Interkingdom multi-omics analysis reveals the effects of nitrogen application on growth and rhizosphere microbial community of Tartary buckwheat

Tartary buckwheat (Fagopyrum tataricum Gaertn.) is an important pseudocereal crop with excellent edible, nutritional and medicinal values. However, the yield of Tartary buckwheat (TB) is very low due to old-fashioned cultivation techniques, particularly unreasonable application of nitrogen fertilizer. To improve the understanding on the theories of nitrogen use in TB, the effects of nitrogen application on growth, as well as chemical properties and microbial community of rhizosphere soil were investigated in this study. Nitrogen application could promote the plant height, stem diameter, nitrogen accumulation and yield of TB. The relative abundance and diversity of bacteria and fungi in the rhizosphere soil of TB were improved by nitrogen fertilizer. Nitrogen application increased the abundance of beneficial bacteria such as Lysobacter and Sphingomonas in rhizosphere soil, and decreased the abundance of pathogenic fungi such as Fusarium and Plectosphaerella. The results indicated that nitrogen application changed the distribution of microbial communities in TB rhizosphere soil. Furthermore, the specific enriched or depleted microorganisms in the rhizosphere soil of four TB varieties were analyzed at OTU level. 87 specific nitrogen-responsive genes with sequence variation were identified in four varieties by integrating genomic re-sequencing and transcriptome analysis, and these genes may involve in the recruitment of specific rhizosphere microorganisms in different TB varieties. This study provided new insights into the effects of nitrogen application on TB growth and rhizosphere microbial community, and improved the understanding on the mechanisms of TB root–microbe interactions

Identify Bitter Peptides by Using Deep Representation Learning Features

A bitter taste often identifies hazardous compounds and it is generally avoided by most animals and humans. Bitterness of hydrolyzed proteins is caused by the presence of bitter peptides. To improve palatability, bitter peptides need to be identified experimentally in a time-consuming and expensive process, before they can be removed or degraded. Here, we report the development of a machine learning prediction method, iBitter-DRLF, which is based on a deep learning pre-trained neural network feature extraction method. It uses three sequence embedding techniques, soft symmetric alignment (SSA), unified representation (UniRep), and bidirectional long short-term memory (BiLSTM). These were initially combined into various machine learning algorithms to build several models. After optimization, the combined features of UniRep and BiLSTM were finally selected, and the model was built in combination with a light gradient boosting machine (LGBM). The results showed that the use of deep representation learning greatly improves the ability of the model to identify bitter peptides, achieving accurate prediction based on peptide sequence data alone. By helping to identify bitter peptides, iBitter-DRLF can help research into improving the palatability of peptide therapeutics and dietary supplements in the future. A webserver is available, too

Polo-like kinase 1 inhibits the activity of positive transcription elongation factor of RNA Pol II b (P-TEFb).

Polo-like kinase 1 (Plk1) is a highly conserved Ser/Thr kinase in eukaryotes and plays a critical role in various aspects of the cell cycle. Plk1 exerts its multiple functions by phosphorylating its substrates. In this study, we found that Plk1 can interact with cyclin T1/Cdk9 complex-the main form of the positive transcription elongation complex b (P-TEFb), and its C-terminal polo-box domain is responsible for the binding. Further analysis indicated that Plk1 could phosphorylate cyclin T1 at Ser564 and inhibit the kinase activity of cyclin T1/Cdk9 complex on phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. By taking the approach of luciferase assay, we demonstrated that over-expression of both wild type Plk1 and constitutively active form of Plk1 inhibits the P-TEFb dependent HIV-1 LTR transcription, while knockdown of Plk1 increases the HIV-1 LTR transcription. Consistently, the data from the HIV-1 pseudovirus reporter assay indicated that Plk1 blocks the gene expression of HIV-1 pseudovirus. Taken together, our results revealed that Plk1 negatively regulates the RNA polymerase II-dependent transcription through inhibiting the activity of cyclin T1/Cdk9 complex

