Overexpression Research Articles

Dysregulating mTORC1-4E-BP2 signaling in GABAergic interneurons impairs hippocampus-dependent learning and memory.

Memory formation is contingent on molecular and structural changes in neurons in response to learning stimuli-a process known as neuronal plasticity. The initiation step of mRNA translation is a gatekeeper of long-term memory by controlling the production of plasticity-related proteins in the brain. The mechanistic target of rapamycin complex 1 (mTORC1) controls mRNA translation, mainly through phosphorylation of the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) and ribosomal protein S6 kinases (S6Ks). mTORC1 signaling decreases throughout brain development, starting from the early postnatal period. Here, we discovered that in mice, the age-dependent decrease in mTORC1 signaling occurs selectively in excitatory but not inhibitory neurons. Using a gene conditional knockout (cKO) strategy, we demonstrate that either up- or downregulating the mTORC1-4E-BP2 axis in GAD65 inhibitory interneurons, but not excitatory neurons, results in long-term object recognition and object location memory deficits. Our data indicate that the mTORC1 pathway in inhibitory but not excitatory neurons plays a key role in memory formation.

Glycoprofile Comparison of the SARS-CoV-2 Spike Proteins Expressed in CHO and HEK Cell Lines.

Coronavirus SARS-CoV-2 spike protein remains a key focus of research due to a continued need for diagnostic and therapeutic tools to monitor and respond to new variants. Glycosylation of the spike protein is critical for the protein's functions in viral attachment and host cell entry. For scalable and cost-effective production of the spike protein, expression system-driven divergence in glycosylation patterns on recombinant spike proteins needs to be fully understood. This study assessed the N-glycosylation profiles of a full-length trimeric spike protein expressed in either Human Embryonic Kidney (HEK Expi293F) or Chinese Hamster Ovary (CHO-S) cells. Glycopeptide analysis was performed using a tandem mass spectrometry workflow and BioPharma incorporating HEK and CHO glycan databases for protein characterisation. The results outline important differences in the variety and types of N-glycan generated by the two cell lines across the 22 known N-glycosylation sites of the spike protein. A notable increase in terminal sialylation, as well as the presence of the potentially immunogenic N-glycolylneuraminic acid at a functionally key N-glycosylation site, was observed in the CHO-S derived spike protein. With the potential for the relatively vast and more complex CHO glycan repertoire (182 glycans relative to 39 human glycans) to produce functional implications with CHO-S expressed spike protein, this study adds valuable knowledge to aid Quality by Design approaches and enable Multi Attribute Monitoring of specific N-glycosylation sites for proteoform analyses. This can further inform antigen development with future variants in order to devise updated diagnostic tests and therapeutic vaccine designs.

Low Testosterone Concentration Improves Colonisation and Viability in the Co-Cultured Goat Spermatogonial Stem Cell With Sertoli Cells.

Spermatogonial stem cells (SSCs) maintain spermatogenesis through self-renewal and differentiation. The proliferation of SSCs in culture systems can provide a valuable source of germ cells. Several studies have investigated new reproductive technologies, including the production of transgenic animals and recombinant proteins secreted from milk in goats. While studies in other species exist, research on goat SSC culture remains limited. We investigated the impact of different testosterone concentrations on the survival and colonisation of cocultured goat SSCs with Sertoli cells. Cells were isolated from immature goats using two-step enzymatic digestion and enriched by differential exclusion method. DMEM/F12 culture medium containing 1% antibiotic and 5% FBS, supplemented with GDNF (20 ng/mL), EGF, bFGF and LIF (10 ng/mL), was used with different testosterone concentrations (0, 60, 120 and 240 μg/mL) and cultured for 10 days. SC subpopulations were confirmed using PGP9.5 immunocytochemistry, and the expression of germ cell markers (ID-4, UCHL-1, THY-1, β1-integrin, BCL6B, VASA, PLZF and OCT-4) was evaluated through RT-PCR. Alkaline phosphatase activity provided additional SSC presence. The survival rate of SSCs after isolation and the number and area of colonies on Days 4, 7 and 10 were measured using an inverted microscope. The presence of PGP 9.5 antigens and germ cell markers (ID-4, UCHL-1, THY-1, β1-integrin, BCL6B, VASA, PLZF and OCT-4) was confirmed by immunocytochemistry and RT-PCR, respectively. According to the results, the group with 60 μg/mL testosterone had the highest number and area of colonies. The number of colonies in the 60 μg/mL testosterone group was significantly higher than the control group (p < 0.05), but no significant difference was observed compared to other groups (p ≥ 0.05). This study suggests that a low testosterone concentration (60 μg/mL) is optimal for goat SSC colonisation and viability in coculture with Sertoli cells, potentially leading to advancements in goat reproductive technologies.

