Growth Medium Research Articles

Transient electrophoresis of colloidal particles in a salt-free medium

A general theory is developed for the time dependent transient electrophoretic mobility of spherical colloidal particles in a salt-free liquid medium containing only counterions when a step external electric field is suddenly applied to the colloidal suspension. It is found that as in the case of the steady electrophoretic mobility in a salt-free medium, there is a certain critical value of the particle surface charge separating two cases, that is, the low-surface-charge case and the high-surface-charge case. In the latter case the counterion condensation takes place near the particle surface. For the low-surface charge case, the transient electrophoretic mobility agrees with that of a sphere in an electrolyte solution in the limit of very low electrolyte concentrations. For the high-surface-charge case, however, the transient mobility becomes independent of the particle surface charge because of the counterion condensation effects. A simple expression is derived for the ratio of the transient electrophoretic mobility to the steady electrophoretic mobility, which is found to take the same form irrespective of the magnitude of the particle surface charge. Using this equation, it is now possible to predict how the system will approach its final steady state.

Rational Design of Cyanine-Based Fluorogenic Dimers to Reduce Nonspecific Interactions with Albumin and Lipid Bilayers: Application to Highly Sensitive Imaging of GPCRs in Living Cells.

Fluorogenic dimers with polarity-sensitive folding are powerful probes for live-cell bioimaging. They switch on their fluorescence only after interacting with their targets, thus leading to a high signal-to-noise ratio in wash-free bioimaging. We previously reported the first near-infrared fluorogenic dimers derived from cyanine 5.5 dyes for the optical detection of G protein-coupled receptors. Owing to their hydrophobic character, these dimers are prone to form nonspecific interactions with proteins such as albumin and with the lipid bilayer of the cell membrane resulting in a residual background fluorescence in complex biological media. Herein, we report the rational design of new fluorogenic dimers derived from cyanine 5. By modulating the chemical structure of the cyanine units, we discovered that the two asymmetric cyanine 5.25 dyes were able to form intramolecular H-aggregates and self-quenched in aqueous media. Moreover, the resulting original dimeric probes enabled a significant reduction of the nonspecific interactions with bovine serum albumin and lipid bilayers compared with the first generation of cyanine 5.5 dimers. Finally, the optimized asymmetric fluorogenic dimer was grafted to carbetocin for the specific imaging of the oxytocin receptor under no-wash conditions directly in cell culture media, notably improving the signal-to-background ratio compared with the previous generation of cyanine 5.5 dimers.

Rational Design of Cyanine-Based Fluorogenic Dimers to Reduce Nonspecific Interactions with Albumin and Lipid Bilayers: Application to Highly Sensitive Imaging of GPCRs in Living Cells.

Fluorogenic dimers with polarity-sensitive folding are powerful probes for live-cell bioimaging. They switch on their fluorescence only after interacting with their targets, thus leading to a high signal-to-noise ratio in wash-free bioimaging. We previously reported the first near-infrared fluorogenic dimers derived from cyanine 5.5 dyes for the optical detection of G protein-coupled receptors. Owing to their hydrophobic character, these dimers are prone to form nonspecific interactions with proteins such as albumin and with the lipid bilayer of the cell membrane resulting in a residual background fluorescence in complex biological media. Herein, we report the rational design of new fluorogenic dimers derived from cyanine 5. By modulating the chemical structure of the cyanine units, we discovered that the two asymmetric cyanine 5.25 dyes were able to form intramolecular H-aggregates and self-quenched in aqueous media. Moreover, the resulting original dimeric probes enabled a significant reduction of the nonspecific interactions with bovine serum albumin and lipid bilayers compared with the first generation of cyanine 5.5 dimers. Finally, the optimized asymmetric fluorogenic dimer was grafted to carbetocin for the specific imaging of the oxytocin receptor under no-wash conditions directly in cell culture media, notably improving the signal-to-background ratio compared with the previous generation of cyanine 5.5 dimers.

Bioenergy feedstock production in Chlamydomonas reinhardtii (microalgae) cultivated under mixotrophic growth with cellulose hydrolysate from agricultural waste.

