A duplex real-time NASBA assay targeting serotype-specific gene for rapid detection of viable S. enterica serovar Paratyphi C in retail foods of animal origin

Salmonella enterica serovars Paratyphi C is highly adapted to humans and can cause a typhoid-like disease with high mortality rates. In this study, three serovar-specific genes were determined for S. Paratyphi C, SPC_0871，SPC_0872, and SPC_0908, by comparative genomics method. Based on SPC_0908 and xcd gene for testing Salmonella spp., we have developed a duplex real-time nucleic acid sequence-based amplification (real-time NASBA) with molecular beacon approach for simultaneous detection of viable cells of Salmonella spp. and serotype Paratyphi C. The test selectively and consistently detected 53 Salmonella spp. (representing 31 serotypes) and 18 non-Salmonella strains. Additionally, the method showed high resistance to interference by natural background flora in pork and chicken samples. The sensitivity of the established approach was determined to be 4.89 CFU/25 g in artificially contaminated pork and chicken samples after pre-enrichment. We propose this NASBA-based protocol as a potential detection method for Salmonella spp. and serotype Paratyphi C in food of animal origin.

In vitro activities of thiazolidione derivatives combined with daptomycin against clinical Enterococcus faecium strains

Background Previous reports have demonstrated two thiazolidione derivatives (H2-60 and H2-81) can robustly inhibit the planktonic growth and biofilm formation of S. epidermidis and S. aureus by targeting the histidine kinase YycG. Whereas the antibacterial and anti-biofilm activity of these two thiazolidione derivatives (H2-60 and H2-81) against Enterococcus faecium remains elusive. Here, the pET28a-YycG recombinant plasmid were in vitro expressed in E. coli competent cell BL21 (DE3) and induced to express YycG’ protein (conding HisKA and HATPase_c domain) by 0.5 mM IPTG and was purified by Ni – NTA agarose and then for the autophosphorylation test. Antimicrobial testing and time-killing assay were also be determined. Anti-biofilm activity of two derivatives with sub-MIC concentration towards positive biofilm producers of clinical E. faecium were detected using polystyrene microtiter plate and CLSM. Results The MICs of H2-60 and H2-81 in the clinical isolates of E. faecium were in the range from 3.125 mg/L to 25 mg/L. Moreover, either H2-60 or H2-81 showed the excellent bactericidal activity against E. faecium with monotherapy or its combination with daptomycin by time-killing assay. E. faecium planktonic cells can be decreased by H2-60 or H2-81 for more than 3 × log10 CFU/mL after 24 h treatment when combined with daptomycin. Furthermore, over 90% of E. faecium biofilm formation could markedly be inhibited by H2-60 and H2-81 at 1/4 × MIC value. In addition, the frequency of the eradicated viable cells embedded in mature biofilm were evaluated by the confocal laser microscopy, suggesting that of H2-60 combined with ampicillin or daptomycin was significantly high when compared with single treatment (78.17 and 74.48% vs. 41.59%, respectively, P < 0.01). Conclusion These two thiazolidione derivatives (H2-60 and H2-81) could directly impact the kinase phosphoration activity of YycG of E. faecium. H2-60 combined with daptomycin exhibit the excellent antibacterial and anti-biofilm activity against E. faecium by targeting YycG.

Reducing sources of variance in experimental procedures in in vitro research

Background: Lack of reproducibility in preclinical research poses ethical and economic challenges for biomedical science. Various institutional activities by society stakeholders of leading industrialised nations are currently underway with the aim of improving the situation. Such initiatives are usually concerned with high-level organisational issues and typically do not focus on improving experimental approaches per se. Addressing these is necessary in order to increase consistency and success rates of lab-to-lab repetitions. Methods: In this project, we statistically evaluated repetitive data of a very basic and widely applied lab procedure, namely quantifying the number of viable cells. The purpose of this was to assess the impact of different parameters and instrumentations which may constitute sources of variance in this procedure. Conclusion: By comparing the variability of data acquired under two different procedures, featuring improved stringency of protocol adherence, our project attempts to identify the sources and propose guidelines on how to reduce such fluctuations. We believe our work can contribute to tackling the repeatability crisis in biomedical research.

