Efficiency Of Protein Synthesis Research Articles

Computational Strategies to Enhance Cell-Free Protein Synthesis Efficiency

Cell-free protein synthesis (CFPS) has emerged as a powerful tool for protein production, with applications ranging from basic research to biotechnology and pharmaceutical development. However, enhancing the efficiency of CFPS systems remains a crucial challenge for realizing their full potential. Computational strategies offer promising avenues for optimizing CFPS efficiency by providing insights into complex biological processes and enabling rational design approaches. This review provides a comprehensive overview of the computational approaches aimed at enhancing CFPS efficiency. The introduction outlines the significance of CFPS and the role of computational methods in addressing efficiency limitations. It discusses mathematical modeling and simulation-based approaches for predicting protein synthesis kinetics and optimizing CFPS reactions. The review also delves into the design of DNA templates, including codon optimization strategies and mRNA secondary structure prediction tools, to improve protein synthesis efficiency. Furthermore, it explores computational techniques for engineering cell-free transcription and translation machinery, such as the rational design of expression systems and the predictive modeling of ribosome dynamics. The predictive modeling of metabolic pathways and the energy utilization in CFPS systems is also discussed, highlighting metabolic flux analysis and resource allocation strategies. Machine learning and artificial intelligence approaches are being increasingly employed for CFPS optimization, including neural network models, deep learning algorithms, and reinforcement learning for adaptive control. This review presents case studies showcasing successful CFPS optimization using computational methods and discusses applications in synthetic biology, biotechnology, and pharmaceuticals. The challenges and limitations of current computational approaches are addressed, along with future perspectives and emerging trends, such as the integration of multi-omics data and advances in high-throughput screening. The conclusion summarizes key findings, discusses implications for future research directions and applications, and emphasizes opportunities for interdisciplinary collaboration. This review offers valuable insights and prospects regarding computational strategies to enhance CFPS efficiency. It serves as a comprehensive resource, consolidating current knowledge in the field and guiding further advancements.

Tyrosine transfer RNA levels and modifications during blood-feeding and vitellogenesis in the mosquito, Aedes aegypti.

Mosquitoes such as Aedes aegypti must consume a blood meal for the nutrients necessary for egg production. Several transcriptome and proteome changes occur post-blood meal that likely corresponds with codon usage alterations. Transfer RNA (tRNA) is the adapter molecule that reads messenger RNA codons to add the appropriate amino acid during protein synthesis. Chemical modifications to tRNA enhance codon decoding, improving the accuracy and efficiency of protein synthesis. Here, we examined tRNA modifications and transcripts associated with the blood meal and subsequent periods of vitellogenesis in A. aegypti. More specifically, we assessed tRNA transcript abundance and modification levels in the fat body at critical times post blood-feeding. Based on a combination of alternative codon usage and identification of particular modifications, we discovered that increased transcription of tyrosine tRNAs is likely critical during the synthesis of egg yolk proteins in the fat body following a blood meal. Altogether, changes in both the abundance and modification of tRNA are essential factors in the process of vitellogenin production after blood-feeding in mosquitoes.

Towards Self-regeneration: Exploring the Limits of Protein Synthesis in the Protein Synthesis Using Recombinant Elements (PURE) Cell-free Transcription-Translation System.

Self-regeneration is a key function of living systems that needs to be recapitulated in vitro to create a living synthetic cell. A major limiting factor for protein self-regeneration in the PURE cell-free transcription-translation system is its high protein concentration, which far exceeds the system's protein synthesis rate. Here, we were able to drastically reduce the nonribosomal PURE protein concentration up to 97.3% while increasing protein synthesis efficiency. Although crowding agents were not effective in the original PURE formulation, we found that in highly dilute PURE formulations, addition of 6% dextran considerably increased protein synthesis rate and total protein yield. These new PURE formulations will be useful for many cell-free synthetic biology applications, and we estimate that PURE can now support the complete self-regeneration of all 36 nonribosomal proteins, which is a critical step toward the development of a universal biochemical constructor and living synthetic cell.

