Ziyuglycoside II attenuated OVX mice bone loss via inflammatory responses and regulation of gut microbiota and SCFAs.

BACKGROUND AND PURPOSE: Osteoporosis (OP) is a frequent clinical problem for the elderly. Traditional Chinese Medicine (TCM) has achieved beneficial results in the treatment of OP. Ziyuglycoside II (ZGS II) is a major active compound of Sanguisorba officinalis L. that has shown anti-inflammation and antioxidation properties, but little information concerning its anti-OP potential is available. Our research aims to investigate the mechanism of ZGS II in ameliorating bone loss by inflammatory responses and regulation of gut microbiota and short chain fatty acids (SCFAs) in ovariectomized (OVX) mice. METHODS: We predicted the mode of ZGS II action on OP through network pharmacology and molecular docking, and an OVX mouse model was employed to validate its anti-OP efficacy. Then we analyzed its impact on bone microstructure, the levels of inflammatory cytokines and pain mediators in serum, inflammation in colon, intestinal barrier, gut microbiota composition and SCFAs in feces. RESULTS: Network pharmacology identified 55 intersecting targets of ZGS II related to OP. Of these, we predicted IGF1 may be the core target, which was successfully docked with ZGS II and showed excellent binding ability. Our in vivo results showed that ZGS II alleviated bone loss in OVX mice, attenuated systemic inflammation, enhanced intestinal barrier, reduced the pain threshold, modulated the abundance of gut microbiota involving norank_f__Muribaculaceae and Dubosiella, and increased the content of acetic acid and propanoic acid in SCFAs. CONCLUSIONS: Our data indicated that ZGS II attenuated bone loss in OVX mice by relieving inflammation and regulating gut microbiota and SCFAs.

Apathetic Graves' disease with severe hepatic and renal dysfunction induced by COVID-19 infection: Case report and literature review.

RATIONALE: A rare and intractable case of apathetic Graves' disease (GD) with severe liver and kidney damage induced by coronavirus disease 2019 (COVID-19) carries a certain risk of missing diagnosis and delayed treatment during the COVID-19 pandemic. PATIENT CONCERN: A 60-year-old female patient developed anorexia, exhaustion, jaundice, nausea, and vomiting 10 days after COVID-19 infection. She was admitted to the Infectious Diseases Department because of recurring symptoms for more than a month. DIAGNOSIS: Based on the patient's epidemiological history, clinical symptoms, and prior history, she was preliminarily diagnosed with GD induced by COVID-19 with severe hyperthyroid-related liver injury and chronic kidney disease stage 4. Drug-induced and radiation-induced liver injuries occurred sequentially throughout the therapy. INTERVENTION: Methimazole (MMI) (10 mg/d) was administered for 1 week, and the patient's symptoms, thyroid function, and liver and kidney function improved. Nevertheless, the aforementioned symptoms and liver and kidney function deteriorated 20 days after increasing the MMI dose (20 mg/d). Therefore, in the presence of an artificial liver, hemodialysis, and other medical conditions, the treatment schedule was adjusted to individualized 131I anti-hyperthyroidism therapy. OUTCOME: After 131I treatment, the patient's liver function returned to almost normal levels after a month, but worsened when the hepatoprotective drugs were stopped. Renal function did not deteriorate significantly and returned to baseline after 3 months. Thyroid function was restored to normal approximately 4 months later. CONCLUSION: COVID-19 may induce GD. Multidisciplinary collaboration can be initiated as early as possible. Individualized 131I therapy or long-term low-dose MMI (10 mg/d) can be considered to manage hyperthyroidism in GD patients with liver and kidney dysfunction and to prolong liver protection therapy appropriately.

Production of polyunsaturated fatty acids in pork backfat fermented by Mucor circinelloides.

