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We evaluated the inhibitory effects of the probiotic Lactobacillus species on different phases of Candida albicans biofilm development. Quantification of biofilm growth and ultrastructural analyses were performed on C. albicans biofilms treated with Lactobacillus rhamnosus , Lactobacillus casei , and Lactobacillus acidophilus planktonic cell suspensions as well as their supernatants. Planktonic lactobacilli induced a significant reduction ( p  < 0.05) in the number of biofilm cells (25.5–61.8 %) depending on the probiotic strain and the biofilm phase. L. rhamnosus supernatants had no significant effect on the mature biofilm ( p  > 0.05), but significantly reduced the early stages of Candida biofilm formation ( p  < 0.01). Microscopic analyses revealed that L. rhamnosus suspensions reduced Candida hyphal differentiation, leading to a predominance of budding growth. All lactobacilli negatively impacted C. albicans yeast-to-hyphae differentiation and biofilm formation. The inhibitory effects of the probiotic Lactobacillus on C. albicans entailed both cell-cell interactions and secretion of exometabolites that may impact on pathogenic attributes associated with C. albicans colonization on host surfaces and yeast filamentation. This study clarifies, for the first time, the mechanics of how Lactobacillus species may antagonize C. albicans host colonization. Our data elucidate the inhibitory mechanisms that define the probiotic candicidal activity of lactobacilli, thus supporting their utility as an adjunctive therapeutic mode against mucosal candidal infections.

Summary Saffron apocarotenoids, including crocins, picrocrocin and safranal, are valuable metabolites with pharmaceutical and cosmetic potential. However, their natural plant sources are difficult to cultivate, which limits large‐scale production. The identification of carotenoid cleavage dioxygenases (CCDs), which catalyse the first and most critical step in their biosynthesis, has enabled the production of these apocarotenoids in heterologous plant systems. In this study, we aimed to generate plant cell suspensions expressing Crocus sativus CCD2 and Gardenia jasminoides CCD4a, along with a bacterial phytoene synthase to enhance carotenoid biosynthesis and CsUGT93P1, which improves crocin stability. Transgenic cell suspensions were established from Nicotiana benthamiana plants and Nicotiana tabacum cv. BY‐2 cells. In BY‐2 cells expressing GjCCD4a, crocin accumulation reached 770 μg/g DW, which further increased upon methyl jasmonate elicitation. Remarkably, the BY‐2 transgenic cells exhibited an 18,000‐fold increase in β‐cyclocitral content compared to wild‐type cells. The best‐performing N. benthamiana and BY‐2 lines were successfully cultivated in wave bioreactors, demonstrating their potential for saffron apocarotenoid production. In the BY‐2 bioreactor, apart from saffron apocarotenoids, phytoene and notably high amounts of lycopene were produced, adding value to the platform and indicating a remodelling of the carotenoid pathway. This study establishes the viability and lays the foundation for the scalable production of saffron apocarotenoids and carotenoids in plant cell suspensions.

Plant secondary biosynthetic pathways are exceedingly inducible by elicitors and facilitate enhanced metabolite production using plant cell tissue and organ cultures. Elicitors can regulate large number of control points and trigger the expression of key genes with increased cellular activities at biochemical and molecular level involving signal compounds. A large number of chemical elicitors viz: jasmonic acid (JA), methyl jasmonate (MeJA), salicylic acid (SA), acetyl salicylic acid (ASA), ethylene (ET) and ethrel (Ethe), heavy metals (HM), many types of chemical compounds (natural or synthetic) and their combinations are used for elicitation studies. Cell suspensions and hairy roots are commonly used culture systems followed by adventitious roots and multiple shoots for elicitation experiments. Amongst the elicitors and concentrations used 100 µM MeJA was found optimum for secondary metabolite enhancement in majority of experiments compared to SA and JA. Elicitor treatments promoted yield enhancements starting from 1.0 to maximum of 2230-fold across plant species studied. Elicitors singly with media additives, combination of elicitors and elicitors other than signal compounds along with hormones were found beneficial for enhanced secondary metabolite production. Further, a combination of target gene over expression and elicitor treatment also supported higher secondary product yield. The present communication presents information exclusively about the use of chemical elicitors for secondary metabolites production in vitro covering approximately more than a decade of research at one place in one review. Further, this extensive appraisal will be useful for the understanding and manipulation of secondary metabolites for enhanced production in vitro.

