Addition Of Substrate Research Articles

The influence of substrates addition order on colorimetric assay based on MnO2 nanocubes: A novel turn-off H2O2 assay strategy in water-soak foods.

Herein, novel MnO2 nanocubes were facilely synthesized by manipulating the dosage of KMnO4 and l-Dopa with the aid of ultrasound. The as-prepared MnO2 nanocubes exhibited interesting oxidation activity which was influenced by the addition order of substrates (H2O2 and 3,3,5,5-tetramethylbenzidine (TMB)). After the mechanism study, we found that H2O2 and TMB can be competitively oxidized by MnO2 nanocubes, which was different from the peroxidase- and oxidase-like activities. According to the discovery, a novel turn-off H2O2 assay method based on MnO2 nanocubes was established, where H2O2 was firstly incubated with MnO2 nanocubes for 3min and then TMB was added for the instantaneous chromogenic reaction. In addition to the shorter operation time, the colorimetric results were less affected by temperature and unchanged within 30min without terminating reaction. Moreover, the method showed ultra-high sensitivity with low limit of detection (0.027μmol L-1) and acceptable reliability for H2O2 assay in water-soak foods.

Agricultural runoff treatment by constructed wetlands filled with iron-carbon composites in winter: Performance augmentation by organic solids and denitrifying bacteria addition

Iron-carbon composite-filled constructed wetlands (Fe-C CWs) were employed to treat agricultural runoff in the winter season in this study, and organic substrates and phosphate-accumulating denitrifying bacteria were supplemented to improve the treatment performance. Fe-C CWs performed significantly better in pollutant removal than the control system filled with only gravel by effectively driving autotrophic denitrification, Fe-based dephosphorization and organic degradation. Organic substrate and functional bacteria addition further augmented the performance, and immobilized bacterial cells were more effective than free cells. Fe-C and organic substrates decreased the greenhouse gas emission fluxes of the CWs, and denitrifier inoculation alleviated N2O emission. The microbial community in the Fe-C substrates showed a very distinct distribution pattern compared to that in the gravel, with notably higher proportions of Trichococcus, Thauera and Dechloromonas. Bioaugmented Fe-C-based CWs are highly promising for agricultural runoff treatment, especially at low temperatures.

NHC-catalyzed enantioselective access to β-cyano carboxylic esters via in situ substrate alternation and release

A carbene-catalyzed asymmetric access to chiral β-cyano carboxylic esters is disclosed. The reaction proceeds between β,β-disubstituted enals and aromatic thiols involving enantioselective protonation of enal β-carbon. Two main factors contribute to the success of this reaction. One involves in situ ultrafast addition of the aromatic thiol substrates to the carbon-carbon double bond of the enal substrate. This reaction converts almost all enal substrate to a Thiol-click Intermediate, significantly reducing aromatic thiol substrates concentration and suppressing the homo-coupling reaction of enals. Another factor is an in situ release of enal substrate from the Thiol-click Intermediate for the desired reaction to proceed effectively. The optically enriched β-cyano carboxylic esters from our method can be readily transformed to medicines that include γ-aminobutyric acids derivatives such as Rolipram. In addition to synthetic utilities, our control of reaction outcomes via in situ substrate modulation and release can likely inspire future reaction development.

Effects of substrate improvement on winter nitrogen removal in riparian reed (Phragmites australis) wetlands: rhizospheric crosstalk between plants and microbes.

