Growth Medium Research Articles

Enhancing Microalgal Biomass and Chemical Composition for Sustainable Wastewater Treatment

The research explores enhancing microalgal biomass production and its chemical composition for sustainable wastewater treatment. By using the Central Composite Design (CCD) methodology, nutrient concentrations and microbial components in the growth medium were optimized. Adjustments to light exposure, particularly with red and blue frequencies, significantly improved biomass yield, carbon sequestration, and protein content. Optimal light intensity further enhanced these parameters. A process incorporating on-demand CO2 supply and controlled pH levels showed potential for increasing biomass concentrations and nutrient utilization efficiency. Results demonstrated a 28% increase in biomass fixation and a 27% increase in biomass production compared to unoptimized conditions. The study emphasizes the effectiveness of integrated approaches in boosting biomass production and optimizing wastewater treatment, offering promising avenues for sustainable bio-based solutions

In vitro acute gamma radiation on tissue of pink and white lotus (Nelumbo nucifera Gaertn.) in Thailand

This research aimed to investigatethe age of embryos of pink and white lotus for in vitropropagation and examine the sensitivity of young lotus plants derived from tissue culture to acute gamma radiation. The seeds of both lotus varieties were 4-week, 6-week and 8-week-old embryos cultivated on solid MS medium. They were then transferred to solid MS medium supplemented with 3 mg/L BA. After culturing, the 6-week-old pink lotus embryos had an average maximum petiole length of 16.78 ± 1.01 cm and an average of 11.84 ± 0.44 roots/explant, while the 6-week-old white lotus embryos had an average maximum petiole length of 15.48 ± 0.68 cm and an average of 11.76 ± 0.70 roots/explant. Each young lotus plant produced one shoot. After treating the young lotus plants with liquid MS medium supplemented with 3 mg/L BA, both lotus varieties showed increased shoot numbers at 3.54 ± 0.13 and 3.40 ± 0.91 shoots/explant. Young lotus plants of both varieties were exposed to gamma radiation levels of 0, 20, 30 and 40 Gy. The LD50 value for pink lotus was 26.138 Gy, while the LD50 value for the white lotus was 32.031 Gy.The LD50 value will offer vital information for determining the optimal dosage of gamma radiation for both pink and white lotus in vitro.

Micropropagation of Zuccagnia punctata, an argentine medicinal plant, by means of “in vitro” technology to promove its conservation and the sustainable production of bioactive compounds

Zuccagnia punctata Cav. (“jarilla poposa”) is a monotypic argentine native medicinal plant used by indigenous communities for the treatment of several pathologies. The pharmacological properties have been scientifically validated and its potential use in phytocosmetic and pharmaceutic industry as antiaged, hypoglycemic, anti-inflammatory, antitumoral, antioxidant, antibacterial and antimicotic has been previously demonstrated. However, the species has a low propagation rate in natural conditions and has been included in the preliminary red list of endangered plants in category 3. Therefore, it is necessary to develop methodologies leading to its conservation and propagation to achieve its sustainable use. The objective of this work is to perform a micropropagation protocol for Z. punctata species for the sustainable use and conservation of this species. Furthermore, the study evaluated also the impact of “in vitro” propagation on the content of bioactive compounds of ethanolic extracts obtained from Z. punctata plants obtained “in vitro” with collected wild plants. Seeds have been used as mother material. The seed germination to obtain explants were performed on Murashige and Skoog medium (MS) without plant growth regulators (PGRs) containing sucrose, agar, and ascorbic acid. The same medium was employed for the multiplication of shoots. “In vitro” rooting was achieved in liquid and semisolid medium, and a higher yield was obtained in the latter. The plants were transferred into commercial substrate and acclimatized in a greenhouse. The micropropagated plants (MP) had a high content of total phenolic compounds similar to those obtained from wild adult plants (WAP) collected in the Monte Desert. The flavonoid content of MP was higher than that of WAP. The main chalcones identified in WAP, 2′,4′ dihydroxychalcone (DHC) and 2′,4′-dihydroxy-3′-methoxychalcone (DHMC) were also present in the MP with a rate 1/1, and 1/3 in WAP. The extracts obtained from the MP showed antioxidant, antiinflammatory and antimicrobial activity, similar to WAP. The “in vitro” propagation and acclimatization of Z. punctata constitutes a fundamental tool to achieve large-scale multiplication of this species in the future and the sustainable production of bioactive compounds with commercial applications. Reimplantation in the natural habitat could prevent the loss of this medicinal genetic resource.