Cell dielectric property measurement holds significant potential for application in cell detection and diagnosis due to its label-free and noninvasive nature. In this study, we developed a biosensor designed to measure the permittivity of liquid samples, particularly cell suspensions at the nanoliter scale, utilizing microwave and millimeter wave coplanar waveguides in conjunction with a microchannel. This biosensor facilitates the measurement of scattering parameters within a frequency domain ranging from 1 GHz to 110 GHz. The obtained scattering parameters are then converted into dielectric constants using specific algorithms. A cell capture structure within the microchannel ensures that cell suspensions remain stable within the measurement zone. The feasibility of this biosensor was confirmed by comparison with a commercial Keysight probe. We measured the dielectric constants of three different cell suspensions (HepG2, A549, MCF-7) using our biosensor. We also counted the number of cells captured in multiple measurements for each cell type and compared the corresponding changes in permittivity. The results indicated that the real part of the permittivity of HepG2 cells is 0.2–0.8 lower than that of the other two cell types. The difference between A549 and MCF-7 was relatively minor, only 0.2–0.4. The fluctuations in the dielectric spectrum caused by changes in cell numbers during measurements were smaller than the differences observed between different cell types. Thus, the sensor is suitable for measuring cell suspensions and can be utilized for label-free, noninvasive studies in identifying biological cell suspensions.

Background Successful usage of autologous skin cell suspension (ASCS) has been demonstrated in some clinical trials. However, its efficacy and safety have not been verified. This latest systematic review and meta‐analysis aim to examine the effects of autologous epidermal cell suspensions in re‐epithelialization of skin lesions. Methods Relevant articles were retrieved from PubMed, Embase, Cochrane Database, Web of Science, International Clinical Trials Registry Platform, China National Knowledge Infrastructureris, VIP Database for Chinese Technical Periodicals and Wanfang database. The primary output measure was the healing time, and the secondary outputs were effective rate, size of donor site for treatment, size of study treatment area, operation time, pain scores, repigmentation, complications, scar scale scores and satisfaction scores. Data were pooled and expressed as relative risk (RR), mean difference (MD) and standardized mean difference (SMD) with a 95% confidence interval (CI). Results Thirty‐one studies were included in this systematic review and meta‐analysis, with 914 patients who received autologous epidermal cell suspensions (treatment group) and 883 patients who received standard care or placebo (control group). The pooled data from all included studies demonstrated that the treatment group has significantly reduced healing time (SMD = −0.86; 95% CI: −1.59–0.14; p = 0.02, I2 = 95%), size of donar site for treatment (MD = −115.41; 95% CI: −128.74–102.09; p<0.001, I2 = 89%), operation time (MD = 25.35; 95% CI: 23.42–27.29; p<0.001, I2 = 100%), pain scores (SMD = −1.88; 95% CI: −2.86–0.90; p = 0.0002, I2 = 89%) and complications (RR = 0.59; 95% CI: 0.36–0.96; p = 0.03, I2 = 66%), as well as significantly increased effective rate (RR = 1.20; 95% CI: 1.01–1.42; p = 0.04, I2 = 77%). There were no significant differences in the size of study treatment area, repigmentation, scar scale scores and satisfaction scores between the two groups. Conclusion Our meta‐analysis showed that autologous epidermal cell suspensions is beneficial for re‐epithelialization of skin lesions as they significantly reduce the healing time, size of donar site for treatment, operation time, pain scores and complications, as well as increased effective rate. However, this intervention has minimal impact on size of treatment area, repigmentation, scar scale scores and satisfaction scores.

