Protein Output Research Articles

A generic cell-based biosensor converts bacterial infection signals into chemoattractants for immune cells.

Bacterial infections are a major challenge to human health. Although various potent antibiotics have emerged in the last decades, current challenges arise from an increasing number of multi-drug-resistant species. Infections associated with implants represent a particular challenge since they are usually diagnosed at an advanced state, and are difficult to treat with antibiotics due to the formation of protecting biofilms. In this study, we designed and explored a synthetic biology-inspired, cell-based bio-sensor/actor for the detection and counteraction of bacterial infections. The system is generic as it senses diverse types of infections and acts by enhancement of the endogenous immune system. The strategy is based on genetically engineered sensor/actor cells that can sense type I interferons (IFNs), which are released by immune cells at the early stages of infections. IFN signalling activates a synthetic circuit to induce reporter genes with a sensitivity of only 5 pg/ml of IFN and leads to a therapeutic protein output of 100ng/ml, resulting in theranostic cells that visualize and fight infections. Robustness and resilience were achieved by the implementation of a positive feedback loop. We show that diverse gram-positive and gram-negative implant-associated pathogenic bacteria activate the cascade in co-culture systems in a dose-dependent manner. Finally, we show that this system can be used to secrete chemoattractants facilitating the infiltration of immune cells in response to bacterial triggers. Together, the system is not only universal to bacterial infections but at the same time hypersensitive allowing the sensing of infections at initial stages.

Development of CadR-based cadmium whole cell biosensor for visual detection of environmental Cd2+

BackgroundAs a threat to human health and public health, cadmium (Cd) pollution has received widespread social concern. Our previously constructed CadR-based bacterial whole cell biosensor (WCB) epCadR5 showed high sensitivity and specificity in cadmium detection. However, the application of the sensor is still hindered by the need for laboratory equipment to read the fluorescence signal output. In this study, we aimed to optimizing the sensor to make it available for visual detection of environmental cadmium and simplify the detection process to advance practical application of the sensor. ResultsBy replacing the constitutive promoter with J110, the fluorescence signal output of the sensor was significantly increased and the fluorescence leakage was decreased. In addition, the fluorescence signal output of green fluorescence protein (GFP) was enhanced by the addition of a 5′ untranslated region (5′-UTR) mlcR10. The fluorescence signal output of the WCB is sufficiently robust to be visible and distinguishable to the naked eye, which is of paramount importance for visual detection. The sensor readout can be conveniently recorded by mobile phone camera and quantified. For ease of on-site application, the WCB's visual detection procedures and conditions were further optimized and simplified. The WCB demonstrated good linearity and detection limit (1.81 μg/L) for visual detection of Cd2+ without the assistance of bulky laboratory equipment. For the detection of real environmental samples, the WCB visual detection results were close to those of WCB-flow cytometry (FACS) and graphite furnace atomic absorption spectroscopy (GFAAS). SignificanceIn this work, we developed an easy-to-use, on-site and visual detection biosensor for monitoring environmental Cd2+. It will advance the utilization of cadmium WCBs in practical settings. The optimization and simplification strategy in the study also provide new insights into the visualization of other bacterial biosensors, and will advance the practical application of WCBs.

Decoded cardiopoietic cell secretome linkage to heart repair biosignature.

