Addition Of Amino Acids Research Articles

Sequence variation and regulatory variation in acetylcholinesterase genes contribute to insecticide resistance in different populations of Leptinotarsa decemlineata.

Although insect herbivores are known to evolve resistance to insecticides through multiple genetic mechanisms, resistance in individual species has been assumed to follow the same mechanism. While both mutations in the target site insensitivity and increased amplification are known to contribute to insecticide resistance, little is known about the degree to which geographic populations of the same species differ at the target site in a response to insecticides. We tested structural (e.g., mutation profiles) and regulatory (e.g., the gene expression of Ldace1 and Ldace2, AChE activity) differences between two populations (Vermont, USA and Belchow, Poland) of the Colorado potato beetle, Leptinotarsa decemlineata in their resistance to two commonly used groups of insecticides, organophosphates, and carbamates. We established that Vermont beetles were more resistant to azinphos‐methyl and carbaryl insecticides than Belchow beetles, despite a similar frequency of resistance‐associated alleles (i.e., S291G) in the Ldace2 gene. However, the Vermont population had two additional amino acid replacements (G192S and F402Y) in the Ldace1 gene, which were absent in the Belchow population. Moreover, the Vermont population showed higher expression of Ldace1 and was less sensitive to AChE inhibition by azinphos‐methyl oxon than the Belchow population. Therefore, the two populations have evolved different genetic mechanisms to adapt to organophosphate and carbamate insecticides.

The bifunctional 6-phosphofructokinase-2/fructose-2,6-bisphosphatase from the shrimp Litopenaeus vannamei: Molecular characterization and down-regulation of expression in response to severe hypoxia

Hypoxia is a frequent stressor in marine environments with multiple adverse effects on marine species. The white shrimp Litopenaeus vannamei withstands hypoxic conditions by activating anaerobic metabolism with tissue-specific changes in glycolytic and gluconeogenic enzymes. In animal cells, glycolytic/gluconeogenic fluxes are highly controlled by the levels of fructose-2,6-bisphosphate (F-2,6-P2), a signal metabolite synthesized and degraded by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFK-2/FBPase-2 has been studied in vertebrates and some invertebrates, but as far as we know, there are no reports on PFK-2/FBPase-2 from crustaceans. In the present work, we obtained cDNA nucleotide sequences corresponding to two mRNAs for PFK-2/FBPase-2 and named them PFKFBP1 (1644 bp) and PFKFBP2 (1566 bp), from the white shrimp L. vannamei. The deduced PFKFBP1 and PFKFBP2 are 547 and 521 amino acids long, respectively. Both proteins share 99.23% of identity, and only differ in 26 additional amino acids present in the kinase domain of the PFKFBP1. The kinase and phosphatase domains are highly conserved in sequence and structure between both isoforms and other proteins from diverse taxa. Total expression of PFKFBP1-2 is tissue-specific, more abundant in gills than in hepatopancreas and undetectable in muscle. Moreover, severe hypoxia (1 mg/L of DO) decreased expression of PFKFBP1-2 in gills while anaerobic glycolysis was induced, as indicated by accumulation of cellular lactate. These results suggest that negative regulation of PFKFBP1-2 at expression level is necessary to set up anaerobic glycolysis in the cells during the response to hypoxia.

Apparent homozygosity for a novel splicing variant in EPS8 causes congenital profound hearing loss

Autosomal recessive deafness-102 (DFNB102), a new profound prelingual non-syndromic hearing loss, is caused by mutations in the EPS8 gene. To date, only three such consanguineous families with three different homozygous variants in EPS8 have been reported. Here, we report the fourth case from a non-consanguineous Chinese family, an 11-month-old male infant presented with congenital profound non-syndromic hearing loss. Trio whole-exome sequencing initially identified the patient with a novel seemingly homozygous splicing variant NM_004447.5: c.1435-2A > T in intron 14 of the EPS8 gene and was inherited from his father; further CNVs analysis identified a novel 65.9 kb intragenic deletion and was inherited from his mother. The deletion is covering intron 14 that could account for the apparent homozygosity of the patient. In vitro splicing assay showed the variant c.1435-2A > T creates a new donor site at position c.1443, which is predicted to produce a stop codon after 14 additional amino acids (p.His479Cysfs*14). Furthermore, quantitative allele-specific expression assay showed that relative EPS8 gene expression in the patient significantly decreased (0-fold for the wild-type transcript and 0.25–0.27-fold for the mutant transcript) compared to the control (P < 0.05), indicating the pathogenicity of the identified variants. Overall, our study provides additional evidence that EPS8 is a causative gene for DFNB102 and highlights the clinical utility of simultaneous analysis of CNVs and SNVs to avoid potential errors in the diagnosis and interpretation of patients with apparent homozygosity.