Resolution of ribosomal stalling by EF-P and ABCF ATPases YfmR and YkpA/YbiT.

Efficiency of protein synthesis on the ribosome is strongly affected by the amino acid composition of the assembled amino acid chain. Challenging sequences include proline-rich motifs as well as highly positively and negatively charged amino acid stretches. Members of the F subfamily of ABC ATPases (ABCFs) have been long hypothesised to promote translation of such problematic motifs. In this study we have applied genetics and reporter-based assays to characterise the four housekeeping ABCF ATPases of Bacillus subtilis: YdiF, YfmM, YfmR/Uup and YkpA/YbiT. We show that YfmR cooperates with the translation factor EF-P that promotes translation of Pro-rich motifs. Simultaneous loss of both YfmR and EF-P results in a dramatic growth defect. Surprisingly, this growth defect can be largely suppressed though overexpression of an EF-P variant lacking the otherwise crucial 5-amino-pentanolylated residue K32. Using in vivo reporter assays, we show that overexpression of YfmR can alleviate ribosomal stalling on Asp-Pro motifs. Finally, we demonstrate that YkpA/YbiT promotes translation of positively and negatively charged motifs but is inactive in resolving ribosomal stalls on proline-rich stretches. Collectively, our results provide insights into the function of ABCF translation factors in modulating protein synthesis in B. subtilis.

Effects of different concentrate levels in AGRI-002E sorghum silage-based diets on nutrient intake and digestibility, ruminal pH and ammonia concentration, ruminal degradability, and microbial efficiency in beef cattle

This study aimed to evaluate the effect of increasing concentrate levels in AGRI-002E sorghum silage (SS)-based diets on nutrient intake and digestibility, ruminal pH and ammonia concentration, Nitrogen (N) balance, efficiency of microbial protein synthesis, and in situ degradability of complete diets. Five rumen-cannulated Nellore bulls (age = 8 ± 1.0 months; initial BW = 242 ± 5 kg) received five dietary treatments in a 5×5 Latin square experimental design. The dietary treatments consisted of five concentrate levels (0, 200, 400, 600, and 800 g of concentrate/kg on a DM basis) in SS-based diets. The experiment lasted 120 d, with five periods of 24 d. Each period consisted of 17 d for dietary adaptation, and 7 d for data collection. In situ degradability assays were conducted to estimate ruminal degradability. Total feces and urine collection were performed to estimate nutrient intake and digestibility and estimate N balance. Omasal and ruminal digesta collection were performed to estimate ruminal digestibility and ruminal parameters. Increasing concentrate levels in SS-based diets led to linear increases (P<0.001) in the intake of dry matter (DM), organic matter (OM), crude protein (CP), and ether extract (EE). Neutral detergent fiber corrected for ash and protein contamination (apNDF) and starch intake showed quadratic responses (P≤0.008). Ruminal digestibility of DM, OM and CP responded quadratically (P≤0.040), while apNDF exhibited linear decrease (P=0.003). Starch ruminal digestibility exhibited a cubic effect (P=0.016). Apparent total-tract digestibility of DM, OM, and EE increased linearly (P≤0.001), whereas apNDF, and CP digestibility decreased linearly (P≤0.012), and starch presented a quadratic effect (P=0.029). In situ ruminal degradation parameters increased linearly with higher concentrate levels (P<0.001). Ruminal pH exhibited a quadratic pattern (P=0.006), ammonia concentration linearly decreased (P=0.003). Total volatile fatty acids, and butyrate showed linear increases (P<0.001), acetate and propionate had quadratic effects (P<0.001), while the ratio Acetate to Propionate decreased linearly (P<0.001). N intake, fecal N excretion, retained N, total digestible nutrients, digestible organic matter, and microbial production increased linearly (P<0.001), although urine N excretion, urinary urea, and blood urea concentration decreased linearly (P<0.028). Therefore, increasing concentrate levels in AGRI-002E sorghum silage-based diets improve TDN intake, microbial protein synthesis efficiency, in situ ruminal degradability parameters, and nitrogen utilization. Moreover, our findings indicate that AGRI-002E sorghum silage demonstrates potential as a fiber source for high-concentrate diets. However, its effectiveness is limited without concentrate supplementation, emphasizing the importance of balanced dietary composition for optimal utilization in beef cattle.

