In situ imaging of mRNA transcripts based on split-aptamer and split protein in living cells.

Messenger RNA (mRNA) is an essential component of cell development and growth. However, the detection of endogenous mRNA in living cells is currently limited. To address this issue, we have developed a novel strategy that comprises split-aptamer and split fluorescent protein dual-color, "turn-on" probes that specifically target mRNA with complementary sequences. Our split-aptamer and split-protein-initiated fluorescence complementation (sAPiFC) approach for live-cell imaging has demonstrated selectivity, stability, and capability for targeting various mRNAs.

Analysis of genetic and chemical variability of five Curcuma species based on DNA barcoding and HPLC fingerprints.

The rhizomes of Curcuma species have a long medicinal history in Asia. In China, Curcuma species mainly be utilized to make pharmaceutical products, including C. phaecocaulis, C. aromatica, C. wenyujin, C. kwangsiensis and C. longa. In this study, twenty-four samples were selected to study the genetic and chemical variability among five Curcuma species. The ITS2 and trnK intron gene fragment were used to identify the five Curcuma species, the differences in chemical composition were computed using the Euclidean distance based on the data of HPLC characteristic peak areas and the content of six key components, and agronomic characteristics were analyzed including morphological and volatile oil characteristics. The ITS2 and trnK intron gene fragment could distinguish the five Curcuma species clearly. The genetic distance between Curcuma species ranged from 0.0085 to 0.0767 based on the data of ITS2 gene sequences with 32 variation sites, and the genetic distance between Curcuma species ranged from 0.0003 to 0.0194 based on the data of trnK intron gene sequences with 39 variation sites. Five Curcuma species showed otherness chemical composition characteristics, with the Euclidean distance ranging from 3.373 to 6.998. The C. longa showed the biggest variation compared with other species, with the Euclidean distance above 6.239. Among the samples of the original plants of Ezhu, the volatile oil yield of W1 was the highest, reached to 105.75 mL per single plant. Among all the samples, J6 showed the highest yield of volatile oil, reached to 149.42 mL per single plant. The results showed that chemical composition similarity of the medicinal plants was the primary proof for the selection of the original plants of the Curcuma medicinal materials. The genetic distance and chemical variability were important references for discovering new medicinal plant resources.

Polo-like kinase 1 as a biomarker predicts the prognosis and immunotherapy of breast invasive carcinoma patients.

Background: Invasive breast carcinoma (BRCA) is associated with poor prognosis and high risk of mortality. Therefore, it is critical to identify novel biomarkers for the prognostic assessment of BRCA. Methods: The expression data of polo-like kinase 1 (PLK1) in BRCA and the corresponding clinical information were extracted from TCGA and GEO databases. PLK1 expression was validated in diverse breast cancer cell lines by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Single sample gene set enrichment analysis (ssGSEA) was performed to evaluate immune infiltration in the BRCA microenvironment, and the random forest (RF) and support vector machine (SVM) algorithms were used to screen for the hub infiltrating cells and calculate the immunophenoscore (IPS). The RF algorithm and COX regression model were applied to calculate survival risk scores based on the PLK1 expression and immune cell infiltration. Finally, a prognostic nomogram was constructed with the risk score and pathological stage, and its clinical potential was evaluated by plotting calibration charts and DCA curves. The application of the nomogram was further validated in an immunotherapy cohort. Results: PLK1 expression was significantly higher in the tumor samples in TCGA-BRCA cohort. Furthermore, PLK1 expression level, age and stage were identified as independent prognostic factors of BRCA. While the IPS was unaffected by PLK1 expression, the TMB and MATH scores were higher in the PLK1-high group, and the TIDE scores were higher for the PLK1-low patients. We also identified 6 immune cell types with high infiltration, along with 11 immune cell types with low infiltration in the PLK1-high tumors. A risk score was devised using PLK1 expression and hub immune cells, which predicted the prognosis of BRCA patients. In addition, a nomogram was constructed based on the risk score and pathological staging, and showed good predictive performance. Conclusions: PLK1 expression and immune cell infiltration can predict post-immunotherapy prognosis of BRCA patients.

A nomogram model predicting the risk of postpartum stress urinary incontinence in primiparas: A multicenter study.