With continued anthropogenic inputs of nitrogen (N) into the environment, non-point source N pollutants produced in winter cannot be ignored. As the water-soil interface zones, riparian wetlands play important roles in intercepting and buffering N pollutants. However, winter has the antagonistic effect on the N removal. Substrate improvement has been suggested as a strategy to optimize wetland performance and there remain many uncertainties about the inner mechanism. This study explores the effects of substrate improvement on N removal in winter and rhizospheric crosstalk between reed (Phragmites australis) and microbes in subtropical riparian reed wetlands. The rates of wetland N removal in winter, root metabolite profiles, and rhizosphere soil microbial community compositions were determined following the addition of different substrates (gravel, gravel + biochar, ceramsite + biochar, and modified ceramsite + biochar) to natural riparian soil. The results showed that the addition of different substrates to initial soil enhanced N removal from the microcosms in winter. Gravel addition increased NH4+-N removal by 8.3% (P < 0.05). Gravel + biochar addition increased both TN and NH4+-N removals by 8.9% (P < 0.05). The root metabolite characteristics and microbial community compositions showed some variations under different substrate additions compared to the initial soil. The three treatments involving biochar addition decreased lipid metabolites and enhanced the contents and variety of carbon sources in rhizosphere soil, while modified ceramsite + biochar addition treatment had a greater impact on the microbial community structure. There was evidence for a complex crosstalk between plants and microbes in the rhizosphere, and some rhizosphere metabolites were seen to be significantly correlated with the bacterial composition of the rhizospheric microbial community. These results highlighted the importance of rhizospheric crosstalk in regulating winter N removal in riparian reed wetland, provided a scientific reference for the protection and restoration of riparian reed areas and the prevention and control of non-point source pollution.

Effect of zeolite–sponge iron combined substrate on microbial community in ecological floating bed

Substrate has been proven to be a useful way to enhance the purification effect of ecological floating bed (EFB), and it also affected the microbial community. While at cold temperature, there was a lack of research on the effect of substrates on plant growth and microbial communities. In this study, three EFBs, namely plant EFB (EFB-P), substrate EFB (EFB-S) and plant–substrate EFB (EFB-PS), were constructed. The effects of zeolite–sponge iron combined substrate on pollutant removal, plant growth and microbial community in the EFBs were investigated at low temperature (3.1 ∼ 11.7 °C). Results showed that the removal efficiencies of total nitrogen, total phosphorus and nitrate nitrogen by EFB-PS (60.35%, 95.09% and 64.43%) was significantly better than that of EFB-P (47.71%, 88.59% and 51.65%), but the relative growth of the aboveground biomass of plants (50.41%) was less than that of EFB-P (81.16%). In addition, the microbial biomass in EFB-PS (69.14 mg/(g DW·day)) and EFB-S (68.76 mg/(g DW·day)) was much larger than that in EFB-P (50.69 mg/(g DW·day)). High-throughput sequencing showed that the microbial diversity (Shannon (6.06 ∼ 8.33) and Simpson (0.88 ∼ 0.97)) and richness (Chao1 (1621.7 ∼ 2556.2) and Observed _ species (1437 ∼ 2423.3)) of EFB with adding zeolite–sponge iron combined substrate were also higher than those of EFB-P. In addition, the addition of combined substrates changed the microbial community structure, some genera with denitrification and nitrogen fixation functions, including Rhodobacter (36.03 ∼ 48.59%), Hydrogenopha (2.21 ∼ 5.89%), were abundantly enriched in the EFB with adding zeolite-sponge iron combined substrate. These results improved the effects of substrate low temperature operation on plant growth and microbial characteristics in EFB, and provide a theoretical basis for strengthening the operation of the low temperature conditions of the EFB.

Utilization of Lignocellulosic Substrates on Oyster Mushroom (Pleurotus citrinopileatus) Cultivation

Background: Mushroom cultivation increased throughout the world with the utilization of crop residues. Oyster mushroom (Pleurotus spp.) is suitable for most of the area and less expensive for cultivation with the availability of paddy straw. Increasing demand of paddy straw compels to search alternate source of substrate for mushroom production. Based on this information efforts were taken to compare many locally available leaf substrates for Pleurotus citrinopileatus production. Methods: Leaf substrates viz., teak, bamboo, maize, banana, coconut, mango, sugarcane bagasse and grass (Dactyloctenium aegyptium) were tested for the cultivation of P. citrinopileatus. Grain substrates viz., paddy, cumbu, bengal gram, green gram, sorghum, black gram, ragi and the above leaf substrates were studied for their effect on radial mycelial growth and dry weight of P. citrinopileatus. Result: All the leaf substrates were on par with each other for radial mycelial growth. Highest mycelial dry weight of 724.67 mg was recorded in paddy straw. Among the different leaf substrates used for mushroom bed preparation, banana leaf substrates showed highest yield of 115.00 g than paddy straw. Weight of mushroom spent bed was recorded to observe the utilization of substrates in which bamboo was decomposed at the maximum. Addition of grain substrates enhanced the growth of mycelium.

Optimal transport of active particles induced by substrate concentration oscillations.

