Cell Suspension Research Articles

Bacterial Strains Isolated from Stingless Bee Workers Inhibit the Growth of Apis mellifera Pathogens.

Apis mellifera bees are an important resource for the local economy of various regions in Argentina and the maintenance of natural ecosystems. In recent years, different alternatives have been investigated to avoid the reduction or loss of colonies caused by pathogens and parasites such as Ascosphaera apis, Aspergillus flavus, and Paenibacillus larvae. We focused on bacterial strains isolated from the intestine of native stingless bees, to elucidate their antagonistic effect on diseases of A. mellifera colonies. For this purpose, worker bees of the species Tetragonisca fiebrigi, Plebeia spp., and Scaptotrigona jujuyensis were captured from the entrance to tree hives and transported to the laboratory, where their intestines were extracted. Twenty bacterial colonies were isolated from the intestines, and those capable of inhibiting enterobacteria in vitro and producing organic acids, proteases, and chitinases were selected. Four genera, Levilactobacillus, Acetobacter, Lactiplantibacillus, and Pantoea, were selected and identified by the molecular marker that codes for the 16S rRNA gene. For inhibition assays, cell suspensions and cell-free suspensions were performed. All treatments showed significant antibacterial effects, in comparison with the controls, against P. larvae and antifungal effects against A. apis and A. flavus. However, the mechanisms by which these bacteria inhibit the growth of these pathogens were not studied.

ITGB6 promotes pancreatic fibrosis and aggravates the malignant process of pancreatic cancer via JAK2/STAT3 signaling pathway.

Integrin β6 (ITGB6) is upregulated in multiple tumor types and elevated ITGB6 levels have been detected in patients with chronic pancreatitis. However, the role of ITGB6 in pancreatic fibrosis and cancer remains to be elucidated. In the present study, ITGB6 expression was assessed using western blotting and qRT-PCR. Besides, cell proliferation, cycling, migration, and invasion were evaluated using CCK-8, flow cytometry, wound healing, and transwell assays, respectively. The expression of fibrosis and JAK2/STAT3 signaling markers was detected by western blotting and immunofluorescence analysis. Moreover, nude mice were subcutaneously injected with co-cultured cell suspensions to establish an in vivo model. The results showed that ITGB6 was highly expressed in pancreatic cancer tissues and TGF-β-induced pancreatic stellate cells (PSCs). Inhibition of ITGB6 expression in PSCs resulted in clear inhibition of activated PSC proliferation, migration, and fibrogenesis. Additionally, reduced ITGB6 expression inhibits the JAK2/STAT3 signaling pathway. Interestingly, activators of the JAK2/STAT3 signaling pathway reversed the effects of ITGB6 disruption on PSCs. Activated PSCs notably promoted the proliferation, invasion, and migration of pancreatic cancer cells in a co-culture assay. In contrast, activated PSCs with low ITGB6 expression failed to significantly affect the malignancy of pancreatic cancer cells. Moreover, in vivo results showed that interference with ITGB6 inhibited the activation of PSCs and promoted the development of pancreatic cancer. Silencing ITGB6 inhibited the proliferation, migration, and fibrosis-like effects of activated PSCs and indirectly inhibited the metastasis and malignant process of pancreatic cancer by inhibiting the JAK2/STAT3 signaling pathway. Therefore, ITGB6 is a potential candidate target for pancreatic cancer prevention and treatment.

Anti-Staphylococcus aureus activity of Lactobacillus rhamnosus and Lactobacillus acidophilus on clinical isolates- an in vitro study

