Transcriptomic-metabolomic analysis reveals the effect of copper toxicity on fermentation properties in Saccharomyces cerevisiae.

Copper is one of the unavoidable heavy metals in wine production. In this study, the effects on fermentation performance and physiological metabolism of Saccharomyces cerevisiae under copper stress were investigated. EC1118 was the most copper-resistant among the six strains. The ethanol accumulation of EC1118 was 26.16-20 mg/L Cu2+, which was 1.90-3.15 times higher than that of other strains. The fermentation rate was significantly reduced by copper, and the inhibition was relieved after 4-10 days of adjustment. Metabolomic-transcriptomic analysis revealed that amino acid and nucleotide had the highest number of downregulated and upregulated differentially expressed metabolites, respectively. The metabolism of fructose and mannose was quickly affected, which then triggered the metabolism of galactose in copper stress. Pathways such as oxidative and organic acid metabolic processes were significantly affected in the early time, resulting in a significant decrease in the amount of carboxylic acids. The pathways related to protein synthesis and metabolism under copper stress, such as translation and peptide biosynthetic process, was also significantly affected. In conclusion, this study analyzed the metabolite-gene interaction network and molecular response during the alcohol fermentation of S. cerevisiae under copper stress, providing theoretical basis for addressing the influence of copper stress in wine production.

Research on workflow recognition for liver rupture repair surgery.

Liver rupture repair surgery serves as one tool to treat liver rupture, especially beneficial for cases of mild liver rupture hemorrhage. Liver rupture can catalyze critical conditions such as hemorrhage and shock. Surgical workflow recognition in liver rupture repair surgery videos presents a significant task aimed at reducing surgical mistakes and enhancing the quality of surgeries conducted by surgeons. A liver rupture repair simulation surgical dataset is proposed in this paper which consists of 45 videos collaboratively completed by nine surgeons. Furthermore, an end-to-end SA-RLNet, a self attention-based recurrent convolutional neural network, is introduced in this paper. The self-attention mechanism is used to automatically identify the importance of input features in various instances and associate the relationships between input features. The accuracy of the surgical phase classification of the SA-RLNet approach is 90.6%. The present study demonstrates that the SA-RLNet approach shows strong generalization capabilities on the dataset. SA-RLNet has proved to be advantageous in capturing subtle variations between surgical phases. The application of surgical workflow recognition has promising feasibility in liver rupture repair surgery.

COX2-ATP Synthase Regulates Spine Follicle Size in Hedgehogs.

Skin evolves essential appendages with adaptive patterns that synergistically insulate the body from environmental insults. How similar appendages in different animals generate diversely-sized appendages remain elusive. Here we used hedgehog spine follicles and mouse hair follicles as models to investigate how similar follicles form in different sizes postnatally. Histology and immunostaining show that the spine follicles have a significantly greater size than the hair follicles. By RNA-sequencing analysis, we found that ATP synthases are highly expressed in hedgehog skin compared to mouse skin. Inhibition of ATP synthase resulted in smaller spine follicle formation during regeneration. We also identified that the mitochondrial gene COX2 functions upstream of ATP synthase that influences energy metabolism and cell proliferation to control the size of the spine follicles. Our study identified molecules that function differently in forming diversely-sized skin appendages across different animals, allowing them to adapt to the living environment and benefit from self-protection.

Effects of Seven Sterilization Methods on the Functional Characteristics and Color of Yan 73 (Vitis vinifera) Grape Juice.