We show the existence of a stochastic resonant regime in the transport of active colloidal particles under confinement. The periodic addition of substrate to the system causes the spectral amplification to exhibit a maximum for an optimal noise level value. The consequence of this is that particles can travel longer distances with lower fuel consumption. The stochastic resonance phenomenon found allows the identification of optimal scenarios for the transport of active particles, enabling them to reach regions that are otherwise difficult to access, and may therefore find applications in transport in cell membranes and tissues for medical treatments and soil remediation.

The Effect of Addition of Various Media Substrates of Local Microorganisms (MOL) to Nutrient Content and Cyanide Acid Level Anaerobic Fermentation of Cassava Peels (Manihot utilissima)

ABSTRAK Keterbatasan hijauan pakan menyebabkan perlunya pakan alternatif untuk memenuhi kebutuhan ternak, salah satu pakan alternatif yang dapat digunakan adalah kulit singkong terfermentasi. Penelitian ini bertujuan untuk mengetahui pengaruh penambahan berbagai media substrat mikroorganisme lokal (MOL) terhadap kandungan nutrien dan asam sianida fermentasi anaerobik kulit singkong. Penelitian dilaksanakan pada bulan September-Desember 2022 di Laboratorium Fakultas Pertanian Universitas Tidar, Laboratorium CV. Chemmix, Bantul, Yogyakarta, dan di Laboratorium Institut Pertanian Bogor. Penelitian menggunakan metode rancangan acak lengkap. Kulit singkong diberi 4 perlakuan dan 5 ulangan, yang terdiri dari (P0) kulit singkong + 1% Effective micro-organism EM4 1%; (P1) kulit singkong + 1% MOL1; (P2) kulit singkong + 1% MOL2; (P3) kulit singkong + MOL3; dan (P4) kulit singkong + MOL4. Data dari hasil penelitian dianalisis menggunakan analisis ragam, jika hasilnya berbeda nyata dilanjutkan menggunakan uji Dunnet. Hasil penelitian menunjukkan bahwa penambahan berbagai MOL memberikan pengaruh nyata (P&lt;0,05) terhadap kandungan protein kasar dan HCN, namun tidak memberikan pengaruh (P&gt;0,05) terhadap kadar air dan abu pada fermentasi kulit singkong. Kata Kunci: Kulit singkong, air cucian beras, air rebusan kedelai, air limbah ampas tahu ABSTRACT Limited forage causes the need for alternative feed to meet the needs of livestock, one of the alternative feeds that can be used is fermented cassava peels. This study aimed to determine the effect of adding various media substrates of local microorganisms (MOL) on the nutrient and cyanide acid content of cassava peels anaerobic fermentation. The research was conducted in September-December 2022 at the Laboratory of the Faculty of Agriculture, Tidar University, CV. Chemmix Laboratory, Bantul, Yogyakarta, Bogor Agricultural Institute Laboratory. The study used a completely Randomize Method. Cassava peels were given 4 treatments and 5 replications, namely (P0) cassava peels + 1% EM4; (P1) cassava peels + 1% MOL1; (P2) cassava peels + 1% MOL 2; (P3) cassava peels + 1% MOL3. Data were analyzed using a Completely Randomized Design (CRD) and continued with the Dunnet test. The results showed that the addition of MOL had a significant effect (P&lt;0.05) on crude protein content and HCN content but had no significant effect (P&gt;0.05) on water content and ash content in the anaerobic fermentation of cassava peels. Keywords: Cassava peels, rice-washing water, soybean-boiled water, tofu wastewater

Bioconversion of vitamin D3 into calcitriol by Actinomyces hyovaginalis isolate CCASU- A11-2