Characteristic flavor formation of thermally processed N-(1-deoxy-α-d-ribulos-1-yl)-glycine: Decisive role of additional amino acids and promotional effect of glyoxal

The role of amino acids and α-dicarbonyls in the flavor formation of Amadori rearrangement product (ARP) during thermal processing was investigated. Comparisons of the volatile compounds and their concentrations when N-(1-deoxy-α-d-ribulos-1-yl)-glycine reacted with different amino acids or glyoxal (GO) at 100 °C were executed. Additional amino acids, such as glycine (Gly), in ARP models contributed to the diversity of furanoids by the chain elongation of the derived formaldehyde. Whereas the monoanion of additional glutamic acid acted as nucleophile, favored 2-ethyl-3,5-dimethylpyrazine and 2,5-dimethylpyrazine formation; the nonionized amino group of additional lysine were involved in α-dicarbonyls formation, causing pyrazine and methylpyrazine accumulation in the ARP model. Moreover, the high dosage and pH stabilization of additional GO probably promoted the ARP degradation and deoxyosones retro-aldol cleavage, resulting in methylpyrazine rather than furanoids formation. The present work provided the guidance for the controlled flavor formation of ARP in industrial application.

Charcot-Marie-Tooth disease type 2CC due to NEFH variants causes a progressive, non-length-dependent, motor-predominant phenotype

ObjectiveNeurofilaments are the major scaffolding proteins for the neuronal cytoskeleton, and variants in NEFH have recently been described to cause axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC).MethodsIn this large observational study,...

TCRpair: prediction of functional pairing between HLA-A*02:01-restricted T-cell receptor α and β chains.

The ability of a T cell to recognize foreign peptides is defined by a single α and a single β hypervariable complementarity determining region (CDR3), which together form the T-cell receptor (TCR) heterodimer. In ∼30-35% of T cells, two α chains are expressed at the mRNA level but only one α chain is part of the functional TCR. This effect can also be observed for β chains, although it is less common. The identification of functional α/β chain pairs is instrumental in high-throughput characterization of therapeutic TCRs. TCRpair is the first method that predicts whether an α and β chain pair forms a functional, HLA-A*02:01 specific TCR without requiring the sequence of a recognized peptide. By taking additional amino acids flanking the CDR3 regions into account, TCRpair achieves an AUC of 0.71. TCRpair is implemented in Python using TensorFlow 2.0 and is freely available at https://www.github.com/amoesch/TCRpair. Supplementary data are available at Bioinformatics online.

Genome-informed characterisation of antigenic drift in the haemagglutinin gene of equine influenza strains circulating in the United States from 2012 to 2017.

Equine influenza virus (EIV) is a major infectious pathogen causing significant respiratory signs in equids worldwide. Voluntary surveillances in the United States recently reported EIV detection in horses with respiratory signs even with adequate vaccine protocols and biosecurity programs and posed a concern about suboptimal effectiveness of EIV vaccine in the United States. This study aims to determine the genetic characteristics of 58 field EIV H3N8 strains in the United States from 2012 to 2017 using the phylogenetic analysis based on the haemagglutinin (HA) gene. Amino acid substitution and acquisition of N-glycosylation of the HA gene were also evaluated. Phylogenetic analysis identified that almost all US field strains belonged to the Florida clade 1 (FC1) except one Florida clade 2 strain from a horse imported in 2014. US EIV strains in 2017 shared 11 fixed amino acid substitutions in the HA gene, compared to the vaccine strain (A/equine/Ohio/2003), and two additional amino acid substitutions were detected in 2019. The introduction of foreign EIV strains into the United States was not detected, but antigenic drift without acquisition of N-glycosylation in the HA gene was observed in US field strains until 2017. Considering the global dominance of FC1 strains, subsequent antigenic drift of US EIV strains should be monitored for better effectiveness of the EIV vaccine in the United States and global equine industries.