Yan 73 (Vitis vinifera) is a dyed grape variety cultivated in China. Currently, most studies have focused on the mechanism of anthocyanins or the impact of anthocyanins as auxiliary color varieties on wine color. There is little research on its direct use or direct processing of products such as juice. In order to investigate the effects of different processing methods on the juice of Yan 73 grapes, the physicochemical and functional properties, as well as the sensory indexes of the juice, were analyzed by using thermal pasteurization (TP), thermosonication (TS), TS combined with nisin (TSN), TS combined with ε-Polylysine (TSε), irradiation (IR), and high hydrostatic pressure (HHP). The physicochemical indexes, functional properties, and sensory indexes of Smoke 73 grape juice were determined and analyzed. The results of the study showed that among the seven sterilization methods, total polyphenol content (TPC) in juice was significantly increased in all treatments except HHP. TPC was the highest in TP (3773.33 mg GAE/L). Total anthocyanin content (TAC) was increased except IR5, and TSN (1202.67 mg/L) had the highest TAC. In terms of color, TP (a* = 36.57, b* = 19.70, L* = 14.81, C* = 41.55, h° = 28.30, ΔE = 5.9) promotes the dissolution of anthocyanins because of high temperatures, which basically improves all the color indicators of grape juice and makes the color of grape juice more vivid. After HHP treatment, the color (ΔE = 1.72) and aroma indicators are closer to the grape juice itself. The Entropy weight-TOPSIS, CRITIC-Topsis, and PCA integrated quality evaluation models showed that all selected TP as the best integrated quality.

Multi-scale structural characteristics of black Tartary buckwheat resistant starch by autoclaving combined with debranching modification.

The impact of autoclaving or autoclave-debranching treatments on the multi-scale structure of resistant starch (RS) and the relationship with starch digestion remains unclear, despite their widespread use in its preparation. This work investigated the relationship between RS structure in black Tartary buckwheat and its digestibility by analyzing the effects of autoclaving and autoclave-debranching combined treatments on the multi-scale structure of RS. The results showed that black Tartary buckwheat RS exhibited a more extensive honeycomb-like network structure and enhanced thermal stability than either black Tartary buckwheat native starch (BTBNS) or common buckwheat native starch (CBNS). Autoclaving and autoclaving-debranching converted A-type native starch to V-type and possibly the formation of flavonoid-starch complexes. Autoclaving treatment significantly increased the proportion of short A chain (DP 6-12) and the amylose (AM) content, reduced the viscosity and the total crystallinity. Notably, the autoclave-debranching co-treatment significantly enhanced the resistance of starch to digestion, promoted the formation of perfect microcrystallines, and increased the AM content, short-range ordered degree, and the proportion of long B2 chain (DP 25-36). This study reveals the relationship between the multi-scale structure and digestibility of black Tartary buckwheat RS by autoclaving combined with debranching modification.

Investigation of B-atp6-orfH79 distributing in Chinese populations of Oryza rufipogon and analysis of its chimeric structure.

BACKGROUND: The cytoplasmic male sterility (CMS) of rice is caused by chimeric mitochondrial DNA (mtDNA) that is maternally inherited in the majority of multicellular organisms. Wild rice (Oryza rufipogon Griff.) has been regarded as the ancestral progenitor of Asian cultivated rice (Oryza sativa L.). To investigate the distribution of original CMS source, and explore the origin of gametophytic CMS gene, a total of 427 individuals with seventeen representative populations of O. rufipogon were collected in from Dongxiang of Jiangxi Province to Sanya of Hainan Province, China, for the PCR amplification of atp6, orfH79 and B-atp6-orfH79, respectively. RESULTS: The B-atp6-orfH79 and its variants (B-atp6-GSV) were detected in five among seventeen populations (i.e. HK, GZ, PS, TL and YJ) through PCR amplification, which could be divided into three haplotypes, i.e., BH1, BH2, and BH3. The BH2 haplotype was identical to B-atp6-orfH79, while the BH1 and BH3 were the novel haplotypes of B-atp6-GSV. Combined with the high-homology sequences in GenBank, a total of eighteen haplotypes have been revealed, only with ten haplotypes in orfH79 and its variants (GSV) that belong to three species (i.e. O. rufipogon, Oryza nivara and Oryza sativa). Enough haplotypes clearly demonstrated the uniform structural characteristics of the B-atp6-orfH79 as follows: except for the conserved sequence (671 bp) composed of B-atp6 (619 bp) and the downstream followed the B-atp6 (52 bp, DS), and GSV sequence, a rich variable sequence (VS, 176 bp) lies between the DS and GSV with five insertion or deletion and more than 30 single nucleotide polymorphism. Maximum likelihood analysis showed that eighteen haplotypes formed three clades with high support rate. The hierarchical analysis of molecular variance (AMOVA) indicated the occurrence of variation among all populations (FST = 1; P < 0.001), which implied that the chimeric structure occurred independently. Three haplotypes (i.e., H1, H2 and H3) were detected by the primer of orfH79, which were identical to the GVS in B-atp6-GVS structure, respectively. All seventeen haplotypes of the orfH79, belonged to six species based on our results and the existing references. Seven existed single nucleotide polymorphism in GSV section can be translated into eleven various amino acid sequences. CONCLUSIONS: Generally, this study, indicating that orfH79 was always accompanied by the B-atp6, not only provide two original CMS sources for rice breeding, but also confirm the uniform structure of B-atp-orfH79, which contribute to revealing the origin of rice gametophytic CMS genes, and the reason about frequent recombination of mitochondrial DNA.