Production of Norovirus VLPs of the Nine Representative Genotypes Widely Distributed in Japan using the Silkworm-Baculovirus Expression Vector System

Norovirus (NoV) is one of the major causes of acute viral gastroenteritis in humans. Genetic variation is abundant, and prevalent genotypes vary from year to year and region to region. Since NoVs are difficult to amplify in cultured cells, genome RNA-free virus-like particles (VLPs) that mimic the capsid structure of the virus are promising vaccine candidates for the prevention of NoVs infection, and the development of multivalent VLP vaccines is required to prevent NoV infection in a wide range of genotypes. In this study, we attempted to produce NoV VLPs of the top nine genotypes that have a history of epidemics in Japan using the silkworm-baculovirus expression vector system (silkworm-BEVS), which has a proven track record in the mass production of recombinant proteins. In silkworm pupae infected with recombinant baculoviruses constructed to express VP1s, the major protein that forms VLP, the NoV VP1 protein was expressed in large amounts. Most genotypes of VP1 accumulated in the cytoplasm as soluble proteins, but solubility was reduced for that of two genotypes. VP1s of five genotypes could be purified in large quantities (>0.9mg per pupa) by a two-step purification process, and gel filtration chromatography analysis confirmed the formation of VLPs. This study demonstrates the utility of silkworm-BEVS in producing NoV VLPs of multiple genotypes and provides the basis for the development of a multivalent vaccine against genetically diverse NoV infections.

Aldosterone, mitochondria and regulation of cardiovascular metabolic disease.

Aldosterone is a mineralocorticoid hormone involved in controlling electrolyte balance, blood pressure, and cellular signaling. It plays a pivotal role in cardiovascular and metabolic physiology. Excess aldosterone activates mineralocorticoid receptors, leading to subsequent inflammatory responses, increased oxidative stress, and tissue remodeling. Various mechanisms have been reported to link aldosterone with cardiovascular and metabolic diseases. However, mitochondria, responsible for energy generation through oxidative phosphorylation, have received less attention regarding their potential role in aldosterone-related pathogenesis. Excess aldosterone leads to mitochondrial dysfunction, and this may play a role in the development of cardiovascular and metabolic diseases. Aldosterone has the potential to affect mitochondrial structure, function, and dynamic processes, such as mitochondrial fusion and fission. In addition, aldosterone has been associated with the suppression of mitochondrial DNA, mitochondria-specific proteins, and ATP production in the myocardium through mineralocorticoid receptor, nicotinamide adenine dinucleotide phosphate oxidase, and reactive oxygen species pathways. In this review, we explore the mechanisms underlying aldosterone-induced cardiovascular and metabolic mitochondrial dysfunction, including mineralocorticoid receptor activation and subsequent inflammatory responses, as well as increased oxidative stress. Furthermore, we review potential therapeutic targets aimed at restoring mitochondrial function in the context of aldosterone-associated pathologies. Understanding these mechanisms is vital, as it offers insights into novel therapeutic strategies to mitigate the impact of aldosterone-induced mitochondrial dysfunction, thereby potentially improving the outcomes of individuals affected by cardiovascular and metabolic disorders.

Data-Independent Acquisition Proteome Technology for Analysis of Antifungal and Anti-aflatoxigenic Properties of Eugenol to Aspergillus flavus.