Amino Acid Carriers of the Solute Carrier Families 7 (SLC7) and 38 (SLC38) Are Involved in Leucine Sensing in the Brain of Atlantic Salmon (Salmo salar)

Sensing of amino acids in fish brain, especially branched-chain amino acids (BCAA) like leucine, is involved in regulation of feed intake through different mechanisms. However, there is limited information regarding the possible involvement of mechanisms dependent on amino acid carriers of the solute carrier families (SLC) known to be key regulators of intracellular leucine concentration, namely L-type amino acid transporter 1 (LAT1), and sodium-dependent neutral amino acid transporter 2 (SNAT2) and 9,(SNAT9), for which evidence of their participation is available in mammals. Comparative analysis amongst sequences revealed a complex pattern of paralogues in Atlantic salmon, for LAT1 (slc7a5aa, slc7a5ab, slc7a5ba, slc7a5bb, slc7a5ca, and slc7a5cb), SNAT2 (slc38a2a and slc38a2b) and SNAT9 (slc38a9). After establishing phylogenetic relationships of the different paralogues evaluated, samples of the selected brain areas were taken from Atlantic salmon to assess tissue distribution of transcripts. In an additional experiment, fish were fed two diets with different levels of leucine (high leucine: 35 g/kg vs. control leucine: 27.3 g/kg). The high leucine diet resulted in lower feed intake and increased mRNA abundance of specific paralogues of LAT1 (slc7a5aa, slc7a5ab, and slc7a5bb) and SNAT2 (slc38a2a and slc38a2b) though apparently not for SNAT9 in brain areas like hypothalamus and telencephalon involved in food intake regulation. The results obtained suggest a role for members of the SLC family in the anorectic effect of leucine and thus their involvement as additional amino acid sensing mechanism not characterised so far in fish regulation of feed intake.

Abstract 1534: Mesothelin isoform 2 is a novel target for allogenic CARγδT cell therapy in solid tumors

Abstract Mesothelin (MSLN) is a tumor-associated antigen over-expressed on the cell surface of various malignant tumor cells, including majority of mesothelioma and ovarian cancer. MSLN-directed therapy has been intensively studied in preclinical and clinical settings. However, the clinical efficacy and safety of mesothelin-targeted therapy remains to be demonstrated. To identify a safe and effective target for CAR-T cell therapy in solid tumors, we used artificial intelligent platform developed with public and private RNA transcription database to identify cancer-associated antigens caused by alternative spicing. One of interesting targets is an alternative spicing isoform from MSLN. Human MSLN transcript has at least three isoforms. The isoform 1 is the predominant transcript detected in normal and tumor tissues and has been a promising target for cancer immunotherapy. The isoform 2 is the minor transcript using alternatively spliced exons producing 8 additional amino acids insertion compared to isoform 1. The isoform 3 produces a truncated and soluble MSLN. Our data demonstrated that MSLN isoform 2 is specifically expressed in ovarian cancers but not normal tissues, confirmed by Q-PCR studies. Furthermore, we generated mouse hybridoma antibodies specifically targeting MSLN isoform 2. The antibody specificity was screened and confirmed by ELISA-based reaction to MSLN isoform 2-specific peptide and flow cytometry-based binding to 293T cells overexpressing MSLN isoform 2 but not isoform 1 proteins. We further detected the endogenous MSLN isoform 2 expression in human mesothelioma cell line NCI H226 and further confirmed MSLN isoform 2 expression on human primary mesothelioma and ovarian cancer tissue but not normal tissues by immunohistochemistry staining. To study if targeting MSLN isoform 2 with chimeric antigen receptor (CAR) can control the tumor growth, we made CAR retrovirus construct and showed that genetically modified allogenic gamma-delta T cells expressing CAR for MSLN isoform 2 can kill human mesothelioma cells in vitro and in vivo. In summary, we have demonstrated that MSLN isoform 2 is a tumor-specific antigen, which can be targeted for CAR-T cell therapy. Citation Format: Xiaohong Wang, Yibin Chen, Anupama Gopisetty, Leonardo Mirandola, Lucia Piccotti, Quynh Nguyen, Maurizio Chiriva-Internati. Mesothelin isoform 2 is a novel target for allogenic CARγδT cell therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1534.