Activated SOX9+ renal epithelial cells promote kidney repair through secreting factors.

A broad spectrum of lethal kidney diseases involves the irreversible destruction of the tubular structures, leading to renal function loss. Following injury, a spectrum of tissue-resident epithelial stem/progenitor cells are known to be activated and then differentiate into mature renal cells to replace the damaged renal epithelium. Here, however, we reported an alternative way that tissue-resident cells could be activated to secrete multiple factors to promote organ repair. At single-cell resolution, we showed that the resident SOX9+ renal epithelial cells (RECs) could expand in the acutely injured kidney of both mouse and human. Compared to other cells, the SOX9+ RECs overexpressed much more secretion related genes, whose functions were linked to kidney repair pathways. We also obtained long-term, feeder-free cultured SOX9+ RECs from human urine and analysed their secretory profile at both transcriptional and proteomic levels. Engraftment of cultured human SOX9+ RECs or injection of its conditional medium facilitated the regeneration of renal tubular and glomerular epithelium, probably through stimulating endogenous REC self-activation and mediating crosstalk with other renal cells. We also identified S100A9 as one of the key factors in the SOX9+ REC secretome. Altogether, the abilities to extensively propagate SOX9+ RECs in culture whilst concomitantly maintaining their intrinsic secretory capacity suggest their future application in cell-free therapies and regeneration medicine.

Summer dormancy of Myricaria laxiflora to escape flooding stress: Changes in phytohormones and enzymes induced by environmental factors.

Dormancy is an adaptation mechanism of plants to environmental stress. Myricaria laxiflora undergoes a long period of flooding stress every year. In order to determine whether this species escapes flooding stress through dormancy, young branches and leaves were collected at different time points before the onset of flooding, and changes in the content/activity of hormones/enzymes that are closely involved in plant growth were monitored. The inducing environmental factors of summer dormancy were identified. The branches and leaves of M. laxiflora showed the following trends as summer flooding approached: (1) gradual increase in the abscisic acid content; (2) gradual decrease in the gibberellin and cytokinin contents; and (3) a continuous decrease in the activities of malate dehydrogenase (MDH), ribulose diphosphate carboxylase (RuBisCo), and glycolate oxidase (GLO). Pearson correlation analysis revealed (1) daylight duration was highly correlated with the hormone content and enzyme activity; (2) the daily mean air temperature (DMAT) was significantly correlated with the cytokinin content. These findings suggest that daylight duration was the main environmental factor leading to changes in the phytohormone content and enzyme activity as well as leading to summer dormancy. M. laxiflora undergoes dormancy before the onset of summer flooding to escape summer flooding stress. Our data indicate that summer flooding does not impede the survival and growth of M. laxiflora.

First Report of flower rot Caused by Fusarium spp. on White kiwifruit in China.