In the present study, cellulose purified from finger millet agricultural waste is subjected to enzymatic hydrolysis, and the hydrolysate (predominantly glucose) is used as a carbon source supplement in the media for the mixotrophic growth of Chlamydomonas reinhardtii. Interestingly, a switch between excess starch production and excess lipid (triacylglycerols, TAG) production occurs by a small change in hydrolysate concentration in the media. Starch production increased 4.5-fold with respect to the photoautotrophic control, with a glucose concentration of 3mg/mL in the media after hydrolysate addition. This culture had TAG production enhancement by 1.5-fold. However, mixotrophic cultivation with 4mg/mL glucose concentration in the media with hydrolysate addition resulted in TAG productivity enhancement by 4.2-fold compared to control and starch amount increase of 1.3-fold. The organic carbon source (glucose) and the inorganic carbon source (citrate ions) in the hydrolysate together played a role in this delicate switching between starch and lipid pathways. Proteins, starch, and TAG molecules are analyzed in the microalgal cells grown under different conditions with FTIR spectroscopy, a rapid, high-throughput method of biomolecular estimation. High-resolution single-cell AFM studies of the cell wall structure reveal enhanced corrugations in surface morphology during mixotrophic growth with cellulose hydrolysate, illustrating an adaptive mechanism with improved mechanical stress management. Lipid droplet morphology at the single-cell level points to two distinct mechanisms of lipid accumulation: one in which the lipids are segregated as droplets, and the other in which lipid molecules are uniformly dispersed in the cytosol as unresolved, ultra-small droplets. The present study therefore analyzes both the bulk and the single-cell level changes when cellulose hydrolysate is used as a carbon source for Chlamydomonas reinhardtii mixotrophic cultivation, which serves a four-fold purpose: value from waste, fixation of atmospheric CO2, production of lipids for biodiesel, and starch for bioethanol.

Development of a Reference Installation for Metrological Support of the Delta Value Measurements of the Ratio of Carbon and Oxygen Isotopes in Exhaled Air

Breath tests in domestic and foreign medicine are recognized as an effective and safe way to identify pathogens in the gastrointestinal tract, in particular the Helicobacter Pylori bacteria. At the end of the 20th century, a urease breath test was introduced into clinical practice to diagnose diseases of the gastrointestinal tract; it was based on the use of urea labeled with a stable isotope 13C ( 13C-urease breath test). The essence was to measure the delta value of the ratio of carbon and oxygen isotopes in exhaled air.Over decades of clinical practice of using this test, a wealth of experience related to the diagnosis of diseases caused, including by the Helicobacter Pylori bacteria has been accumulated. A significant amount of knowledge has also been accumulated in terms of metrological support of the carbon and oxygen isotope delta in exhaled air.The article describes the procedure and results of developing a development of a measurement standard of carbon and oxygen isotope delta in exhaled air, implemented within the R&D a/c No. «Sistema-2020-М» at the D. I. Mendeleyev Institute for Metrology (VNIIM). The authors presented the theoretical study, test methodology, their sequence and results. The article also contains a report on the international comparisons CCQM-P204 “Isotope ratios of CO2 (δ13VPDB and δ18VPDB) in pure CO2” with the application of the measurement standard developed by VNIIM. The metrological characteristics of the measurement standard obtained during the comparisons correspond to the most accurate measurements of the ratio of carbon and oxygen isotopes, which guarantees the recognition of the measurement results at the international level. Considering the high demand for breath tests in medical practice and obvious prospects of this type of diagnostics, further research will be aimed at creating a State Primary Standard for the delta value unit of the ratio of isotopes of carbon, oxygen, hydrogen based on the created measurement standard and the development of certified reference materials of the ratio of isotopes of carbon, oxygen, hydrogen in liquid, solid, and gaseous media, including for the purpose of replacing foreign-made materials.

Release of Exosomal PD-L1 in Bone and Soft Tissue Sarcomas and Its Relationship to Radiotherapy.

(1) Background: Exosomal PD-L1 has garnered attention owing to its role in instigating systemic immune suppression. The objective of this study is to elucidate whether bone and soft tissue sarcoma cells possess the capacity to secrete functionally active exosomal PD-L1 and whether radiotherapy (RT) induces the exosomal PD-L1 release. (2) Methods: Human osteosarcoma cell line 143B and human fibrosarcoma cell line HT1080 were utilized. Exosomes were isolated from the culture medium and blood via ultracentrifugation. The expression of PD-L1 on both tumor cells and exosomes was evaluated. The inhibitory effect on PBMC was employed to assess the activity of exosomal PD-L1. Post radiotherapy, changes in PD-L1 expression were compared. (3) Results: Exosomal PD-L1 was detected in the culture medium of tumor cells but was absent in the culture medium of PD-L1 knockout cells. Exosomal PD-L1 exhibited an inhibitory effect on PBMC activation. In tumor-bearing mice, human-derived exosomal PD-L1 was detected in the bloodstream. Following radiotherapy, tumor cells upregulated PD-L1, and human-derived exosomal PD-L1 were detected in the bloodstream. (4) Conclusions: Exosomal PD-L1 emanates from bone and soft tissue sarcoma cells and is disseminated into the circulatory system. The levels of PD-L1 in tumor cells and the release of exosomal PD-L1 were augmented after irradiation with RT.