Examining the Effects of Temperature on the Evolution of Bacterial tRNA Pools.

The genetic code consists of 61 codons coding for 20 amino acids. These codons are recognized by transfer RNAs (tRNAs) that bind to specific codons during protein synthesis. All organisms utilize less than all 61 possible anticodons due to base pair wobble: the ability to have a mismatch with a codon at its third nucleotide. Previous studies observed a correlation between the tRNA pool of bacteria and the temperature of their respective environments. However, it is unclear if these patterns represent biological adaptations to maintain the efficiency and accuracy of protein synthesis in different environments. A mechanistic mathematical model of mRNA translation is used to quantify the expected elongation rates and error rate for each codon based on an organism's tRNA pool. A comparative analysis across a range of bacteria that accounts for covariance due to shared ancestry is performed to quantify the impact of environmental temperature on the evolution of the tRNA pool. We find that thermophiles generally have more anticodons represented in their tRNA pool than mesophiles or psychrophiles. Based on our model, this increased diversity is expected to lead to increased missense errors. The implications of this for protein evolution in thermophiles are discussed.

239 Variation in muscle protein synthesis in growing gilts is partially associated with insulin resistance and sensitivity

Abstract Pigs with similar body weight (BW), same genetic background and sex vary in protein deposition (PD) overgrowth. We aimed to study metabolic factors influencing PD in 65 kg BW gilts. Gilts were fed ad libitum a diet balanced to maximize PD. Dual X-rays were conducted to estimate body protein mass on d 1 and 21, allowing calculation of PD by difference. On d 23, the gilts were categorized into Low (157 g/d) and High (219 ± 9.97 g/d) PD groups. In each group, eight pigs underwent jugular vein cannulation. After a 48-h recovery period, where they were fed at 90% of the measured feed intake, they were fasted for 8 h. All pigs were then given a 300 g meal at time 0, and blood samples were collected at 10, 15, 20, 30, 40, 50, 60, 75, 90, 120, 150, 178, and 178 min, to study insulin and glucose responses. At 180 min post-meal, a solution of L-13C valine was administered i.v. (18 ape – flooding dose), and liver, duodenum, and longissimus dorsi muscle samples were collected within 5 minutes of slaughter. The fractional synthesis rate (FSR, %/d), absolute synthesis rate (ASR, g/d), ribosomal capacity for protein synthesis (Cs), and the efficiency of protein synthesis (KRNA) based on total RNA concentration in skeletal muscle were calculated. The insulin resistance index (HOMA-B) and insulin sensitivity index (QUICKI) were also determined. The difference between Low and High PD gilts was compared using a F-test. The longissimus dorsi muscle exhibited a 31% greater (P = 0.01) FSR in High compared with Low PD group. No significant differences were observed in the FSR of the jejunum and liver. High PD gilts showed a 55% greater ASR in whole-body muscles (P = 0.003) and 42% greater ASR in the liver (P = 0.04) compared with Low PD gilts, with no difference for the ASR in the jejunum. The KRNA in the longissimus dorsi was 48% greater in High PD gilts (P = 0.01), with no differences in KRNA for the jejunum or liver. Cs did not differ between Low and High PD gilts. The QUICKI index tended to be greater in High PD gilts (P = 0.10). Spearman’s correlation revealed that longissimus dorsi FSR was negatively correlated with the HOMA-B index (r = -0.53; P = 0.04). Increases in the HOMA-B index tended to be associated with greater KRNA (r = -0.49; P = 0.06). These correlations highlight the complex interplay of metabolic and hormonal factors involved in the observed differences in protein synthesis and efficiency in growing gilts. Insulin resistance and sensitivity may play a role in influencing skeletal muscle PD variations.

