubMed for potential studies. Studies had to report tibial nerve CSA at the ankle and diabetes status (DM, DPN, or healthy) to be included. A random-effect meta-analysis was applied to calculate pooled tibial nerve CSA and mean differences across the groups. Subgroup and correlational analyses were conducted to study the potential covariates. Results: The analysis of 3295 subjects revealed that tibial nerve CSA was 13.39 mm2 (CI: 10.94−15.85) in DM patients and 15.12 mm2 (CI: 11.76−18.48) in DPN patients. The CSA was 1.93 mm2 (CI: 0.92−2.95, I2 = 98.69%, p < 0.01) larger than DPN-free diabetic patients. The diagnostic criteria of DPN and age were also identified as potential moderators of tibial nerve CSA. Conclusions: Although tibial nerve CSA at the ankle was significantly larger in the DPN patients, its clinical usefulness is limited by the overlap between groups and the inconsistency in the criteria used to diagnose DPN.

Phytosterols and their derivatives: Potential health-promoting uses against lipid metabolism and associated diseases, mechanism, and safety issues.

Consumption of phytosterols (PSs), the plant-based analogs of cholesterol, can reduce serum cholesterol levels. This review discusses the current state of the art into the research of the structural features and dietary sources of PSs and their derivatives. The effect of PSs on individual lipid metabolites is summarized in the present review. PS-related nonalcoholic fatty liver disease (NAFLD), obesity, and the alleviation of inflammatory bowel diseases are discussed. PSs reduce the risk of having NAFLD by improving the blood biochemical parameters related to lipid transport and metabolism. However, current research on the circulating PSs indicates its safety concern regarding fatty liver disease induction. In addition, PS oxidation products exhibit pro-atherogenic properties, cytotoxicity oxidative stress, apoptosis, and pro-inflammatory properties. Further research is needed to investigate the bioavailability and safety issues of PSs and their derivatives in animal models and clinical trials.

Enhancing stability and bioaccessibility of chlorogenic acid using complexation with amylopectin: A comprehensive evaluation of complex formation, properties, and characteristics.

Chlorogenic acid (CA) performs numerous bioactivities; however, its usage is currently limited because of low stability and poor bioaccessibility. In this study, a amylopectin-CA complex was formed. FTIR studies confirmed that the new complex formed via hydrogen and CH-π bonding, and was involved with the reorganization of the skeletal α-1,4 glucosidic linkages of amylopectin. DSC and XRD studies suggested that complexation affected starch crystallinity and increased the size of the amorphous region. Under high temperature, complex degradation followed first-order reaction kinetics whereas under low acidity, the complex retained maximum CA content at pH 2.5. In vitro gastrointestinal (GI) digestion studies showed that maximum digestion of the complex took place during the gastric phase (39%). In addition, 81.14% of the retained CA was absorbed after GI digestion. In conclusion, amylopectin complexation may improve the stability of CA during digestion and under various food-processing operations.

Recovery of lotus (Nelumbo nucifera Gaertn.) seedpod flavonoids using polar macroporous resins: The updated understanding on adsorption/desorption mechanisms and the involved intermolecular attractions and bonding.

Macroporous resins have been employed in the effective recovery of flavonoids from plants. In this study, S8 polar resins were used to recover flavonoids and procyanidins from lotus seedpods. Adsorption kinetics, isotherms, and thermodynamics studies revealed that the adsorption process involved physico-chemical interactions, including flavonoid-resin and flavonoid-flavonoid electrostatic interactions, π-π aromatic stacking, moderate and strong hydrogen bonding, and repulsive forces. These forces worked complementarily in adsorption, except for the repulsive force, which opposed the adsorption. Further, adsorption temperature determined the adsorption behavior, with multilayer adsorption enhancing adsorption capacity. In dynamic desorption tests, an acetone/water/acetic acid mixture (58.77: 39.34: 1.89) designed by the D-optimal design method was able to desorb 95.57% and 89.85% of total flavonoids and procyanidins, respectively, using less than two bed volumes of solvent. Ultra-performance liquid chromatography triple-time of flight/mass spectrometry (UPLC-TOF/MS) analysis showed that 26 flavonoids, including 5 procyanidins, were detected after the recovery.