Cardiopoiesis-primed human stem cells exert sustained benefit in treating heart failure despite limited retention following myocardial delivery. To assess potential paracrine contribution, the secretome of cardiopoiesis conditioned versus naïve human mesenchymal stromal cells was decoded by directed proteomics augmented with machine learning and systems interrogation. Cardiopoiesis doubled cellular protein output generating a distinct secretome that segregated the conditioned state. Altering the expression of 1035 secreted proteins, cardiopoiesis reshaped the secretome across functional classes. The resolved differential cardiopoietic secretome was enriched in mesoderm development and cardiac progenitor signaling processes, yielding a cardiovasculogenic profile bolstered by upregulated cardiogenic proteins. In tandem, cardiopoiesis enhanced the secretion of immunomodulatory proteins associated with cytokine signaling, leukocyte migration, and chemotaxis. Network analysis integrated the differential secretome within an interactome of 1745 molecules featuring prioritized regenerative processes. Secretome contribution to the repair signature of cardiopoietic cell-treated infarcted hearts was assessed in a murine coronary ligation model. Intramyocardial delivery of cardiopoietic cells improved the performance of failing hearts, with undirected proteomics revealing 50 myocardial proteins responsive to cell therapy. Pathway analysis linked the secretome to cardiac proteome remodeling, pinpointing 17 cardiopoiesis-upregulated secretome proteins directly upstream of 44% of the cell therapy-responsive cardiac proteome. Knockout, in silico, of this 22-protein secretome-dependent myocardial ensemble eliminated indices of the repair signature. Accordingly, in vivo, cell therapy rendered the secretome-dependent myocardial proteome of an infarcted heart indiscernible from healthy counterparts. Thus, the secretagogue effect of cardiopoiesis transforms the human stem cell secretome, endows regenerative competency, and upregulates candidate paracrine effectors of cell therapy-mediated molecular restitution.

Transfer RNA levels are tuned to support differentiation during Drosophila neurogenesis.

Neural differentiation requires a multifaceted program to alter gene expression along the proliferation to differentiation axis. While critical changes occur at the level of transcription, post-transcriptional mechanisms allow fine-tuning of protein output. We investigated the role of tRNAs in regulating gene expression during neural differentiation by quantifying tRNA abundance in neural progenitor-biased and neuron-biased Drosophila larval brains. We found that tRNA profiles are largely consistent between progenitor-biased and neuron-biased brains but significant variation occurs for 10 cytoplasmic isodecoders (individual tRNA genes) and this establishes differential tRNA levels for 8 anticodon groups. We used these tRNA data to investigate relationships between tRNA abundance, codon optimality-mediated mRNA decay, and translation efficiency in progenitors and neurons. Our data reveal that tRNA levels strongly correlate with codon optimality-mediated mRNA decay within each cell type but generally do not explain differences in stabilizing versus destabilizing codons between cell types. Regarding translation efficiency, we found that tRNA expression in neural progenitors preferentially supports translation of mRNAs whose products are in high demand in progenitors, such as those associated with protein synthesis. In neurons, tRNA expression shifts to disfavor translation of proliferation-related transcripts and preferentially support translation of transcripts tied to neuron-specific functions like axon pathfinding and synapse formation. Overall, our analyses reveal that changes in tRNA levels along the neural differentiation axis support optimal gene expression in progenitors and neurons.

Improvement of dairy characters in farms and breeding value of the breeding bulls when assessing the quality of offspring

The article presents studies on comparison of the levels of precocity and milk productivity indicators of the daughters of Holstein bulls, who are evaluated for the quality of offspring simultaneously in two breeding plants of the Moscow region. According to the official ratings of breeding values, the bulls were divided into groups of "improving" and "worsening". The ratio of the daughters of the "best" and "worst" breeding bulls was 511:568 and 160:127 heads over the farms. It was determined which values of the differences in the "daughter – mother" indicators were increased in groups of officially evaluated bulls that received ratings of "improvers" and "deteriorators" according to the characteristics in these two breeding plants. The bulls being tested were fixed individually, and according to the number of pairs as the main ones. At the first insemination, the daughters of the "improving" group of bulls had 6.5–8.5 kg increased live weight of, but were relatively older by 0.36–0.6 months. According to the milk yield trait, the total yield of milk fat and protein, highly rated fathers in breeding plants were significantly inferior to competitors with low breeding value in milk yield by 688–1136 kg, according to the difference "mother – daughter" in the total yield of milk fat and protein per lactation – by 43.1–97.3 kg. In one of the farms, the "daughter – mother" differences in milk yield in all bull clusters increased by 599–1275 kg of milk; according to mass fat fraction (MFF) — by 0.02–0.20% and for mass protein fraction (MPF) — by 0.19–0.31%; in another farm, the increase in milk yield was by 43.8–1551 kg of milk and 0.05–0.13% according to the MPF. The daughters of the "worst" fathers in terms of MFF and MPF significantly outperformed the group of daughters of the "best" fathers in terms of "daughter–mother" differences by 35.6 and 61.7 kg of total milk protein and fat output over 305 days of lactation. At the same time, there were no significant differences between daughters and peers. The relativity of the official definition of the values of the breeding value of breeding bulls has been proved. It is proposed, regardless of the "commercial" and previously conducted estimates of breeding bulls, to take into account the indices of pedigrees and types of selection when breeding them. Conclusions are drawn about the significant influence of the factor and selection methods on the values of milk production traits; in order to save growing costs, it is proposed to optimize the live weight and age of insemination of heifers in farms.