Vitamin D3 is a fat-soluble prohormone that is activated inside the liver to produce 25-hydroxyvitamin D3 (calcidiol), and in the kidney to produce the fully active 1α, 25-dihydroxy vitamin D3 (calcitriol). A previous work piloted in our laboratory, resulted in a successful recovery of a local soil-promising Actinomyces hyovaginalis isolate CCASU-A11-2 capable of converting vitamin D3 into calcitriol. Despite the rising amount of research on vitamin D3 bioconversion into calcitriol, further deliberate studies on this topic can significantly contribute to the improvement of such a bioconversion process. Therefore, this work aimed to improve the bioconversion process, using the study isolate, in a 14 L laboratory fermenter (4 L fermentation medium composed of fructose (15 g/L), defatted soybean (15 g/L), NaCl (5 g/L), CaCO3 2 g/L); K2HPO4, (1 g/L) NaF (0.5 g/L) and initial of pH 7.8) where different experiments were undertaken to investigate the effect of different culture conditions on the bioconversion process. Using the 14 L laboratory fermenter, the calcitriol production was increased by about 2.5-fold (32.8 µg/100 mL) to that obtained in the shake flask (12.4 µg/100 mL). The optimal bioconversion conditions were inoculum size of 2% v/v, agitation rate of 200 rpm, aeration rate of 1 vvm, initial pH of 7.8 (uncontrolled); addition of vitamin D3 (substrate) 48 h after the start of the main culture. In conclusion, the bioconversion of vitamin D3 into calcitriol in a laboratory fermenter showed a 2.5-fold increase as compared to the shake flask level where, the important factors influencing the bioconversion process were the aeration rate, inoculum size, the timing of substrate addition, and the fixed pH of the fermentation medium. So, those factors should be critically considered for the scaling-up of the biotransformation process.

Effect of acclimatization rate on biogas production from anaerobic digestion of biodiesel waste products

AbstractDigester acclimatization can improve the efficiency of anaerobic digestion (AD), a potentially sustainable process for generating biogas from the biological decomposition of organic byproducts from biodiesel production. This study examined the impact of acclimatization rate on biogas production and conversion efficiency in laboratory digesters using inocula from a wastewater treatment plant digester and from an agro‐industrial waste digester. The results showed that co‐digestion of crude glycerol and biodiesel wastewater at high organic loading rates, up to 6.8 g chemical oxygen demand (COD) L−1 day−1, is possible without the addition of other substrates or pretreatment. Higher organic loading rates were achieved in the digesters using inocula from the agro‐industrial waste digester than in the digesters using the wastewater treatment plant digester, indicating that an inoculum with similar physical characteristics may be most appropriate for the AD of biodiesel waste. Of the two acclimatization rates studied, the slower rate improved the cumulative biogas production for both inocula. Both inocula also exhibited higher maximum daily efficiencies with a slower rate of acclimatization, up to a maximum average daily biogas yield of 621 mL biogas g−1 COD added. This implied that the rate of acclimatization can impact the ability of the digester to adapt efficiently to a new substrate.

Structural basis of bidirectional allostery across the heme in a cytochrome P450 enzyme

Cytochromes P450 (CYPs) are heme-containing enzymes that are present in all kingdoms of life and share a structurally homologous, globular protein fold. CYPs utilize structures distal to the heme to recognize and coordinate substrates, while the necessary interactions with redox partner proteins are mediated at the opposite, proximal surface. In the current study, we investigated the functional allostery across the heme for the bacterial enzyme CYP121A1, which utilizes a non-polar distal-to-distal dimer interface for specific binding of its dicyclotyrosine substrate. Fluorine-detected Nuclear Magnetic Resonance (19F-NMR) spectroscopy was combined with site-specific labeling of a distal surface residue (S171C of the FG-loop), one residue of the B-helix (N84C) and two proximal surface residues (T103C and T333C) with a thiol-reactive fluorine label. Adrenodoxin was used as a substitute redox protein and was found to promote a closed arrangement of the FG-loop, similar to the addition of substrate alone. Disruption of the protein-protein interface by mutagenesis of two CYP121 basic surface residues removed the allosteric effect. Moreover, 19F-NMR spectra of the proximal surface indicate that ligand-induced allostery modulates the environment at the C-helix, but not the meander region of the enzyme. In light of the high degree of structural homology in this family of enzymes, we interpret the findings from this work to represent a conserved allosteric network in CYPs.