Strep Easy Kit; a bio-enrichment dual ICG-strip test for simultaneous detection of Streptococcus agalactiae serotypes Ia and III in fish samples.

The Strep Easy Kit, a bio-enrichment dual ICG-strip test, is a diagnostic tool designed for the detection of Streptococcus agalactiae, an important pathogenic bacterium in tilapia farming. The kit can simultaneously identify two different serotypes of S. agalactiae, serotype Ia and serotype III. This capability is crucial for the collection of valuable epidemiological data and facilitates strategic planning for effective vaccine development and deployment. The Strep Easy Kit consists of two main steps: pathogen enrichment and pathogen detection. The enrichment step increases the concentration of bacteria so that the bacterial load is raised to the level reliably detectable by the subsequent ICG strip test. This is achieved by incubating the fish samples in a suitable liquid medium under specified temperature and time conditions. The second step involves the use of the dual-ICG strip test. This strip test consists of two monoclonal antibodies and one polyclonal antibody that are specific to S. agalactiae and can distinguish between S. agalactiae serotype Ia and S. agalactiae serotype III. This dual capability enables the ICG strip test to simultaneously detect both serotypes of S. agalactiae in a single test kit. The detection limit of the test kit, which consists of a dual ICG-Strip test combined with an enrichment step, is 100 CFU/mL. The kit can be used to detect S. agalactiae in both live and dead fish samples, making it versatile for various testing scenarios. The test results obtained using the Strep Easy Kit have shown a 94.4% correlation with the standard method (Thai Agricultural Standard; TAS 10453-2010), with 90.2% sensitivity and 100% specificity. Significant advantages of the Strep Easy Kit lie in its simplicity and portability, allowing farmers to perform the test by themselves and on-site. This makes it a practical and accessible tool for the tilapia farming industry.

Towards sustainable biocontrol: inhibition of soil borne fungi by microalgae from harsh environments

Using microorganisms as biocontrol agents against soilborne plant pathogens is a promising alternative to chemical pesticides. However, only some biocontrol agents have proven effective under field conditions. This study explores the potential of highly resilient microalgae isolated from harsh environments, such as Biological Soil Crusts and agricultural fields in semi-arid regions, as a novel and sustainable approach to biocontrol. Fifty-nine microalgal strains, including thirteen cyanobacteria and forty-six green algae, were isolated and identified. Dual-culture plate assays and toxicity tests of microalgal growth media were conducted to evaluate the antifungal activity of the isolates against eight representative soilborne pathogens. The results showed that many microalgae strains exhibited significant inhibitory effects on the growth of specific fungal pathogens, although the activity varied among different microalgal strains and pathogen species. Some strains even promoted the growth of certain fungi. The lack of a clear pattern in the antifungal activity highlights the complexity and specificity of the interactions between microalgae and soilborne pathogens. An “Inhibition Effectiveness” metric was developed to quantify biocontrol potential based on fungal growth inhibition. The green algal genus Desmodesmus, particularly Desmodesmus subspicatus isolates, showed higher antifungal efficacy than other genera. While the inhibitory mechanisms remain unclear, the results demonstrate the promising biocontrol capabilities of microalgae from extreme environments like BSCs. Further research could unlock novel opportunities for sustainable disease management by harnessing specific microalgal strains or synergistic strain combinations targeting soilborne pathogens.

Effect of Tomato Grafting onto Novel and Commercial Rootstocks on Improved Salinity Tolerance and Enhanced Growth, Physiology, and Yield in Soilless Culture

Grafting high-yielding tomato varieties onto stress-tolerant rootstocks can mitigate the adverse effects of saline water irrigation on plant tomato productivity in arid regions like Saudi Arabia. This study investigates the efficacy of grafting tomatoes onto both novel and commercial rootstocks to enhance salinity tolerance and its impact on growth, physiological parameters, and yield in a soilless culture system. The experiment involved two water quality levels, 2 (S1) and 4 (S2) dS m−1, two growth media types, volcanic rock (M1) and sand (M2), and six grafting treatments: Tone Guitar F1 non-grafted (G1) (commercial scion), grafted onto itself (G2), Tone Guitar F1* Maxifort F1 (G3) (commercial rootstock), and grafting the scion onto three novel rootstocks, X-218 (G4), X-238 (G5), and Alawamiya365 (G6). Growth, physiology, photosynthetic pigments, and yield improved with lower salinity (2 dS m−1) in volcanic rock and with the grafting treatments (G2–G6) compared to the non-grafted treatment (G1). The best results were achieved with the S1M1G5 treatment, where yield increased by 53% compared to the lowest yield in non-grafted plants grown in sand under higher salinity (S2M2G1). All studied traits were adversely affected under high salinity (S2) in sandy media, with the G1 treatment resulting in the lowest values for these traits.