Background: This study evaluated the in vitro antimicrobial and antagonistic activity of both neat and pH-adjusted whole-cell suspensions of L. rhamnosus and L. acidophilus towards S. aureus, as an alternative to control the spread of MRSA, which is endemic in India. Materials and Methods: The study was conducted at School of Medical Education, Kerala, India, between October 2022 and May 2023. Anti-Staphylococcus aureus activity of Lactobacillus rhamnosus MTCC 1408 and Lactobacillus acidophilus MTCC 10307 grown on MRS agar, was evaluated on a total of 76 clinical isolates of S. aureus from Kerala, India, by the Agar overlay method. The study involved a comparison of probiotic activities by both neat and pH-adjusted whole-cell suspensions of L. rhamnosus and L. acidophilus. Results: The study results indicate that the mean value of inhibition zones produced by L. rhamnosus was greater than that of L. acidophilus, and statistically significant data was obtained in which the pH-adjusted whole cell suspensions of L. rhamnosus exhibited larger inhibition zones than that of neat suspensions of the same. Also, the mean value of activity by pH-adjusted L. acidophilus suspension was slightly greater than its neat suspension. Conclusions: L. rhamnosus and L. acidophilus were found to possess in vitro anti-Staphylococcus aureus activity. The antimicrobial and antagonistic activity of pH-adjusted live suspensions suggests the possible use of L. acidophilus and L. rhamnosus as probiotic treatment options in sites other than the gastrointestinal tract, supporting their use as local administration and topical applications, in particular for the treatment and decolonization of S. aureus.

Red Blood Cell Storage with Xenon: Safe or Disruption?

Xenon, an inert gas commonly used in medicine, has been considered as a potential option for prolonged preservation of donor packed red blood cells (pRBCs) under hypoxic conditions. This study aimed to investigate how xenon affects erythrocyte parameters under prolonged storage. In vitro model experiments were performed using two methods to create hypoxic conditions. In the first method, xenon was introduced into bags of pRBCs which were then stored for 42 days, while in the second method, xenon was added to samples in glass tubes. The results of our experiment showed that the presence of xenon resulted in notable alterations in erythrocyte morphology, similar to those observed under standard storage conditions. For pRBC bags, hemolysis during storage with xenon exceeded the acceptable limit by a factor of six, whereas the closed-glass-tube experiment showed minimal hemolysis in samples exposed to xenon. Notably, the production of deoxyhemoglobin was specific to xenon exposure in both cell suspension and hemolysate. However, this study did not provide evidence for the purported protective properties of xenon.

Uncovering the Role of Hydroxycinnamoyl Transferase in Boosting Chlorogenic Acid Accumulation in Carthamus tinctorius Cells under Methyl Jasmonate Elicitation.

Chlorogenic acids (CGAs) are bioactive compounds widely used in the food, pharmaceutical, and cosmetic industries. Carthamus tinctorius is an important economic crop, and its suspension cells are rich in CGAs. However, little is known about the biosynthesis and regulation of CGAs in Carthamus tinctorius cells. This study first elucidated the regulatory mechanism of CGA biosynthesis in methyl jasmonate (MeJA)-treated Carthamus tinctorius cells and the role of the MeJA-responsive hydroxycinnamoyl transferase (HCT) gene in enhancing their CGA accumulation. Firstly, temporal changes in intracellular metabolites showed that MeJA increased the intracellular CGA content up to 1.61-fold to 100.23 mg·g-1. Meanwhile, 31 primary metabolites showed significant differences, with 6 precursors related to increasing CGA biosynthesis. Secondly, the transcriptome data revealed 3637 new genes previously unannotated in the Carthamus tinctorius genome and 3653 differentially expressed genes. The genes involved in the plant signaling pathway and the biosynthesis of CGAs and their precursors showed a general up-regulation, especially the HCT gene family, which ultimately promoted CGA biosynthesis. Thirdly, the expression of a newly annotated and MeJA-responsive HCT gene (CtHCT, CtNewGene_3476) was demonstrated to be positively correlated with CGA accumulation in the cells, and transient overexpression of CtHCT enhanced CGA accumulation in tobacco. Finally, in vitro catalysis kinetics and molecular docking simulations revealed the ability and mechanism of the CtHCT protein to bind to various substrates and catalyze the formation of four hydroxycinnamic esters, including CGAs. These findings strengthened our understanding of the regulatory mechanism of CGA biosynthesis, thereby providing theoretical support for the efficient production of CGAs.