Herbal medicines for fatty liver diseases

Background Fatty liver disease is potentially a reversible condition that may lead to end-stage liver disease. Since herbal medicines such as Crataegus pinnatifida and Salvia miltiorrhiza have increasingly been used in the management of fatty liver disease, a systematic review on herbal medicine for fatty liver disease is needed. Objectives To assess the beneficial and harmful effects of herbal medicines for people with alcoholic or non-alcoholic fatty liver disease. Search methods We searched The Cochrane Hepato-Biliary Group Controlled Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL) (Issue 3, 2012), MEDLINE, EMBASE, and Science Citation Index Expanded to 1 March 2012. We also searched the Chinese BioMedical Database, Traditional Chinese Medical Literature Analysis and Retrieval System, China National Knowledge Infrastructure, Chinese VIP Information, Chinese Academic Conference Papers Database and Chinese Dissertation Database, and the Allied and Complementary Medicine Database to 2 March 2012. Selection criteria We included randomised clinical trials comparing herbal medicines with placebo, no treatment, a pharmacological intervention, or a non-pharmacological intervention such as diet or lifestyle, or Western interventions in participants with fatty liver disease. Data collection and analysis Two review authors extracted data independently. We used the 'risk of bias' tool to assess the risk of bias of the included trials. We assessed the following domains: random sequence generation, allocation concealment, blinding, incomplete outcome data, selective outcome reporting, and other sources of bias. We presented the effects estimates as risk ratios (RR) with 95% confidence intervals (CI) or as mean differences (MD) with 95% CI, depending on the variables of the outcome measures. Main results We included 77 randomised clinical trials, which included 6753 participants with fatty liver disease. The risks of bias (overestimation of benefits and underestimation of harms) was high in all trials. The mean sample size was 88 participants (ranging from 40 to 200 participants) per trial. Seventy-five different herbal medicine products were tested. Herbal medicines tested in the randomised trials included single-herb products (Gynostemma pentaphyllum, Panax notoginseng, and Prunus armeniaca), proprietary herbal medicines commercially available, and combination formulas prescribed by practitioners. The most commonly used herbs were Crataegus pinnatifida, Salvia miltiorrhiza, Alisma orientalis, Bupleurum chinense, Cassia obtusifolia, Astragalus membranaceous, and Rheum palmatum. None of the trials reported death, hepatic-related morbidity, quality of life, or costs. A large number of trials reported positive effects on putative surrogate outcomes such as serum aspartate aminotransferase, alanine aminotransferase, glutamyltransferase, alkaline phosphatases, ultrasound, and computed tomography scan. Twenty-seven trials reported adverse effects and found no significant difference between herbal medicines versus control. However, the risk of bias of the included trials was high. The outcomes were ultrasound findings in 22 trials, liver computed tomography findings in eight trials, aspartate aminotransferase levels in 64 trials, alanine aminotransferase activity in 77 trials, and glutamyltransferase activities in 44 trials. Six herbal medicines showed statistically significant beneficial effects on ultrasound, four on liver computed tomography, 42 on aspartate aminotransferase activity, 49 on alanine aminotransferase activity, three on alkaline phosphatases activity, and 32 on glutamyltransferase activity compared with control interventions. Authors' conclusions Some herbal medicines seemed to have positive effects on aspartate aminotransferase, alanine aminotransferase, ultrasound, and computed tomography. We found no significant difference on adverse effects between herbal medicine and control groups. The findings are not conclusive due to the high risk of bias of the included trials and the limited number of trials testing individual herbal medicines. Accordingly, there is also high risk of random errors

An Integrative Analysis of the Immune Features of Inactivated SARS-CoV-2 Vaccine (CoronaVac)