Pork backfat (PB) contains excessive saturated fatty acids (SFAs), but lacks polyunsaturated fatty acids (PUFAs). Excessive SFAs can be used as a substrate for the growth of certain microorganisms that convert them into PUFAs and monounsaturated fatty acids (MUFAs), and the added value of PB can be enhanced. In this study, Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189 were co-cultured for conversion of PB into fermented pork backfat (FPB) with high level of PUFAs. Our results showed that the content of γ-linolenic acid (GLA) and linoleic acid (LA) in the surface of FPB reached 9.04 ± 0.14 mg/g and 107.31 ± 5.16 mg/g for 7-day fermentation, respectively. To convert the internal SFAs of PB, ultrasound combined with papain was used to promote the penetrative growth of M. circinelloides into the internal PB, and the GLA level in the third layer of fat reached 2.58 ± 0.31 mg/g FPB. The internal growth of M. circinelloides in PB was promoted by adjusting the oxygen rate and ventilation rate through the wind velocity sensor. When the oxygen rate is 2 m/s and the ventilation rate is 18 m3/h, the GLA level in the third layer of fat reached 4.13 ± 1.01 mg/g FPB. To further improve the level of PUFAs in PB, FPB was produced by M. circinelloides at 18 °C. The GLA content on the surface of FPB reached 15.73 ± 1.13 mg/g FPB, and the GLA yield in the second and third layers of fat reached 8.68 ± 1.77 mg/g FPB and 6.13 ± 1.28 mg/g FPB, the LA yield in the second and third layers of fat reached 105.45 ± 5.01 mg/g FPB and 98.46 ± 4.14 mg/g FPB, respectively. These results suggested that excessive SFAs in PB can be converted into PUFAs and provided a new technique for improving PUFAs in FPB. KEY POINTS: • This article achieved the conversion of PUFAs in pork backfat by Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189. • This article solved the internal growth of M. circinelloides CBS277.49 in pork backfat by ultrasound combined with papain. • This article proposed an innovative of promoting the internal growth of M. circinelloides and increasing the PUFAs production by oxygen ventilation in pork backfat.

A global land cover training dataset from 1984 to 2020.

State-of-the-art cloud computing platforms such as Google Earth Engine (GEE) enable regional-to-global land cover and land cover change mapping with machine learning algorithms. However, collection of high-quality training data, which is necessary for accurate land cover mapping, remains costly and labor-intensive. To address this need, we created a global database of nearly 2 million training units spanning the period from 1984 to 2020 for seven primary and nine secondary land cover classes. Our training data collection approach leveraged GEE and machine learning algorithms to ensure data quality and biogeographic representation. We sampled the spectral-temporal feature space from Landsat imagery to efficiently allocate training data across global ecoregions and incorporated publicly available and collaborator-provided datasets to our database. To reflect the underlying regional class distribution and post-disturbance landscapes, we strategically augmented the database. We used a machine learning-based cross-validation procedure to remove potentially mis-labeled training units. Our training database is relevant for a wide array of studies such as land cover change, agriculture, forestry, hydrology, urban development, among many others.

Spatial and Temporal Availability of Cloud-free Optical Observations in the Tropics to Monitor Deforestation.

State-of-the-art methodologies to monitor deforestation rely mostly on optical satellite observations. High-density optical time series can enable the detection of deforestation almost as soon as it occurs. However, deforestation monitoring in the tropics can be hindered by high cloud coverage, and thus the responsiveness of managers, enforcement agencies, and scientists. To understand the implications of cloud contamination in freely available optical data we analyzed combined time series from Landsat 7, 8, and Sentinel-2 over the tropics from 2017-2021. Datasets derived for each 30 m × 30 m of the 59.4 M km2 domain include a) number of cloud-free observations per year, b) maximum consecutive days without clear imagery within a year, and c) final date of the longest waiting period. The datasets reflect where and when data gaps in optical time series exist due to cloud contamination. Scripts to access and extend the datasets are shared and documented. The datasets can be used to prioritize areas where complementary observations, such as radar imagery, are needed for implementing effective deforestation alert systems.

The glucosinolate profiles of Brassicaceae vegetables responded differently to quick-freezing and drying methods.

Glucosinolates (GLS) are bioactive compounds found in Brassicaceae vegetables. Industrial food processing treatments, such as blanching, quick-freezing (QF), vacuum freeze-drying (VFD), vacuum-drying (VD) and oven-drying (OD), significantly affect the degradation and ingestion of GLS. Here, the effects of these treatments, followed by boiling, on the GLS content and mimicking ingestion level of isothiocyanate from Brassicaceae vegetables (broccoli, cauliflower, white and red cabbages, Chinese and baby cabbages, white and red radish roots) were investigated. The results showed that blanching-QF maintained or increased the GLS content as well as preserved the ingestion level of isothiocyanate, an optimum treatment for GLS preservation. Blanching-VFD was recommended for these vegetables, while blanching-VD and blanching-OD caused relatively high GLS losses and low isothiocyanate production. Additionally, stabilities of individual GLS during processing rely on their chemical structures and species. Generally, aliphatic GLS from Brassicaceae showed lower loss than indole GLS, indicating differences in their stabilities during processing.