Modification of Growth Media for Scenedesmus and Nitchzer Species for Fatty Acid Methyl Esters Production in Raceway Pond

The aim of this study was to modify the growth media for Scenedesmus and Nitchzia species for fatty acid methyl esters production in raceway pond. Native Scenedesmus and Nitchzia species were cultivated in raceway pond in three different growth media (Nitrogen-sufficient, Nitrogen-deficient and Blue Green 11) media at different periods to assess their quantitative parameters (biomass concentration (g/L), biomass productivity (g/L/d), lipid content (%), and lipid productivity (mg/L/d) as well as their fatty acid methyl esters composition. The quantitative parameters were determined following standard methods and the fatty acid methyl esters composition was analysed by Gas Chromatography Mass Spectrometry (GC-MS). The results of the quantitative parameters obtained from Scenedesmus species were highest in the Blue Green 11 (control) media as compared to the experimental media. Statistically, there is significant difference (p<0.05) in the lipid content obtained from Scenedesmus species between the Blue Green 11 media and the experimental media. The biomass concentration and lipid content obtained from Nitchzia species were also found to be highest in Blue Green 11 media as compared to the other two media. However, the lipid content obtained from Nitchzia species in the three different media showed insignificant difference (p>0.05). Scenedesmus and Nitchzia species did not respond to nitrogen deficiency condition. The Gas Chromatography Mass Spectrometry analysis of the fatty acid methyl esters composition revealed the presence of similar compounds in different proportion. The dominant compound of the fatty acid methyl esters of the two species in the three different media are Hexadecanoic acid methyl ester ((palmitic acid)), 9-octadecenoic acid (Z) methyl ester ((oleic acid) and 9, 12-octadecadienoic acid methyl ester (linolenic acid) among others. The Carbon16-Carbon18 (C16-C18) chain in Scenedesmus and Nitchzia species ranges from 74.14-61.84% and 63.81-61.98% respectively. These could suggest that both species have a great potential for the production of fatty acid methyl esters in large scale cultivation.

Evaluation of the effect of clonazepam and its metabolites on the life cycle of Megaselia scalaris (Loew) (Diptera: Phoridae)

Fly colonization patterns and development are crucial in estimating the post-mortem interval (PMI) of decomposing corpses. Understanding the potential effects of xenobiotics on species development in cadaveric entomofauna is essential for accurate PMI estimation, given their presence in decomposing bodies. Benzodiazepines, commonly prescribed for their anxiolytic, hypnotic, and muscle relaxant effects, are of forensic interest due to their potential for abuse, dependence, intoxication, and overdose-related deaths. This study aimed to explore the effect of clonazepam and its metabolites on Megaselia scalaris, a species commonly used to estimate PMI, the alteration of which could impact the accuracy of said estimation. The S9 biotransformation fraction, an in vitro model consisting of an array of metabolic enzymes, was used to generate phase I and II metabolites for evaluating their effect on M. scalaris development, representing an innovative approach to this type of study. Megaselia scalaris larvae were reared in synthetic growth media under controlled conditions. The study compared different groups: control, clonazepam, and clonazepam with S9 fraction. Larvae were measured daily to determine growth rate, and clonazepam concentrations were analyzed using HPLC-DAD. Results showed that larvae grown in media containing clonazepam or clonazepam with S9 fraction developed faster than control larvae, reaching their pupal stage earlier. Growth rates were also altered in treated groups. In conclusion, the presence of clonazepam and its metabolites accelerated the life cycle of M. scalaris, potentially impacting the accuracy of PMI estimation. These findings underscore the importance of considering xenobiotics in forensic entomological studies for precise post-mortem interval determination.

