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With the aim to discuss the similarities and differences of phytochemicals in Moringa oleifera leaves collected from China (CML) and India (IML) in mind, comparative ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MS) analysis was performed in this study. A screening analysis based on a UNIFI platform was first carried out to discuss the similarities. Next, untargeted metabolomic analysis based on multivariate statistical analysis was performed to discover the differences. As a result, a total of 122 components, containing 118 shared constituents, were characterized from CML and IML. The structure types included flavonoids, alkaloids, glyosides, organic acids and organic acid esters, iridoids, lignans, and steroids, etc. For CML, 121 compounds were characterized; among these, 18 potential biomarkers with higher contents enabled differentiation from IML. For IML, 119 compounds were characterized; among these, 12 potential biomarkers with higher contents enabled differentiation from CML. It could be concluded that both CML and IML are rich in phytochemicals and that CML is similar to IML in the kinds of the compounds it contains, except for the significant differences in the contents of some compounds. This comprehensive phytochemical profile study provides a basis for explaining the effect of different growth environments on secondary metabolites and exists as a reference for further research into or applications of CML in China.

Boron is an essential trace element with roles in growth, development, and physiological functions; however, its mechanism of action is still unclear. In this study, the regulatory roles of the PI3K/Akt signaling pathway on boron-induced changes in barrier function, proliferation, and apoptosis in rat intestinal epithelial cells were evaluated. Occludin levels, the proportion of cells in the G2/M phase, cell proliferation rate, and mRNA and protein expression levels of PCNA were higher, while the proportions of cells in the G0/G1 and S phases, apoptosis rate, and caspase-3 mRNA and protein expression levels were lower in cells treated with 0.8 mmol/L boron than in control IEC-6 cells ( P  < 0.01 or P  < 0.05). However, 40 mmol/L boron decreased ZO-1 and Occludin levels, the proportion of cells in the G2/M phase, cell proliferation rate, and mRNA and protein levels of PCNA and increased the apoptosis rate and caspase-3 mRNA expression ( P  < 0.01 or P  < 0.05). After specifically blocking PI3K and Akt signals (using LY294002 and MK-2206 2HCL), 0.8 mmol/L boron had no effects on Occludin, PCNA level, apoptosis rates, and caspase-3 levels ( P  < 0.05); however, the proliferation rate and PCNA levels decreased significantly (P  < 0.01 or P  < 0.05). The addition of 40 mmol/L boron did not affect ZO-1 and Occludin levels and did not affect the apoptosis rate or PCNA and caspase-3 levels. These results suggested that the PI3K/Akt signaling pathway mediates the effects of low-dose boron on IEC-6 cells.

Background We aimed to determine the potential predictive value of the intra-tumoral microbiome as a marker of the response to external beam radiation therapy (EBRT) in cervical cancer (CC). Methods A prospective longitudinal trial of 36 CC patients receiving pelvic radiotherapy was designed to investigate microbial characteristic signatures and diversity (alpha and beta) of multiple sites (tumor, vaginal, gut, urethral, and oral) in the superior response (SR) and inferior response (IR) groups of CC patients by 16 S rRNA sequencing. Utilized the least absolute shrinkage and selection operator (LASSO) logistic regression method to analyze clinicopathological factors that potentially influenced the efficacy of EBRT. LEfSe analysis highlighted the microbiome features that best distinguished the categorized patient samples. Selected parameters were validated with Spearman correlation analysis, receiver operating characteristic (ROC) area under the curve (AUC) analysis and Kaplan-Meier survival analysis. Results Firstly, in our cohort, LASSO logistic regression analysis revealed no association between clinicopathological factors and EBRT efficacy. Subsequently, we employed 16 S rRNA sequencing to compare microbiome differences across multiple sites and their correlations with major clinicopathological factors. We discovered that the intra-tumoral microbiome was independent of clinicopathologic features and represented the most direct and reliable reflection of the microbial differences between the SR and IR groups. We found lower alpha diversity in the tumor microbiome of SR group and identified the most relevant microbiome taxa ( Bifidobacteriaceae , Beijerinckiaceae , and Orbaceae ) associated with the efficacy of the response to EBRT in CC patients. We then conducted ROC analysis, finding that specific microbial taxa had an AUC of 0.831 (95% CI, 0.667–0.995), indicating the potential of these taxa as biomarkers for predicting EBRT efficacy. Kaplan-Meier survival analysis showed a better prognosis for patients with lower alpha diversity and higher relative abundance of Bifidobacteriaceae . Conclusions Our data suggested that intra-tumoral specific microbiome taxa and lower alpha diversity may play an important role in the CC patient sensitivity to EBRT and offer novel potential biomarkers for predicting the response to EBRT efficacy.