Several toxicogenic Aspergilli, such as Aspergillus flavus and A. parasiticus, could biosynthesize aflatoxin B1 (AFB1) and other mycotoxins. Chemical fungicides are commonly used to control fungal contamination, but chemical residues may pose significant risks to human health and environmental stability. Consequently, natural antifungal and aflatoxin-inhibiting agents could be sustainable alternatives. Eugenol has been used as an inhibitor of aflatoxins (AFs), which is a common essential oil. Nevertheless, the definite mechanism by which eugenol exerts its inhibitory effect on Aspergillus remains unclear. This research demonstrates that eugenol significantly suppressed fungi growth and AF production as the dose increased (40.9 to 100%). With the proteomics approach, the inhibition pathway of eugenol was investigated. The production of proteins involved in cell wall integrity was notably reduced under eugenol treatment, indicating that eugenol destroys the cell wall integrity of A. flavus. Furthermore, exposure to eugenol downregulated several fungal developmental regulators and subsequently inhibited A. flavus development. Energy metabolism in A. flavus is closely related to its secondary metabolism. Under eugenol treatment, the synthesis of proteins relevant to the pentose phosphate pathway was significantly enhanced, leading to a decrease in the availability of acetyl-CoA, a precursor for AF biosynthesis. Simultaneously, the valine, leucine, and isoleucine biosynthesis pathways were enhanced, further reducing the content of acetyl-CoA. This might be the primary factor in the inhibition of AF biosynthesis by eugenol. Ribosome biogenesis was the most dysregulated pathway based on KEGG data, indicating that eugenol disturbed ribosome biogenesis and affected its normal function in A. flavus. In conclusion, eugenol inhibits the cellular integrity, energy metabolism, and protein synthesis and then suppresses A. flavus development and AF biosynthesis, which provides a clearer grasp of the inhibitory mechanism meaningful for A. flavus and AF contamination control.

Anemarrhena asphodeloides Bunge polysaccharides alleviate lipoteichoic acid-induced lung inflammation and modulate gut microbiota in mice

Pneumonia remains a prevalent infection primary ailment characterized by severe lung inflammation, leading to respiratory distress and significant mortality rates, particularly affecting young children in less developed regions. This study explores the therapeutic potential of low and high-molecular weight polysaccharides derived from Anemarrhena asphodeloides in a murine model of lipoteichoic acid (LTA)-induced pneumonia, which represents bacterial-induced lung inflammation. Administration of Anemarrhena asphodeloides polysaccharides effectively alleviated LTA-induced symptoms, including decreased lung and colon inflammation, and restored dysbiosis of gut microbiota. Polysaccharide treatment notably increased mucin-2 expression, reduced serum cytokine levels (IL-10, TNF-α), and increased tight junction protein production (ZO-1, Occludin, Claudin). Additionally, polysaccharides promoted a significant recovery in gut microbiota composition, indicating potential prebiotic effects. These findings highlight the therapeutic capability of Anemarrhena asphodeloides polysaccharides against LTA-induced pneumonia through gut microbiota modulation and restored intestinal homeostasis.

A cell-free system for functional studies of small membrane proteins

Numerous small proteins have been discovered across all domains of life, among which many are hydrophobic and predicted to localize to the cell membrane. Based on a few that are well-studied, small membrane proteins are regulators involved in various biological processes, such as cell signaling, nutrient transport, drug resistance, and stress response. However, the function of most identified small membrane proteins remains elusive. Their small size and hydrophobicity make protein production challenging, hindering function discovery. Here, we combined a cell-free system with lipid sponge droplets and synthesized small membrane proteins in vitro. Lipid sponge droplets contain a dense network of lipid bilayers, which accommodates and extracts newly synthesized small membrane proteins from the aqueous surroundings. Using small bacterial membrane proteins MgrB, SafA, and AcrZ as proof of principle, we showed that the in vitro produced membrane proteins were functionally active, for example, modulating the activity of their target kinase as expected. The cell-free system produced small membrane proteins, including one from human, up to micromolar concentrations, indicating its high level of versatility and productivity. Furthermore, AcrZ produced in this system was used successfully for in vitro co-immunoprecipitations to identify interaction partners. This work presents a robust alternative approach for producing small membrane proteins, which opens a door to their function discovery in different domains of life.