Microbes: A Hidden Treasure of Polyunsaturated Fatty Acids.

Microbes have gained a lot of attention for their potential in producing polyunsaturated fatty acids (PUFAs). PUFAs are gaining scientific interest due to their important health-promoting effects on higher organisms including humans. The current sources of PUFAs (animal and plant) have associated limitations that have led to increased interest in microbial PUFAs as most reliable alternative source. The focus is on increasing the product value of existing oleaginous microbes or discovering new microbes by implementing new biotechnological strategies in order to compete with other sources. The multidisciplinary approaches, including metabolic engineering, high-throughput screening, tapping new microbial sources, genome-mining as well as co-culturing and elicitation for the production of PUFAs, have been considered and discussed in this review. The usage of agro-industrial wastes as alternative low-cost substrates in fermentation for high-value single-cell oil production has also been discussed. Multidisciplinary approaches combined with new technologies may help to uncover new microbial PUFA sources that may have nutraceutical and biotechnological importance.

Post-harvest Application of Methyl Jasmonate or Prohydrojasmon Affects Color Development and Anthocyanins Biosynthesis in Peach by Regulation of Sucrose Metabolism.

The roles of methyl jasmonate (MeJA) and prohydrojasmon (PDJ) in postharvest color development and anthocyanins biosynthesis in the skin of peach fruit remain unclear. In this study, peach fruit were infiltrated with MeJA (200 µM) or PDJ (40 µM) and stored at 22°C for 7 days. The results showed that treatment with MeJA or PDJ had a positive effect on red color formation in peach fruits due to anthocyanins accumulation (∼120% increase). This was attributed to increased enzyme activities, and enhanced transcript abundance of the genes associated with anthocyanins biosynthesis, induced by MeJA or PDJ. Both MeJA and PDJ promoted sucrose biosynthesis, and the subsequently elevated levels of the sucrose during storage were positively correlated with anthocyanins accumulation (0.49) and the activities of key biosynthesis enzymes (0.42-0.79). Based on these findings, we proposed that MeJA or PDJ treatments promote anthocyanins biosynthesis by regulating sucrose metabolism during the postharvest storage of peach fruit.

Overexpression of Shinorhizobium meliloti flavohemoglobin improves cell growth and fatty acid biosynthesis in oleaginous fungus Mucor circinelloides.

Oxygen availability is a limiting factor for lipid biosynthesis in eukaryotic microorganisms. Two bacterial hemoglobins from Vitreoscilla sp. (VHb) and Shinorhizobium meliloti (SHb), which deliver oxygen to the respiratory chain to produce more ATP, were introduced into Mucor circinelloides to alleviate oxygen limitation, thereby improving cell growth and fatty acid production. The VHb and SHb genes were integrated into the M. circinelloides MU402 genome by homologous recombination. VHb and SHb protein expression was verified by carbon monoxide difference spectrum analysis. The biomass was increased by ~ 50% in the strain expressing SHb compared with VHb. The total fatty acid (TFA) content of the strain expressing SHb reached 15.7% of the dry cell weight (~ 40% higher than that of the control strain) during flask cultivation. The biomass and TFA content were markedly increased (12.1 g/L and 21.1% dry cell weight, respectively) in strains expressing SHb than strains expressing VHb during fermenter cultivation. VHb and SHb expression also increased the proportion of polyunsaturated fatty acids. Overexpressed bacterial hemoglobins, especially SHb, increased cell growth and TFA content in M. circinelloides at low and high aeration, suggesting that SHb improves fatty acid production more effectively than VHb in oleaginous microorganisms.

Effects of in-package atmospheric cold plasma treatment on the qualitative, metabolic and microbial stability of fresh-cut pears.

BACKGROUND: The greatest hurdle to commercial marketing of fresh-cut fruits and vegetables is limited shelf life due to microbial hazards and quality deterioration. Atmospheric cold plasma (ACP) is an emerging non-thermal technology with significant potential to improve the safety and storability of fresh products. The objective of this study was to evaluate the effects of ACP, generated in sealed packaging, on the qualitative, metabolic and microbial stability of fresh-cut pears during simulated cold storage. RESULTS: ACP treatments were effective in inhibiting the growth of mesophilic aerobic bacteria, yeast and mold, particularly CP3 (65 kV, 1 min), which could prolong shelf life to the greatest extent. While decontamination was not always associated with an increase in plasma intensity. Moreover, at 65 kV for 1 min, ACP treatment had the potential to retard respiration, and maintain organoleptic properties and other quality attributes. Additionally, peroxidase and pectin methylesterase (PME) activities were reduced immediately after treatments. These effects were dependent on treatment voltage and time, while a subsequent recovery in activity was only observed for PME. CONCLUSION: The results obtained from this study will contribute to an understanding of the effects of in-package ACP treatments on the storability and microbial safety of fresh-cut pears. This knowledge could be beneficial in reducing quality losses for fresh-cut pears and the preservation of other products. © 2021 Society of Chemical Industry.