Rice cell suspension culture (RCSC) is one of the most widely studied plant cell culture systems next to tobacco and carrot. Simple cell culture techniques, scalability and high genetic transformation potential make RCSC an ideal platform to produce high-value recombinant proteins and plant specialized metabolites (PSM). Our understanding of the rice genome and its genetic regulation makes RCSC amenable to efficient genetic engineering with precision genome editing tools such as CRISPR/Cas. Further, the metabolic pool of RCSC can be harnessed and bioengineered to produce recombinant proteins and PSM. This review highlights the studies performed on transgenic RCSC and potential of this platform to synthesize PSM. Recent advancements in RCSC-mediated production and yield enhancement of bioactives using precision molecular biology tools such as CRISPR/Cas, media optimization and challenges associated with the establishment of RCSC are also summarized. This effort is to put the spotlight back on RCSC, which can become an attractive alternative to existing transgenic plant cell suspension culture systems.Key MessageNew developments in transgenic rice cell suspension culture and subsequent bioprocess optimization have potential to advance the field of plant-based biopharmaceutical production. Further use of precise genetic engineering tools can leverage the prospects of rice cell suspension culture for molecular pharming.

The bacterium Aeromonas salmonicida is the causative agent of furunculosis, a systemic, ubiquitous disease of fish in the salmon family, characterized by high mortality and morbidity. Probiotics are a promising approach for prevention of furunculosis in aquaculture. A bacterial strain with anti- A. salmonicida properties, Bacillus velezensis V4, was isolated and the mechanisms underlying these properties were investigated. Anti- A. salmonicida compounds present in cell-free supernatant of V4 were purified and structurally identified as members of the iturin, macrolactin, and difficidin groups. The compounds contributed jointly to inhibition of A. salmonicida , and the diversity of the compounds was related to the versatility of their mode of action. Addition of the compounds to A. salmonicida cell suspensions reduced cell density. Analyses by confocal microscopy and scanning electron microscopy revealed cell membrane disruption, deletion of cellular content, and cell lysis of A. salmonicida . The V4 genome was sequenced, and gene clusters involved in synthesis of anti- Aeromonas compounds were detected and identified. A possible probiotic effect on growth performance of Oncorhynchus mykiss (rainbow trout) was investigated by addition of 0, 1, and 3 % ( v / w ) V4. Relative to control, mortality was reduced 27.25 % in the 1 % addition group and 81.86 % in the 3 % addition group. Feed coefficient ratio was reduced 19.49 % and weight gain ratio was increased 71.22 % in the 1 % addition group. Our findings demonstrate that V4 is an effective probiotic strain in O. mykiss and has clear potential for both control of furunculosis and growth promotion of aquaculture animals.

To address the high demand for Pueraria candollei var. mirifica (PM) used as the active ingredient in health products and its difficulty to cultivate in the field, the growth and production of deoxymiroestrol (DME) and isoflavonoid (ISF) phytoestrogens in PM cell suspensions were studied. In a 125-mL shake flask, the cell suspension produced DME [78.7 ± 8.79–116 ± 18.2 μg/g dry weight (DW)] and ISF (140 ± 6.83–548 ± 18.5 μg/g DW), which are the predominant ISF glycosides. While ISF aglycones accumulated in the PM cell suspension cultured in the airlift bioreactor. The DME content was increased to 976 ± 79.6 μg/g DW when the PM cell suspension was cultured in the 5-L scale bioreactor. The production of DME and ISF was enhanced by elicitors including methyl jasmonate (MJ), yeast extract (YE), and chitosan (CHI). MJ produced the highest induction of DME accumulation, while ISF accumulation was the highest with YE treatment. Analysis of catalase activity implied that the elicitors enhanced ROS production, which resulted in the enhancement of DME and ISF production and accumulation in PM cell suspension cultures. PM cell suspension culture is a promising source of beneficial PM phytoestrogens that exhibit bioactivity that may useful for the treatment of menopausal symptoms.