Zebrafish tumour xenograft models: a prognostic approach to epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the gynaecological malignancy with highest mortality. Although adjuvant treatment with carboplatin and paclitaxel leads to an objective response in ~80% of these patients, a majority will relapse within two years. Better methods for assessing long-term treatment outcomes are needed. To address this, we established safe and efficacious doses of carboplatin and paclitaxel using IGROV-1 zebrafish-CDX models. Then fluorescently-labelled cell suspensions from 83 tumour biopsies collected at exploratory laparotomy of women with suspected EOC were generated and 37 (45%) were successfully implanted in zebrafish larvae. Among these 19 of 27 pathology-confirmed EOC samples (70%) engrafted. These zebrafish patient-derived tumour xenograft (ZTX) models were treated with carboplatin or paclitaxel and tumour growth/regression and metastatic dissemination were recorded. In a subgroup of nine patients, four ZTX models regressed during carboplatin treatment. All four corresponding patients had >24 months PFS. Furthermore, both ZTX models established from two patients having <24 months PFS failed to regress during carboplatin treatment. Seven of eight models seeding <6 metastatic cells were established from patients having >24 months PFS. In eleven of fourteen patients, FIGO stage I + II or III tumours gave rise to ZTX models seeding <4 or >4 metastatic cells, respectively. In conclusion, ZTX models predicted patients having >24 or <24 months PFS, based on response/no response to carboplatin. Furthermore, high metastatic dissemination in ZTX models correlated to shorter PFS and more advanced disease at diagnosis. These preliminary results suggest that ZTX models could become a useful prognostic tool in EOC treatment planning.

Suspension cell cultures of Panax vietnamensis as a biotechnological source of ginsenosides: growth, cytology, and ginsenoside profile assessment.

Panax vietnamensis is a valuable medicinal plant and a source of a broad spectrum of biologically active ginsenosides of different structural groups. Overexploitation and low adaptability to planation cultivation have made this species vulnerable to human pressure and prompted the development of cell cultivation in vitro as a sustainable alternative to harvesting wild plants for their bioactive components. Despite high interest in biotechnological production, little is known about the main factors affecting cell growth and ginsenoside biosynthesis of this species under in vitro conditions. In this study, the potential of cell cultures of P. vietnamensis as a biotechnological source of ginsenosides was was assessed. Six suspension cell lines that were developed from different sections of a single rhizome through a multi-step culture optimization process and maintained for over 3 years on media with different mineral salt base and varying contents of auxins and cytokinins. These cell lines were evaluated for productivity parameters and cytological characteristics. Ginsenoside profiles were assessed using a combination of the reversed-phase ultra-high-performance liquid chromatography-Orbitrap-tandem mass spectrometry (UHPLC-Orbitrap-MS/MS) and ultra-performance liquid chromatography-time of flight-mass spectrometry (UPLC-TOF-MS). All lines demonstrated good growth with a specific growth rate of 0.1-0.2 day-1, economic coefficient of 0.31-0.70, productivity on dry weight (DW) of 0.30-0.83 gDW (L·day)-1, and maximum biomass accumulation varying from 10 to 22 gDW L-1. Ginsenosides of the protopanaxadiol (Rb1, Rb2/Rb3, malonyl-Rb1, and malonyl-Rb2/Rb3), oleanolic acid (R0 and chikusetsusaponin IV), and ocotillol (vinaginsenoside R1) groups and their isomers were identified in cell biomass extracts. Chikusetsusaponin IV was identified in P. vietnamensis cell culture for the first time. These results suggest that suspension cell cultures of Vietnamese ginseng have a high potential for the biotechnological production of biomass containing ginsenosides, particularly of the oleanolic acid and ocotillol groups.

Negevirus Piura Suppresses Zika Virus Replication in Mosquito Cells.