A data-driven estimation of the ribosome drop-off rate in S. cerevisiae reveals a correlation with the genes length.

Ribosomes are the molecular machinery that catalyse all the fundamental steps involved in the translation of mRNAs into proteins. Given the complexity of this process, the efficiency of protein synthesis depends on a large number of factors among which ribosome drop-off (i.e. the premature detachment of the ribosome from the mRNA template) plays an important role. However, an in vitro quantification of the extent to which ribosome drop-off occurs is not trivial due to difficulties in obtaining the needed experimental evidence. In this work we focus on the study of ribosome drop-off in Saccharomyces cerevisiae by using 'Ribofilio', a novel software tool that relies on a high sensitive strategy to estimate the ribosome drop-off rate from ribosome profiling data. Our results show that ribosome drop-off events occur at a significant rate also when S. cerevisiae is cultured in standard conditions. In this context, we also identified a correlation between the ribosome drop-off rate and the genes length: the longer the gene, the lower the drop-off rate.

One-pot ligation of multiple peptide segments via N-terminal thiazolidine deprotection chemistry.

Peptide ligation chemistries have revolutionized the synthesis of proteins with site-specific modifications or proteomimetics through assembly of multiple peptide segments. In order to prepare polypeptide chains consisting of 100-150 amino acid residues or larger generally assembled from three or more peptide segments, iterative purification process that decreases the product yield is usually demanded. Accordingly, methodologies for one-pot peptide ligation that omit the purification steps of intermediate peptide segments have been vigorously developed so far to improve the efficiency of chemical protein synthesis. In this chapter, we first outline the concept and recent advances of one-pot peptide ligation strategies. Then, the practical guideline for the preparation of peptide segments for one-pot peptide ligation is described with an emphasis on diketopiperazine thioester synthesis. Finally, we disclose the explicit protocols for one-pot four segment ligation via repetitive deprotection of N-terminal thiazolidine by a 2-aminobenzamide type aldehyde scavenger.

Evaluation Additional of Synbiotic Powder in Feed to Improve Milk Production and Quality of Dairy Cattle

Evaluation Additional of Synbiotic Powder in Feed to Improve Milk Production and Quality of Dairy Cattle

Effect of degradable protein in cow diet on body systems’ physiological status

The relationship between indicators of enzymatic and microbiological processes in the rumen and metabolism in the body was studied in lactating cows when the content of soluble protein in the diet was changed. The change in digestible protein content was provided by different ratios of natural feed and synthetic protein additives. The study was conducted on 3 lactating first-calf Holstein cows with rumen and 12 intestinal cannulas. The rate of fractional efflux from the complex stomach was evaluated by chromium oxide and microbial synthesis by purine bases. Experimental data were obtained to improve feeding rates, evaluate diets for high-yielding dairy cattle and develop rates of readily available carbohydrates in diets. At increase of share of soluble and degradable protein fractions in feed there is a natural increase of ammonia formation in rumen and urea content in blood and at achievement of ratio of degradable protein fractions and easily available carbohydrates in feed in the ratio of 0.44, there is maximum efficiency of microbial synthesis and supply with available amino acids of synthesis of milk components, which is proved by increase of concentration of amino acids in blood and formation of milk protein and promotes more complete realization of productive capacity of dairy cattle. Application to lactating cows of a ration with a higher ratio of degradable protein and DHC (higher than 0.44) reduces the efficiency of feed nitrogen utilization by reducing the efficiency of microbial protein synthesis by 10%.

The balance of rumen degradable protein and readily available carbohydrate in sheep rations on in vitro fermentability.