Fruit and vegetable waste used as bacterial growth media for the biocementation of two geomaterials

This paper investigates the feasibility of using randomly collected fruit and vegetable (FV) waste as a cheap growing medium of bacteria for biocementation applications. Biocementation has been proposed in the literature as an environmentally-friendly ground improvement method to increase the stability of geomaterials, prevent erosion and encapsulate waste, but currently suffers from the high costs involved, such as bacteria cultivation costs. After analysis of FV waste of varied composition in terms of sugar and protein content, diluted FV waste was used to grow ureolytic (S. pasteurii, and B.licheniformis) and also an autochthonous heterotrophic carbonic anhydase (CA)-producing B.licheniformis strain, whose growth in FV media had not been attempted before. Bacterial growth and enzymatic activity in FV were of appropriate levels, although reduced compared to commercial media. Namely, the CA-producing B.licheniformis had a maximum OD600 of 1.799 and a CA activity of 0.817 U/mL in FV media. For the ureolytic pathway, B. licheniformis reached a maximum OD600 of 0.986 and a maximum urease activity of 0.675 mM urea/min, and S. pasteurii a maximum OD600 = 0.999 and a maximum urease activity of 0.756 mM urea/min. Biocementation of a clay and locomotive ash, a geomaterial specific to UK railway embankments, using precultured bacteria in FV was then proven, based on recorded unconfined compressive strengths of 1–3 MPa and calcite content increases of up to 4.02 and 8.62 % for the clay and ash respectively. Scanning Electron Microscope (SEM) and energy dispersive X-ray spectroscopy (EDS), attested the formation of bioprecipitates with characteristic morphologies and elementary composition of calcite crystals. These findings suggest the potential of employing FV to biocement these problematic geomaterials and are of wider relevance for environmental and geoenvironmental applications involving bioaugmentation. Such applications that require substrates in very large quantities can help tackle the management of the very voluminous fruit and vegetable waste produced worldwide.

Genomic sequencing reveals convergent adaptation during experimental evolution in two budding yeast species

Convergent evolution is central in the origins of multicellularity. Identifying the basis for convergent multicellular evolution is challenging because of the diverse evolutionary origins and environments involved. Haploid Kluyveromyces lactis populations evolve multicellularity during selection for increased settling in liquid media. Strong genomic and phenotypic convergence is observed between K. lactis and previously selected S. cerevisiae populations under similar selection, despite their >100-million-year divergence. We find K. lactis multicellularity is conferred by mutations in genes ACE2 or AIM44, with ACE2 being predominant. They are a subset of the six genes involved in the S. cerevisiae multicellularity. Both ACE2 and AIM44 regulate cell division, indicating that the genetic convergence is likely due to conserved cellular replication mechanisms. Complex population dynamics involving multiple ACE2/AIM44 genotypes are found in most K. lactis lineages. The results show common ancestry and natural selection shape convergence while chance and contingency determine the degree of divergence.

Characterization and Enhanced Production of Fungal Melanin from Aspergillus Species Isolated from Soil

This study aimed to isolate and identify fungi from soil samples, focusing on their ability to produce melanin. Eleven different genera of fungi were isolated and identified using lactophenol cotton blue staining and microscopic examination, referencing H.L. Barnett and Barry B. Hunter. Among these, only Aspergillus was found to produce melanin. Optimal melanin production conditions were determined to be a 1.5% tyrosine supplement in the growth medium, under shaking conditions (120 rpm) and dark incubation for three weeks, resulting in a yield of 21.08 mg/100 ml. Physiochemical characterization revealed that the extracted melanin was insoluble in organic solvents but soluble in alkaline solutions (NaOH, KOH), and partially soluble in DMSO. Spectrometric analysis using UV-Visible spectroscopy showed characteristic absorption peaks. FTIR indicated functional groups and Scanning Electron Microscopy (SEM) images displayed a granular and heterogeneous surface topology. The study also assessed the effects of different carbon and nitrogen sources, and trace elements on melanin production. Maltose and sucrose were the most effective carbon sources, while peptone was the most effective nitrogen source. Among trace elements, calcium significantly enhanced melanin yield, while copper and zinc had moderate effects. These findings provide valuable insights into optimizing fungal melanin production and its potential industrial applications. The study highlights the sustainable and scalable production of melanin from Aspergillus, contributing to the broader understanding of fungal metabolites and their commercial exploitation. Future research should focus on genetic and metabolic pathways to further enhance melanin biosynthesis and explore its diverse applications.