Systematically quantitative proteomics and metabolite profiles offer insight into fruit ripening behavior in Fragaria × ananassa.

Profound metabolic and proteomic changes involved in the primary and the secondary metabolism are required for the ripeness of fleshy fruit such as strawberries (Fragaria × ananassa). Here we present the quantitative proteomic profiling in parallel with metabolic and transcriptional profiling at five developmental stages of strawberry fruit ripening, and correlations between changes in representative metabolites and the abundance of related proteins were analyzed. Hierarchical clustering analysis of the quantitative proteomic profiling identified 143 proteins in strawberry fruit across five developmental stages. Meanwhile, both protein abundance and gene expression spanned a wide range of roles, such as the primary and the secondary metabolism, defense system, and response to stress stimuli. The decreased abundance of proteins contributed to the carbohydrate metabolism and the up-regulated expression of secondary biosynthetic proteins was found to be positively correlated with the accumulation of primary and secondary metabolites during strawberry development. Moreover, with the same annotations and high homology, the gene function of key genes involved in primary and secondary metabolism (FaTPI, FaPAL, FaMDH and FaME) was confirmed in Nicotiana via the transient expression assay, which provides further evidence for the role of those genes in metabolism of strawberry fruit. The results of the present study may serve as an important resource for the functional analysis of the proteome and offer new perspectives on regulation of fruit quality.

Improvement of phenolic compounds extraction from high-starch lotus (Nelumbo nucifera G.) seed kernels using glycerol: New insights to amylose/amylopectin - Phenolic relationships.

Lotus seed kernel (LSK) has high starch content and possesses many bioactivities, which are mainly attributed to its phenolics. In the present study, LSK phenolics were extracted using aqueous glycerol, and the obtained phenolics and its starch properties were observed for their correlations. Response surface methodology indicated significant quadratic effect of temperature and significant linear effect of glycerol on LSK phenolics (p < 0.05). Temperature also had a significant effect on LSK starch solubility and swelling power and % leaching amylose (p < 0.05). Leaching amylose in the presence of glycerol and swelling power showed positive correlations with LSK phenolics in a linear relationship (r = 0.825, p < 0.01) below 50 °C and a monotonic relationship (rs = 0.933, p < 0.01) above 60 °C, respectively. A building block backbone model was adopted to illustrate the interaction between amylose/amylopectin-glycerol, and most importantly, their relationship to LSK phenolics.

Effects of elevated CO2 on energy metabolism and γ-aminobutyric acid shunt pathway in postharvest strawberry fruit.

The elevated CO2 was applied to the strawberry fruit during storage at 0 °C, in which its effects on energy metabolism and the γ-aminobutyric acid (GABA) shunt pathway were investigated. 10% and 20% CO2 maintained quality of the fruit. The energy charge of 10% and 20% CO2-treated fruit was decreased by 7.58% and 23.93% on day 12, respectively, compared with the control fruit, which was associated with the decline of NADH/NAD+. The GABA in 10% and 20% CO2-treated fruit was increased by 1.2-fold and 1.6-fold compared with the control fruit on day 12, respectively. The accumulation of GABA resulted from the decrease of GABA transaminase activity and gene expression, which may partially contribute to the decrease of NADH at later storage. These results indicated that 20% CO2 is an optimal concentration for strawberry fruit to prolong the storage to 12 days.

Lotus Flavonoids and Phenolic Acids: Health Promotion and Safe Consumption Dosages.

Nelumbo nucifera Gaertn., also known as the sacred lotus, is extensively cultivated in Southeast Asia, primarily for food and as an herbal medicine. This article reviews studies published between 1995 and 2017, on flavonoid and phenolic acid profiles and contents of 154 different cultivars of lotus. So far, some 12 phenolic acids and 89 to 90 flavonoids (47 flavonols, 25 to 26 flavons, 8 flavan-3-ols, 4 flavanons, and 5 anthocyanins) have been isolated from different parts of the lotus plant, including its leaves (whole leaf, leaf pulp, leaf vein, and leaf stalk), seeds (seedpod, epicarp, coat, kernel, and embryo), and flowers (stamen, petal, pistil, and stalk), although not all of them have been quantified. Factors affecting flavonoids and phenolic acid profiles, including types of tissues and extracting factors, are discussed in this review, in order to maximize the application of the lotus and its polyphenols in the food industry. Health promotion activities, attributed to the presence of flavonoids and phenolic acids, are described along with toxicology studies, illustrating appropriate usage and safe consumption dosages of lotus extracts. This review also presents the controversies and discusses the research gaps that limit our ability to obtain a thorough understanding of the bioactivities of lotus extracts.