An integrated multitool analysis contributes elements to interpreting unclassified factor IX missense variants associated with hemophilia B

BackgroundDissection of genotype–phenotype relationships in hemophilia B (HB) is particularly relevant for challenging (mild HB) or for HB-associated but unclassified factor (F)IX missense variants. ObjectiveTo contribute elements to interpret unclassified HB-associated FIX missense variants by a multiple-level approach upon identification of a reported, but uncharacterized, FIX missense variant associated with mild HB. MethodsMolecular modeling of wild-type and V92A FIX variants, expression studies in HEK293 cells with evaluation of protein (ELISA, western blotting) and activity (activated partial thromboplastin time-based/chromogenic assays) levels after recombinant expression, and multiple prediction tools. ResultsThe F9(NM_000133.4):c.275T>C (p.V92A) variant was found in a mild HB patient (antigen, 45.4 U/dL; coagulant activity, 23.6 IU/dL; specific activity, 0.52). Newly generated molecular models showed alterations in Gla/EGF1–EGF2 domain conformation impacting Ca++ affinity and protein–protein interactions with activated factor XI (FXIa). Multitool analysis indicated a moderate impact on protein structure/function of the valine-to-alanine substitution, in accordance with patient and modeling data. Expression studies on the V92A variant showed a specific activity (0.49 ± 0.07; wild-type, 1.0 ± 0.1) recapitulating that of the natural variant, and pointed toward a moderate activation impairment as the main determinant underlying the p.V92A defect. The validated multitool approach, integrated with evidence-based data, was challenged on a panel (n = 9) of unclassified FIX missense variants, which resulted in inferred protein (secretion/function) outputs and HB severity. ConclusionThe rational integration of multitool and multiparameter analyses contributed elements to interpret genotype/phenotype relationships of unclassified FIX missense variants, with implications for diagnosis, management, and treatment of HB patients, and potentially translatable into other human disorders.

Effects of isoleucine, lysine, valine, and a group of non-essential amino acids on mammary amino acid metabolism in lactating dairy cows.

Intracellular amino acids (AA) regulate milk protein synthesis within the mammary glands by modifying mammary plasma flow (MPF) and AA transporter activity. Amino acid transporters catalyze translocation using Na+-gradient, substrate gradient (uniporters), and exchange mechanisms; further, they exhibit specificity for individual AA or groups of AA with similar side-chain properties within each transport system. Non-essential AA are actively transported through Na+-dependent transporters and, thus, are often utilized as intracellular currencies for EAA transport through exchange transporters. Therefore, it was hypothesized that individual EAA supplementation would compete with other EAA for shared transporters, and supplementation with Ala, Gln, and Gly would stimulate EAA transport through exchange transporters. Ten primiparous lactating dairy cows were divided into 2 groups based on milk production and were randomly assigned to treatment sequences within 2 balanced 5 × 5 Latin Squares by group. Period length was 14 d. Treatments were 9-d jugular infusions of 1) saline; 2) 34.5 g Val/d; 3) 32.7 g Ala/d: 40 g Gln/d: 26.7 g Gly/d (AQG); 4) 43 g Lys/d; or 5) 33.5 g Ile/d. All cows were fed a common base diet formulated to contain 15.0% CP. Ile, Lys, or AQG infusions did not affect milk protein or milk production; however, Val infusion decreased both. The effects of Val infusion on milk protein production appeared to be partially driven by decreased DMI. The decline in milk protein percentage indicated that milk lactose production was also affected. Additionally, Val infusion increased MPF efficiency (MPF/Milk; L/L) by approximately 44%. Val infusion tended to decrease or decreased mammary net uptakes of Lys, Leu, Met, and total AA. Ile infusion tended to increase its mammary net uptakes but did not affect any other AA. Lys and AQG infusions did not affect any mammary net uptakes. Val infusion tended to decrease Phe and total NEAA mammary clearance rates. AQG infusion stimulated Tyr clearance rates and tended to decline System N mammary clearance rates. Mammary uptake to milk protein output ratios (U:O) of BCAA did not differ from 1 for Val-infused cows, which indicated that little intramammary catabolism was occurring. Additionally, the average NEAA U:O in response to all treatments except Val was 0.70, but Val-infused cows had NEAA U:O that averaged 0.09 indicating increased synthesis within the glands. The effects of Val on mammary net clearance rates of multiple EAA support the incorporation of AA limitations in ration optimizers to prevent AA imbalances. It is possible that over-supplementation of EAA other than Val may also decrease DMI and mammary activity. Identifying efficiency apexes for each of the EAA will allow more precise diet formulation and supplementation, leading to improved production efficiency.