Currently, an inactivated vaccine has been widely used with encouraging results as a prophylactic agent against COVID-19 infection, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. However, in vitro SARS-CoV-2 vaccine-specific immune features remain elusive, hindering the promotion of a third dose of the vaccine. Here, we present a detailed in vitro immune cellular response and large-scale multi-omics analysis for peripheral blood mononuclear cells (PBMCs) from participants vaccinated with CoronaVac (Sinovac Life Sciences, Beijing, China) and recovered participants from COVID-19. The mean titers of SARS-CoV-2 serum-neutralizing antibodies were significantly increased after the boosting immunization (Day 45) compared to the unimmunized state. We observed that type-1 helper T cells (Th1) tended to dominate after the first dose of vaccine, while humoral immune responses became dominant after the second dose due to the activation of type-2 helper T cell (Th2), memory B cells, and plasmablasts. T follicular helper cells (Tfh) involved in antibody production were activated after the first dose and were maintained for the observed time points. Single-cell RNA sequencing of PBMCs revealed specific changes in cell compositions and gene expression in immunized participants. Multi-omics analysis also demonstrated that CoronaVac-specific serum proteins, plasma metabolites, and plasma lipid changes were skewed to those changes in convalescent patients. Collectively, we provide a comprehensive understanding of CoronaVac-specific in vitro immune features

IUP-BERT: Identification of Umami Peptides Based on BERT Features

Umami is an important widely-used taste component of food seasoning. Umami peptides are specific structural peptides endowing foods with a favorable umami taste. Laboratory approaches used to identify umami peptides are time-consuming and labor-intensive, which are not feasible for rapid screening. Here, we developed a novel peptide sequence-based umami peptide predictor, namely iUP-BERT, which was based on the deep learning pretrained neural network feature extraction method. After optimization, a single deep representation learning feature encoding method (BERT: bidirectional encoder representations from transformer) in conjugation with the synthetic minority over-sampling technique (SMOTE) and support vector machine (SVM) methods was adopted for model creation to generate predicted probabilistic scores of potential umami peptides. Further extensive empirical experiments on cross-validation and an independent test showed that iUP-BERT outperformed the existing methods with improvements, highlighting its effectiveness and robustness. Finally, an open-access iUP-BERT web server was built. To our knowledge, this is the first efficient sequence-based umami predictor created based on a single deep-learning pretrained neural network feature extraction method. By predicting umami peptides, iUP-BERT can help in further research to improve the palatability of dietary supplements in the future

Plk1 inhibites the kinase activity of the P-TEFb complex.

<p>(A) <i>In vitro</i> kinase assay. HCT116 cells transfected with pCMV FLAG-cyclin T1 were synchronized into different phases as described in Material and Method. The synchronization of the cells was detected by FACS. Cell lysates were immunoprecipated with FLAG antibody. Half of the immunoprecipates were subjected to immunoblotting with Cdk9 and FLAG antibody. The other half of the immunoprecipates were then incubated with GST-RNA Pol II CTD in the presence of [γ-³²P] ATP for the <i>in vitro</i> kinase assay. (B) FLAG-cyclin T1 over-expressed in 293T cells were immunoprecipated with FLAG antibody. The immunoprecipitated cyclin T1 complexes were preincubated with the lysates from HeLa cells either asynchronized or synchronized in M phase with cold ATP for the <i>in vitro</i> kinase assay, and washed with kinase buffer. Then the FLAG-cyclin T1 complexes were incubated with GST-RNA Pol II CTD and [γ-³²P] ATP for a second round of <i>in vitro</i> kinase assay. The expression level of Plk1, phosphorylation of histone H3 in HeLa cells and the immunoprecipitated FLAG-cyclin T1 after incubating with Hela cell extracts and washed were detected by immunoblotting with the indicated antibody. (C) 293T cells were transfected with pCMV FLAG-cyclin T1 or pCMV FLAG-Cdk9 respectively. The cell lysates were immunoprecipitated with FLAG antibody and the immunoprecipitated complexes were subjected to <i>in vitro</i> kinase assay in the presence or absence of His-Plk1 TD or His-Plk1 KD with GST- RNA Pol II CTD as the substrate. (D) pCMV FLAG-cyclin T1 transfected 293T cells were treated with or without BI2536(1µM) for 3.5 h before harvest. The FLAG-cyclin T1 complexes were immunoprecipated with FLAG antibody and subjected to <i>in vitro</i> kinase assay with GST-RNA Pol II CTD as the substrate. Equal amount of the immunoprecipated FLAG-cyclin T1 and Cdk9 was shown. (E) FLAG-tagged cyclin T1, cyclin T1 S564A, cyclin T1 S564D over-expressed in 293T cells were immunoprecipitated with FLAG antibody and incubated with GST- RNA Pol II CTD and [γ-³²P] ATP for the <i>in vitro</i> kinase assay. The immunoprecipated FLAG-cyclin T1 and Cdk9 were subjected to immunoblotting.</p