FVIII Proteins with a Modified Immunodominant T-Cell Epitope Exhibit Reduced Immunogenicity and Normal Procoagulant Activity

Factor VIII (FVIII) neutralizing antibodies (inhibitors) are a serious complication in hemophilia A (HA). The peptide FVIII2194-2213 (legacy FVIII numbering, sequence: SYFTNMFATWSPSKARLHLQ) has been shown to contain an immunodominant HLA-DRA*01-DRB1*01:01 (DRB1*01:01)-restricted epitope recognized by CD4+ T-effector cells isolated from HA subjects. The aim of this study was to identify amino acid substitutions to de-immunize this epitope while retaining pro-coagulant function and expression levels comparable to those of wild-type (WT) FVIII proteins. The minimal DRB1*01:01-binding motif was mapped to FVIII2194-2205 by quantitative peptide-MHC Class II (MHCII) binding assays. Further binding assays with sequence-modified FVIII peptides established that the residues important for DRB1*01:01 affinity were F2196, M2199, A2201, and S2204, corresponding to MHCII anchor positions 1, 4, 6 and 9. T-cell proliferation experiments with Ala-substituted FVIII2194-2205 peptides identified F2196A as a substitution that abrogated proliferation and cytokine secretion of five clones specific for FVIII2194-2213. The substitution F2196A was then engineered into a recombinant (r)FVIII-C2 protein. T-cell clones that were strongly stimulated by wild-type rFVIII-C2 did not proliferate when cultured with rFVIII-C2-F2196A, indicating the immunogenic synthetic peptide includes a naturally processed T-cell epitope. The measured and predicted effects of additional amino acid substitutions within this binding motif were further evaluated by peptide-MHCII binding assays, CD4+ T-cell proliferation assays, and MHCII-binding prediction algorithms. Amino acid homologs within this binding motif were evaluated with sequence homologies to FVIII and FV proteins from different species. On the basis of all these results, six rB-domain-deleted (BDD)-FVIII proteins with amino acid substitutions F2196A, F2196L, F2196K, M2199A, M2199W or M2199R, respectively, were designed and produced; all but the F2196A variant had specific activities comparable to that of rWT-BDD-FVIII. The F2196A variant also had the lowest expression level among the rBDD-FVIIII proteins produced. Peptide-MHCII binding assays confirmed that FVIII2194-2205 peptides with substitutions F2196A, F2196K, M2199A and M2199R did not bind to DRB1*01:01 or to an additional 9 HLA-DR proteins tested, with the exception of the M2199A peptide binding to DRB1*09:01. The predicted effects of these FVIII sequence modifications on binding to additional MHCII alleles included in reference sets representing ~99% of the human population HLA diversity were evaluated using ProPred and Immune Epitope Database algorithms. These programs correctly predicted that the substitutions would abrogate binding to DRB1*01:01 and also indicated that affinities for additional MHCII would be reduced by each substitution. The substitutions were predicted to increase peptide-MHCII affinities for a smaller number of MHCII alleles, indicating a possible risk of creating neo-epitopes restricted to these specific HLA alleles. Importantly, proliferation of patient-derived T-cell clones in response to rBDD-FVIII-F2196K and rBDD-FVIII-M2199A proteins was dramatically reduced compared with responses to rWT-BDD-FVIII. We have previously demonstrated that a severe HA inhibitor subject had an oligoclonal T-cell response to only one T-cell epitope within the FVIII A2, C1 and C2 domains and that one epitope was FVIII2194-2213 (Ettinger et al., Blood 2106; 128(16):2043). This observation, together with the results presented here, further suggests that modification of only a small number of immunodominant epitopes may be sufficient to significantly de-immunize FVIII and decrease FVIII inhibitors in HA patients. The present results provide proof-of-principle for the design of less immunogenic FVIII proteins targeted to specific subsets of HA patients. DisclosuresEttinger:Bloodworks NW: Patents & Royalties: Inventor on FVIII patents. James:Bloodworks NW: Patents & Royalties: inventor on FVIII patent(s). Pratt:Bloodworks NW: Patents & Royalties: inventor on FVIII patents.