Protein and carbohydrates are substances needed by ruminants, especially sheep. Providing protein and carbohydrates must pay attention to their degradation. In addition, balancing nutrients to meet the nutritional needs of rumen microbes is very important because the unbalanced availability of rumen degradable protein (RDP) and readily available carbohydrate (RAC) at one time can cause suboptimal microbial protein synthesis efficiency. Completely randomized design with a nonfactorial pattern of five treatments with three replications. Treatment includes RDP and RAC ratios, namely R1 = 2.30, R2 = 2.00, R3 = 1.70, R4 = 1.50, and R5 = 1.30. The results showed that the balance of RDP and RAC in sheep ration formulation in vitro had a very significant effect (p < 0.01) on NH3, microbial protein synthesis, total gas, total microbes, and organic matter digestibility (OMD) but had no significant effect (p > 0.05) on rumen pH and dry matter digestibility (DMD). The optimal balance of RDP and RAC in the formulation of sheep rations in vitro was obtained at a ratio of 2.30 with NH3 (mM) 8.47, rumen pH 5.97, microbial protein synthesis (mg/100 ml) 123, gas (ml/g of material) 145, total microbes (cells/ml) 2.012 × 106, (log CFU cells/ml) 6.3025, DMD 61.0%, and OMD 63.1%.

Effect of pregnancy and feeding level on voluntary intake, digestion, and microbial nitrogen synthesis in Zebu beef cows.

The objective of this research was to evaluate how pregnancy and feeding regimens affect the feed intake, digestibility, and efficiency of microbial nitrogen (N) synthesis in beef cows. Forty-four multiparous Nellore cows, comprising 32 gestating and 12 non-gestating cows, with an average weight of 451 ± 10kg, were assigned to either a HIGH (ad libitum) or LOW (limited feeding at 1.2 times maintenance based on the NRC) feeding regimen during the gestational period. The dry matter intake (DMI) in kg/d was significantly greater (P < 0.01) in HIGH-fed cows. The DMI reduced (P < 0.05) in proportion to the shrunk body weight (SBW) as days of pregnancy (DOP) increased. The interaction between feeding level and DOP was significant (P < 0.05) for the digestibility of dry matter (DM), organic matter (OM), N compounds, ether extract (EE), ash- and protein-free neutral detergent fiber (NDFap), gross energy (GE), and total digestible nutrients (TDN). Except for DM and TDN digestibility, there was a reduced nutrient digestibility as gestation progressed in HIGH-fed cows. In contrast, digestibility increased as a function of DOP in LOW-fed cows. Microbial N synthesis (g/day) was significantly higher in HIGH-fed cows (P < 0.001) compared to LOW-fed cows. The efficiency of microbial N production per g of N intake and kg of digestible OM intake was (P = 0.021) and tended (P = 0.051) to be greater in LOW-fed cows compared to HIGH-fed cows. In summary, HIGH-fed Nellore cows reduce feed intake and digestibility with advancing gestation, affecting feed utilization. In addition, LOW-fed cows, showed higher microbial protein synthesis efficiency, potentially making them more nutrient-efficient under challenging nutritional conditions.

Assessment of Nutritional Potential and Methane Mitigation Efficacy of Napier Grass Cultivars

Napier grass is cultivated as a high-yielding forage crop and widely utilized in the nutrition of grazing animals such as cattle and goats due to its rapid growth rate, expansive leaf surface, and ability to thrive even in low-quality soils, making it a valuable animal feed option. In this study, the chemical compositions, in vitro gas (GP) and methane (CH4) production, metabolizable energy (ME), organic matter digestibility (OMD), true digestible dry matter (TDDM), microbial protein (MP), partitioning factor (PF), microbial protein synthesis efficiency (MPSE, and relative feed values (RFV) of the grasses Packhong1 (Penisetum purpureum x Penisetum americanum), Chinese Pennisetum (Penisetum purpureum x Penisetum alopecurides), and Dwarf Elephant (Pennisetum purpureum cv. Mott) were investigated. Significant differences were found in the chemical contents of napier varieties used in the study (p

Multi-Attribute Monitoring of Therapeutic mRNA by Liquid Chromatography–Mass Spectrometry

Therapeutic mRNA is receiving growing interest in various therapeutic applications such as genome editing, cancer immunotherapy and prophylactic vaccines. As with other drugs, it is essential to guarantee product quality. Among the critical quality attributes of therapeutic mRNA, characterization of the capping and poly(A) tail are of the greatest importance because of their involvement in mRNA stability and in the efficiency of protein synthesis. This article presents a method for the simultaneous characterization of both attributes in a single sample preparation workflow. The method involves lipid extraction, various RNAse enzymes, purification steps and LC–MS to analyze the capping and poly(A) tailing.