Quantification of Soluplus for Dissolution Tests: SEC Method Development and Validation

The quantification of both polymer and drug during the dissolution of an amorphous solid dispersion (ASD) in aqueous media arouses great interest and may aid in the formulation. However, the available quantification methods for polymer excipients are limited, expensive, and challenging compared to drugs. In this work, a size exclusion chromatography method (HPLC-SEC) was developed and validated to determine the concentration of a frequently used polymer excipient, Soluplus® (Sol). In order to develop a method for the quantification of dissolved Soluplus®, two methods (SEC-UV and SEC-RID) with two injection volumes were tested with standard solutions of three different batches of Soluplus. The developed HPLC-SEC-UV method showed acceptable linearity (R2 > 0.9990) for all batches of Soluplus, good accuracies above a concentration of 0.1 mg/mL (coefficient of variation < 2 %), relatively good precision at a concentration of 0.1 mg/mL (coefficient of variation < 2.5 %), and high recoveries at a concentration of 0.75 mg/mL (coefficient of variation < 0.5 %). The presence of Felodipine (Fel) and Lumefantrine (Lum) in the liquid media did not interfere with Soluplus quantification. The use of various surfactants, such as Tween® 80, Tween® 20, Span® 80, Span® 20, Kolliphor® TPGS, and sodium lauryl sulphate at a low concentration (0.005 mg/mL) did not show any effect on Soluplus® and did not interfere with Soluplus® quantification with any of the Soluplus batches. The addition of lithium bromide (LiBr) to the mobile phase within a concentration range of 0.05–1.0 M did not improve Soluplus® quantification.

Comparison of the diversity of anaerobic-cultured gut bacterial communities on different culture media using 16S rDNA sequencing

The gut microbiome is a dense and diverse community of different microorganisms that deeply influence human physiology and that have important interactions with pathogens. For the correct antibiotic treatment of infections, with its twin goals of effective inhibition of the pathogen and limitation of collateral damage to the microbiome, the identification of infectious organisms is key. Microbiological culturing is still the mainstay of pathogen identification, and anaerobic species are among the most demanding bacterial communities to culture. This study aimed to evaluate the impact of growth media on the culture of an-aerobic bacteria from human stool samples. Stool samples from eight human subjects were cultured each on a yeast extract cysteine blood agar (HCB) and a modified peptone-yeast extract-glucose (MPYG) plate and subjected to Illumina NGS analysis after DNA extraction and amplification. The results showed tight clustering of sequencing samples belonging to the same human subject. Various differences in bacterial richness and evenness could be observed between the two media, with HCB plates supporting the growth of a more diverse microbial community, and MPYG plates improving the growth rates of certain taxa. No statistical significance was observed between the groups. This study highlights the importance of choosing the appropriate growth media for anaerobic bacterial culture and adjusting culture conditions to target specific pathological conditions. HCB plates are suitable for standard microbiological diagnostics, while MPYG plates may be more appropriate for targeting specific conditions. This work emphasizes the role of next-generation sequencing in supporting future research in clinical microbiology.

Overcoming the inconsistent cell culture performance caused by single-use bioreactors during scaling-up the manufacturing process for a therapeutic bispecific antibody

Single use technology has been widely used in modern biopharmaceutical process development and manufacturing. However, there are concerns about the adverse effects of disposable materials on bioprocess, product quality and patient safety. Recently, we observed slow cell growth, increased lactate accumulation, and decreased production titer during scaling up a bispecific antibody (BsAb) upstream process from 3L to 500L bioreactor. To investigate the phenomenon, we conducted medium incubation and leachable spike-in experiments. We found that cell culture medium incubated in CX5-14 bag caused poor cell culture performance, which was correlated with the concentration of bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP), a breakdown product of the antioxidant Irgafos®168 in polyethylene film. In addition, when the media were spiked with 0.15μg/ml or a greater concentration of bDtBPP, slow cell growth and reduced BsAb expression level were observed. In contrast, Aegis5-14 film produced bDtBPP at a level of below detection during medium incubation. Thus, the bispecific-expressing CHO cell line restores normal cell culture performance and production titer when Aegis5-14 single-use bioreactor was employed in large-scale manufacturing. Our results reveal the importance of evaluating leachable profiles from disposable materials during process development of bispecifics and other novel therapeutic modalities.