Plk1 represses P-TEFb-dependent transcription.

<p>(A) Schematic map of G5-83-HIV-Luc reporter. (B) 293T cells were co-transfected with Plk1, Plk1 TD or Plk1 KD expression plasmids and HIV-1 LTR luciferase reporter for 36 h. The cell lysates were harvested for luciferase assay. The expression level of Plk1 and its mutants was monitored by immunoblotting with FLAG antibody with β-actin as the internal control. (C) 293T cells were transfected with different doses of pCMV FLAG-Plk1 (150ng, 300ng and 600ng) and empty vector to keep an equal amount of co-tranfected DNA in each group, and with G5-83-HIV-luc luciferase reporter. After 36 h, the cell lysates were subjected to luciferase assay. The expression level of Plk1 was monitored by immunoblotting with FLAG antibody with β-actin as the internal control. (D) 293T cells were transfected with Plk1-specific siRNA or control siRNA followed by transfection with G5-83-HIV-luc luciferase reporter. The luciferase assay was performed 24 h after transfection. Immunoblotting was performed to detect the expression level of Plk1 and β-actin. (E) 293T cells were transfected with HIV-1 LTR luciferase reporter for 24 h and then treated with BI2536(100nM,500nM,1µM), Nocodazole or DMSO for 16hr before harvest. The cell lysates were subjected to luciferase assay and immunoblotting with Plk1 and β-actin. (F) NIH3T3 cells were transfected with human cyclin T1, cyclin T1 S564A, or cyclin T1 S564D expression plasmids with HIV-1 LTR reporter in the presence or absence of pCMV Tat for 24 h. The cell lysates were subjected to luciferase assay and immunoblotting with FLAG antibody and β-actin. The luciferase activity was normalized to the amount of luciferase DNA in transfected cells which was quantified by real-time PCR. The data are shown as the mean ± SD from three independent experiments. Statistical significance was determined by Students t-test (* <i>p value</i> < 0.05). “C” indicates that empty vector was used as the negative control.</p

Plk1 interacts with P-TEFb independent of it kinase activity.

<p>(A) 293T cells were transfected with pCMV myc-Plk1 and pCMV FLAG-Cdk7 or FLAG-Cdk9, or empty vector as the control. The total cell extracts (TCE) were immunoprecipitated (IP) with myc antibody and immunoblotted (IB) with FLAG antibody. Asterisk indicates cross-reacting unrelated band. (B) 293T cells were transfected with empty vector or pCMV FLAG-Plk1. The total cell extracts were immunoprecipitated with FLAG antibody and immunoblotted with Cdk9 and cyclin T1 antibodies. The empty vector was used as the negative control. (C) HeLa cells were synchronized in M phase by Nocodazole treatment. The cell lysates were harvested and immunoprecipitated with normal mouse serum or Plk1 antibody, and then immunoblotted with Cdk9, cyclin T1 and Plk1 antibodies respectively. (D) GST pull-down assay. Purified His-Plk1 was incubated with immobilized GST, GST-cyclin T1 or GST-Cdk9 respectively. The bound protein was detected by immunoblotting with Plk1 antibody. <i>CBB</i>, Coomassie Brilliant Blue. (E) 293T cells were transfected with pCMV FLAG-Plk1, pCMV FLAG-Plk1 KD (K82R) or empty vector as the control. The cell lysates were immunoprecipitated with FLAG antibody and immunoblotted with Cdk9 and cyclin T1 antibodies.</p