Revisiting soil microbial biomass: Considering changes in composition with growth rate

Soil microbial biomass is assumed to have stable chemical composition. Various components of the biomass such as DNA, ATP, or chloroform-labile organic carbon are measured in soil and converted into total microbial biomass using experimentally derived conversion factors, which are also assumed to be constant. However, several observations suggest the opposite. The composition of soil microbial biomass is likely changing with specific growth rate as observed in pure cultures of single microbial species. In this study, we define a “sub-Microbial” model that explicitly represents changes in composition of soil microbial biomass associated with changes in specific growth rate. We calibrate the model with published data and compare its performance with the simpler Monod and Pirt models, which consider microbial biomass as a single pool with invariant chemical composition. The model explains well the variability in chloroform-labile content of microbial biomass following organic substrate additions as well as variability in ratios of different components of microbial biomass. Changes in composition of soil microbial biomass are quantitatively significant and occur over hours and days resulting in our sub-Microbial model outperforming both the Monod and Pirt models. Our results further indicate that the composition of soil microbial biomass changes consistently with growth rate across various soils. Here, we provide a methodological recommendation how to determine total soil microbial biomass and its physiological characteristics such as growth rate, turnover rate and substrate use efficiency as accurately as possible. In light of the presented results, we would like to initiate a discussion about the methodological issues associated with measurement of soil microbial biomass as these measurements are expected to inform a new generation of microbially-explicit soil biogeochemical models predicting development of terrestrial ecosystems under various scenarios.

The D-amino acid oxidase-carbon nanotubes: evaluation of cytotoxicity and biocompatibility of a potential anticancer nanosystem

The ‘enzyme prodrug therapy’ represents a promising strategy to overcome limitations of current cancer treatments by the systemic administration of prodrugs, converted by a foreign enzyme into an active anticancer compound directly in tumor sites. One example is D-amino acid oxidase (DAAO), a dimeric flavoenzyme able to catalyze the oxidative deamination of D-amino acids with production of hydrogen peroxide, a reactive oxygen species (ROS), able to favor cancer cells death. A DAAO variant containing five aminoacidic substitutions (mDAAO) was demonstrated to possess a better therapeutic efficacy under low O2 concentration than wild-type DAAO (wtDAAO). Recently, aiming to design promising nanocarriers for DAAO, multi-walled carbon nanotubes (MWCNTs) were functionalized with polyethylene glycol (PEG) to reduce their tendency to aggregation and to improve their biocompatibility. Here, wtDAAO and mDAAO were adsorbed on PEGylated MWCNTs and their activity and cytotoxicity were tested. While PEG-MWCNTs-DAAOs have shown a higher activity than pristine MWCNTs-DAAO (independently on the DAAO variant used), PEG-MWCNTs-mDAAO showed a higher cytotoxicity than PEG-MWCNTs-wtDAAO at low O2 concentration. In order to evaluate the nanocarriers’ biocompatibility, PEG-MWCNTs-DAAOs were incubated in human serum and the composition of protein corona was investigated via nLC-MS/MS, aiming to characterize both soft and hard coronas. The mDAAO variant has influenced the bio-corona composition in both number of proteins and presence of opsonins and dysopsonins: notably, the soft corona of PEG-MWCNTs-mDAAO contained less proteins and was more enriched in proteins able to inhibit the immune response than PEG-MWCNTs-wtDAAO. Considering the obtained results, the PEGylated MWCNTs conjugated with the mDAAO variant seems a promising candidate for a selective antitumor oxidative therapy: under anoxic-like conditions, this novel drug delivery system showed a remarkable cytotoxic effect controlled by the substrate addition, against different tumor cell lines, and a bio-corona composition devoted to prolong its blood circulation time, thus improving the drug’s biodistribution.

The Pannexin 1 Channel and the P2X7 Receptor Are in Complex Interplay to Regulate the Release of Soluble Ectonucleotidases in the Murine Bladder Lamina Propria.