Effects of straw structure and component on feeding efficiency of yellow mealworm for insect protein production

Elucidating the influence of straw structure and component on the feeding efficacy of yellow mealworm is pivotal for improving insect protein production from straw. This research utilized four distinct types of straws—water hyacinth straw (WHS), corn straw (CS), rape straw (RAS), and rice straw (RIS)—as the sole substrate for larvae. Results indicated that the straw utilization rate and fresh larval weight gain rate followed the order of WHS > CS > RAS > RIS. Analysis of straw structural characteristics demonstrated that decreasing straw hardness and cellulose crystallinity, while enhancing straw chewability, facilitated the ingestion of larvae. Feeding efficiency of yellow mealworm was positively correlated with the hemicellulose and crude protein content, and inversely correlated with lignin content in the straw. Additionally, the structural characteristics and components of straw significantly influenced the composition of the gut microbiota. These results offer valuable insights for optimizing yellow mealworm feeding on straw.

Preventive effects of coixol, an active compound of adlay seed, in NGF-differentiated PC12 cells against beta-amyloid25-35-induced neurotoxicity.

The health benefits of coixol, an active compound of adlay seed, have attracted certain attention. Adlay seed is often adopted in traditional Chinese medicine for the treatment of various inflammatory disorders. Thus, it is hypothesized that coixol could protect neuronal cells. The preventive effects of coixol against Abeta25-35-induced damage in nerve growth factor-differentiated PC12 cells were explored. Differentiated PC12 cells were treated with coixol at 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, and 2 μM for 48 h. Then, cells were further exposed to Abeta25-35 at 20 μM for 24 h. Coixol treatments at 0.25-2 μM exhibited antiapoptotic effect via increasing Bcl-2 mRNA expression, mitochondrial membrane potential, and Na+-K+ ATPase activity as well as decreasing Bax mRNA expression, caspase-3 activity, and intracellular Ca2+ release. In addition, coixol treatments at 0.25-2 μM alleviated oxidative and inflammatory responses via lowering reactive oxygen species level, increasing glutathione content, promoting the activity of glutathione peroxidase, glutathione reductase, and catalase, decreasing the generation of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and prostaglandin E2. Furthermore, coixol treatments at 0.25-2 μM diminished intracellular Ca2+ release, and restricted nuclear factor kappa B-binding activity and phosphorylation of p65 and p38. Coixol treatments at 0.5-2 μM increased protein generation of nuclear factor E2-related factor 2, and limited protein production of inducible nitric oxide synthase and receptor of advanced glycation end product. Our novel findings suggested that coixol was a compelling agent against beta-amyloid peptide-induced neurotoxicity.

Economic optimization of sustainable soil management: a Dutch case study

Soil quality is pivotal for crop productivity and the environmental quality of agricultural ecosystems. Achieving sufficient yearly income and long-term farm continuity are key goals for farmers, making sustainable soil management an economic challenge. Existing bio-economic models often inadequately address soil quality. In this study, we apply the novel FARManalytics model, which integrates chemical, physical, and biological indicators of soil quality indicator, quantitative rules on how these indicators respond to farmers’ production management over time, and an economic calculation framework that accurately calculates the contribution of production management decisions towards farm income. This is the first study applying this model on existing arable farms. FARManalytics optimizes crop rotation design, cover crops, manure and fertilizer application and crop residue management. Nine Dutch arable farms were analyzed with a high variation in farm size, soil type, and cultivated crops. First, we assessed farm differences in soil quality and farm economics. Second, we optimized production management to maximize farm income while meeting soil quality targets using farm-specific scenarios. Third, we explored the impact of recent policy measures to preserve water quality and to increase the contribution of local protein production. The results show that the case farms already perform well regarding soil quality, with 75% of the soil quality indicators above critical levels. The main soil quality bottlenecks are subsoil compaction and soil organic matter input. We show that even in front-runner farms, bio-economic modeling with FARManalytics substantially improves economic performance while increasing soil quality. We found that farm income could be increased by up to €704 ha−1 year−1 while meeting soil quality targets. Additionally, we show that to anticipate on stricter water quality regulation and market shift for protein crops, FARManalytics is able to provide alternative production management strategies that ensure the highest farm income while preserving soil quality for a set of heterogenous farms.

Chitosan‐Coated Glass Beads as Stabilizer of Insulin; A Novel Strategy for the Storage of Pharmaceutical Proteins

AbstractBesides the pharmaceutical protein production challenge, storing and transporting them between industrial sectors and the administration site have always been problematic. Insulin, an example of the most widely used therapeutic protein, aggregates into unstable larger particles and, in many cases, into amyloid fibrils under environmental stresses. In this study, chitosan was used to decorate the surface of glass beads (to produce Chi@GB) to increase the structural stability of insulin against environmental stresses. FT‐IR spectroscopy characterized the beads, and HPLC determined the chitosan loading. The results showed that the modified beads could inhibit insulin fibrillation. In addition, the beads can be used in biopharmaceutical containers of insulin or other proteins for long‐term storage of their commercial products.