We investigated the interaction between the insect-specific virus, Piura virus (PIUV), and the arbovirus Zika virus (ZIKV) in Aedes albopictus cells. We performed coinfection experiments in C6/36 cells. Piura virus (Cor 33 strain, Colombia) and ZIKV (PRVABC58 strain, Puerto Rico) were co-inoculated into C6/36 cells using two multiplicity of infection (MOI) combinations: 0.1 for both viruses and 1.0 for ZIKV, 0.1 for PIUV. Wells were infected in triplicate with either PIUV and ZIKV coinfection, ZIKV-only, or PIUV-only. Mock infected cells served as control wells. The cell suspension was collected daily 7 days post-infection. Zika virus load was titrated by TCID50 on Vero 76 cells. The ZIKV-only infection and PIUV and ZIKV coinfection experiments were also quantified by RT-qPCR. We also investigated whether ZIKV interfered in the PIUV replication. PIUV suppressed the replication of ZIKV, resulting in a 10,000-fold reduction in ZIKV titers within 3 days post-infection. PIUV viral loads were not reduced in the presence of ZIKV. We conclude that, when concurrently infected, PIUV suppresses ZIKV in C6/36 cells while ZIKV does not interfere in PIUV replication.

In situ-formed cryomicroneedles for intradermal cell delivery

Cryomicroneedles (cryoMNs) offer a convenient and minimally invasive way to precisely deliver therapeutic cells intradermally for treating local and systemic diseases. cryoMNs are manufactured by shaping and freezing the cell-containing cryogenic media in a microneedle template, which allows cells to be packaged in advance for direct usage in the clinic. However, the current cryoMNs require cold-chain transportation and storage and do not permit the loading of autologous cells in situ. This article introduces the second generation of cryoMNs (S-cryoMNs) that address these limitations. Specifically, S-cryoMNs are made by dipping a porous MN scaffold in the cell suspension before cryopreservation. The porous scaffold can be transported at room temperature, and researchers can load any cells with the optimized cryogenic medium. As a proof-of-concept, we examined the loading and intradermal delivery of three cell types in clinically relevant in vitro and in vivo models, including mesenchymal stem cells for wound healing, melanocytes for vitiligo treatment, and antigen-pulsed dendritic cells for cancer vaccination.

Calcium Chloride Treatment Enhances Antigen Production in Foot-and-Mouth Disease Vaccines for Serotypes SAT1 and SAT3.

Foot-and-mouth disease (FMD) is a highly contagious viral infection causing acute and severe vesicular lesions in cattle and pigs, which has prompted global vaccination policies. This study presents a technique for enhancing antigen yield in SAT1 BOT and SAT3 ZIM by treatment with calcium chloride (CaCl2). We tested changes in cell viability in BHK-21 suspension cells treated with varying concentrations of CaCl2. The optimal CaCl2 concentration was determined based on antigen yield. The timing of CaCl2 supplementation relative to FMD virus inoculation was tested. Finally, the optimal medium for antigen production was identified. We observed a concentration-dependent decrease in BHK-21 cell viability at >7.5 mM CaCl2. A CaCl2 concentration of 3 mM yielded the most antigens. CaCl2 supplementation relative to FMD virus infection was optimal 2 h before or with viral inoculation. CD-BHK 21 medium supplemented with CaCl2 was the most productive medium. Specifically, SAT1 BOT and SAT3 ZIM showed improved antigen production in CD-BHK 21 medium with 3 mM CaCl2, while Provero-1 and Cellvento BHK-200 media showed no significant enhancement. Overall, CaCl2 supplementation enhanced FMD antigen productivity. This study provides a useful framework for enhancing antigen production efficiently in the FMD vaccine industry.

Synergistic suppression of ovarian cancer by combining NRF2 and GPX4 inhibitors: in vitro and in vivo evidence

Ovarian cancer is a significant challenge in women's health due to the lack of effective screening and diagnostic methods, often leading to late detection and the highest mortality rate among all gynecologic tumors worldwide. Recent research has shown that ovarian cancer has an "iron addiction" phenotype which makes it vulnerable to ferroptosis inducers. We tested the combination of NRF2-targeted inhibitors with GPX4-targeted inhibitors in ovarian cancer through in vitro and in vivo experiment. The data showed that combination treatment effectively suppressed adherent cell growth, inhibited suspended cell spheroid formation, and restrained the ability of spheroid formation in 3D-culture. Mechanistically, the combination induced accumulation of ROS, 4-HNE, as well as activation of caspase-3 which indicates that this combination simultaneously increases cell ferroptosis and apoptosis. Notably, inhibition of GPX4 or NRF2 can suppress ovarian cancer spreading and growth in the peritoneal cavity of mice, while the combination of NRF2 inhibitor ML385 with GPX4 inhibitors showed a significant synergistic effect compared to individual drug treatment in a syngeneic mouse ovarian cancer model. Overall, these findings suggest that combining NRF2 inhibitors with GPX4 inhibitors results in a synergy suppression of ovarian cancer in vitro and in vivo, and maybe a promising therapeutic strategy for the treatment of ovarian cancer.