Deconvolution of synthetic mRNA expression: Nucleoside chemistry alters translatability.

Recent technological advances in the production of in vitro transcribed messenger RNA (IVT-mRNA) facilitate its clinical use as well as its application in basic research. In this regard, numerous chemical modifications, which are not naturally observed in endogenous mRNA, have been implemented primarily to address the issue of immunogenicity and improve its biological performance. However, recent findings suggested pronounced differences between expression levels of IVT-mRNAs with different nucleoside modifications in transfected cells. Given the multistep process of IVT-mRNA delivery and subsequent intracellular expression, it is unclear which step is influenced by IVT-mRNA chemistry. Here, we deconvolute this process and show that the nucleoside modification does not interfere with complexation of carriers, their physicochemical properties, and extracellular stability, as exemplified by selected modifications. The immediate effect of mRNA chemistry on the efficiency of ribosomal protein synthesis as a contributor to differences in expression was quantified by in vitro cell-free translation. Our results demonstrate that for the nucleoside modifications tested, translatability was the decisive step in determining overall protein production. Also of special importance for future work on rational selection of tailored synthetic mRNA chemistries, our findings set a workflow to identify potentially limiting, modification-dependent steps in the complex delivery process.

Mitochondrial tRNA Pseudouridylation Regulates Erythropoiesis Via the mTOR Signaling Pathway: Implications for Mlasa and Treatment Strategies

Pseudouridine, the most abundant RNA modification found in various RNA molecules, is crucial for cellular functions. Pseudouridylation is the process of converting uridine to pseudouridine, catalyzed by pseudouridine synthases (PUSs), and abnormal pseudouridylation has been linked to human diseases, including mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA). MLASA is associated with genetic mutations in the PUS1 gene, leading to progressive exercise intolerance, sideroblastic anemia, hyperlactatemia, and muscle weakness. The underlying mechanism of anemia in MLASA remains unclear. In this study, we identified a novel homozygous frameshift deletion (c.523delC, p.P175fs*8) in the PUS1 gene in an MLASA patient with progressive anemia in our hospital. We investigated the role of pseudouridylation in erythropoiesis and its impact on MLASA using in vitro and in vivo approaches. We established an MLASA patient-derived inducible pluripotent stem cell (iPSC) line (referred to as MLASA-iPSCs) with the mutation, as well as a repaired line (MLASA-Res iPSC), and a corresponding mutant mouse model for the investigation. During erythroid differentiation, MLASA-iPSCs showed a significant decrease in the production of erythroblasts compared to the repaired line. Also, MLASA-iPSCs exhibited a lower ratio of mitochondrial membrane potential to mitochondrial mass, decreased oxygen consumption rates, reduced ATP production, and elevated levels of ROS. Attenuated activities of NADH dehydrogenase (Complex I) and cytochrome c reductase (Complex III) were also observed in MLASA-iPSCs, indicating compromised mitochondrial functions. Further analysis revealed a reduction in mitochondrial tRNA levels and abnormal mitochondrial translation in MLASA-iPSCs due to the specific loss of pseudouridylation. Screening of mitochondrial supplements, which could boost mitochondrial function or modulating protein synthesis, revealed that nicotinamide ribose (NR) and mitoquinone (MitoQ) improved mitochondrial function but failed to promote erythroid differentiation in MLASA-iPSCs. Interestingly, rapamycin, an mTOR inhibitor, improved erythroid differentiation in MLASA-iPSCs. Furthermore, MLASA-iPSCs showed activated mTOR signaling, increased protein synthesis and higher translation efficiency of mTOR1 targets, all of which could be rescued by rapamycin treatment, suggesting mTOR-associated translation stress. Consistently, the corresponding PUS1 mutant mouse model exhibited anemia with blocked erythropoiesis at four weeks of age. Serial competitive transplantation experiments revealed an intrinsic impairment in the reconstitution of red blood cells. Mutant erythroblasts showed impaired oxidative phosphorylation and aberrant activation of the mTOR signaling pathway. Rapamycin treatment partially alleviated the anemia phenotype in mice. Significantly, in the MLASA patient, sirolimus (rapamycin) administration under strict supervision and medical guidance effectively alleviated anemia symptoms and improved blood routine profile. In summary, our findings provide insights into the critical role of mitochondrial tRNA pseudouridylation in governing erythropoiesis via mTOR signaling pathway. We propose rapamycin as potential therapeutic strategies for anemia patients experiencing protein translation pressure, such as those with MLASA. #These authors contributed equally: Chu Y, Wang B, Shi D and Lian Y. Correspondence to: wpyuan@ihcams.ac.cn, shijun@ihcams.ac.cn Fig. 1 Schematic illustration of erythropoiesis regulated by PUS1-mediated pseudouridylation. Under physiological conditions (left), PUS1 regulates mt-tRNA abundance by catalyzing the pseudouridine synthesis of specific mt-tRNAs, thereby regulating mitochondrial translation, and ultimately affecting mitochondrial function and erythroid differentiation. In MLASA disease states (right), PUS1 deficiency causes the loss of pseudouridine modifications of specific tRNAs and thus affects the amount of PUS1 targeted tRNAs and its corresponding mitochondrial protein products, leading to mitochondrial dysfunction. The defects resulted in aberrant activation of the mTOR signaling pathway and accelerated global translation rate, ultimately leading to a blockage in erythroid differentiation, which could be relieved with rapamycin treatment.