Taxol is a rare secondary metabolite that accumulates considerably in Taxus species under salicylic acid (SA) and methyl jasmonate treatment. However, the molecular mechanism of its accumulation remains unclear. We investigated TcWRKY33 , a nuclear-localized group I WRKY transcription factor, as an SA-responsive regulator of taxol biosynthesis. Overexpression and RNA interference of TcWRKY33 confirmed that TcWRKY33 regulates the expression of most taxol biosynthesis genes, especially 10-deacetylbaccatin III-10-O-acetyltransferase ( DBAT ) and taxadiene synthase ( TASY ), which were considered as key enzymes in taxol biosynthesis. Transient overexpression of TcWRKY33 in Taxus chinensis leaves resulted in increased taxol and 10-deacetylbaccatin accumulation by 1.20 and 2.16 times compared with the control, respectively. Furthermore, TcWRKY33, DBAT , and TASY were confirmed to respond positively to SA signals. These results suggested that TcWRKY33 was the missing component of taxol biosynthesis that responds to SA. The sequence analysis identified two W-box motifs in the promoter of DBAT but not in the TASY . Yeast one-hybrid and dual-luciferase activity assays confirmed that TcWRKY33 can bind to the two W-boxes in the promoter of DBAT , upregulating its expression level. Hence, DBAT is a direct target of TcWRKY33 . Furthermore, TcERF15 , encoding a TASY activator, also contains two W-boxes in its promoter. Yeast one-hybrid and dual-luciferase activity assays further confirmed that TcWRKY33 can upregulate TASY expression through the activation of TcERF15 . In summary, TcWRKY33 transmits SA signals and positively regulates taxol biosynthesis genes in two ways: directly and through the activation of other activators. Therefore, TcWRKY33 is an excellent candidate for genetically engineering regulation of taxol biosynthesis in Taxus plants.

Enterotoxigenic (ETEC) is the main diarrhea-causing pathogen in children and young animals and has become a global health concern. Berberine is a type of \"medicine and food homology\" and has a long history of use in China, particularly in treating gastrointestinal disorders and bacterial diarrhea. In this study, we explored the effects of berberine on growth performance, intestinal inflammation, oxidative damage, and intestinal microbiota in a weaned piglet model of ETEC infection. Twenty-four piglets were randomly divided into four groups-a control group (fed a basal diet [BD] and infused with saline), a BD+ETEC group (fed a basal diet and infused with ETEC), a LB+ETEC group (fed a basal diet with 0.05% berberine and infused with ETEC infection), and a HB+ETEC group (fed a basal diet with 0.1% berberine and infused with ETEC). Berberine significantly improved the final body weight (BW), average daily gain (ADG), and average daily feed intake (ADFI) ( <0.05) of piglets, and effectively decreased the incidence of diarrhea among the animals ( <0.05). Additionally, berberine significantly downregulated the expression levels of the genes encoding TNF-α, IL-1β, IL-6, IL-8, TLR4, MyD88, NF-κB, IKKα, and IKKβ in the small intestine of piglets ( <0.05). ETEC infection significantly upregulated the expression of genes coding for Nrf2, CAT, SOD1, GPX1, GST, NQO1, HO-1, GCLC, and GCLM in the small intestine of the animals ( <0.05). Berberine significantly upregulated 12 functional COG categories and 7 KEGG signaling pathways. A correlation analysis showed that berberine significantly increased the relative abundance of beneficial bacteria (Gemmiger, Pediococcus, Levilactobacillus, Clostridium, Lactiplantibacillus, Weissella, Enterococcus, Blautia, and Butyricicoccus) and decreased that of pathogenic bacteria (Prevotella, Streptococcus, Parabacteroides, Flavonifractor, Alloprevotella) known to be closely related to intestinal inflammation and oxidative stress in piglets. In conclusion, ETEC infection disrupted the intestinal microbiota in weaned piglets, upregulating the TLR4/MyD88/NF-κB and Nrf2 signaling pathways, and consequently leading to intestinal inflammation and oxidative stress-induced damage. Our data indicated that berberine can optimize intestinal microbiota balance and modulate the TLR4/MyD88/NF-κB and Nrf2 signaling pathways, thus helping to alleviate intestinal inflammation and oxidative damage caused by ETEC infection in weaned piglets.