Enhancement of protein production in Aspergillus niger by engineering the antioxidant defense metabolism

BackgroundResearch on protein production holds significant importance in the advancement of food technology, agriculture, pharmaceuticals, and bioenergy. Aspergillus niger stands out as an ideal microbial cell factory for the production of food-grade proteins, owing to its robust protein secretion capacity and excellent safety profile. However, the extensive oxidative folding of proteins within the endoplasmic reticulum (ER) triggers ER stress, consequently leading to protein misfolding reactions. This stressful phenomenon results in the accelerated generation of reactive oxygen species (ROS), thereby inducing oxidative stress. The accumulation of ROS can adversely affect intracellular DNA, proteins, and lipids.ResultIn this study, we enhanced the detoxification of ROS in A. niger (SH-1) by integrating multiple modules, including the NADPH regeneration engineering module, the glutaredoxin system, the GSH synthesis engineering module, and the transcription factor module. We assessed the intracellular ROS levels, growth under stress conditions, protein production levels, and intracellular GSH content. Our findings revealed that the overexpression of Glr1 in the glutaredoxin system exhibited significant efficacy across various parameters. Specifically, it reduced the intracellular ROS levels in A. niger by 50%, boosted glucoamylase enzyme activity by 243%, and increased total protein secretion by 88%.ConclusionThe results indicate that moderate modulation of intracellular redox conditions can enhance overall protein output. In conclusion, we present a strategy for augmenting protein production in A. niger and propose a potential approach for optimizing microbial protein production system.

Computational design of Periplasmic binding protein biosensors guided by molecular dynamics.

Periplasmic binding proteins (PBPs) are bacterial proteins commonly used as scaffolds for substrate-detecting biosensors. In these biosensors, effector proteins (for example fluorescent proteins) are inserted into a PBP such that the effector protein's output changes upon PBP-substate binding. The insertion site is often determined by comparison of PBP apo/holo crystal structures, but random insertion libraries have shown that this can miss the best sites. Here, we present a PBP biosensor design method based on residue contact analysis from molecular dynamics. This computational method identifies the best previously known insertion sites in the maltose binding PBP, and suggests further previously unknown sites. We experimentally characterise fluorescent protein insertions at these new sites, finding they too give functional biosensors. Furthermore, our method is sufficiently flexible to both suggest insertion sites compatible with a variety of effector proteins, and be applied to binding proteins beyond PBPs.