Establishment of Physalis alkekengi cell suspension culture: time-dependent behavior of genes related to the steroidal compounds, key enzymes, and physalins under static magnetic field.

Cell suspension culture has the potential to be a valuable source for the bioactive compound productions. In this study, an optimized procedure was established for callus and cell suspension culture of Physalis alkekengi for the first time, and the impact of static magnetic field (SMF, 6 mT) was studied on the high-value metabolic compounds through investigation of signaling molecules and gene expressions at the late log-to-stationary phase. Results showed that the growth regulators of 6-benzyl amino purine (BAP, 1.5mg-1 L) and 1-naphthaleneacetic acid (NAA, 0.4mg-1 L) induced the highest fresh weight, callus rate, callus index, and total withanolides. Cell suspension culture was established in the liquid MS medium supplied with BAP (1.5mg-1 L) and NAA (0.1mg-1 L). SMF application decreased slightly the cell growth and viability and enhanced the number of round-shaped cells. The hydrogen peroxide (H2O2) and nitric oxide (NO) levels increased at an all-time series after SMF exposure, and their maximum contents were observed after 12h. A significant alteration of malondialdehydecontent was also identified after 12h of SMF exposure. The expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), 1-deoxyD-xylulose 5-phosphate synthase (DXS), squalene synthase (SQS), sterol Δ7-reductase (DWF5), and C-7,8 sterol isomerase (HYD1) genes was upregulated significantly after 24 and 48h. An increase in the total withanolides was related to more activity of HMGR and DXS enzymes in SMF-exposed cells and the maximum physalin A (12.8mgg-1 DW) and physalin B (1.92mgg-1 DW) obtained after 24h compared to controls. Findings suggest that SMF can play a supportive factor in inducing steroidal compounds in P. alkekengi through modulating H2O2 and NO levels and the related-gene expressions.

Twenty Years in EUCAST Anti-Fungal Susceptibility Testing: Progress & Remaining Challenges.

Since its inception in 2002, the EUCAST Antifungal Susceptibility Testing Subcommittee (AFST) has developed and refined susceptibility testing methods for yeast, moulds and dermatophytes, and established epidemiological cut-off values and breakpoints for antifungals. For yeast, three challenges have been addressed. Interpretation of trailing growth in fluconazole susceptibility testing, which has been proven without impact on efficacy if below the 50% endpoint. Variability in rezafungin MIC testing due to laboratory conditions, which has been solved by the addition of Tween 20 to the growth medium in E.Def 7.4. And third, interpretation of MICs for rare yeast with no breakpoints, where recommendations have been established for MIC-based clinical advice. For moulds, refinements include the validation of spectrophotometer reading for A. fumigatus to facilitate objective MIC determination, and for dermatophytes the establishment of a microdilution method with automated reading and a selective medium to minimise the risk of contaminations. Recent initiatives involve development and validation of agar-based screening assays for detection of potential azole and echinocandin resistance in A. fumigatus and Aspergillus species, respectively, and of terbinafine resistance in Trichophyton species. Moreover, the development of a EUCAST guidance document for molecular resistance testing represents an advancement, particularly for identifying target gene alterations associated with resistance. In summary, EUCAST AFST continues to play a pivotal role in standardizing AFST and facilitating accurate interpretation of susceptibility data for clinical decision-making. Adoption of EUCAST breakpoints for commercial test methods, however, requires thorough validation to ensure concordance with EUCAST reference testing species-specific MIC distributions.

Nicotinamide riboside Induced Energy Stress and Metabolic Reprogramming in BEAS-2B Cells.

Nicotinamide riboside (NR), a NAD+ precursor, has received attention due to several health benefits it has induced in experimental models. Studies in cultured cells, animals, and humans consistently show increased NAD+ availability after NR supplementation, which is considered the only mode of NR action that leads to health benefits. In the present study, we show that a persistently low NR concentration (1 μM) in the growth medium of BEAS-2B human cells, grown in a monolayer, induces energy stress, which precedes a cellular NAD+ increase after 192 h. NR concentrations greater than 1 μM under the specified conditions were cytotoxic in the 2D cell culture model, while all concentrations tested in the 3D cell culture model (BEAS-2B cell spheroids exposed to 1, 5, 10, and 50 μM NR) induced apoptosis. Shotgun proteomics revealed that NR modulated the abundance of proteins, agreeing with the observed effects on cellular energy metabolism and cell growth or survival. Energy stress may activate pathways that lead to health benefits such as cancer prevention. Accordingly, the premalignant 1198 cell line was more sensitive to NR cytotoxicity than the phenotypically normal parent BEAS-2B cell line. The role of a mild energy stress induced by low concentrations of NR in its beneficial effects deserves further investigation. On the other hand, strategies to increase the bioavailability of NR require attention to toxic effects that may arise.