Stress urinary incontinence (SUI) is a common gynecological urinary system disease, and globally, 200 million or more people suffer from it. However, the existing literature mostly focuses on postpartum urinary incontinence (UI) or UI in middle-aged and elderly people, with little focus on primiparas. To analyse urinary incontinence prevalence and its risk factors in primiparas and establish a nomogram prediction model, 360 parturients were recruited from three hospitals between April and September 2021. A homemade electronic questionnaire was used to investigate the general demographic and perinatal characteristics of primiparas. The SUI was diagnosed by the physicians. Logistic regression analysis of independent risk factors for SUI and a nomogram prediction model were established. Ninety people were diagnosed as SUI. The number of pregnancies (OR = 3.322, 95% CI = 1.473-7.492), residence (OR = 5.451, 95% CI = 2.725-10.903), occupation (OR = 3.393, 95% CI = 1.144-10.064), education level (OR = 3.551, 95% CI = 1.223-10.308), delivery method (OR = 10.270, 95% CI = 4.090-25.789), and oxytocin use (OR = 2.166, 95% CI = 1.142-4.109) were independent risk factors for SUI. The C-index of the nomogram prediction model was 0.798 (95% CI = 0.749-0.846). The POPDI score, CRADI score, UDI score, and PFDI scores of women with SUI were significantly higher than those of non-SUI women, while I-QOL scores were significantly lower than those of non-SUI women. In conclusion, the prevalence of SUI among primiparas in Fuyang, China, was 25.00%, which exhibited a large impact on the quality of life of puerperae. The present study successfully established an individualized nomogram prediction model of SUI for primiparas with good discrimination and diagnostic efficiency, which was helpful for the early clinical identification of high-risk primiparas with SUI.

Case Report: A PD-L1-Positive Patient With Pleomorphic Rhabdomyosarcoma Achieving an Impressive Response to Immunotherapy.

There is currently a lack of effective systemic treatment for patients with advanced pleomorphic rhabdomyosarcoma (PRMS). Although programmed death protein 1 (PD-1) inhibitors have shown efficacy in various solid tumors, their effects on PRMS have not been well established. Here, we present a case of a 12-year-old Chinese male adolescent with metastatic PRMS who benefited from the PD-1 inhibitor nivolumab. The patient initially underwent primary tumor resection but failed to respond to subsequent first-line chemotherapy and second-line pazopanib treatment. Pathological examination showed positive PD-L1 expression and tumor-infiltrating lymphocytes in the tumor tissue, and the patient was administered nivolumab as a posterior-line treatment. After attaining a clinically partial response (PR), surgical resection was performed, which was followed by adjuvant nivolumab. At the time of the submission of this manuscript, the patient achieved recurrence-free survival (RFS) lasting 45 months and counting. This is the first clinical evidence that a patient with refractory PRMS was controlled by anti-PD-1 antibody, with an RFS lasting more than 3 years. This case suggests that PD-L1 expression and T-cell infiltration could be used as potential biomarkers for PRMS immunotherapy.

The mediating role of self-acceptance in the relationship between loneliness and subjective well-being among the elderly in nursing home: A cross-sectional study.

ABSTRACT: The purpose of this study was to investigate the mediating effects of self-acceptance on loneliness and subjective well-being (SWB) among elderly subjects living in Chinese nursing homes.This cross-sectional study was conducted between October 2019 and March 2020. A total of 415 elderly participants aged 60 to 97 years (mean 81.12 ±â€Š8.90 years) from 3 medical and nursing homes in Fuyang city, Anhui province, were selected using a convenience sampling method. Data were collected using a general information questionnaire, the Memorial University of Newfoundland Scale of Happiness, the self-acceptance scale, and the UCLA Loneliness scale. Correlations, regressions, and structural equation models were used for the analyses. Multiple linear regression analysis was performed to confirm the factors influencing the SWB. Bootstrapping was performed to confirm the mediation effect.The loneliness of elderly subjects in nursing homes was significantly correlated with self-acceptance and SWB (r = -0.338, P < .01; r = -0.383, P < .01), and self-acceptance was significantly correlated with SWB (r = 0.401, P < .01). Multiple linear regression revealed that the relationship with children, loneliness, residence time in nursing homes, income, marital status, self-acceptance, original residence, and frequency of children's visits were the main factors affecting SWB. Bootstrapping showed that the mediating role of self-acceptance was statistically significant.The SWB of elderly individuals living in Chinese nursing homes was moderate. Low-income people, subjects from rural areas, and those newly admitted to nursing homes should be emphasized in interventions, and appropriate measures should be taken to harmonize the relationships between elderly residents and their children. Self-acceptance partially mediated the relationship between loneliness and SWB. Consequently, self-acceptance should be the focus of improving the SWB of elderly nursing home residents.

The reductive carboxylation activity of heterotetrameric pyruvate synthases from hyperthermophilic archaea.