ALS-associated VRK1 R321C mutation causes proteostatic imbalance and mitochondrial defects in iPSC-derived motor neurons

Vaccinia-related kinase 1 (VRK1) is a gene which has been implicated in the pathological process of a broad range of neurodevelopmental disorders as well as neuropathies, such as Amyotrophic Lateral Sclerosis (ALS). Here we report a family presenting ALS in an autosomal recessive mode of inheritance, segregating with a homozygous missense mutation located in VRK1 gene (p.R321C; Arg321Cys). Proteomic analyses from iPSC-derived motor neurons identified 720 proteins eligible for subsequent investigation, and our exploration of protein profiles revealed significant enrichments in pathways such as mTOR signaling, E2F, MYC targets, DNA repair response, cell proliferation and energetic metabolism. Functional studies further validated such alterations, showing that affected motor neurons presented decreased levels of global protein output, ER stress and downregulation of mTOR signaling. Mitochondrial alterations also pointed to decreased reserve capacity and increased non-mitochondrial oxygen consumption. Taken together, our results present the main pathological alterations associated with VRK1 mutation in ALS.

Long-duration environmental biosensing by recording analyte detection in DNA using recombinase memory.

Microbial biosensors that convert environmental information into real-time visual outputs are limited in their sensing abilities in complex environments, such as soil and wastewater, due to optical inaccessibility. Biosensors that could record transient exposure to analytes within a large time window for later retrieval represent a promising approach to solve the accessibility problem. Here, we test the performance of recombinase-memory biosensors that sense a sugar (arabinose) and a microbial communication molecule (3-oxo-C12-L-homoserine lactone) over 8 days (~70 generations) following analyte exposure. These biosensors sense the analyte and trigger the expression of a recombinase enzyme which flips a segment of DNA, creating a genetic memory, and initiates fluorescent protein expression. The initial designs failed over time due to unintended DNA flipping in the absence of the analyte and loss of the flipped state after exposure to the analyte. Biosensor performance was improved by decreasing recombinase expression, removing the fluorescent protein output, and using quantitative PCR to read out stored information. Application of memory biosensors in wastewater isolates achieved memory of analyte exposure in an uncharacterized Pseudomonas isolate. By returning these engineered isolates to their native environments, recombinase-memory systems are expected to enable longer duration and in situ investigation of microbial signaling, cross-feeding, community shifts, and gene transfer beyond the reach of traditional environmental biosensors.IMPORTANCEMicrobes mediate ecological processes over timescales that can far exceed the half-lives of transient metabolites and signals that drive their collective behaviors. We investigated strategies for engineering microbes to stably record their transient exposure to a chemical over many generations through DNA rearrangements. We identify genetic architectures that improve memory biosensor performance and characterize these in wastewater isolates. Memory biosensors are expected to be useful for monitoring cell-cell signals in biofilms, detecting transient exposure to chemical pollutants, and observing microbial cross-feeding through short-lived metabolites within cryptic methane, nitrogen, and sulfur cycling processes. They will also enable in situ studies of microbial responses to ephemeral environmental changes, or other ecological processes that are currently challenging to monitor non-destructively using real-time biosensors and analytical instruments.

Cattle production strategies to deliver protein with less land and lower environmental impact

Global land resources are over-exploited and natural habitats are declining, often driven by expanding livestock production. In Ireland, pastureland for grazing cattle and sheep account for circa 60% of terrestrial land use. The agriculture, forestry and other land use sector (AFOLU) is responsible for 44% of national greenhouse gas (GHG) emissions. A new Grassland Animal response Model (GLAM) was developed to relate livestock-cohort grass and feed requirements to farm-grassland system areas, enhancing environmental assessment of prospective AFOLU configurations. Although land conversion targets are often well-defined, they tend to lack a clear definition of where land sparing can occur. Through analyses of 10 scenarios of milk and beef production and management strategies, we found that displacing beef cows with dairy cows can increase national protein output while sparing up to 0.75 million ha (18%) of grassland (albeit with a minor increase in overseas land requirement for additional concentrate feed). Reducing slaughter age, increasing exports of male dairy calves and increasing grassland use efficiency on beef farms each achieved between 0.19 and 0.32 million ha of land sparing. Sexed semen to achieve more favourable male-female birth ratios had a minor impact. GHG emissions, ammonia emissions and nutrient leaching were only reduced substantially when overall cattle numbers declined, confirming the need for cattle reductions to achieve environmental objectives. Nonetheless, application of GLAM shows potential for improved grass and cattle management to spare good quality land suitable for productive forestry and wetland restoration. This change is urgently needed to generate scalable carbon dioxide removals from the land sector in Ireland, and globally.

Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo.

Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am─a common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap-m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6Am in mRNA.

MRNAid, an open-source platform for therapeutic mRNA design and optimization strategies.

Recent COVID-19 vaccines unleashed the potential of mRNA-based therapeutics. A common bottleneck across mRNA-based therapeutic approaches is the rapid design of mRNA sequences that are translationally efficient, long-lived and non-immunogenic. Currently, an accessible software tool to aid in the design of such high-quality mRNA is lacking. Here, we present mRNAid, an open-source platform for therapeutic mRNA optimization, design and visualization that offers a variety of optimization strategies for sequence and structural features, allowing one to customize desired properties into their mRNA sequence. We experimentally demonstrate that transcripts optimized by mRNAid have characteristics comparable with commercially available sequences. To encompass additional aspects of mRNA design, we experimentally show that incorporation of certain uridine analogs and untranslated regions can further enhance stability, boost protein output and mitigate undesired immunogenicity effects. Finally, this study provides a roadmap for rational design of therapeutic mRNA transcripts.

Patients with oral lichen planus display lower levels of salivary acidic glycoproteins than individuals without oral mucosal disease

ObjectivesSalivary proteins, acidic glycoproteins, and free calcium might take part in oral mucosal defence against inflammation in oral lichen planus (OLP). The study aimed to investigate whether the levels of sulfated and sialylated glycoproteins, total protein, and free calcium in saliva from patients with OLP differ from those of individuals without oral mucosal diseases.Material and MethodsPatients diagnosed with OLP (n = 25) and two control groups without any oral mucosal disease; age- and gender-matched controls (n = 25, 65.6 ± 2.9 years), and younger controls (n = 25, 41.8 ± 2.5 years) were included. Subjective dry mouth (xerostomia) was assessed by asking a single-item question. Chew-stimulated whole saliva was collected to measure sulfated and sialylated glycoproteins by the Alcian Blue method. The total protein was determined spectrophotometrically, and the free calcium measured using an electrode.ResultsThe output of salivary sulfated and sialylated glycoproteins in the OLP group (21.8 ± 2.4 µg/min) was lower than in the age- and gender-matched controls (43.0 ± 2.9 µg/min, p = 0.0002), whereas the total protein and calcium output did not differ between the three groups (p > 0.05). The prevalence of xerostomia was significantly higher in the OLP group compared to both control groups (p = 0.038).ConclusionsPatients with OLP showed a high prevalence of xerostomia and lower levels of salivary acidic type glycoproteins compared to the individuals without oral mucosa disease.Clinical relevanceIt is relevant to investigate the role of acidic glycoproteins in the pathogenesis of OLP.

Abstract C051: Alteration of protein translation by eIF4A1 inhibition exquisitely primes lymphoma cells to induction of ferroptosis