As an important cultivated germplasm, cultivar 'White' (Actinidia eriantha) is appreciated by kiwifruit breeders because of its long shelf life, richness in ascorbic acid and peelable skin. In May 2020, about 1% to 3% of cultivated 'White' plants displayed typical symptoms of flower rot at farms in Hefei (117°25'E, 31°86'N) and Lujiang (117°27'E, 31°48'N), Anhui Province of China (Fig.1a&b). The infected flowers were yellowish at first, gradually turned brown, withered and shrunk, and finally died without blossoming. Infected flowers were surface sterilized in 70% alcohol for 30 s and 1% NaOCl for 3 min, then washed with double distilled water (ddH2O) for 5 times, and finally incubated in potato dextrose agar at 25 ± 2°C in the dark. Twenty fungal isolates were obtained and their colonies showed slightly raised center with dense and cotton-like mycelium. Colonies from Hefei (HF1~HF9) appeared pale yellow (Fig.1c), and those from Lujiang (LJ1~LJ11) showed purplish-red on PDA (Fig.1d). Two types of colonies grown on oatmeal agar were flat with few aerial hyphae (Fig.1e&f). On carnation leaf agar (CLA), isolate of HF1 produced abundant slightly curved macroconidia with 3 to 6 septa, 4.3-5.5×20.7-42.5 µm in size (n=100) (Fig.1g&h), without microconidia and chlamydospores observed. By contrast, macroconidia derived from LJ1 isolate were straight to slightly curved with 3 to 5 septa, 4.0-6.58×21.70-71.10 µm (n=100) in size (Fig.1i); Its chlamydospores were globose to subglobose (5.1 to 9.5 µm) on CLA (Fig.1j). Pathogenicity tests were performed on A. eriantha cv. 'White' flowers. The conidia suspension (105 spore/ml, 30 µL/flower) derived from the HF1 and LJ1 were separately dripped on flowers (n=100). Control flowers were treated with ddH2O. Two-week post-inoculation, all inoculated flowers were turned brown and withered (Fig.1k&m), whereas no symptoms were observed on the controls (Fig.1l&n). This experiment was repeated three times. All isolated and re-isolated pathogens from diseased flowers were subjected to molecular identification. Different molecular markers, including internal transcribed spacers (ITS), translation elongation factor (TEF-1α), calmodulin (CaM), RNA polymerase II subunit 1 (RPB1) gene and RNA polymerase II largest subunit (RPB2) gene, were amplified and sequenced to validate species identification (White et al. 1990; O'Donnell, et al. 1998; O'Donnell, et al. 2012). Based on sequence analysis, the re-isolated strains were identical to the inoculated individuals. Sequences of HF1 and HF2 or LJ1 and LJ2 were deposited in GenBank under accession numbers OK310710 to OK310713 (ITS), OK334291 to OK334294 (TEF-1α), OK412973 to OK412976 (CaM), OK412977 to OK412980 (RPB1), and OK484317 to OK484320 (RPB2), respectively. The BLAST search showed that the sequences of HF1 and HF2 showed 99 to 100% identity with ITS (NR_164594), TEF-1α (MK289601), CaM (MK289698), RPB1 (HM347158), and RPB2 (MK289754) of Fusarium luffae isolates. The sequences of LJ1 and LJ2 also revealed 99 to 100% identity with ITS (NR_121320), TEF-1α (AF212452), RPB1 (JX171459), and RPB2 (MW233412) of F. asiaticum isolates. F. asiaticum species-specific primers were used to detect LJ isolates (Yin et al. 2009), and the correct fragments were amplified (Fig.1o). Phylogenetic trees were constructed based on the tandem nucleotide sequences. Thus, both morphological and molecular criteria supported identification of HF group as F. Luffae (Fig.2a) and LJ group as F. asiaticum (Fig.2b). Fusarium spp. causing flower rot on many hosts have been previously reported (W. Elmer, et al. 2019; Liu, et al. 2021), but this is the first report of F. luffae and F. asiaticum on 'White' kiwifruit in China.

Single-cell analysis defines the lineage plasticity of stem cells in cervix epithelium.

Information about the dynamic change and post-injury regeneration of cervical epithelium is relatively rare, even though it is tightly related to gynecologic malignancy. Here, using a feeder cell-based culturing system, we stably cloned mouse and human P63 and KRT5 expressing cells from the adult cervix as putative cervical stem/progenitor cells (CVSCs). When subjected to differentiation, the cultured cells gave rise to mature cervical epithelium by differentiating into squamous or glandular cells. The ability of endogenous mouse CVSCs to reconstitute cervical epithelium after injury was also evident from the genetic lineage tracing experiments. Single-cell transcriptomic analysis further classified the CVSCs into three subtypes and delineated their bi-lineage differentiation roadmap by pseudo-time analysis. We also tracked the real-time differentiation routes of two representing single CVSC lines in vitro and found that they recapitulated the predicted roadmap in pseudo-time analysis. Signaling pathways including Wnt, TGF-beta, Notch and EGFR were found to regulate the cervical epithelial hierarchy and implicated the different roles of distinct types of cells in tissue homeostasis and tumorigenesis. Collectively, the above data provide a cloning system to achieve stable in vitro culture of a bi-lineage stem/progenitor cell population in the cervix, which has profound implications for our understanding of the cervix stem/progenitor cell function in homeostasis, regeneration, and disease and could be helpful for developing stem cell-based therapies in future.