ISOLATION OF LIVE CELLS FROM DIFFERENT MICE TISSUES UP TO NINE DAYS AFTER DEATH

Some limited reports suggest that cells can survive in the cadavers for much longer than it was previously thought. In our study we explored how time after death, tissue type (muscle, brain and adipose tissue), storage temperature of cadavers (4 °C or at room temperature) and form of tissue storage (stored as cadavers or tissue pieces in phosphate buffered saline) affect the success of harvesting live cells from mice after death. Cells were isolated from dead tissues and grown in standard conditions. Some cells were used for RNA extraction and RT² Profiler™ PCR Array for cell lineage identification was performed to establish which lineages the cells obtained from post mortem tissues belong to. Results of our study showed that viable cells can be regularly isolated from muscle and brain tissue 3 days post mortem and with difficulty up to 6 days post mortem. Viable cells from brain tissue can be isolated up to 9 days post mortem. No cells were isolated from adipose tissue except immediately after death. In all instances viable cells were isolated only when tissues were stored at 4 °C. Tissue storage did not affect cell isolation. Isolated cells were progenitors from different germ layers. Our results show that live cells could be obtained from mouse cadavers several days after death.Key words: mouse; cadaver; stem cells; brain; muscle; adipose tissue IZOLACIJA ŽIVIH CELIC IZ RAZLIČNIH TKIV MIŠI DO DEVET DNI PO SMRTI Izvleček: Nekatere raziskave kažejo, da je preživetje celic v truplih precej daljše, kot je bilo znano do sedaj. V naši raziskavi smo proučevali, kako na uspešnost izolacije živih celic po smrti miši vplivajo različen čas izolacije po smrti, vrsta tkiva (mišično, možgansko in maščobno), temperatura shranjevanja trupel ter oblika shranjenega tkiva (kot koščki tkiv ali kot celi kadavri). Izolacija in gojenje celic iz tkiv mrtvih miši sta potekali pod standardnimi pogoji. Da bi ugotovili, katerim celičnim linijam pripadajo izolirane celice, je bil del celic uporabljen za izolacijo RNK in nadaljno uporabo v sistemu identifikacije izvornih celičnih linij z verižno reakcijo s polimerazo v realnem času. Rezultati naše raziskave so pokazali, da je žive celice mogoče izolirati iz mišičnega in možganskega tkiva 3 dni po smrti, pogojno tudi do 6 dni po smrti. Iz možganskega tkiva je bilo žive celice mogoče izolirati tudi do 9 dni po smrti. Iz maščobnega tkiva je bilo celice mogoče izolirati zgolj takoj po smrti, ne pa tudi v kasnejših časovnih intervalih. V vseh primerih so bile celice izolirane samo v primeru shranjevanja tkiv pri 4°C. Oblika shranjenega tkiva na izolacijo celic ni vplivala. Izolirane celice so pripadale različnim zarodnim plastem. Rezultati raziskave so pokazali, da je žive celice iz mišjih trupel mogoče izolirati tudi več dni po smrti.Ključne besede: miš; truplo; matične celice; možgansko tkivo; mišično tkivo; maščobno tkivo

The Ceramide Synthase Subunit Lac1 Regulates Cell Growth and Size in Fission Yeast

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.

Effect of Temperature and Cell Viability on Uranium Biomineralization by the Uranium Mine Isolate Penicillium simplicissimum

The remediation of heavy-metal-contaminated sites represents a serious environmental problem worldwide. Currently, cost- and time-intensive chemical treatments are usually performed. Bioremediation by heavy-metal-tolerant microorganisms is considered a more eco-friendly and comparatively cheap alternative. The fungus Penicillium simplicissimum KS1, isolated from the flooding water of a former uranium (U) mine in Germany, shows promising U bioremediation potential mainly through biomineralization. The adaption of P. simplicissimum KS1 to heavy-metal-contaminated sites is indicated by an increased U removal capacity of up to 550 mg U per g dry biomass, compared to the non-heavy-metal-exposed P. simplicissimum reference strain DSM 62867 (200 mg U per g dry biomass). In addition, the effect of temperature and cell viability of P. simplicissimum KS1 on U biomineralization was investigated. While viable cells at 30°C removed U mainly extracellularly via metabolism-dependent biomineralization, a decrease in temperature to 4°C or use of dead-autoclaved cells at 30°C revealed increased occurrence of passive biosorption and bioaccumulation, as confirmed by scanning transmission electron microscopy. The precipitated U species were assigned to uranyl phosphates with a structure similar to that of autunite, via cryo-time-resolved laser fluorescence spectroscopy. The major involvement of phosphates in U precipitation by P. simplicissimum KS1 was additionally supported by the observation of increased phosphatase activity for viable cells at 30°C. Furthermore, viable cells actively secreted small molecules, most likely phosphorylated amino acids, which interacted with U in the supernatant and were not detected in experiments with dead-autoclaved cells. Our study provides new insights into the influence of temperature and cell viability on U phosphate biomineralization by fungi, and furthermore highlight the potential use of P. simplicissimum KS1 particularly for U bioremediation purposes.Graphical Abstract

EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis

We found that cells from Mycobacterium smegmatis , a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l- tryptophan, l -phenylalanine, and l -tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs.