The bladder urothelium releases ATP into the lamina propria (LP) during filling, which can activate P2X receptors on afferent neurons and trigger the micturition reflex. Effective ATP concentrations are largely dependent on metabolism by membrane-bound and soluble ectonucleotidases (s-ENTDs), and the latter are released in the LP in a mechanosensitive manner. Pannexin 1 (PANX1) channel and P2X7 receptor (P2X7R) participate in urothelial ATP release and are physically and functionally coupled, hence we investigated whether they modulate s-ENTDs release. Using ultrasensitive HPLC-FLD, we evaluated the degradation of 1,N6-etheno-ATP (eATP, substrate) to eADP, eAMP, and e-adenosine (e-ADO) in extraluminal solutions that were in contact with the LP of mouse detrusor-free bladders during filling prior to substrate addition, as an indirect measure of s-ENDTS release. Deletion of Panx1 increased the distention-induced, but not the spontaneous, release of s-ENTDs, whereas activation of P2X7R by BzATP or high concentration of ATP in WT bladders increased both. In Panx1-/- bladders or WT bladders treated with the PANX1 inhibitory peptide 10Panx, however, BzATP had no effect on s-ENTDS release, suggesting that P2X7R activity depends on PANX1 channel opening. We concluded, therefore, that P2X7R and PANX1 are in complex interaction to regulate s-ENTDs release and maintain suitable ATP concentrations in the LP. Thus, while stretch-activated PANX1 hinders s-ENTDS release possibly to preserve effective ATP concentration at the end of bladder filling, P2X7R activation, presumably in cystitis, would facilitate s-ENTDs-mediated ATP degradation to counteract excessive bladder excitability.

Super-Resolution Imaging of Voltages in the Interior of Individual, Vital Mitochondria.

We present super-resolution microscopy of isolated functional mitochondria, enabling real-time studies of structure and function (voltages) in response to pharmacological manipulation. Changes in mitochondrial membrane potential as a function of time and position can be imaged in different metabolic states (not possible in whole cells), created by the addition of substrates and inhibitors of the electron transport chain, enabled by the isolation of vital mitochondria. By careful analysis of structure dyes and voltage dyes (lipophilic cations), we demonstrate that most of the fluorescent signal seen from voltage dyes is due to membrane bound dyes, and develop a model for the membrane potential dependence of the fluorescence contrast for the case of super-resolution imaging, and how it relates to membrane potential. This permits direct analysis of mitochondrial structure and function (voltage) of isolated, individual mitochondria as well as submitochondrial structures in the functional, intact state, a major advance in super-resolution studies of living organelles.

Soil Enzyme Activity Response to Substrate and Nutrient Additions on Undisturbed Forest Subsoil Samples

Previous studies have found that C turnover is bound to hotspots of microbial activity. The objective of this study was to analyze the effects of pure energy substrate (glucose), nutrient (mineral N or P) and combined substrate and nutrient (glucose + N, glucose + P, sterile DOC, artificial root exudate extract) additions to enzyme activity inside and outside hotspots as a proxy for microbial C turnover in a subsoil. By means of different substrate and nutrient additions, we tested how the limitations of our site were distributed on a small scale and depth-dependently to contribute to an increase in knowledge of subsoil mechanistics. The study site is a sandy Dystric Cambisol under an over 100-year-old beech forest stand in Lower Saxony, Germany. Forty-eight undisturbed soil samples from two depth increments (15–27 cm and 80–92 cm) of three profiles were sprayed homogeneously with easily available C, N and P sources to investigate the impacts of substrates and nutrients on three enzyme activities (acid phosphatase, β-glucosidase and N-acetylglucosaminidase) by using the soil zymography approach. Comparisons of upper and lower subsoils showed significantly fewer and smaller hotspots in the lower subsoil but with a high degree of spatial variation in comparison to the upper subsoil. Different patterns of enzyme distribution between upper and lower subsoil suggest microbial communities with a lower diversity are found in deeper soil regions of the site. Both substrate and nutrient additions stimulated enzyme activities significantly more outside the initial hotspots than within. Because of this, we conclude that microorganisms in the initial hotspots are less limited than in the surrounding bulk soil. Changes in enzyme activities owing to both substrate and nutrient addition were stronger in the lower subsoil than in the upper subsoil, showing differences in limitations and possible changes in microbial community structure with increasing depth. The results of our study emphasize the need to consider spatial factors in microbial turnover processes, especially in lower subsoil regions where stronger substrate and nutrient limitations occur.

Loss of cholinergic receptor muscarinic 1 impairs cortical mitochondrial structure and function: implications in Alzheimer's disease.