Green-Nano Manganese and Its Impact on the Growth, Yield, and Fruit Properties of Flame Seedless Grapes

The present work aimed to evaluate green-nano manganese produced at the Microbial Biotechnology Department, National Research Centre, Egypt, and assess its impact on the growth, yield, and fruit properties of grapevines. To this end, two experiments were conducted. The first was microbiological, where several incorporation strategies were utilized to enrich the yeast with manganese, as follows: (1) manganese was added to the liquid medium (non-growth phase), and (2) manganese was added after 24 h of incubation (growth phase). The results showed that the non-growth phase had a reduced possibility of medium contamination. The manganese concentration in the yeast cells was increased due to manganese sulfate in the medium. The manganese incorporation in yeast cells was 99.93% (1.205 × 105) higher than that of the medium at 0.0195 g/L of manganese. Although the concentration of manganese in the medium raised the optical density (OD) of the yeast cell biomass, manganese sulfate had no passive influence on it. The second experiment was horticultural, where Flame Seedless grapevines were sprayed with frozen and active fresh yeast enriched with manganese that resulted from the microbiological experiment. Grapevines were sprayed twice a year at 10 or 20 cm3/L, and the results were compared with those for the mineral and chelate forms. The results demonstrated that yeast extracts in both forms showed positive effects on grapevine. The most effective treatment with regard to growth, yield, and fruit properties was frozen yeast enriched with manganese at 20 cm3/L, which yielded 10.14 and 12.6 kg/vine, compared with the control, which recorded 3.7 and 5.6 kg/vine in the two seasons, respectively.

Green-Nano Manganese and Its Impact on the Growth, Yield, and Fruit Properties of Flame Seedless Grapes

The present work aimed to evaluate green-nano manganese produced at the Microbial Biotechnology Department, National Research Centre, Egypt, and assess its impact on the growth, yield, and fruit properties of grapevines. To this end, two experiments were conducted. The first was microbiological, where several incorporation strategies were utilized to enrich the yeast with manganese, as follows: (1) manganese was added to the liquid medium (non-growth phase), and (2) manganese was added after 24 h of incubation (growth phase). The results showed that the non-growth phase had a reduced possibility of medium contamination. The manganese concentration in the yeast cells was increased due to manganese sulfate in the medium. The manganese incorporation in yeast cells was 99.93% (1.205 × 105) higher than that of the medium at 0.0195 g/L of manganese. Although the concentration of manganese in the medium raised the optical density (OD) of the yeast cell biomass, manganese sulfate had no passive influence on it. The second experiment was horticultural, where Flame Seedless grapevines were sprayed with frozen and active fresh yeast enriched with manganese that resulted from the microbiological experiment. Grapevines were sprayed twice a year at 10 or 20 cm3/L, and the results were compared with those for the mineral and chelate forms. The results demonstrated that yeast extracts in both forms showed positive effects on grapevine. The most effective treatment with regard to growth, yield, and fruit properties was frozen yeast enriched with manganese at 20 cm3/L, which yielded 10.14 and 12.6 kg/vine, compared with the control, which recorded 3.7 and 5.6 kg/vine in the two seasons, respectively.

3D- printed modular spr platform integrated with ellipsometer for in-situ label-free optical sensing applications

The present work focuses on advancing optical sensing abilities through the instrumentation of Surface Plasmon Resonance Spectroscopy (SPR) with a commercial ellipsometer. This approach involves the incorporation of an in-house-built 3D-printed SPR module to circumvent the complexity of optical alignments with the ellipsometer. Currently, no commercially available SPR-enhanced Ellipsometry (SPRE) system exists, and previous systems are mostly custom-designed. Our work addresses this gap by introducing an in-house-built SPR module compatible with Ellipsometry and constructed using 3D printing technology. The 3D-printed SPR module is user-friendly, durable, lightweight, and cost-effective, incorporating a flow cell crucial for liquid media operation. This integrated tool provides a highly surface-sensitive, non-invasive, and label-free optical sensing platform. The developed SPR-Ellipsometry platform demonstrates excellent performance in detecting biochemical analytes and multilayer adsorption of poly-electrolytes. The bulk refractive index sensitivity measurement was performed to calibrate the Au sensor chip. The Wavelength (SPR mode), amplitude (Ψ), and phase interrogation (∆) exhibit bulk refractive index sensitivities of 3098 ± 177 nm/RIU, 835 ± 22 °/RIU and 5839 ± 229 °/RIU, respectively. We obtained the refractive index resolution around 1.712 × 10−7 RIU. Protein-protein (BSA/Anti-BSA) interactions were also conducted, achieving a detection limit of 3 pM for Anti-BSA. This work will significantly expand the functionality of SPRE sensors by offering enhanced sensitivity, rich information, and high precision, as well as opening avenues for more efficient and accessible optical sensing technologies. Moreover, the study establishes a foundational framework for researchers, shedding light on the potential of 3D printers in laboratories and expediting the development of cost-effective integrated sensing platforms.