Single-cell analysis identified lung progenitor cells in COVID-19 patients.

OBJECTIVES: The high mortality of severe 2019 novel coronavirus disease (COVID-19) cases is mainly caused by acute respiratory distress syndrome (ARDS), which is characterized by increased permeability of the alveolar epithelial barriers, pulmonary oedema and consequently inflammatory tissue damage. Some but not all patients showed full functional recovery after the devastating lung damage, and so far there is little knowledge about the lung repair process. We focused on crucial roles of lung progenitor cells in alveolar cell regeneration and epithelial barrier re-establishment and aimed to uncover a possible mechanism of lung repair after severe SARS-CoV-2 infection. MATERIALS AND METHODS: Bronchoalveolar lavage fluid (BALF) of COVID-19 patients was analysed by single-cell RNA-sequencing (scRNA-seq). Transplantation of a single KRT5+ cell-derived cell population into damaged mouse lung and time-course scRNA-seq analysis was performed. RESULTS: In severe (or critical) COVID-19 patients, there is a remarkable expansion of TM4SF1+ and KRT5+ lung progenitor cells. The two distinct populations of progenitor cells could play crucial roles in alveolar cell regeneration and epithelial barrier re-establishment, respectively. The transplanted KRT5+ progenitors could long-term engraft into host lung and differentiate into HOPX+ OCLN+ alveolar barrier cell which restored the epithelial barrier and efficiently prevented inflammatory cell infiltration. CONCLUSIONS: This work uncovered the mechanism by which various lung progenitor cells work in concert to prevent and replenish alveoli loss post-severe SARS-CoV-2 infection.

Genetically engineered distal airway stem cell transplantation protects mice from pulmonary infection.

Severe pulmonary infection is a major threat to human health accompanied by substantial medical costs, prolonged inpatient requirements, and high mortality rates. New antimicrobial therapeutic strategies are urgently required to address the emergence of antibiotic resistance and persistent bacterial infections. In this study, we show that the constitutive expression of a native antimicrobial peptide LL-37 in transgenic mice aids in clearing Pseudomonas aeruginosa (PAO1), a major pathogen of clinical pulmonary infection. Orthotopic transplantation of adult mouse distal airway stem cells (DASCs), genetically engineered to express LL-37, into injured mouse lung foci enabled large-scale incorporation of cells and long-term release of the host defense peptide, protecting the mice from bacterial pneumonia and hypoxemia. Further, correlates of DASCs in adult humans were isolated, expanded, and genetically engineered to demonstrate successful construction of an anti-infective artificial lung. Together, our stem cell-based gene delivery therapeutic platform proposes a new strategy for addressing recurrent pulmonary infections with future translational opportunities.

[Design and application of medical electric leg-raising machine].

OBJECTIVE: Passive leg raising is widely used in clinic, but it lacks of specialized mechanical raise equipment. It requires medical staff to raise leg by hand or requires a multi-functional bed to raise leg, which takes time and effort. Therefore we have developed a new medical electric leg-raising machine. The equipment has the following characteristics: simple structure, stable performance, easy operation, fast and effective, safe and comfortable. The height range of the lifter is 50-120 cm, the range of the angle of raising leg is 10degree angle-80degree angle, the maximum supporting weight is 40 kg. Because of raising the height of the lower limbs and making precise angle, this equipment can completely replace the traditional manner of lifting leg by hand with multi-functional bed to lift patients' leg and can reduce the physical exhaustion and time consumption of medical staff. It can change the settings at any time to meet the needs of the patient; can be applied to the testing of PLR and dynamically assessing the hemodynamics; can prevent deep vein thrombosis and some related complications of staying in bed; and the machine is easy to be cleaned and disinfected, which can effectively avoid hospital acquired infection and cross infection; and can also be applied to emergency rescue of various disasters and emergencies.