Introduction: Cholinergic Receptor Muscarinic 1 (CHRM1) is a G protein-coupled acetylcholine (ACh) receptor predominantly expressed in the cerebral cortex. In a retrospective postmortem brain tissues-based study, we demonstrated that severely (≥50% decrease) reduced CHRM1 proteins in the temporal cortex of Alzheimer's patients significantly correlated with poor patient outcomes. The G protein-mediated CHRM1 signal transduction cannot sufficiently explain the mechanistic link between cortical CHRM1 loss and the appearance of hallmark Alzheimer's pathophysiologies, particularly mitochondrial structural and functional abnormalities. Therefore, the objective of this study was to analyze the molecular, ultrastructural, and functional properties of cortical mitochondria using CHRM1 knockout (Chrm1-/-) and wild-type mice to identify mitochondrial abnormalities. Methods: Isolated and enriched cortical mitochondrial fractions derived from wild-type and Chrm1-/- mice were assessed for respiratory deficits (oxygen consumption) following the addition of different substrates. The supramolecular assembly of mitochondrial oxidative phosphorylation (OXPHOS)-associated protein complexes (complex I-V) and cortical mitochondrial ultrastructure were investigated by blue native polyacrylamide gel electrophoresis and transmission electron microscopy (TEM), respectively. A cocktail of antibodies, specific to Ndufb8, Sdhb, Uqcrc2, Mtco1, and Atp5a proteins representing different subunits of complexes I-V, respectively was used to characterize different OXPHOS-associated protein complexes. Results: Loss of Chrm1 led to a significant reduction in cortical mitochondrial respiration (oxygen consumption) concomitantly associated with reduced oligomerization of ATP synthase (complex V) and supramolecular assembly of complexes I-IV (Respirasome). Overexpression of Chrm1 in transformed cells (lacking native Chrm1) significantly increased complex V oligomerization and respirasome assembly leading to enhanced respiration. TEM analysis revealed that Chrm1 loss led to mitochondrial ultrastructural defects and alteration in the tinctorial properties of cortical neurons causing a significant increase in the abundance of dark cortical neurons (Chrm1-/- 85% versus wild-type 2%). Discussion: Our findings indicate a hitherto unknown effect of Chrm1 deletion in cortical neurons affecting mitochondrial function by altering multiple interdependent factors including ATP synthase oligomerization, respirasome assembly, and mitochondrial ultrastructure. The appearance of dark neurons in Chrm1-/- cortices implies potentially enhanced glutamatergic signaling in pyramidal neurons under Chrm1 loss condition. The findings provide novel mechanistic insights into Chrm1 loss with the appearance of mitochondrial pathophysiological deficits in Alzheimer's disease.

Evaluating the prebiotic effect of oligosaccharides on gut microbiome wellness using in vitro fecal fermentation

We previously proposed the Gut Microbiome Wellness Index (GMWI), a predictor of disease presence based on a gut microbiome taxonomic profile. As an application of this index for food science research, we applied GMWI as a quantitative tool for measuring the prebiotic effect of oligosaccharides. Mainly, in an in vitro anaerobic batch fermentation system, fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), inulin (IN), and 2’-fucosyllactose (2FL), were mixed separately with fecal samples obtained from healthy adult volunteers. To find out how 24 h prebiotic fermentation influenced the GMWI values in their respective microbial communities, changes in species-level relative abundances were analyzed in the five prebiotics groups, as well as in two control groups (no substrate addition at 0 h and for 24 h). The GMWI of fecal microbiomes treated with any of the five prebiotics (IN (0.48 ± 0.06) > FOS (0.47 ± 0.03) > XOS (0.33 ± 0.02) > GOS (0.26 ± 0.02) > 2FL (0.16 ± 0.06)) were positive, which indicates an increase of relative abundances of microbial species previously found to be associated with a healthy, disease-free state. In contrast, the GMWI of samples without substrate addition for 24 h (–0.60 ± 0.05) reflected a non-healthy, disease-harboring microbiome state. Compared to the original prebiotic index (PI) and α-diversity metrics, GMWI provides a more data-driven, evidence-based indexing system for evaluating the prebiotic effect of food components. This study demonstrates how GMWI can be applied as a novel PI in dietary intervention studies, with wider implications for designing personalized diets based on their impact on gut microbiome wellness.