First Report of bacterial wilt disease of banana caused by Klebsiella variicola in Malaysia.

In Malaysia, bananas (Musa spp.) are the second most cultivated fruit and the fourth most cultivated fruit in terms of export revenue. In October 2018, about 5.0 out of 6.6 hectares of a banana plantation located in Teluk Intan, Malaysia, was impacted by an outbreak of banana disease. The onset of bacterial wilt symptoms is characterized by initial leaf wilting, followed by the subsequent withering of the entire plant during later stages, fruit stalk and fruit pulp discoloration, fruit rotting, and pseudostem necrosis. The diseased banana's symptomatic pseudostems and fruit pulps were surface-sterilised in 70% ethanol for 30 s, followed by 2% NaClO for 3 min, rinsed three times in sterilised water, and cut into small pieces approximately 5 mm2 in size. The tissues were macerated in a sterilised 0.85% NaCl solution for 5 min, and the resulting suspension was streaked onto nutrient agar, followed by incubation at 28°C for 2 days. After incubation, bacterial colonies with five unique morphological characteristics were observed. Two colonies of each unique morphological type were randomly chosen and subjected to preliminary bacterial identification by 16S rRNA gene sequencing. Based on BLASTn analysis, the five unique morphological types of bacteria were preliminarily identified as Enterobacter cloacae, Citrobacter farmeri, Klebsiella variicola, Kosakonia radicincitans, and Phytobacter ursingii. Previous reports identified K. variicola and K. radicincitans as banana pathogens, but Malaysia has yet to report the former. The amplified partial 16S rDNA sequences of both K. variicola isolates (designated as UTAR-BC1 and UTAR-BC2; GenBank accession numbers: PP531448 and PP531460, respectively), which were chosen to be the focus of this study, exhibited complete similarity to each other and were 100% identical (1426/1426 identity and 1420/1420 identity, respectively) to K. variicola (CP026013.1). To verify the identity of the bacterial isolate, three housekeeping genes, namely, infB(PP538994), rpoB (PP538995), and gyrB (PP538996) of UTAR-BC1, were amplified, sequenced, and subjected to multilocus phylogenetic analysis via the neighbour-joining method (1,000 bootstrap values). Phylogenetic analysis revealed that UTAR-BC1 belongs to the K. variicola clade. A pathogenicity assay of UTAR-BC1 was conducted on 4-month-old healthy banana plantlets (cv. Nangka) using the pseudostem injection method (Tripathi et al., 2008). First, UTAR-BC1 was grown overnight in nutrient broth and then adjusted to 108 CFU/ml in a sterile 10 mM MgCl2 solution. A total volume of 100 µL of the bacterial suspension was injected into the pseudostem of five healthy banana plantlets via a syringe with a needle. Control plants were mock-inoculated with a sterile 10 mM MgCl2 solution. The experiments were replicated thrice and inoculated plants were maintained at room temperature with natural sunlight and humidity, which resembled the field conditions. Two months after inoculation, all of the UTAR-BC1 inoculated spots of banana plantlets showed severe necrosis, while the banana leaves showed symptoms of wilted appearance, whereas the control plants remained symptomless. The reisolated pathogen from 90% of the symptomatic pseudostems and leaf blades shares the same morphological and molecular features as UTAR-BC1, thus fulfilling Koch's postulates. Previously, K. variicola has been reported to be a banana pathogen causing rhizome rot in India (Loganathan et al., 2021), plantain soft rot in Haiti (Fulton et al. 2020), and sheath rot and bulb rot in China (Sun et al., 2023; Jiang et al., 2024). To the best of our knowledge, this is the first report of bacterial wilt disease in bananas attributed to K. variicola in Malaysia. This finding will facilitate the surveillance of K. variicola as an emerging pathogen in banana plants in this region, thereby safeguarding the country's food security and promoting socio-economic growth.

Cultivation of Diverse Novel Marine Bacteria from Deep Ocean Sediment Using Spent Culture Supernatant of Ca. Bathyarchaeia Enrichment.