Transcriptional Changes in Damask Rose Suspension Cell Culture Revealed by RNA Sequencing.

Damask roses (Rosa x damascena) are widely used in cosmetics and pharmaceutics. Here, we established an in vitro suspension cell culture for calli derived from damask rose petals. We analyzed rose suspension cell transcriptomes obtained at two different time points by RNA sequencing to reveal transcriptional changes during rose suspension cell culture. Of the 580 coding RNAs (1.3%) highly expressed in the suspension rose cells, 68 encoded cell wall-associated proteins. However, most RNAs encoded by the chloroplasts and mitochondria are not expressed. Many highly expressed coding RNAs are involved in translation, catalyzing peptide synthesis in ribosomes. Moreover, the amide metabolic process producing naturally occurring alkaloids was the most abundant metabolic process during the propagation of rose suspension cells. During rose cell propagation, coding RNAs involved in the stress response were upregulated at an early stage, while coding RNAs associated with detoxification and transmembrane transport were upregulated at the late stage. We used transcriptome analyses to reveal important biological processes and molecular mechanisms during rose suspension cell culture. Most non-coding (nc) RNAs were not expressed in the rose suspension cells, but a few ncRNAs with unknown functions were highly expressed. The expression of ncRNAs and their target coding RNAs was highly correlated. Taken together, we revealed significant biological processes and molecular mechanisms occurring during rose suspension cell culture using transcriptome analyses.

The energy-converting hydrogenase Ech2 is important for the growth of the thermophilic acetogen Thermoanaerobacter kivui on ferredoxin-dependent substrates.

Acetogens thrive by converting H2+CO2 to acetate. Under environmental conditions, this allows for only very little energy to be conserved (∆G'<-20 kJ mol-1). CO2 serves as a terminal electron acceptor in the ancient Wood-Ljungdahl pathway (WLP). Since the WLP is ATP neutral, energy conservation during growth on H2 + CO2 is dependent on the redox metabolism. Two types of acetogens can be distinguished, Rnf- and Ech-type. The function of both membrane-bound enzyme complexes is twofold-energy conversion and redox balancing. Ech couples the Fd-dependent reduction of protons to H2 to the formation of a proton gradient in the thermophilic bacterium Thermoanaerobacter kivui. This bacterium may be utilized in gas fermentation at high temperatures, due to very high conversion rates and the availability of genetic tools. The physiological function of an Ech hydrogenase in T. kivui was studied to contribute an understanding of its energy and redox metabolism, a prerequisite for future industrial applications.

Evaluation of engraftment and growth dynamics of orthotopic and heterotopic in vivo models of human breast cancer

Purpose of the study. This work was to assess the engraftment and growth dynamics of breast cancer xenografts during orthotopic and subcutaneous injection using various types of biological material, as well as to develop an adequate model of breast cancer for further research.Materials and methods. We used a disaggregated fragment of a tumor obtained from the patient, a certified breast cancer cell line VT20 – human breast carcinoma; a primary human breast carcinoma cell line. Female immunodeficient mice of the Balb/c Nude line in the amount of 36 animals were used as recipient animals. The subcutaneous and orthotopic models of breast cancer were developed in this project. Tumor growth was observed for 28 days from the moment of injection and tumor nodes were measured 2 times a week until the end of the experiment. Results were assessed using medians and percentiles. The nonparametric Mann-Whitney test was used to assess the significance of differences.Results. The dynamics of the growth of tumor cells when injected into various sites was determined in the process of this work. The most successful in terms of a subcutaneous injection was the injection of tumor cells of the certified VT20 line. By the end of the experiment, the median tumor node of this group was 100.32 mm³. The analysis revealed tumor dynamics with orthotopic injection of tumor material, and the median volume of the tumor node in the group with the passport culture cell VT20 and the primary culture cell reached the same value – 149.22 and 148.25. mm³. It was found that both the cell line and the cell suspension were injected into tumor nodes that reached a significantly larger volume when injected orthotopically.Conclusion. We have obtained a tumor model of breast cancer using various methods of material implantation and with the possibility of further use in testing new pharmacological substances.