Impact of mtDNA-encoded proteins arising from cytosolic translation (mPACT)

Mitochondria are multifunctional organelles that regulate energy homeostasis, apoptosis, cell signaling and many biosynthetic pathways. Mitochondria have their own genomes that reportedly contain 37 well-known genes, including 13 protein-coding genes, 2 genes encoding ribosomal RNAs (rRNAs) and 22 genes encoding transfer RNAs (tRNAs). RNA is essential for most biological functions which can act as a translation template for protein production or perform the function itself to directly regulate cellular processes. In addition to those functioning inside mitochondria, mitochondria can regulate many important biological processes through exporting RNAs including long non-coding mitochondrial RNAs (ncmtRNAs), circRNAs, rRNAs, tRNAs and mRNAs.

Loss of Hdac4 in Osteoprogenitors Impairs Postnatal Trabecular and Cortical Bone Formation, Resulting in a Dwarfism and Osteopenia Phenotype in Mice

HDAC4 is a class II histone deacetylation protein with a well-characterized role in chondrocyte differentiation and skeletal development, and dysregulated expression or haploinsufficiency of Hdac4 leads to skeletal formation and malformation disorders. The early lethality of hdac4 ablation mice hindered further investigation of its role in postnatal bone growth and development. Therefore, this study aims to investigate the significant role of Hdac4 in postnatal endochondral bone development using two mouse models with conditional deletion of Hdac4 in Sp7-expressing osteoprogenitors or chondrocytes and monitored postnatal bone development. The phenotype of Acan-CreERT2; Hdac4fl/fl mice largely resembled that of conventional Hdac4-/- mice. But phenotypic characterizations of mice with Hdac4 inactivation in Sp7-expressing osteoprogenitors (Sp7-Cre; Hdac4fl/fl) showed dwarfism with body and limb shortening and remarkable skeletal defects. Micro-computed tomography analysis of tibias further demonstrated that loss of Hdac4 expression impaired bone formation and microarchitecture, mainly characterized by dysplasia of trabecular and cortical bone in young mice. Our in vivo and in vitro data support a crucial role for Hdac4 in regulating osteoblast proliferation and differentiation, bone matrix protein production, angiogenesis, and ultimately trabecular and cortical bone formation. Moreover, RNA-seq analysis implicated Hdac4 in the regulation of key genes and pathways necessary to affect the accumulation of extracellular matrix, biological processes related to signal transduction, and skeletal growth. Collectively, our data show that postnatal expression of Hdac4 in Sp7-expressing osteoprogenitors provides essential regulatory oversight of endochondral bone formation, bone morphology, and homeostasis.

A comparison of animal and plant-based proteins from an economic, environmental, and nutritional perspective in the Republic of Ireland

CONTEXTProtein is a central component of health and nutrition. The current protein production system is unlikely to sustainably meet the growing global demand for protein. Therefore, alternative sources of protein must be considered. OBJECTIVEThis study uniquely compares animal-based (milk, beef meat, sheep meat) and plant-based (cereals and legumes) protein production in terms of land usage. The comparison is carried out across a suite of economic, environmental, and nutritional metrics. As land use decisions are taken at the farm level, the analysis adopts a farm gate approach. Focusing on the supply side, consumption is not considered in the analysis. METHODSEconomic performance is measured through the gross and net margins. Environmental performance is evaluated through farm-level greenhouse gas (GHG) emissions and nutrient (nitrogen and phosphorus) balances. Nutritional performance is measured through the gross protein yield, the protein yield corrected for digestibility and land-use efficiency, i.e., the land needed to generate one kg of (digestible) protein. Results are expressed per hectare and per 100 g of gross and digestible protein to allow for a holistic comparison. The analysis focuses on the Republic of Ireland, a relevant country given the importance of the strong agricultural export focus, the dominance of the livestock sector and the country's ambitious environmental emission reduction targets. RESULTS AND CONCLUSIONSFindings indicate that, on a per hectare basis, milk production on dairy farms has the strongest economic performance and highest GHG emissions amongst the products considered. Crops' gross and net margins are less than half that of dairy farms, but they also show the strongest environmental performance generally. Cereals and legumes have the best nutritional performance, from a protein perspective, whereas sheep meat production returned the lowest gross and digestible protein yields per hectare. Arable crops also show the lowest GHG emissions and nutrient balances on a per unit of protein basis. SIGNIFICANCELand-use policies need to be designed holistically, given the complexity of the agricultural sector. Agricultural policy design currently focuses on income support and on the environmental impact of the agricultural sector, but farmers' livelihood and food and protein security need to be safeguarded. Diversifying agricultural production and increasing plant-based protein production in Ireland, a country with a livestock-focused agricultural sector, could contribute to achieving the climate change targets of the country, provided the necessary policy levers are in place.