The Enigmatic N-Terminal Domain of Proglucagon; A Historical Perspective.

Enteroglucagon refers to the predominant peptide with glucagon-like immunoreactivity (GLI) that is released by the intestine into the circulation in response to nutrients. Development of a radioimmunoassay for glucagon revealed issues that were not apparent in applications of the insulin radioimmunoassay. The fact that some antisera raised against glucagon recognized glucagon-related peptides in extracts of both pancreas and gut whereas others recognized only components in the pancreas remained a mystery until it was realized that the “gut GLI cross-reactive” antisera were directed against an epitope in the N-terminal to central region of glucagon whereas the “pancreatic glucagon specific” antisera were directed against an epitope in the C-terminal region. Unlike the cross-reactive antisera, the glucagon specific antisera did not recognize components in which glucagon was extended from its C-terminus by additional amino acids. Initial attempts to purify enteroglucagon from porcine ileum led to the erroneous conclusion that enteroglucagon comprised 100 amino acids with an apparent molecular mass of 12,000 Da and was consequently given the name glicentin. Subsequent work established that the peptide constituted residues (1-69) of proglucagon (Mr 8128). In the 40 years since the structural characterization of glicentin, attempts to establish an unambiguous physiological function for enteroglucagon have not been successful. Unlike the oxyntomodulin domain at the C-terminus of enteroglucagon, the primary structure of the N-terminal domain (glicentin-related pancreatic peptide) has been poorly conserved among mammals. Consequently, most investigations of the bioactivity of porcine glicentin may have been carried out in inappropriate animal models. Enteroglucagon may simply represent an inactive peptide that ensures that the intestine does not release equimolar amounts of a hyperglycemic agent (glucagon) and a hypoglycemic agent (GLP-1) after ingestion of nutrients.

Effect of 3-Aminobenzoic Acid Ethyl Ester Methanesulfonate (MS-222) on Quality of Marine Cultured Turbot (Scophthalmus maximus) during Simulated Transport in Water

This study evaluated the effect of different concentrations (20, 40 and 60 mg/L) of 3-aminobenzoic acid ethyl ester methanesulfonate (MS-222) on the quality changes in turbot during simulated transport in water. The results showed that the ammonia nitrogen content in the transportation water of each sample increased significantly, and the dissolved oxygen level decreased. The dissolved oxygen content in MS-222-treated samples was higher than that of control group (CK) samples. For turbot flesh quality, simulated transport in water led to a decrease in moisture, fat and protein contents in all samples. The MS-222-treated turbot samples showed higher pH values, glycogen contents, springiness and chewiness values and lower lactic acid contents comparing with the CK samples during simulated transport in water. In addition, the fresh and bitter amino acids in the muscle of turbot increased in each treatment group compared to the non-transported fish at the end of the simulated transport. The results showed that MS-222 treatment could retard the turbot transport stress and improve the quality of turbot during simulated transport in water.

Enhanced protein and amino acids of corn-ethanol co-product by Mucor indicus and Rhizopus oryzae.

Upcycle of co-products from corn-ethanol plant into protein-rich animal feed with balanced key amino acids via solid-state fermentation is a promising approach to economically support both biofuel and animal feed industries. However, there are multiple types of solid-state fermentation microorganisms and growth conditions that have not been tested. In this study, Mucor indicus and Rhizopus oryzae were used to ferment corn-based wet distiller's grains with solubles (WDGS). The effects of fermentation conditions (temperature, agitation, and moisture) and supplementations (extraneous carbon and nitrogen sources) were evaluated on protein production and amino acids profiles before and after fermentation. The study established best fermentation conditions (23°C, static incubation for 4days at 70% initial moisture content) to improve protein content for both R. oryzae and M. indicus. Moreover, urea supplied to R. oryzae and M. indicus improved protein concentration by 35 and 38%, and total amino acids content by 28 and 18%, respectively. The amount of 693.1 and 451.8mg of additional total amino acids including 262.8 and 227.7mg of key amino acids (lysine, methionine, tryptophan, and arginine) was synthesized by R. oryzae and M. indicus, respectively, per supply of 536mg urea in 25g of WDGS. This study demonstrated the feasibility of urea as a low-cost nitrogen source for amino acid biosynthesis in fungal fermentation of WDGS, which could contribute to the increasing demand for high-value monogastric animal feed.