MicroRNA-513b-5p inhibits epithelial mesenchymal transition of colon cancer stem cells through IL-6/STAT3 signaling pathway

ObjectiveTo reveal the mechanisms by which miR-513b-5p inhibits metastasis of colon cancer stem cells (CCSCs) through IL-6/STAT3 in HCT116 cells.MethodsSphere formation media and magnetic cell sorting were used to enrich and screen CCSCs. We used a colony formation assay, cell proliferation and viability assays, and a nude mouse transplantation tumor assay to identify CCSCs. ELISA was performed to identify IL-6 in the cell culture medium, and the growth, viability, wound healing, and transwell migration of distinct cell groups were compared to differentiate them. Dual-luciferase reporter assay, RT-PCR, and/or Western Blot analysis were conducted to determine the correlation between them.ResultsCD133+CD44+ HCT116 cells were shown to have higher cloning efficiency, greater proliferation ability and viability, and stronger tumorigenicity. A dual-luciferase reporter assay revealed that miR-513b-5p negatively affected STAT3 expression. RT-PCR and/or Western Blot analysis suggested that miR-513b-5p negatively affected STAT3 and Vimentin, while positively affecting E-cadherin expression. The STAT3 overexpression vector + miR-513b-5p inhibitor cell group had the highest efficiency, greatest proliferation ability and viability, and the highest IL-6 level in the experiments.ConclusionsMir-513b-5p inhibited the epithelial-mesenchymal transition (EMT) of CCSCs through IL-6/STAT3. This potential mechanism may provide a new therapeutic target for colon cancer.

Piggybacking functionalized DNA nanostructures into live-cell nuclei.

DNA origami nanostructures (DOs) are promising tools for applications including drug delivery, biosensing, detecting biomolecules, and probing chromatin substructures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing, visualizing, and controlling biomolecular processes within live cells. We present an approach to deliver DOs into live-cell nuclei. We show that these DOs do not undergo detectable structural degradation in cell culture media or cell extracts for 24 hours. To deliver DOs into the nuclei of human U2OS cells, we conjugated 30-nanometer DO nanorods with an antibody raised against a nuclear factor, specifically the largest subunit of RNA polymerase II (Pol II). We find that DOs remain structurally intact in cells for 24 hours, including inside the nucleus. We demonstrate that electroporated anti-Pol II antibody-conjugated DOs are piggybacked into nuclei and exhibit subdiffusive motion inside the nucleus. Our results establish interfacing DOs with a nuclear factor as an effective method to deliver nanodevices into live-cell nuclei.

In vitro cultivation of adherent cells on microscope slides for downstream applications

In vitro studies with cultured cells are often conducted as an important part of basic research. Adherent cells are typically cultivated in flasks or trays, for which cell staining and subsequent visualization become impractical. We here present a simple step-by-step method for growing adherent cells directly on glass microscope slides, using low-cost equipment readily available in most laboratories. Most parameters such as type of microscope slide (e.g. surface coating), cell seeding concentrations and incubation times can be adjusted according to cell line characteristics and experimental aims, reflecting the methods’ flexibility. Through our experiments, microscope slides proved to provide an acceptable surface for cell adhesion and growth of the tested cell lines, as well as being robust and functional with respect to downstream procedures. The method can potentially be combined with different techniques for visualization of experimental effects, such as histological staining methods, fluorescent staining, and immunochemistry. In our method development we have successfully cultivated three different cell lines directly on microscope slides – Atlantic salmon kidney cells (ASK), rainbow trout gill cells (RTgill-W1), and human cancerous lung cells (A549) – and subjected them to various experimental treatments. Finally, as proof-of-concept we provide examples of successful histological staining of the fixed cells.Experimental design in short:• Cultivate cells and calculate cell concentration• Seed a small volume of growth medium with an appropriate number of cells on microscope slide in an area confined by hydrophobic marker• Let cells adhere over night before adding more growth medium or directly conducting experiments and fixing cells for downstream applications