Effect of Exogenous Nitro Oxide on Chilling Tolerance, Polyamine, Proline, and γ-Aminobutyric Acid in Bamboo Shoots (Phyllostachys praecox f. prevernalis).

The effects of exogenous nitro oxide (NO) on chilling resistance and the metabolism of polyamine, proline, and γ-aminobutyric acid of bamboo shoots were investigated. Bamboo shoots were dipped in 0.07 mM sodium nitroprusside (SNP) and stored at 1 °C for 56 days. During the storage, the development of chilling injury of SNP treated bamboo shoots was inhibited with decreased accumulation of malonaldehyde and electrical leakage. At the end of storage, the chilling injury incidence of treated bamboo shoots decreased by 37.9% while their malonaldehyde content and electrical leakage were 8.8% and 18.6% lower than that of the control, respectively. Interestingly, the endogenous NO, polyamines, γ-aminobutyric acid, and proline contents of treated bamboo shoot also significantly increased. Consistently, the metabolisms of these nitrogenous compounds were stimulated in treated bamboo shoots, according to their higher (20.2%-49.8%) related enzyme activities, including nitric oxide synthase, arginine decarboxylase, ornithine decarboxylase, glutamate decarboxylase, orn-δ-aminotransferase, and Δ1-pyrroline-5-carboxylate synthetase. The results indicated that the SNP treatment enhanced chilling tolerance of bamboo shoots, which might associate with the activated metabolism of polyamines, γ-aminobutyric acid, and proline. SNP treatment might be an alternative technology to avoid chill injury during cold storage of bamboo shoots.

Developmental and stress regulation on expression of a novel miRNA, Fan-miR73, and its target ABI5 in strawberry.

Abscisic acid (ABA) is a critical plant hormone for fruit ripening and adaptive stress responses in strawberry. Previous high-throughput sequencing results indicated that ABA-insensitive (ABI)5, an important transcription factor in the ABA signaling pathway, was a target for a novel microRNA (miRNA), Fan-miR73. In the present study, exogenous ABA treatment was found to accelerate fruit ripening through differentially regulating the transcripts of ABA metabolism and signal transduction related genes, including NCED1, PYR1, ABI1, and SnRK2.2. Expression of Fan-miR73 was down-regulated in response to exogenous ABA treatment in a dosage-dependent manner, which resulted in an accumulation of ABI5 transcripts in the ripening-accelerated fruits. In addition, both UV-B radiation and salinity stress reduced the transcript levels of Fan-miR73, whereas promoted ABI5 expression. Furthermore, high negative correlations between the transcriptional abundance of Fan-miR73 and ABI5 were observed during ripening and in response to stress stimuli. These results enriched the possible regulatory role of miRNA involved in the post-transcriptional modification of ABI5 during strawberry ripening, as well as responses to environmental stresses.

Involvement of energy metabolism to chilling tolerance induced by hydrogen sulfide in cold-stored banana fruit.

In this study, the effect of hydrogen sulfide (H2S) on energy metabolism in postharvest banana fruit under chilling stress was investigated. Banana fruit, fumigated with optimal concentration (0.5mM) of aqueous sodium hydrosulfide (NaHS) solution for 24h, were initially stored at 7°C for 14d and 20°C for another 6d. H2S treated banana fruit showed both higher value of firmness and Hue angle, as well as lower value of electrolyte leakage, malondialdehyde (MDA) content and ethylene production. These indicated slower development of chilling injury compared with the control. Decrease in adenosine triphosphate (ATP) and energy charge was not noticeable in H2S treated banana fruit. Moreover, the activity of H(+)-ATPase, Ca(2+)-ATPase, cytochrome C oxidase (CCO) and succinate dehydrogenase (SDH), associated with energy metabolism, were significantly enhanced by H2S treatment. Therefore, it can be deduced that H2S can potentially alleviate chilling development in banana fruit by increasing enzymes activities, involved in energy metabolism, to maintain energy charge.