Abstract Background: Ferroptosis induction as a cancer therapy faces challenges establishing clinical therapeutic windows. Cells employ diverse mechanisms to prevent iron-mediated oxidation of membrane polyunsaturated fatty acids that causes ferroptosis, but populations with low rates of protein synthesis such as hematopoietic stem cells are more susceptible. Therefore, we hypothesized protein synthesis inhibitors might enhance therapeutic ferroptosis induction. Rocaglates, inhibitors targeting eIF4A1, have entered clinical trials with promising potential against diffuse large B-cell lymphoma (DLBCL), which also exhibits pre-clinical susceptibility to ferroptosis induction. Up to 40% of patients with DLBCL, the most common lymphoma type, have poor outcomes requiring new approaches to address these persistent unmet clinical needs. Our results reveal striking synergy of rocaglates, including the clinical compound zotatifin (eFT226), with ferroptosis inducers. TMT-pSILAC and RNA-seq analyses revealed profound deregulation of ferroptotic protection resulting from rocalate effects on protein synthesis. Methods: We used RNA-seq and TMT-pSILAC followed by mass spectrometry to analyze transcriptome and protein output in SU-DHL-10 DLBCL cells treated with zotatifin or DMSO for 24h. Enrichment analyses (Enrichr platform, ChIP-X Enrichment Analysis 3, GSEA) identified differentially expressed genes, proteins and transcription factors. We explored synergy between zotatifin and ferroptosis modulators in DLBCL cell lines and measured a wide variety of associated cellular endpoints including lipid peroxidation, glutathione levels, and glutathione peroxidase 4 (GPX4) expression. Results: TMT-pSILAC translatome assessment during rocaglate exposure revealed expression alteration(log2FC ≥ 1.5) of numerous key proteins involved in oxidative stress (i.e. SDHB, SDHA, TRAP1, SCO1, UQCRH, ETFA, COX7C) and enrichment of factors indicating induction of an antioxidant response (CD98HC, NRF1, ATF2, SP2, and NFE2L). These changes led to high synergy of zotatifin in combination with a wide variety of ferroptosis inducers (Bliss δ synergy score > 10) and antagonism with ferroptosis inhibitors (Bliss δ synergy score < - 10). These results were consistent across multiple DLBCL mouse and human experimental systems, and preliminarily appear present also in models of T-cell lymphomas. Lipid peroxidation levels were significantly enhanced when zotatifin was combined with erastin (p-value < 0.0005). Moreover, zotatifin treatment resulted in elevated levels of reduced glutathione (GSH), an effect overcome by addition of ferroptosis inducers (p-value < 0.005). Consistently, GPX4 expression was also impaired. Conclusions: Rocaglates induce novel alterations in oxidative stress factors and associated response pathways, sensitizing B and T lymphomas to therapeutic ferroptosis induction. These findings provide valuable insights for using rocaglates in combination with ferroptosis induction to overcome heterogeneous resistance pathways in high-risk lymphoma patients. Citation Format: Paola Manara, Alexa Marie Barroso, Abdessamad Youssfi Alaoui, Olivia Barbara Lightfuss, Tyler Andrew Cunningham, Kyle Hoffman, Caroline Alice Coughlin, Jonathan Harry Schatz. Alteration of protein translation by eIF4A1 inhibition exquisitely primes lymphoma cells to induction of ferroptosis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C051.

Subcytoplasmic location of translation controls protein output

The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined mRNA enrichment in TIS granules (TGs) and the rough endoplasmic reticulum (ER) through particle sorting and isolated cytosolic mRNAs by digitonin extraction. When focusing on genes that encode non-membrane proteins, we observed that 52% have transcripts enriched in specific compartments. Compartment enrichment correlates with a combinatorial code based on mRNA length, exon length, and 3' UTR-bound RNA-binding proteins. Compartment-biased mRNAs differ in the functional classes of their encoded proteins: TG-enriched mRNAs encode low-abundance proteins with strong enrichment of transcription factors, whereas ER-enriched mRNAs encode large and highly expressed proteins. Compartment localization is an important determinant of mRNA and protein abundance, which is supported by reporter experiments showing that redirecting cytosolic mRNAs to the ER increases their protein expression. In summary, the cytoplasm is functionally compartmentalized by local translation environments.

PSIX-10 Influence of Graded Levels of Microbially Enhanced Protein on Nutrient Digestibility of Extruded Cat Foods

Abstract Pet food ingredients that bring extra nutrition and health benefits are much better accepted than being simply an economic choice by the manufacturer. Microbially enhanced protein (MEP) from soybeans is a fermented ingredient that is believed to promote nutrient utilization. The objective of this study was to determine the effect of increasing levels of MEP on nutrient digestibility of extruded cat food. Four extruded dietary treatments differing by level of MEP in exchange for soybean meal (15%; SBM) at 5, 10 and 15% (5MEP, 10MEP and 15MEP, respectively) were randomly assigned to 12 individually housed adult domestic shorthair cats (6 castrated males and 6 spayed females) of similar age (0.9 ± 0.23 months) and body weight (4.35 ± 0.87). The study was designed as a 4 × 4 replicated Latin square with 9-day adaptation followed by 5-day total fecal collection for each period. Data were analyzed using a mixed model through SAS (version 9.4, SAS Institute, Inc., Cary, NC) with treatment as a fixed effect and square (i.e., replicate), period, and cat(square) as random effects. Treatment means were separated by Tukey’s test. The 15MEP increased fecal moisture content (P<0.05) while 5MEP and 10MEP made no difference compared with SBM. No differences were observed for food intake, fecal dry matter output, fecal score, fecal pH or apparent total tract digestibility of dry matter, organic matter, crude protein or gross energy for MEPs compared with SBM. However, crude fat digestibility of cats fed 5MEP was lower (P<0.05) than cats fed with10MEP and 15MEP but not different from cats fed with SBM. These results suggest that MEP did not have adverse effects on nutrient digestibility when added to extruded diets at up to 15% in healthy adult cats.