Genome-Wide Identification of the Vacuolar H+-ATPase Gene Family in Five Rosaceae Species and Expression Analysis in Pear (Pyrus bretschneideri).

Vacuolar H+-ATPases (V-ATPase) are multi-subunit complexes that function as ATP hydrolysis-driven proton pumps. They play pivotal roles in physiological processes, such as development, metabolism, stress, and growth. However, there have been very few studies on the characterisation of V-ATPase (VHA) genes in Rosaceae species. Therefore, in the present study, we performed a genome-wide analysis and identified VHA gene family members in five Rosaceae species (Pyrus bretschneideri, Malus domestica, Prunus persica, Fragaria vesca, and Prunus mume). A total of 159 VHA genes were identified, and were classified into 13 subfamilies according to the phylogenetic analysis. The structure of VHA proteins revealed high similarity among different VHA genes within the same subgroup. Gene duplication event analysis revealed that whole-genome duplications represented the major pathway for expansion of the Pyrus bretschneideri VHA genes (PbrVHA genes). The tissue-specific expression analysis of the pear showed that 36 PbrVHA genes were expressed in major tissues. Seven PbrVHA genes were significantly downregulated when the pollen tube growth stopped. Moreover, many PbrVHA genes were differentially expressed during fruit development and storage, suggesting that VHA genes play specific roles in development and senescence. The present study provides fundamental information for further elucidating the potential roles of VHA genes during development and senescence.

Physiological and transcriptomic analysis of 'Whangkeumbae' pear core browning during low-temperature storage.

Core browning of 'Whangkeumbae' pear has become an urgent problem in the Chinese pear industry, which often appears after several months of low-temperature storage. However, little is known regarding the crosstalk between physiology and molecular mechanisms regulating the core browning process of the pear. In this study, the physiological and genetic responses of the core were identified during storage. The results showed that the malonyldialdehyde (MDA) content, electrolyte leakage, hydrogen peroxide (H2O2) content and superoxide anion (O2·-) production rate progressively increased during the browning process. Polyphenoloxidase (PPO), phospholipase D (PLD) and lipoxygenase (LOX) activity initially slightly increased but then sharply increased during the later storage stage. A total of 33,265 unigenes was generated via high-throughput sequencing, and 5121 differentially expressed genes (DEGs) were identified. These DEGs were functionally annotated and some core browning-related DEGs involved in the redox reaction, membrane lipid metabolism and enzymatic browning were also determined. We found that the changes in the gene expression accorded with the physiological variation, indicating the close crosstalk between physiological and genetic response during storage. Our study provides a basis for future research on the core browning mechanism during pear storage.

Generation of lycopene-overproducing strains of the fungus Mucor circinelloides reveals important aspects of lycopene formation and accumulation.

OBJECTIVES: To generate lycopene-overproducing strains of the fungus Mucor circinelloides with interest for industrial production and to gain insight into the catalytic mechanism of lycopene cyclase and regulatory process during lycopene overaccumulation. RESULTS: Three lycopene-overproducing mutants were generated by classic mutagenesis techniques from a ß-carotene-overproducing strain. They carried distinct mutations in the carRP gene encoding lycopene cyclase that produced loss of enzymatic activity to different extents. In one mutant (MU616), the lycopene cyclase was completely destroyed, and a 43.8% (1.1 mg/g dry mass) increase in lycopene production was observed in comparison to that by the previously existing lycopene overproducer. In addition, feedback regulation of the end product was suggested in lycopene-overproducing strains. CONCLUSIONS: A lycopene-overaccumulating strain of the fungus M. circinelloides was generated that could be an alternative for the industrial production of lycopene. Vital catalytic residues for lycopene cyclase activity and the potential mechanism of lycopene formation and accumulation were identified.

[Characterization of a malic enzyme isoform V from Mucor circinelloides].