Changes in sialic acid binding associated with egg adaptation decrease live attenuated influenza virus replication in human nasal epithelial cell cultures

Live Attenuated Influenza Virus (LAIV) is administered to and replicates in the sinonasal epithelium. Candidate LAIV vaccine strains are selected based on their ability to replicate to a high titer in embryonated hen’s eggs, a process that can lead to mutations which alter the receptor binding and antigenic structure of the hemagglutinin (HA) protein. In the 2012–2013 northern hemisphere vaccine, the H3N2 HA vaccine strain contained three amino acid changes - H156Q, G186V and S219Y – which altered HA antigenic structure and thus presumably decreased vaccine efficacy. To determine if these mutations also altered LAIV replication, reabcombinant viruses were created that encoded the wild-type (WT) parental HA of A/Victoria/361/2011 (WT HA LAIV), the egg adapted HA (EA HA LAIV) from the A/Victoria/361/2011 vaccine strain and an HA protein with additional amino acid changes to promote α2,3 sialic acid binding (2,3 EA HA LAIV). The WT HA LAIV bound α2,6 sialic compared to the EA HA LAIV and 2,3 EA HA LAIV which both demonstrated an increased preference for α2,3 sialic acid. On MDCKs, the WT HA and EA HA LAIVs showed similar replication at 32 °C but at 37 °C the EA HA LAIV replicated to lower infectious virus titers. The 2,3 EA HA LAIV replicated poorly at both temperatures. This replication phenotype was similar on human nasal epithelial cell (hNEC) cultures, however the WT HA LAIV induced the highest amount of IFN-λ and infected more nasal epithelial cells compared to the other viruses. Together, these data indicate that egg adaption mutations in the HA protein that confer preferential α2,3 sialic acid binding may adversely affect LAIV replication and contribute to reduced vaccine efficacy.

118 Increased Growth and Carcass Attributes in Improvest®-treated Gilts Do Not Require Additional Dietary Lysine

Abstract Numerous studies have shown that gilts treated with Improvest® have greater carcass weights and increased ADG compared with untreated gilts. To develop the optimum nutritional program for Improvest-treated gilts, a randomized 5×2 factorial design of dietary lysine levels (90, 100, 110, 120, and 130% of NRC recommendations) with or without Improvest was performed. Gilts were housed in 120 pens (4 pigs/pen) at 8 weeks of age (day 0). Gilts and feed were weighed immediately prior to each dietary phase change (days 0, 21, 42, 70, 91, and 105). Improvest was administered at 9 and 19 weeks of age (4 weeks pre-harvest). There was no diet × treatment × day (P &amp;gt; 0.78) nor diet × treatment (P &amp;gt; 0.11) interactions for any variables. Gilts had similar BWT, ADG, and ADFI until after the 2nd dose of Improvest, when Improvest-treated gilts were heavier (123.62 vs. 121.59 ± 0.68 and 138.16 vs. 133.97 ± 0.71 kg, days 91 and 105; P &amp;lt; 0.01), had increased ADG (1.19 vs. 1.09 ± 0.01 and 1.03 vs. 0.88 ± 0.02 kg/day days 91 and 105; P &amp;lt; 0.01) and consumed more feed (2.99 vs. 2.84 ± 0.03 and 3.19 vs. 2.68 ± 0.04 kg/pig/day; days 91 and 105; P &amp;lt; 0.01) compared with untreated gilts. Carcass evaluation was conducted on 120 pigs (2 pigs/60 pens). No significant structures were present on ovaries of Improvest-treated gilts. Improvest-treated gilts were heavier (market and HCW; P ≤ 0.02) than controls. Improvest-treated gilts tended (P ≤ 0.08) to have heavier bone-in butt and bone-in ham weights. Belly weights were heavier (kg and %HCW; P ≤ 0.05) in Improvest-treated vs control gilts and were thicker (P = 0.01) but were similar (P &amp;gt; 0.3) in length and width. While IV was similar (P &amp;gt; 0.2) in belly fat, loin intramuscular fat was increased (P &amp;lt; 0.01) from Improvest-treated gilts. Without additional dietary amino acids, Improvest-treated gilts delivered greater gain after the 2nd dose, yielding significantly heavier carcasses and primal cuts, including bellies which were larger as a percentage of HCW, and increased loin intramuscular fat.