The impact of participatory action research and endogenous integrated soil fertility management on farm-gate dietary outputs in northern Tanzania

In most developing countries, although agricultural extension and research devolved since 1980s to promote relevance, cost-effectiveness, ownership, and sustainability, participatory action research (PAR) have been run, albeit with limited empirical evidence on their impacts on farmers livelihoods. The study use a three-stage extended regression model (ERM) to estimate the effect PAR and the promoted agronomic practices on crop produce dietary outputs considering potential endogenous engagement, omitted variable bias, reverse causality, endogenous covariates, and factor simultaneity. Using a sample of 607 small family farms with varying levels of research engagement in the Northern Highlands of Tanzania, the study found that PAR was associated with increased farm-gate dietary outcomes: per-capita calories, proteins, and minerals produced by 139, 216, and 143 %, respectively, and consumption of farm-produced minerals by 74–200 %. The organic manure application was associated with increased the dietary outputs by 62–113 % while the application of inorganic fertilizer with increased protein and mineral outputs by 68 and 105 %, respectively. The crop diversification was associated with increased dietary outputs by 11–25 % while pest and diseases control with increased dietary outputs by 12–17 % but with reduced consumption by 13–14 %. Soil and water conservation measures including terracing were associated with decreased dietary outputs. These findings indicate that PAR contributes to nutritional outcomes of smallholder farmers contingent on the promotion of low-cost input sources and crop diversification which can be leveraged to inform upscaling of participatory policies, strategies, and technologies.

81 Creating a More Efficient Production System (Feed Efficiency in the Real World)

Abstract Modern food production is the subject of much scrutiny, especially with regard to potential environmental impact and sustainability. To accurately assess impact, both costs and benefits should be evaluated, and inputs and outputs should be standardized. Emphasis is commonly placed on efficiency when comparing production of various animal proteins. Efficiency of a process can be described as a ratio. This ratio is simply: 1) input to output or 2) output to input. The relationship of input to output such as feed input to gain (i.e., body weight, carcass weight, or retail cut weight) output is termed feed to gain (F:G) and the relationship of output to input is termed gain to feed (G:F). Production efficiency is optimal when animals are depositing lean tissue and poorest when the animal fattens and approaches chemical maturity. Because of the energy density of tissues deposited, energetic (calorie:calorie) efficiency can look very different than production (feed) efficiency. Factors related to animal health are also important as a reduction in death loss can offer a substantial boost to efficiency. Feed efficiency should be expressed on a dry matter basis. Failure to account for moisture content in a feedstuff can make feed efficiency appear poorer, especially when cattle are compared with non-ruminants, which typically consume drier diets. Should the environmental impact of beef production be lifetime or feedyard only? Much, if not all, of what a cow eats has no other value to mankind. The authors believe that the creation of human available calories from cellulose must be accounted for. For a more just comparison, efficiency could be measured using inputs that could have been used for human consumption, when making across species comparisons. With this in mind, non-ruminants are not nearly as attractive for large-scale protein production. There would also be value in evaluating efficiency of input to ideal protein output, rather than just weight gain or pounds of food produced. In that scenario plant-based foods would fare poorly. How best can cattle be used to supply the bioavailable nutrients for a growing population? Modern cattle eat large amounts of feed, get very large and have the ability to fatten and still be very efficient. Continual improvement and advancement is critical; but more meaningful assessments of efficiency are possible when the appropriate input:ouput comparisons are evaluated.