OBJECTIVE: We aimed at characterizing a malic enzyme isoform V from Mucor circinelloides. METHODS: me1 gene encoding malic enzyme isoform V was amplified and cloned into expression vector pET28a. High-purity recombinant protein BLME1 was obtained by affinity chromatography using. Ni-NTA column and characterized subsequently. RESULTS: The optimum conditions were pH at 8.0 and temperature at 33 degrees C. Under optimum conditions, BLME1 activity achieved 92.8 U/mg. The K(m) for L-malate and NADP+ were 0.74960 ± 0.06120 mmol/L and 0.22070 ± 0.01810 mmol/L, the V(max) for L-malate and NADP+ were 72.820 ± 1.077 U/mg and 86.110 ± 1.665 U/mg, respectively. In addition, ions played important roles in BLME1 activity; several ions such as Mn2+, Mg2+, Co2+, Ni2+ could activate BLME1, whereas Ca2+, Cu2+ could be used as inhibitors. Additionally, the metabolic intermediates such as oxaloacetic acid and α-ketoglutaric acid inhibited the activity of BLME1, whereas succinic acid activated it. CONCLUSION: A malic enzyme isoform V from Mucor circinelloides was characterized, providing the references for further studies on this enzyme.

A new regulatory mechanism controlling carotenogenesis in the fungus Mucor circinelloides as a target to generate ß-carotene over-producing strains by genetic engineering.

BACKGROUND: Carotenoids are natural pigments with antioxidant properties that have important functions in human physiology and must be supplied through the diet. They also have important industrial applications as food colourants, animal feed additives and nutraceuticals. Some of them, such as ß-carotene, are produced on an industrial scale with the use of microorganisms, including fungi. The mucoral Blakeslea trispora is used by the industry to produce ß-carotene, although optimisation of production by molecular genetic engineering is unfeasible. However, the phylogenetically closely related Mucor circinelloides, which is also able to accumulate ß-carotene, possesses a vast collection of genetic tools with which to manipulate its genome. RESULTS: This work combines classical forward and modern reverse genetic techniques to deepen the regulation of carotenoid synthesis and generate candidate strains for biotechnological production of ß-carotene. Mutagenesis followed by screening for mutants with altered colour in the dark and/or in light led to the isolation of 26 mutants that, together with eight previously isolated mutants, have been analysed in this work. Although most of the mutants harboured mutations in known structural and regulatory carotenogenic genes, eight of them lacked mutations in those genes. Whole-genome sequencing of six of these strains revealed the presence of many mutations throughout their genomes, which makes identification of the mutation that produced the phenotype difficult. However, deletion of the crgA gene, a well-known repressor of carotenoid biosynthesis in M. circinelloides, in two mutants (MU206 and MU218) with high levels of ß-carotene resulted in a further increase in ß-carotene content to differing extents with respect to the crgA single-null strain; in particular, one strain derived from MU218 was able to accumulate up to 4 mg/g of ß-carotene. The additive effect of crgA deletion and the mutations present in MU218 suggests the existence of a previously unknown regulatory mechanism that represses carotenoid biosynthesis independently and in parallel to crgA. CONCLUSIONS: The use of a mucoral model such as M. circinelloides can allow the identification of the regulatory mechanisms that control carotenoid biosynthesis, which can then be manipulated to generate tailored strains of biotechnological interest. Mutants in the repressor crgA and in the newly identified regulatory mechanism generated in this work accumulate high levels of ß-carotene and are candidates for further improvements in biotechnological ß-carotene production.

Annotation and analysis of malic enzyme genes encoding for multiple isoforms in the fungus Mucor circinelloides CBS 277.49.

Based on the newly-released genomic data of Mucor circinelloides CBS 277.49, we have annotated five genes encoding for malic enzyme: all code for proteins that contain conserved domains/motifs for malic acid binding, NAD(+) binding and NAD(P)(+) binding. Phylogenetic analysis for malic enzyme genes showed that genes ID 78524 and 11639 share ~80% amino acid identity and are grouped in cluster 1; genes ID 182779, 186772 and 116127 share ~66% amino acid identity are grouped in cluster 2. Genes ID 78524, 11639 and 166127 produce proteins that are localized in the mitochondrion, while the products from genes 182779 and 186772 are localized in the cytosol. Based on the comparative analysis published previously by Song et al. (Microbiology 147:1507-1515, 2001), we propose that malic enzyme genes ID 78524, 166127, 182779, 186772, 11639, respectively, represent protein isoforms I, II, III/IV, V, and VI.