Uses of plasma in medicine

Plasma is the liquid portion of whole blood, It is composed largely of water and proteins, and it provides a medium for red blood cells, white blood cells and platelets to circulate through the body. The plasma contains an important amount of antibiotics capable of strengthening the patient's immunity in the face of the threat of the virus. When plasma is injected into the scalp, it renews cells and stimulates the production of collagen and protein, two substances that make up the second middle layer of the skin, which give the skin freshness and vitality. One of the most important benefits of plasma for the face is that it uses platelets to inject the face and it plays several roles inside the skin once it is injected under it. Plasma can be used as a local injection as well to treat tissue ruptures such as knee ligament tears. platelet-rich plasma (PRP) can improve wound-healing processes in orthopedic surgery, dermatology, ophthalmology, gynecology and plastic surger.

Характеристика нового штамма гриба Schizophyllum commune EO22 и его способности разлагать полиэтилен

The widespread and possessing extensive biotechnological potential xylotrophic basidiomycete Schizophyllum commune Fr. has a significant genetic variability. This actualizes the study of the fungus various strains to solve a complex of environmental problems, in particular, the disposal of plastic waste. The S. commune strain EO22 used in the work was isolated from rotting Acer negundo wood in the southern taiga subzone of the European Northeast (N58о35´, E49о69´, vicinity of Kirov). It is maintained in mycelial culture in laboratory conditions. The species identification was performed based on the morphology and sequencing result of the ITS1–5.8S–ITS2 fragment nucleotide sequence. The features of the growth and fruiting of mycelial culture EO22 on potato-sucrose and malt agar are described. The degradation potential of the S. commune strain EO22 was established in the experiment on co-incubation with low-pressure polyethylene (HDPE) film. HDPE is one of the most common environmental pollutants (64% of the total plastics production). The process of HDPE biodegradation was carried out exclusively by the dikaryotic type of mycelium and was accompanied by the forming of the fungus fruiting bodies. The results obtained are of interest in connection with the development of technologies for food protein production in the process of plastic waste utilization.

Grifola frondosa extract as a fetal bovine serum supplement for the culture of bovine muscle satellite cells under low serum conditions

Expensive fetal bovine serum (FBS) is a major obstacle to the production of cultivated meat. However, because FBS substitutes do not sufficiently induce cell proliferation, a good alternative is to reduce the amount of FBS and use ingestible additives to promote cell proliferation. In this study, Grifola frondosa extract (GFE) was used to investigate its potential as an additive to promote myogenesis of bovine muscle satellite cells from Hanwoo cattle under low serum conditions (10 % FBS). GFE treated with 10 % FBS only during the proliferation period not only increased cell proliferation and related biomarkers in a concentration-dependent manner (0.78–12.5 μg/mL), but also increased cell differentiation. Additionally, differentiation was promoted when cells were with GFE treated only during the differentiation period. Especially GFE at 12.5 µg/mL induced significantly higher proliferation and differentiation rates than 20 % FBS medium. In particular, compared to treatment alone in the proliferation or differentiation periods, GFE treatment in both periods contributed to an increase in the differentiation rate and significantly enhanced total protein production. The integration of GFE into cultivated meat production presents a promising approach to reducing FBS dependence, lowering costs, and enhancing scalability, aligning with sustainability and consumer acceptance goals.