Objectives The aim of this study was to characterize the microbiome of multiple mucosal organs in cervical cancer (CC) patients. Methods We collected oral, gut, urinary tract, and vaginal samples from enrolled study participants, as well as tumor tissue from CC patients. The microbiota of different mucosal organs was identified by 16S rDNA sequencing and correlated with clinical-pathological characteristics of cervical cancer cases. Results Compared with controls, CC patients had reduced α-diversity of oral and gut microbiota ( p Oral_Sob < 0.001, p Oral_Shannon = 0.049, p Oral_Simpson = 0.013 p Fecal_Sob = 0.030), although there was an opposite trend in the vaginal microbiota ( p Vaginal_Pielou = 0.028, p Vaginal_Simpson = 0.006). There were also significant differences in the β-diversity of the microbiota at each site between cases and controls ( p Oral = 0.002, p Fecal = 0.037, p Urine = 0.001, p Vaginal = 0.001). The uniformity of urine microbiota was lower in patients with cervical squamous cell carcinoma ( p Urine = 0.036) and lymph node metastasis ( p Urine_Sob = 0.027, p Urine_Pielou = 0.028, p Urine_Simpson = 0.021, p Urine_Shannon = 0.047). The composition of bacteria in urine also varied among patients with different ages ( p  = 0.002), tumor stages ( p  = 0.001) and lymph node metastasis ( p  = 0.002). In CC cases, Pseudomonas were significantly enriched in the oral, gut, and urinary tract samples. In addition, Gardnerella , Anaerococcus , and Prevotella were biomarkers of urinary tract microbiota; Abiotrophia and Lautropia were obviously enriched in the oral microbiota. The microbiota of tumor tissue correlated with other mucosal organs (except the gut), with a shift in the microflora between mucosal organs and tumors. Conclusions Our study not only revealed differences in the composition and diversity of the vaginal and gut microflora between CC cases and controls, but also showed dysbiosis of the oral cavity and urethra in cervical cancer cases.

Aromatic rice ( Oryza sativa ) fetches a premium price due to the pleasant aroma. The major aroma compound 2-acetyl-1-pyrroline (2AP) has been found to be enhanced under stress. This condition can be considered to study the genes, precursors, enzymes, and metabolites involved in elevated levels of 2AP biosynthesis. In the present study, 100 mM salt treatment was given to two aromatic rice cultivars Ambemohar-157 (A-157) and Basmati-370 (B-370) at the vegetative stage (VS 3 ). After salt treatment, in the leaves, 2AP contents were elevated by 2.2 and 1.8 fold in A-157 and B-370, respectively. Under these elevated 2AP conditions, the precursor amino acids (glutamate, putrescine, ornithine, and proline), their related genes, enzymes, and metabolites (methylglyoxal and γ-aminobutyric acid (GABA) related to 2AP biosynthesis were analyzed. In addition, agronomic characters were also studied. It was observed that the proline content was enhanced in both the cultivars by 29% (A-157) and 40% (B-370) as compared to control. The Δ 1 -pyrroline-5-carboxylate synthetase (P5CS) enzyme activity was increased in salt-treated plants leaf tissue by 31% (A-157) and 40% (B-370) compared to control. The P5CS gene expression was enhanced by A-157 (1.8 fold) and B-370 (2.2 fold) compared to control, putrescine content in A-157 and B-370 decreased by 2.5 and 2.7 fold respectively as compared to control. The ornithine decarboxylase (ODC) activity was enhanced in A-157 (12%) and B-370 (35%) over control. Further, ODC gene expression was enhanced in both the cultivars A-157 (1.5 fold) and B-370 (1.3 fold). The diamino oxidase (DAO) enzyme activity was increased by 28% (A-157) and 35% (B-370) respectively over control. The GABA content marginally increased over control in both the cultivars namely, A-157 (1.9%) and B-370 (9.5%). The methylglyoxal levels were enhanced by 1.4 fold in A-157 and 1.6 fold in B-370. Interestingly, the enhancement in 2AP in the vegetative stage also helped to accumulate it in mature grains (twofold in A-157 and 1.5 fold in B-370) without test weight penalty. The study indicated that the ornithine and proline together along with methylglyoxal contribute towards the enhancement of 2AP under salt stress.