Electrosprayed Ethyl Cellulose Core-Shell Microcapsules for the Encapsulation of Probiotics

Electrosprayed ethyl cellulose core–shell microcapsules were produced for the encapsulation of probiotic Bifidobacterium animalis subsp. lactis (Bifido). Ethyl cellulose (ETC) was used as a shell material with different core compounds (concentrated Bifido, Bifido–maltodextrin and Bifido–glycerol). The core–shell microcapsules have an average diameter between 3 µm and 15 µm depending on the core compounds, with a distinct interface that separates the core and the shell structure. The ETC microcapsules displayed relatively low water activity (aw below 0.20) and relatively high values of viable cells (109–1011 CFU/g), as counted post-encapsulation. The effect of different core compounds on the stability of probiotics cells over time was also investigated. After four weeks at 30 °C and 40% RH the electrospray encapsulated samples containing Bifido–glycerol in the core showed a loss in viable cells of no more than 3 log loss CFU/g, while the non-encapsulated Bifido lost about 7.57 log CFU/g. Overall, these results suggest that the viability of the Bifido probiotics encapsulated within the core–shell ETC electrosprayed capsules can be extended, despite the fact that the shell matrix was prepared using solvents that typically substantially reduce their viability.

Comparison of Harvesting and Processing Technique for Adipose Tissue Graft: Evaluation of Cell Viability

Background: Clinical studies demonstrated the efficacy of therapies based on the autologous grafting of adult mesenchymal stem cells to accelerate the healing and regenerative processes of the skin and mesenchymal tissues; therefore, it is considered a valuable approach in the aesthetic rejuvenation treatment to give volumization and skin regeneration effects. Objective: The aim of the project consisted of the control of cell viability of adipose tissue (AT) harvested using the two types of cannulas having 0.8 mm and 1 mm side port holes. The results were compared with tissue harvested with a standard liposuction technique and processed with a standard procedure consisting of enzymatic digestion (collagenase). Methods: This study was performed on adipose tissues harvested from 7 patients (6 females and 1 male) with an average age of 48.5 years with 3 different techniques. We compared the cell vitality of every sample at T0 and T72. Results: Lipoaspirate tissue-derived by 0.8- and 1 mm cannula from all samples proved to be vital and possess viable cells. The average absorbance was similar immediately after plating (T0) and 72 hours after (T72) for the two cannulas, 0.8- and 1 mm cannula. The two systems proved to equally harvest vital tissue. An increase in cell viability was observed in all samples for each condition (0.8-, 1 mm and enzymatic digestion). Conclusion: This study proved that guided harvested adipose tissue with small cannulas with small side port holes yields a comparable amount of viable cells compared to adipose tissue harvested with a liposuction system and processed with enzymatic digestion (collagenase). This study confirms that the minimally invasive technique and minimal manipulation of the adipose tissue could yield a tissue with a good amount of viable cells. This micro fragmented adipose tissue is a promising source for regenerative treatments.

Green Tea (Camellia sinensis) Extract Induces p53-Mediated Cytotoxicity and Inhibits Migration of Breast Cancer Cells

Green tea (GT) has been shown to play an important role in cancer chemoprevention. However, the related molecular mechanisms need to be further explored, especially regarding the use of GT extract (GTE) from the food matrix. For this study, epigallocatechin gallate (EGCG) and epigallocatechin (EGC) were identified in GTE, representing 42 and 40% of the total polyphenols, respectively. MDA-MB-231 (p53-p.R280K mutant) and MCF-7 (wild-type p53) breast tumor cells and MCF-10A non-tumoral cells were exposed to GTE for 24–48 h and cell viability was assessed in the presence of p53 inhibitor pifithrin-α. GTE selectively targeted breast tumor cells without cytotoxic effect on non-tumoral cells and p53 inhibition led to an increase in viable cells, especially in MCF-7, suggesting the involvement of p53 in GTE-induced cytotoxicity. GTE was also effective in reducing MCF-7 and MDA-MD-231 cell migration by 30 and 50%, respectively. An increment in p53 and p21 expression stimulated by GTE was observed in MCF-7, and the opposite phenomenon was found in MDA-MB-231 cells, with a redistribution of mutant-p53 from the nucleus and no differences in p21 levels. All these findings provide insights into the action of GTE and support its anticarcinogenic potential on breast tumor cells.