BTN3A1 expressed in cervical cancer cells promotes Vγ9Vδ2 T cells exhaustion through upregulating transcription factors NR4A2/3 downstream of TCR signaling

BackgroundClinical trials have shown that immunotherapy based on Vγ9Vδ2 T cells (Vδ2 T cells) is safe and well-tolerated for various cancers including cervical cancer (CC), but its overall treatment efficacy remains limited. Therefore, exploring the mechanisms underlying the suboptimal efficacy of Vδ2 T cell-based cancer immunotherapy is crucial for enabling its successful clinical translation.MethodsTumor samples from CC patients and CC cell line-derived xenograft (CDX) mice were analyzed using flow cytometry to examine the exhausted phenotype of tumor-infiltrating Vδ2 T cells. The interrelationship between BTN3A1 expression and Vδ2 T cells in CC, along with their correlation with patient prognosis, was analyzed using data from The Cancer Genome Atlas (TCGA) database. CC cell lines with BTN3A1 knockout (KO) and overexpression (OE) were constructed through lentivirus transduction, which were then co-cultured with expanded Vδ2 T cells, followed by detecting the function of Vδ2 T cells using flow cytometry. The pathways and transcription factors (TFs) related to BTN3A1-induced Vδ2 T cells exhaustion and the factors affecting BTN3A1 expression were identified by RNA-seq analysis, which was confirmed by flow cytometry, Western Blot, and gene manipulation.ResultsTumor-infiltrating Vδ2 T cells exhibited an exhausted phenotype in both CC patients and CDX mice. BTN3A1 expressed in CC is highly enhancing exhaustion markers, while reducing the secretion of effector molecules in Vδ2 T cells. Blocking TCR or knocking down nuclear receptor subfamily 4 group A (NR4A) 2/3 can reverse BTN3A1-induced exhaustion in Vδ2 T cells. On the other hand, IFN-γ secreted by Vδ2 T cells promoted the expression of BTN3A1 and PD-L1.ConclusionsThrough binding γδ TCRs, BTN3A1 expressed on tumor cells, which is induced by IFN-γ, can promote Vδ2 T cells to upregulate the expression of TFs NR4A2/3, thereby affecting their activation and expression of exhaustion-related molecules in the tumor microenvironment (TME). Therefore, targeting BTN3A1 might overcome the immunosuppressive effect of the TME on Vδ2 T cells in CC.

Sulfamethoxazole removal and ammonium conversion in microalgae consortium: Physiological responses and microbial community changes

Microalgae (Mychonastes sp.) consortium was investigated for nutrient and antibiotics removal and its responses to varying sulfamethoxazole (SMX) concentrations (0–1000 μg/L) in ammonia-rich wastewater. The results showed that the introduction of SMX (100–1000 μg/L) slightly improved ammonium nitrogen removal efficiency instead of inhibition. Swift SMX degradation was observed across all SMX-treated systems, with the highest SMX removal efficiency (96 %) at an SMX concentration of 100 μg/L. Biodegradation remained the dominant SMX removal mechanism, contributing 78 % of SMX removal at an SMX concentration of 800 μg/L, while adsorption and photolysis played minor roles. Addition of SMX augmented biomass and lipid productivity, but decreased chlorophyll contents in the microalgae consortium. Furthermore, extracellular polymeric substance (EPS) production correlated positively with SMX input concentration, with the microalgae consortium exposed to 800 μg/L SMX displaying the most pronounced stimulation of protein production (51.5 ± 2.0 mg/g DCW) and polysaccharides production (74.8 ± 3.9 mg/g DCW). In response to an increase in SMX concentrations, enzyme activities associated with antioxidant defense, such as superoxide dismutase (SOD), peroxidase (POD) and malondialdehyde (MDA) increased, the catalase (CAT) decreased, indicating an initial defense mechanism. Concurrently, the relative abundance of Mychonastes sp. within the consortium rose from 87 % at 300 μg/L SMX to 99.9 % at 800 μg/L SMX. while Shannon indices of the bacterial community increased from 1.415 to 2.867. This shift inhibited the initially dominant Saprospiraceae bacteria, facilitating the profound increase of adapted Aquimonas. These findings demonstrate the feasibility of the simultaneous removal of antibiotics and nutrients from wastewater with a microalgae consortium system.