Developing a Robust and High Throughput Assay for Screening Innate Immune Training Molecules

Abstract Innate immune training molecules have several potential applications in the biomedical field. We developed an assay to screen for new training molecules by generating HiBiT tagged TNF cells in the THP1 monocytic cell line through CRISPR knock-in. The HiBiT tag, an 11 amino acid long peptide, was fused to the TNF C-terminus to produce luminescence upon addition of the LgBiT substrate. To optimize the assay, we first titrated different concentrations of multiple TLR ligands including pIC, p(dAdT), PGN, FLGN, LPS, R848, and P3C. LPS, PGN, and P3C induced the strongest signal to noise (S/N) ratio in the pool of TNF-HiBiT cells. From the TNF-HiBiT pool, we then single cell cloned and characterized them for optimum TNF response. The clones D3 and D6 had significantly higher S/N ratio compared to the pools, especially in response to P3C and PGN. To optimize the TNF-HiBiT cells for a training screen, we used previously known training molecules such as Syk kinase inhibitor, beta-Glucan and Rutaecarpine, to train multiple TNF-HiBiT cell clones. We found that the clones that induced a lower TNF signal upon acute TLR activation exhibited a more robust training phenotype compared to the clones that induced a more robust acute TNF signal. We found therefore that the optimal protocol to assess trained immunity is to use lower concentrations of activating TLR ligands (0.1, 0.1, and 0.0001 ng/mL of P3C, LPS and PGN) in the trained cells. This may be due to saturation of the TNF-HiBit reporter signal when cells are challenged with higher TLR ligand concentrations (10, 100, LPS, and 10 ng/mL of P3C, LPS and PGN). In brief, we have developed and optimized a robust assay for screening novel training molecules. This work was supported by the Intramural Research Program of NIAID, NIH Supported by grants from Intramural Research Program of NIAID, NIH and the Office of AIDS Research at NIH.

Alterations of Mitochondrial Respiratory Function in Isolated Nephron Segments of Dahl Salt-Sensitive Rats in Salt-Induced Hypertension

Rationale: Mammalian kidneys actively reabsorb nearly 99% of the glomerular filtrate to maintain body fluid and solute homeostasis which requires a great amount of ATP generation and O2 consumption to meet these demands. A high salt diet increases glomerular filtration of Na+ and places a high metabolic demand on the renal tubules to reabsorb more Na+in order for the organism to maintain Na+ balance. Dahl salt-sensitive (SS) rats which mimic the human condition become hypertensive when fed a high salt diet. They are also known to reabsorb greater amounts of Na+ in the proximal tubules (PT) and medullary thick ascending limbs (mTAL) thereby requiring greater ATP production and O2consumption. We hypothesized that the mitochondrial respiratory function of the PT and mTAL would be altered when SS rats are fed a high salt diet in ways that reduce the ability of the kidney to function efficiency thereby leading to salt-induced hypertension. Method: Agilent Seahorse XF96 Extracellular Flux Analyzer was used to assess mitochondrial respiratory function in freshly bulk isolated PT and mTAL from age-matched SS rats during the development of salt-induced hypertension fed with 0.4% NaCl diet (low salt; LS) or 4.0% NaCl diet (high salt; HS) for 7, 14, and 21 days. The Seahorse “Cell Mito Stress Test Protocol” was slightly modified with a substrate addition step to assess the segment-specific and substrate-dependent tubular O2 consumption rate (OCR) under different perturbations, which was used to determine alterations in different mitochondrial respiratory parameters, including H+ leak and ATP production dependent OCR, in the PT and mTAL during the development of salt-induced hypertension. Results: Renal tubular OCR data show that PT of SS rats preferred to use lactate, glutamine, and palmitate, while mTAL preferred to use glucose, pyruvate, and lactate for ATP production under LS diet condition. HS diet resulted in significantly reduced substrate-induced OCR in PT but increased OCR in mTAL. In addition, HS induced a higher level of OCR in the baseline as well as when oligomycin (an inhibitor of F1F0-ATP synthase) was added to both PT and mTAL suspensions indicating that an increased proton leakage/uncoupling of mitochondria occurs in SS rats during the development of salt-induced hypertension. Conclusion: Our emergent data demonstrated that the mitochondrial respiratory function is differentially altered in the PT and mTAL of SS rats in response to different circulatory substrates during the development of salt-induced hypertension. These changes in metabolism appear to be necessary to meet the increased tubular workloads and need for greater ATP production and O2 utilization in both the PT and mTAL of SS rats. NIH R01-HL151587. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.