Enicostema axillare (Poir. ex Lam.) A. Raynal is an immense medicinal plant that has been extensively used to treat malaria, diabetes, stomachache, and fever in traditional medicine. This plant contains high amounts of secoiridoid glycosides (gentiopicroside and swertiamarin), which have numerous health benefits. However, no leaf callus or cell suspension culture has been established for E. axillare for the production of beneficial metabolites. As a result, the goal of this work was to standardize an efficient and robust method for callus induction, callus and cell biomass, as well as swertiamarin and gentiopicroside accumulation. The highest callus induction frequency (85.40 ± 0.51%) and formation of leaf into callus with a mean callus biomass (1.283 ± 0.02 g/explant fresh weight (FW) and 0.176 ± 0.00 g/explant dry weight (DW)) and metabolite contents (swertiamarin 0.53 ± 0.01 mg/g and gentiopicroside 0.64 ± 0.05 mg/g) were obtained on Murashige and Skoog (MS) medium augmented with 2,4-D (1.0 mg/L) + Kin (0.5 mg/L) after 12 weeks of culture. The induced leaf callus was studied for cell suspension growth kinetics (biomass) and metabolite production. After 60th days of culture, the maximum of the leaf cell suspension (5.25 ± 0.05 g FW (50 mL medium) and 0.39 ± 0.00 g DW (50 mL medium) and secoiridoid glycosides (gentiopicroside, 1.44 ± 0.05 mg/g, and swertiamarin, 0.88 ± 0.01 mg/g DW) were obtained, which corresponded to 4.19 and 36 times greater than the lag phase (after 10th days). Cultured leaf cell suspensions were treated with methyl jasmonate (MJ) and salicylic acid (SA) at 50, 100 and 200 μM after 60th day of cell growth and evaluated for swertiamarin and gentiopicroside contents after 24 h of treatment. The greatest swertiamarin (2.43 ± 0.05 mg/g DW, 2.76-fold) and gentiopicroside (2.41 ± 0.09 mg/g DW, 1.67-fold) levels were observed in the treatments with 200 and 100 μM SA elicitor, respectively. These findings can lead to the use of this plant as an alternative source to improve the commercial production of secoiridoid glycosides in large-scale cultures.Key messageThis is the first report on leaf callus induction in Enicostema axillare and enhanced production of swertiamarin and gentiopicroside in leaf cell suspension culture elicited by methyl jasmonate and salicylic acid.

Summary The snow lotus species Saussurea involucrata (Kar. & Kir.) Sch.Bip., an endangered traditional Chinese herb, belongs to a genus of the Asteraceae family. Syringin present in S. involucrata stands as one of the predominant bioactive compounds. However, the biosynthetic pathway of syringin remains largely elusive. Here, S. involucrata suspension cell culture was subjected to methyl jasmonate (MeJA) treatment, which stimulated the synthesis of syringin, increasing its content by up to 3.9‐fold. Comparative transcriptome analysis revealed that genes involved in syringin biosynthesis were generally upregulated in response to MeJA. Furthermore, two candidate UDP‐glycosyltransferase genes, SiUGT72BZ2 and SiUGT72CY1, were identified through phylogenetic tree and expression profiling analyses. Overexpression of SiUGT72BZ2 (BZ2_OE) and SiUGT72CY1 (CY1_OE) in S. involucrata suspension cell cultures led to 15.2‐ and 5.9‐fold higher syringin levels than empty vector control cultures, respectively. Notably, upregulation of SiUGT72BZ2 enhanced the biosynthesis of coniferin as well. In contrast, only trace amounts of coniferin were present in control and CY1_OE cell cultures. Subsequent anti‐inflammatory assays using lipopolysaccharide (LPS)‐stimulated RAW264.7 cells demonstrated that the extracts from these cell cultures possessed remarkable anti‐inflammatory properties. Most strikingly, the BZ2_OE cultures exhibited superior anti‐inflammatory effects compared to the control and CY1_OE. In conclusion, our research has not only identified the key enzymes in syringin synthesis but also, through genetic engineering, has generated novel cell culture resources enriched with syringin and coniferin, and enhanced anti‐inflammatory activities, highlighting the potential of S. involucrata cell culture as an alternative for wild snow lotus resources.