Fermentation of Whole Grain Sorghum (Sorghum bicolor (L.) Moench) with Different Dry Matter Concentrations: Effect on the Apparent Total Tract Digestibility of Energy, Crude Nutrients and Minerals in Growing Pigs.

Simple SummaryDue to climate change and pests as result of maize monoculture, the need of diversification of crop rotation forces researchers to look for alternative grains for animal nutrition. Furthermore, grain fermentation may increase the nutritional value of feed and, simultaneously, decrease costs of feed conservation because the necessity for grain drying and associated energy costs are reduced. In this context, the cultivation and integration of early harvested and fermented sorghum grain in pig diets might be an interesting strategy for the substitution of maize. Therefore, we tested the nutritional value of three varieties of fermented sorghum grains with gradual differences in total dry matter, in a Latin-Square experiment comprising growing pigs. Results indicated there is a potential for improving the nutrient digestibility of sorghum-based pig diets by using early harvested and fermented whole sorghum grain with lower dry matter concentration. Especially the need for inorganic phosphorus supplementation and, hence, the fecal phosphorus emissions were significantly reduced.This study investigated the effects of sorghum ensiled as whole grains with different dry matter concentrations on the apparent total tract digestibility (ATTD) of energy, crude nutrients and minerals in growing pigs. Whole grain sorghum batches with varying dry matter (DM) concentrations of 701 (S1), 738 (S2) and 809 g kg−1 (S3) due to different dates of harvest from the same arable plot, were stored in air-tight kegs (6 L) for 6 months to ensure complete fermentation. Subsequently, 9 crossbred barrows (34.6 ± 1.8 kg; (Duroc x Landrace) × Piétrain)) were used in a 3 × 3 Latin square feeding experiment. Diets were based on the respective sorghum grain silage and were supplemented with additional amino acids, minerals and vitamins to meet or exceed published feeding recommendations for growing pigs. The ATTD of gross energy, dry matter, organic matter, nitrogen-free extracts, and crude ash were higher in S1 compared to S3 treatments (p ≤ 0.05), while S2 was intermediate. Pigs fed S1 showed significantly higher ATTD of phosphorus (P) compared to all other groups while ATTD of calcium was unaffected irrespective of the feeding regime. In conclusion, growing pigs used whole grain sorghum fermented with a DM concentration of 701 g kg−1 (S1) most efficiently. In particular, the addition of inorganic P could have been reduced by 0.39 g kg−1 DM when using this silage compared to the variant with the highest DM value (809 g kg−1).

The Tryptophan-Induced tnaC Ribosome Stalling Sequence Exposes High Amino Acid Cross-Talk That Can Be Mitigated by Removal of NusB for Higher Orthogonality.

A growing number of engineered synthetic circuits have employed biological parts coupling transcription and translation in bacterial systems to control downstream gene expression. One such example, the leader sequence of the tryptophanase (tna) operon, is a transcription-translation system commonly employed as an l-tryptophan inducible circuit controlled by ribosome stalling. While induction of the tna operon has been well-characterized in response to l-tryptophan, cross-talk of this modular component with other metabolites in the cell, such as other naturally occurring amino acids, has been less explored. In this study, we investigated the impact of natural metabolites and E.coli host factors on induction of the tna leader sequence. To do so, we constructed and biochemically validated an experimental assay using the tna operon leader sequence to assess differential regulation of transcription elongation and translation in response to l-tryptophan. Operon induction was then assessed following addition of each of the 20 naturally occurring amino acids to discover that several additional amino acids (e.g., l-alanine, l-cysteine, l-glycine, l-methionine, and l-threonine) also induce expression of the tna leader sequence. Following characterization of dose-dependent induction by l-cysteine relative to l-tryptophan, the effect on induction by single gene knockouts of protein factors associated with transcription and/or translation were interrogated. Our results implicate the endogenous cellular protein, NusB, as an important factor associated with induction of the operon by the alternative amino acids. As such, removal of the nusB gene from strains intended for tryptophan-sensing utilizing the tna leader region reduces amino acid cross-talk, resulting in enhanced orthogonal control of this commonly used synthetic system.