Background The eating and cooking qualities (ECQs) of rice ( Oryza sativa L.) are key characteristics affecting variety adoption and market value. Starch viscosity profiles tested by a rapid visco analyzer (RVA) offer a direct measure of ECQs and represent the changes in viscosity associated with starch gelatinization. RVA profiles of rice are controlled by a complex genetic system and are also affected by the environment. Although Waxy ( Wx ) is the major gene controlling amylose content (AC) and ECQs, there are still other unknown genetic factors that affect ECQs. Results Quantitative trait loci (QTLs) for starch paste viscosity in rice were analyzed using chromosome segment substitution lines (CSSLs) developed from the two cultivars 9311 and Nipponbare, which have same Wx-b allele. Thus, the effect of the major locus Wx was eliminated and the other locus associated with the RVA profile could be identified. QTLs for seven parameters of the starch RVA profile were tested over four years in Nanjing, China. A total of 310 QTLs were identified (from 1 to 55 QTLs per trait) and 136 QTLs were identified in more than one year. Among them, 6 QTLs were stalely detected in four years and 26 QTLs were detected in at least three years including 13 pleiotropic loci, controlling 2 to 6 RVA properties simultaneously. These stable QTL hotspots were co-located with several known starch synthesis-related genes (SSRGs). Sequence alignments showed that nucleotide and amino acid sequences of most SSRGs were different between the two parents. Finally, we detected stable QTLs associated with multiple starch viscosity traits near Wx itself, supporting the notion that additional QTLs near Wx control multiple characteristic values of starch viscosity. Conclusions By eliminating the contribution from the major locus Wx , multiple QTLs associated with the RVA profile of rice were identified, several of which were stably detected over four years. The complexity of the genetic basis of rice starch viscosity traits might be due to their pleiotropic effects and the multiple QTL hot spots. Minor QTLs controlling starch viscosity traits were identified by using the chromosome segment substitution strategy. Allele polymorphism might be the reason that QTLs controlling RVA profile characteristics were detected in some known SSRG regions.

Bulky residual tumor after external beam radiotherapy (EBRT) presents a major technical challenge for brachytherapy (BT) and is a significant prognostic risk factor in locally advanced cervical cancer (LACC). The use of chemotherapy combined with an immune checkpoint inhibitor to reduce tumor volume (TV) during the interval between EBRT completion and BT initiation has been rarely explored. We present two cases of stage IIIC1r cervical squamous cell carcinoma with residual tumors larger than 5 cm following EBRT. Patient characteristics, examination findings, laboratory and imaging data, treatment responses, and adverse events were recorded. After sequential treatment with albumin-bound paclitaxel plus cisplatin combined with zimberelimab, both patients achieved marked tumor volume reduction and subsequently underwent reduced-volume BT. Neither patient experienced recurrence at 36-month and 25-month follow-ups, respectively. Sequential chemo-immunotherapy followed by reduced-volume BT may represent an alternative treatment option for patients with bulky residual disease after EBRT. Further clinical studies are warranted to validate the effectiveness of this combined regimen.

Starch is the most important food energy source in cereals. Many of the known enzymes involved in starch biosynthesis are partially or entirely granule-associated in the endosperm. Studying the proteome of rice starch granules is critical for us to further understand the mechanisms underlying starch biosynthesis and packaging of starch granules in rice amyloplasts, consequently for the improvement of rice grain quality. In this article, we developed a protocol to purify starch granules from mature rice endosperm and verified the quality of purified starch granules by microscopy observations, I2 staining, and Western blot analyses. In addition, we found the phenol extraction method was superior to Tris-HCl buffer extraction method with respect to the efficiency in recovery of starch granule associated proteins. LC-MS/MS analysis showed identification of already known starch granule associated proteins with high confidence. Several proteins reported to be involved in starch synthesis in prior genetic studies in plants were also shown to be enriched with starch granules, either directly or indirectly, in our studies. In addition, our results suggested that a few additional candidate proteins may also be involved in starch synthesis. Furthermore, our results indicated that some starch synthesis pathway proteins are subject to protein acetylation modification. GO analysis and KEGG pathway enrichment analysis showed that the identified proteins were mainly located in plastids and involved in carbohydrate metabolism. This study substantially advances the understanding of the starch granule associated proteome in rice and post translational regulation of some starch granule associated proteins.