MiniRead: A simple and inexpensive do-it-yourself device for multiple analyses of micro-organism growth kinetics.

Fitness in micro-organisms can be proxied by growth parameters on different media and/or temperatures. This is achieved by measuring optical density at 600 nm using a spectrophotometer, which measures the effect of absorbance and side scattering due to turbidity of cells suspensions. However, when growth kinetics must be monitored in many 96-well plates at the same time, buying several 96-channel spectrophotometers is often beyond budgets. The MiniRead device presented here is a simple and inexpensive do-it-yourself 96-well temperature-controlled turbidimeter designed to measure the interception of white light via absorption or side scattering through liquid culture medium. Turbidity is automatically recorded in each well at regular time intervals for up to several days or weeks. Output tabulated text files are recorded into a micro-SD memory card to be easily transferred to a computer. We propose also an R package which allows (1) to compute the nonlinear calibration curves required to convert raw readings into cell concentration values, and (2) to analyze growth kinetics output files to automatically estimate proxies of growth parameters such as lag time, maximum growth rate, or cell concentration at the plateau.

Effects of cerium oxide nanoenzyme-gelatin methacrylate anhydride hydrogel in the repair of infected full-thickness skin defect wounds in mice

Objective: To investigate the effects of cerium oxide nanoenzyme-gelatin methacrylate anhydride (GelMA) hydrogel (hereinafter referred to as composite hydrogel) in the repair of infected full-thickness skin defect wounds in mice. Methods: This study was an experimental study. Cerium oxide nanoenzyme with a particle size of (116±9) nm was prepared by hydrothermal method, and GelMA hydrogel with porous network structure and good gelling performance was also prepared. The 25 μg/mL cerium oxide nanoenzyme which could significantly promote the proliferation of human skin fibroblasts and had high superoxide dismutase activity was screened out. It was added to GelMA hydrogel to prepare composite hydrogel. The percentage of cerium oxide nanoenzyme released from the composite hydrogel was calculated after immersing it in phosphate buffer solution (PBS) for 3 and 7 d. The red blood cell suspension of mice was divided into PBS group, Triton X-100 group, cerium oxide nanoenzyme group, GelMA hydrogel group, and composite hydrogel group, which were treated with corresponding solution. The hemolysis of red blood cells was detected by microplate reader after 1 h of treatment. The bacterial concentrations of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli were determined after being cultured with PBS, cerium oxide nanoenzyme, GelMA hydrogel, and composite hydrogel for 2 h. The sample size in all above experiments was 3. Twenty-four 8-week-old male BALB/c mice were taken, and a full-thickness skin defect wound was prepared in the symmetrical position on the back and infected with MRSA. The mice were divided into control group without any drug intervention, and cerium oxide nanoenzyme group, GelMA hydrogel group, and composite hydrogel group applied with corresponding solution, with 6 mice in each group. The wound healing was observed on 3, 7, and 14 d after injury, and the remaining wound areas on 3 and 7 d after injury were measured (the sample size was 5). The concentration of MRSA in the wound exudation of mice on 3 d after injury was measured (the sample size was 3), and the blood flow perfusion in the wound of mice on 5 d after injury was observed using a laser speckle flow imaging system (the sample size was 6). On 14 d after injury, the wound tissue of mice was collected for hematoxylin-eosin staining to observe the newly formed epithelium and for Masson staining to observe the collagen situation (the sample size was both 3). Results: After immersion for 3 and 7 d, the release percentages of cerium oxide nanoenzyme in the composite hydrogel were about 39% and 75%, respectively. After 1 h of treatment, compared with that in Triton X-100 group, the hemolysis of red blood cells in PBS group, GelMA hydrogel group, cerium oxide nanoenzyme group, and composite hydrogel group was significantly decreased (P<0.05). Compared with that cultured with PBS, the concentrations of MRSA and Escherichia coli cultured with cerium oxide nanoenzyme, GelMA hydrogel, and composite hydrogel for 2 h were significantly decreased (P<0.05). The wounds of mice in the four groups were gradually healed from 3 to 14 d after injury, and the wounds of mice in composite hydrogel group were all healed on 14 d after injury. On 3 and 7 d after injury, the remaining wound areas of mice in composite hydrogel group were (29±3) and (13±5) mm2, respectively, which were significantly smaller than (56±12) and (46±10) mm2 in control group and (51±7) and (38±8) mm2 in cerium oxide nanoenzyme group (with P values all <0.05), but was similar to (41±5) and (24±9) mm2 in GelMA hydrogel group (with P values both >0.05). On 3 d after injury, the concentration of MRSA on the wound of mice in composite hydrogel group was significantly lower than that in control group, cerium oxide nanoenzyme group, and GelMA hydrogel group, respectively (with P values all <0.05). On 5 d after injury, the volume of blood perfusion in the wound of mice in composite hydrogel group was significantly higher than that in control group, cerium oxide nanoenzyme group, and GelMA hydrogel group, respectively (P<0.05). On 14 d after injury, the wound of mice in composite hydrogel group basically completed epithelization, and the epithelization was significantly better than that in the other three groups. Compared with that in the other three groups, the content of collagen in the wound of mice in composite hydrogel group was significantly increased, and the arrangement was also more orderly. Conclusions: The composite hydrogel has good biocompatibility and antibacterial effect in vivo and in vitro. It can continuously sustained release cerium oxide nanoenzyme, improve wound blood perfusion in the early stage, and promote wound re-epithelialization and collagen synthesis, therefore promoting the healing of infected full-thickness skin defect wounds in mice.