Native Ubiquitin Structural Changes Resulting from Complexation with β-Methylamino-l-alanine (BMAA).

The objective of this research was to investigate potential changes to unfolding energy barriers for ubiquitin in the presence of the noncanonical amino acid β-methylamino-l-alanine (BMAA). Although BMAA has been implicated in neurodegenerative disease, its specific role remains unclear. We hypothesized that formation of a ubiquitin + BMAA noncovalent complex would alter the protein's unfolding dynamics in comparison with native ubiquitin alone or in noncovalent complexes with other amino acids. Ion mobility-mass spectrometry (IM-MS) revealed that at sufficiently high concentrations BMAA did in fact form a noncovalent complex with ubiquitin, and similar complexes were identified for a range of additional amino acids. Collision-induced unfolding (CIU) was used to interrogate the unfolding of native ubiquitin and these Ubq-amino acid complexes, showing a major transition from its compact native state (∼1200 Å2) to an unfolded state (∼1400 Å2) at activation energies in the range from 8.0 to 9.0 V (entrance grid delta). The Ubq-BMAA complex, on the other hand, was observed to have a significantly higher energy barrier to unfolding, requiring more than 10.5 V. This indicates that the complex remains more stable under native conditions and this may indicate that BMAA has attached to a critical binding location worthy of further study for its potential role in the onset of neurodegenerative disease.

Fluconazole-Resistant Candida glabrata Bloodstream Isolates, South Korea, 2008-2018.

We investigated the clinical outcomes and molecular mechanisms of fluconazole-resistant (FR) Candida glabrata bloodstream infections. Among 1,158 isolates collected during multicenter studies in South Korea during 2008–2018, 5.7% were FR. For 64 patients with FR bloodstream infection isolates, the 30-day mortality rate was 60.9% and the 90-day mortality rate 78.2%; these rates were significantly higher than in patients with fluconazole-susceptible dose-dependent isolates (30-day mortality rate 36.4%, 90-day mortality rate 43.8%; p<0.05). For patients with FR isolates, appropriate antifungal therapy was the only independent protective factor associated with 30-day (hazard ratio 0.304) and 90-day (hazard ratio 0.310) mortality. Sequencing of pleiotropic drug-resistance transcription factor revealed that 1–2 additional Pdr1p amino acid substitutions (except genotype-specific Pdr1p amino acid substitutions) occurred in 98.5% of FR isolates but in only 0.9% of fluconazole-susceptible dose-dependent isolates. These results highlight the high mortality rate of patients infected with FR C. glabrata BSI isolates harboring Pdr1p mutations.

Di-lysine motif-like sequences formed by deleting the C-terminal domain of aquaporin-4 prevent its trafficking to the plasma membrane.

Aquaporin-4 is a transmembrane water channel protein, the C-terminal domain of which is facing the cytosol. In the process of investigating the role of the C-terminal domain of aquaporin-4 with regard to intracellular trafficking, we observed that a derivative of aquaporin-4, in which the C-terminal 53 amino acids had been removed (Δ271-323), was localized to intracellular compartments, including the endoplasmic reticulum, but was not expressed on the plasma membranes. This was determined by immunofluorescence staining and labeling of the cells with monoclonal antibody specifically recognizing the extracellular domain of aquaporin-4, followed by confocal microscopy and flow cytometry. Deletion of additional amino acids in the C-terminal domain of aquaporin-4 led to its redistribution to the plasma membrane. This suggests that the effect of the 53-amino acid deletion on the subcellular localization of aquaporin-4 could be attributed to the formation of a signal at the C terminus that retained aquaporin-4 in intracellular compartments, rather than the loss of a signal required for plasma membrane targeting. Substitution of the lysine at position 268 with alanine could rescue the Δ271-323-associated retention in the cytosol, suggesting that the C-terminal sequence of the mutant served as a signal similar to a di-lysine motif.