Pyruvate synthase (pyruvate:ferredoxin oxidoreductase, PFOR) catalyzes the interconversion of acetyl-CoA and pyruvate, but the reductive carboxylation activities of heterotetrameric PFORs remain largely unknown. In this study, we cloned, expressed, and purified selected six heterotetrameric PFORs from hyperthermophilic archaea. The reductive carboxylation activities of these heterotetrameric PFORs were measured at 70 °C and the ratio of reductive carboxylation activity to oxidative decarboxylation activity (red/ox ratio) were calculated. Four out of six showed reductive decarboxylation activities. Among them, the PFORpfm from Pyrolobus fumarii showed the highest reductive carboxylation activities and the highest red/ox ratio, which were 54.32 mU/mg and 0.51, respectively. The divergence of the reductive carboxylation activities and the red/ox ratios of heterotetrameric PFORs in hyperthermophilic archaea indicate the diversity of the functions of PFOR over long-term evolution. This can help us better understand the autotrophic CO2 fixation process in thermal vent, or in other CO2-rich high temperature habitat.

Synthetic biology for CO2 fixation.

Recycling of carbon dioxide (CO2) into fuels and chemicals is a potential approach to reduce CO2 emission and fossil-fuel consumption. Autotrophic microbes can utilize energy from light, hydrogen, or sulfur to assimilate atmospheric CO2 into organic compounds at ambient temperature and pressure. This provides a feasible way for biological production of fuels and chemicals from CO2 under normal conditions. Recently great progress has been made in this research area, and dozens of CO2-derived fuels and chemicals have been reported to be synthesized by autotrophic microbes. This is accompanied by investigations into natural CO2-fixation pathways and the rapid development of new technologies in synthetic biology. This review first summarizes the six natural CO2-fixation pathways reported to date, followed by an overview of recent progress in the design and engineering of CO2-fixation pathways as well as energy supply patterns using the concept and tools of synthetic biology. Finally, we will discuss future prospects in biological fixation of CO2.

Fermentation and genomic analysis of acetone-uncoupled butanol production by Clostridium tetanomorphum.

In typical acetone-butanol-ethanol (ABE) fermentation, acetone is the main by-product (50 % of butanol mass) of butanol production, resulting in a low yield of butanol. It is known that some Clostridium tetanomorphum strains are able to produce butanol without acetone in nature. Here, we described that C. tetanomorphum strain DSM665 can produce 4.16 g/L butanol and 4.98 g/L ethanol at pH 6.0, and 9.81 g/L butanol and 1.01 g/L ethanol when adding 1 mM methyl viologen. Butyrate and acetate could be reassimilated and no acetone was produced. Further analysis indicated that the activity of the acetate/butyrate:acetoacetyl-CoA transferase responsible for acetone production is lost in C. tetanomorphum DSM665. The genome of C. tetanomorphum DSM665 was sequenced and deposited in DDBJ, EMBL, and GenBank under the accession no. APJS00000000. Sequence analysis indicated that there are no typical genes (ctfA/B and adc) that are typically parts of an acetone synthesis pathway in C. tetanomorphum DSM665. This work provides new insights in the mechanism of clostridial butanol production and should prove useful for the design of a high-butanol-producing strain.

Quantitative analysis of an engineered CO2-fixing Escherichia coli reveals great potential of heterotrophic CO2 fixation.

BACKGROUND: Production of fuels from the abundant and wasteful CO2 is a promising approach to reduce carbon emission and consumption of fossil fuels. Autotrophic microbes naturally assimilate CO2 using energy from light, hydrogen, and/or sulfur. However, their slow growth rates call for investigation of the possibility of heterotrophic CO2 fixation. Although preliminary research has suggested that CO2 fixation in heterotrophic microbes is feasible after incorporation of a CO2-fixing bypass into the central carbon metabolic pathway, it remains unclear how much and how efficient that CO2 can be fixed by a heterotrophic microbe. RESULTS: A simple metabolic flux index was developed to indicate the relative strength of the CO2-fixation flux. When two sequential enzymes of the cyanobacterial Calvin cycle were incorporated into an E. coli strain, the flux of the CO2-fixing bypass pathway accounts for 13 % of that of the central carbon metabolic pathway. The value was increased to 17 % when the carbonic anhydrase involved in the cyanobacterial carbon concentrating mechanism was introduced, indicating that low intracellular CO2 concentration is one limiting factor for CO2 fixation in E. coli. The engineered CO2-fixing E. coli with carbonic anhydrase was able to fix CO2 at a rate of 19.6 mg CO2 L(-1) h(-1) or the specific rate of 22.5 mg CO2 g DCW(-1) h(-1). This CO2-fixation rate is comparable with the reported rates of 14 autotrophic cyanobacteria and algae (10.5-147.0 mg CO2 L(-1) h(-1) or the specific rates of 3.5-23.7 mg CO2 g DCW(-1) h(-1)). CONCLUSIONS: The ability of CO2 fixation was created and improved in E. coli by incorporating partial cyanobacterial Calvin cycle and carbon concentrating mechanism, respectively. Quantitative analysis revealed that the CO2-fixation rate of this strain is comparable with that of the autotrophic cyanobacteria and algae, demonstrating great potential of heterotrophic CO2 fixation.