Comparison of Viability of the Preserved Autologous Aspirated Fat Tissue Transfer in Refrigerated and Frozen Specimens.

Fat graft surgery is one of the most effective procedures in plastic surgery, and since some patients request multiple surgeries and these cases sometimes take hours, it endangers the viability of the fat graft. In this study, we intend to evaluate the viability of adipose tissue aspirated with a syringe at refrigerator (4°C) and freezer (-20 °C) temperatures. This was a cross-sectional study. After receiving the ethics committee's approval (IR.MUMS.MEDICAL.REC.1401.423), 17 volunteers entered the study. The harvested fat tissue sample was divided into 3 parts, and each of them was transferred to 3 separate sterile tubes. The first tube was sent to the laboratory for preliminary examination of fresh fat, and the second tube was transferred to a 4°C refrigerator for 72 hours. The sample from the third tube was first passed through a strainer and after drying, it was transferred to a -20°C freezer for 72 hours. After treatment with trypsin, we placed the sample inside the centrifuge using the Coleman method. Finally, 3 layers were formed, and the white middle layer was extracted as a fat cell suspension. Tissue samples were stained with trypan blue, and the percentage of viable cells was calculated using an optical microscope. There was a significant difference between the mean number and percentage of viable cells in all 3 groups. Samples in the 4°C refrigerator had significantly more cellular viability than those in the -20°C freezer (mean difference, 72.842%; P < 0.001). Our findings showed that after 72 hours at 4°C, adipose tissue has significantly higher survival than at -20°C (98.93% vs 75.31%). Since the survival of fat cells is one of the direct determinants of fat retention, it can affect the results after surgery. The present study recommends fresh adipose tissue for immediate transplantation unless there is an urgent need for cold storage.