Most microorganisms resist pure cultivation under conventional laboratory conditions. One of the primary issues for this un-culturability is the absence of biologically produced growth-promoting factors in traditionally defined growth media. However, whether cultivating microbes by providing spent culture supernatant of pivotal microbes in the growth medium can be an effective approach to overcome this limitation is still an under-explored area of research. Here, we used the spent culture medium (SCM) method to isolate previously uncultivated marine bacteria and compared the efficiency of this method with the traditional cultivation (TC) method. In the SCM method, Ca. Bathyarchaeia-enriched supernatant (10%) was used along with recalcitrant organic substrates such as lignin, humic acid, and organic carbon mixture. Ca. Bathyarchaeia, a ubiquitous class of archaea, have the capacity to produce metabolites, making their spent culture supernatant a key source to recover new bacterial stains. Both cultivation methods resulted in the recovery of bacterial species from the phyla Pseudomonadota, Bacteroidota, Actinomycetota, and Bacillota. However, our SCM approach also led to the recovery of species from rarely cultivated groups, such as Planctomycetota, Deinococcota, and Balneolota. In terms of the isolation of new taxa, the SCM method resulted in the cultivation of 80 potential new strains, including one at the family, 16 at the genus, and 63 at the species level, with a novelty ratio of ~ 35% (80/219). In contrast, the TC method allowed the isolation of ~ 10% (19/171) novel strains at species level only. These findings suggest that the SCM approach improved the cultivation of novel and diverse bacteria.

Rosmarinic Acid Content and Antioxidant Activity in Ehretia asperula Zollinger et Moritzi Cell Suspension Cultures

Background: Ehretia asperula Zollinger et Moritzi is a medicinal tree abundant in rosmarinic acid, the primary phenolic component, with numerous beneficial biological properties, including antioxidant, antibacterial, antiviral, anti-allergy, anti-arthritis, asthma and anti-cancer. Methods: The cell suspension cultures of E. asperula Zollinger et Moritzi derived from the leaf in vitro callus were established in liquid B5 medium added with 0.4 mg/L NAA, 0.1 mg/L BA and 45 g/L glucose. After four weeks, 50 mg/L of chitosan was given to the cell suspension cultures to stimulate rosmarinic acid (RA) production after a 48-hour treatment period. RA content was analyzed using the HPLC and spectrophotometry method after four weeks and 48 hours of chitosan treatment. In addition, the antioxidant capacity of the extracts from E. asperula Zollinger et Moritzi was also tested using DPPH free radical scavenging assay. Result: The RA content and antioxidant capacity of E. asperula Zollinger et Moritzi’ extracts from the leaf in vitro greater than callus derived from the leaf in vitro greater than field-grown leaf greater than biomass of cell suspension cultures. These results suggested a strong correlation between RA concentration and antioxidant capacity. The use of E. asperula Zollinger et Moritzi cell suspension cultures with chitosan as an elicitor for RA production and evaluation of antioxidant activity is presented in this study for the first time. Our results suggest that cell suspension cultures and others may be a good source of RA, an antioxidant compound.

The Use Of Droplet-Based Microfluidic Technologies For Accelerated Selection Of Yarrowia lipolytica and Phaffia rhodozyma Yeast Mutants

Abstract Microorganisms are widely used for the industrial production of various valuable products such as pharmaceuticals, food and beverages, biofuels, enzymes, amino acids, vaccines, etc. Research is constantly carried out to improve their properties, mainly to increase their productivity and efficiency and reduce the cost of the processes. The selection of microorganisms with improved qualities takes a lot of time and resources (both human and material); therefore, this process itself needs optimisation. In the last two decades, microfluidics technology appeared in bioengineering, which allows for manipulating small particles (from tens of microns to nanometer scale) in the flow of liquid in microchannels. The technology is based on small-volume objects (microdroplets from nano to femtoliters), which are manipulated using a microchip. The chip is made of an optically transparent inert to liquid medium material and contains a series of channels of small size (< 1 mm) of certain geometry. Based on the physical and chemical properties of microparticles (like size, weight, optical density, dielectric constant, etc.), they are separated using microsensors. The idea of accelerated selection of microorganisms is the application of microfluidic technologies to separate mutants with improved qualities after mutagenesis. This paper discusses the possible application and practical implementation of microfluidic separation of mutants, including yeasts like Yarrowia lipolytica and Phaffia rhodozyma after chemical mutagenesis will be discussed.

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.