Transcriptomic Profiling to Understand Inhibitor Development in Previously Untreated Patients with Severe Hemophilia a

Background: When starting treatment with factor VIII (FVIII) concentrate in individuals with severe hemophilia A, minimizing the risk of FVIII inhibitor development is an important concern for clinicians, patients and their families. Despite increased understanding of some of the genetic and environmental risk factors for inhibitor formation from epidemiological studies, identification of previously untreated patients (PUPs) at risk of developing inhibitors remains a challenge. Prediction of inhibitor development may help optimize care in PUPs with hemophilia A. RNA sequencing (RNA-Seq) with next-generation sequencing (NGS) could be used to investigate the underlying mechanisms of inhibitor development. Aims: Toevaluate peripheral blood transcriptomic profiles prior to and during early FVIII treatment in severe hemophilia A patients who did or did not develop inhibitors to simoctocog alfain the prospective NuProtect study. Methods: The NuProtect study (NCT01712438; EudraCT 2012-002554-23) was a multicenter, multinational, open-label, non-controlled, phase III study that enrolled male PUPs with severe hemophilia A (FVIII:C &amp;lt;1%) of any age and ethnicity. Patients received simoctocog alfa for prophylaxis or on-demand treatment, as well as for surgical prophylaxis. RNA-Seq was performed on blood samples collected from baseline and every 3-4 exposure days (ED) up to 20 EDs or until inhibitor development to assess messenger RNA (mRNA) expression profiles. Bioinformatics analyses were performed for the following timepoints: baseline, early treatment and a later timepoint on treatment. Principal component analysis (PCA) was performed to identify differences in global mRNA expression profiles between inhibitor and non-inhibitor patients. Gene ontology (GO) analyses were performed to detect differences in GO clusters between inhibitor and non-inhibitor patients. Results: Within this first analysis of the full dataset, transcriptomic profiles from 78 of 108 severe hemophilia A PUPs were analyzed (inhibitors n=22, non-inhibitors n=56). Median (range) age at ED1 was 13 (0.5-146) months; 44.9% of patients had the intron 22 inversion F8 gene mutation. Although the PCA indicated no strong global signal for differential expression profiles between non-inhibitor and inhibitor patients, GO analyses demonstrated differences between these patient groups. Inhibitor patients demonstrated increased gene expression of metabolic processes (GO:0008152) and cellular processes (GO:0009987) at baseline. Upregulation of immune system processes (GO:0002376) was seen in inhibitor patients at the early timepoint compared with non-inhibitor patients. Expression of ribosomal proteins, including RPL17 and RPS8 were significantly upregulated at baseline in inhibitor patients (p&amp;lt;0.05). Inhibitor patients further demonstrated upregulation of proteins involved in adaptive immune responses: IGHV1-69-2 at all timepoints (p&amp;lt;0.01) and IGHV2-70D at baseline and the early timepoint (p&amp;lt;0.01) compared with non-inhibitor patients. Upregulation of the B-cell related factors DPP4 at baseline and the early timepoint (p&amp;lt;0.05) and CD20 at the late timepoint (p&amp;lt;0.05) was observed in inhibitor patients compared with non-inhibitor patients. Conclusion: Differential transcriptomic profiles were seen in severe hemophilia A patients who developed inhibitors compared with those who do not, even before commencing FVIII treatment. Inhibitor patients demonstrated alterations in ribosomal protein expression that could disrupt protein synthesis efficiency, protein folding and quality control processes, and trigger cellular stress responses. Inhibitor patients also showed an upregulation of B-cell mediated factors involved in adaptive immune mechanisms. Additional analyses are ongoing to evaluate dynamic changes in gene expression during commencement of FVIII replacement to further identify patients at risk of inhibitor development.