Review on the Impact of Impeller‐induced Hydrodynamics on Suspension Cell Culture for Production of Biopharmaceuticals

AbstractHydrodynamic effects continue to play a crucial role in the design and operation of bioreactors for cultivating mammalian cells. Despite intensive studies in the past the link between hydrodynamics and biological process behaviour is still not fully understood. Common methods to describe dependencies, which relay mostly on averaged hydrodynamic parameters, does not seem to be suitable, as they do not incorporate the hydrodynamic heterogeneity. Within this review first a brief introduction to hydrodynamic effects is given, followed by an evaluation of common process design approaches with respect to hydrodynamic effects and recommendations for process development.

A novel sorting method for the enrichment of early human spermatocytes from clinical biopsies

To determine if early spermatocytes can be enriched from a human testis biopsy using fluorescence-activated cell sorting (FACS). Potential surface markers for early spermatocytes were identified using bioinformatics analysis of single-cell RNA-sequenced human testis tissue. Testicular sperm extraction samples from three participants with normal spermatogenesis were digested into single-cell suspensions and cryopreserved. Two to four million cells were obtained from each and sorted by FACS as separate biologic replicates using antibodies for the identified surface markers. A portion from each biopsy remained unsorted to serve as controls. The sorted cells were then characterized for enrichment of early spermatocytes. A laboratory study. Three men with a diagnosis of obstructive azoospermia (age range, 30-40 years). None. Sorted cells were characterized for RNA expression of markers encompassing the stages of spermatogenesis. Sorting markers were validated by their reactivity on human testis formalin-fixed paraffin-embedded tissue. Serine protease 50 (TSP50) and SWI5-dependent homologous recombination repair protein 1 were identified as potential surface proteins specific for early spermatocytes. After FACS sorting, the TSP50-sorted populations accounted for 1.6%-8.9% of total populations and exhibited the greatest average-fold increases in RNA expression for the premeiotic marker stimulated by retinoic acid (STRA8), by 23-fold. Immunohistochemistry showed the staining pattern for TSP50 to be strong in premeiotic undifferentiated embryonic cell transcription factor 1-/doublesex and Mab-3 related transcription factor 1-/STRA8+ spermatogonia as well as SYCP3+/protamine 2- spermatocytes. This work shows that TSP50 can be used to enrich early STRA8-expressing spermatocytes from human testicular biopsies, providing a means for targeted single-cell RNA sequencing analysis and invitro functional interrogation of germ cells during the onset of meiosis. This could enable investigation into details of the regulatory pathways underlying this critical stage of spermatogenesis, previously difficult to enrich from whole tissue samples.

A New Biocontrol Agent Bacillus velezensis SF334 against Rubber Tree Fungal Leaf Anthracnose and Its Genome Analysis of Versatile Plant Probiotic Traits.

Natural rubber is an important national strategic and industrial raw material. The leaf anthracnose of rubber trees caused by the Colletotrichum species is one of the important factors restricting the yields of natural rubber. In this study, we isolated and identified strain Bacillus velezensis SF334, which exhibited significant antagonistic activity against both C. australisinense and C. siamense, the dominant species of Colletotrichum causing rubber tree leaf anthracnose in the Hainan province of China, from a pool of 223 bacterial strains. The cell suspensions of SF334 had a significant prevention effect for the leaf anthracnose of rubber trees, with an efficacy of 79.67% against C. siamense and 71.8% against C. australisinense. We demonstrated that SF334 can lead to the lysis of C. australisinense and C. siamense mycelia by causing mycelial expansion, resulting in mycelial rupture and subsequent death. B. velezensis SF334 also harbors some plant probiotic traits, such as secreting siderophore, protease, cellulase, pectinase, and the auxin of indole-3-acetic acid (IAA), and it has broad-spectrum antifungal activity against some important plant pathogenic fungi. The genome combined with comparative genomic analyses indicated that SF334 possesses most genes of the central metabolic and gene clusters of secondary metabolites in B. velezensis strains. To our knowledge, this is the first time a Bacillus velezensis strain has been reported as a promising biocontrol agent against the leaf anthracnose of rubber trees caused by C. siamense and C. australisinense. The results suggest that B. velezensis could be a potential candidate agent for the leaf anthracnose of rubber trees.