A functional recT gene for recombineering of Clostridium.

Recombineering is an efficient genetic manipulation method employing the mechanism of phagenic RecT-mediated homologous recombination. To develop a recombineering method for Clostridium, a putative recT gene (CPF0939) from Clostridium perfringens genome was functionally verified in a clostridial host Clostridium acetobutylicum. We show that a short synthetic oligonucleotide can be introduced into the target site for specific point mutation. This functional recT gene would therefore contribute to development of recombineering tools for Clostridium.

Developing controllable hypermutable Clostridium cells through manipulating its methyl-directed mismatch repair system.

Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution. However, there have not been any similar systems developed for Clostridium, an important bacterial genus. Here we report a novel two-step strategy for developing controllable hypermutable cells of Clostridium acetobutylicum, an important and representative industrial strain. Firstly, the mutS/L operon essential for methyldirected mismatch repair (MMR) activity was inactivated from the genome of C. acetobutylicum to generate hypermutable cells with over 250-fold increased mutation rates. Secondly, a proofreading control system carrying an inducibly expressed mutS/L operon was constructed. The hypermutable cells and the proofreading control system were integrated to form a controllable hypermutable system SMBMutC, of which the mutation rates can be regulated by the concentration of anhydrotetracycline (aTc). Duplication of the miniPthl-tetR module of the proofreading control system further significantly expanded the regulatory space of the mutation rates, demonstrating hypermutable Clostridium cells with controllable mutation rates are generated. The developed C. acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress, indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.

[Direct secretory expression of active microbial transglutaminase in Pichia pastoris].

Direct secretory expression of active microbial transglutaminase (MTG) using heterologous hosts is a promising strategy, although its production level still needs to be improved for industrial production. Pichia pastoris is one of the most efficient expression systems developed in recent years. In this study, secretory expression of active MTG was successfully achieved by co-expressing the pro sequence and mature MTG genes in P. pastoris. Furthermore, we optimized the copy number of pro/MTG expression cassettes and the fermentation conditions. MTG production level reached 7.3 U/mL in 1-liter fermentor through high density fermentation, providing the feasiblity for industrial scale preparation of MTG.

Biobutanol.

China initiated its acetone-butanol-ethanol (ABE) industry in the 1950s; it peaked in the 1980s, and ended at the end of the last century owing to the development of more competitive petrochemical pathways. However, driven by the high price of crude oil and environmental concerns raised by the over-consumption of petrochemical products, biofuels and bio-based chemicals including butanol have garnered global attention again. Currently, butanol produced from ABE fermentation is mainly used as an industrial solvent or a platform chemical for several bulk derivatives, and is also believed to be a potential biofuel. A number of plants have been built or rebuilt in recent years in China for butanol production with the ABE process. Chinese researchers also show great interest in the improvement of the production strains and corresponding processes. They have applied conventional mutagenesis methods to improve butanol-producing strains such as the Clostridium acetobutylicum mutant strains EA2018 (butanol ratio of 70%) and Rh8 (butanol tolerance of 19 g/L). The omics technologies, such as genome sequencing, proteomic and transcriptomic analysis, have been adapted to elucidate the characteristics of different butanol-producing bacteria. Based on the group II intron method, the genetic manipulation system of C. acetobutylicum was greatly improved, and some successful engineering strains were developed. In addition, research in China also covers the downstream processes. This article reviews up-to-date progress on biobutanol production in China.

Combinatorial strategy of sorbitol feeding and low-temperature induction leads to high-level production of alkaline ß-mannanase in Pichia pastoris.

A process for efficient production of an alkaline ß-mannanases from Bacillus sp. N16-5 was established by heterologous expression using Pichia pastoris. A high producing strain was generated by removing the native ß-mannanases signal peptide and increasing the copy number of the mature ß-mannanases gene. High cell density fermentation of this strain in 1-L bioreactor led to a production level of 4164 U/mL after 96 h of induction. Sorbitol co-feeding and temperature-lowering strategies both increased the ß-mannanase production levels. Combined usage of these two strategies achieved the most effective result-the enzyme level reached 6336 U/mL within 84 h, which to our best knowledge is the highest production level reported for the expression of extreme ß-mannanase thus far. The strategy described in this work can also be adapted to express other important industrial enzymes with extreme properties.