Effect of the inclusion of rumen-protected amino acids in the diet of growing beef cattle on animal performance and meat quality

IntroductionThis study investigated the impact of rumen-protected amino acids on beef cattle's growth, fattening performance, and meat quality.MethodsTwo groups of 40 Montbeliard steers (237.8 ± 30 kg body weight) were housed separately and fed, with mono-tunnel feeders, either a control diet (CON) or a diet containing rumen-protected lysine and methionine with 3% less crude protein (APR). Feed consumption and weight were tracked in all animals, and meat quality analysis focused on the longissimus muscle, with 10 steers randomly selected from each treatment.Results and discussionResults for overall performance revealed no significant differences in body weight, average daily gain (ADG), and concentrate conversion ratio between the CON and APR diets (p &amp;gt; 0.05). However, during the final 90 days of the trial, the APR group showed significantly higher ADG (p &amp;lt; 0.05) than the CON group, while the concentrate conversion ratio was significantly higher (p &amp;lt; 0.05) in the CON group compared to the APR group. The hot carcass weight, dressing percentage, and carcass conformation did not differ significantly between the CON and APR diets (p &amp;gt; 0.05). There was a slight tendency (p = 0.06) for higher fatness score in the CON group. Regarding meat composition, the diet did not significantly affect proximate composition, but a tendency (p = 0.059) for higher crude protein content in APR cattle's meat compared to the CON group was observed. The APR meat showed slightly lower levels of C17:1 (0.56% vs. 0.72%) and C18:1n9 (31.7% vs. 34%). There was a tendency for C14:0 to be lower (P = 0.07), and for C16:0, C18:2 and polyunsaturated fatty acids (PUFA) to be greater in meat from cattle fed APR compared to CON. However, meat quality attributes like pH, water-holding capacity, color, and texture were similar in both CON and APR groups (p &amp;gt; 0.05). The findings indicate that utilizing rumen-protected amino acids enables formulating diets with reduced crude protein levels while enhancing nitrogen utilization efficiency for protein synthesis in intensively reared steers. Importantly, these dietary improvements do not adversely affect meat quality.

Glacial secrets uncovered: Revealing the modes of survival of metabolically active microbial communities entrapped in polar glacial ice

Glacial secrets uncovered: Revealing the modes of survival of metabolically active microbial communities entrapped in polar glacial ice