Utilization of exogenous fibrolytic enzymes in fiber fermentation, degradation, and digestions and characteristics of whole legume faba bean and its plant silage.

This article aims to review recent progress and update on utilization of exogenous fibrolytic enzymes in fiber fermentation, degradation, and digestions and nutritive and anti-nutritional characteristics of whole legume faba bean and its silage. The study focused on strategies to improve the utilization and bioavailability of fiber through pre-treating exogenous fibrolytic enzymes. The review includes features of nutrition and anti-nutritional factors and environment impact, forage fiber fermentation, degradation and digestion, legume bean in various diets, use of exogenous enzyme and factor affecting enzyme action in fiber digestion as well as exogenous enzyme response. This review also provides very recent research on effects of fibrolytic enzyme on rumen degradation characteristics of dry matter and fiber of whole plant faba bean silage and effect of exogenous fibrolytic enzyme derived from trichoderma reesei on lactational performance, feeding behavior, rumen fermentation and nutrient digestibility in dairy cows fed whole plant faba bean silage-based diet. This study provides an insight on nutritive and anti-nutritive characteristics of whole legume bean and its plant silage and utilization of exogenous fibrolytic enzymes in fiber fermentation, degradation, and digestions.

Universal Screening for Malnutrition Prior to Total Knee Arthroplasty Is Cost-Effective: A Markov Analysis.

BACKGROUND: Patients undergoing total knee arthroplasty (TKA) who have malnutrition possess an increased risk of periprosthetic joint infection (PJI). Although malnutrition screening and intervention may decrease the risk of PJI, it utilizes healthcare resources. To date, no cost-effectiveness analyses have been performed on the screening and treatment of malnutrition prior to TKA. METHODS: A Markov model projecting lifetime costs and quality-adjusted life years (QALYs) was built to determine the cost-effectiveness of malnutrition screening and intervention for TKA patients from a societal perspective. Costs, health state utilities, and state transition probabilities were obtained from previously published literature, hospital costs at our institution, and expert opinions. Two important assumptions included that 30% of patients would be malnourished and that a malnutrition intervention would be 50% effective. The primary outcome of this study was the incremental cost-effectiveness ratio, with a willingness-to-pay threshold of $100,000 per QALY. One-way and two-way sensitivity analyses were performed to evaluate model parameter assumptions. RESULTS: When using the base case values, universal malnutrition screening and intervention was cost-effective compared to no malnutrition screening or intervention, with an incremental cost-effectiveness ratio of $6,454 per QALY. Universal screening and intervention remained cost-effective, provided the cost of screening remained less than $3,373, the cost of nutritional intervention remained less than $12,042, the prevalence of malnutrition among surgical candidates was higher than 2%, and the risk of PJI among patients with malnutrition was greater than 1%. CONCLUSION: Universal preoperative malnutrition screening and intervention among TKA candidates is cost-effective at parameters encountered in clinical practice. Nutritional optimization programs should be considered to facilitate malnutrition screening and intervention and future studies should evaluate their efficacy at lowering PJI risk.

Incubator type affects human blastocyst formation and embryo metabolism: a randomized controlled trial.

STUDY QUESTION: Does the type of incubator used to culture human preimplantation embryos affect development to the blastocyst stage and alter amino acid utilization of embryos in assisted reproduction? SUMMARY ANSWER: Culturing embryos in a time lapse system (TLS) was associated with a higher Day 5 blastocyst formation rate and altered amino acid utilization when measured from Day 3 to Day 5 compared to the standard benchtop incubator. WHAT IS KNOWN ALREADY: Culture environment is known to be important for the developing preimplantation embryo. TLSs provide a stable milieu allowing embryos to be monitored in situ, whereas embryos cultured in standard benchtop incubators experience environmental fluctuations when removed for morphological assessment. STUDY DESIGN, SIZE, DURATION: A prospective clinical trial randomizing 585 sibling embryos to either the TLS (289 embryos) or the standard benchtop incubator (296 embryos) over a 23-month period in a UK University Hospital Fertility Clinic. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were aged 42 years or under, had an antral follicle count of ≥12 and ≥6 2 pronucleate zygotes. Zygotes were cultured individually in 25 µl of medium. Randomized embryos were graded and selected for transfer or cryopreservation on Day 5. For those embryos produced by women who underwent stimulation with recombinant FSH injections and were triggered with hCG, spent medium was collected on Day 5 for amino acid analysis by high pressure liquid chromatography. Clinical pregnancy was defined as the presence of a foetal heart beat on ultrasound scan at 7 weeks. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, blastocyst formation rate on Day 5 was significantly higher in embryos cultured in the TLS (55%) compared to the standard incubator (45%; P = 0.013). Similarly, there was an increase in the number of blastocysts suitable for cryopreservation in the TLS (31%) compared to the standard incubator (23%; P = 0.032). There was a significant difference in the utilization of 12 amino acids by blastocysts cultured from Day 3 to Day 5 in the TLS compared to the standard incubator. Embryos cultured in the TLS displayed an increased total amino acid utilization (P < 0.001) and reduced amino acid production (P < 0.001) compared to those in the standard incubator. Irrespective of incubator used, embryos fertilized by ICSI depleted significantly more amino acids from the medium compared to those fertilized by conventional IVF. There was no difference in the mean score of blastocysts transferred, or the clinical pregnancy rate after transfer of embryos from either of the incubators. LIMITATIONS, REASONS FOR CAUTION: The study was not powered to discern significant effects on clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: The metabolism and development of preimplantation embryos is impacted by the type of incubator used for culture. Further research is required to investigate the long-term implications of these findings. STUDY FUNDING/COMPETING INTEREST(S): NIHR Southampton Biomedical Research Centre Commercial and Enterprise Incubator Fund funded this study. The TLS was provided on loan for the study by Vitrolife. The authors declare no conflict of interests. TRIAL REGISTRATION NUMBER: ISRCTN73037149. TRIAL REGISTRATION DATE: 12 January 2012. DATE OF FIRST PATIENT'S ENROLMENT: 21 January 2012.

Utilization of synchrotron-based and globar-sourced mid-infrared spectroscopy for faba nutritional research about molecular structural and nutritional interaction.

The traditional wet chemistry analysis is to use combination of specific chemical reactions to quantify a group of compounds with similar chemical and nutritional properties. However, plant cell wall complex is not uniform in terms of chemical, physical or nutritional characteristics and the digestion progress is achieved by a series of enzymatic hydrolysis of specific chemical bonds which cannot be revealed by wet chemistry analysis. Synchrotron-based and globar-sourced mid-infrared spectroscopy instead utilizing the unique absorption of mid-infrared light at different frequencies and more information about specific chemical bonds can be revealed. As a result, taking spectral change during digestion into consideration may give some insight about nutritional utilization features. However, the utilization of synchrotron-based and globar-sourced mid-infrared spectroscopy on feed and food nutritional research is limited. Therefore, the aim of this study is to provide idea about how to systematically study the nutritional and spectral structure feature of faba bean with traditional and advanced synchrotron-based and globar-sourced vibrational molecular spectroscopy. The study reviews (1) Utilization of faba bean for human and animal consumption; (2) Traditional evaluation methods for faba bean nutritional characteristics and (3) Contribution of synchrotron-based and globar-sourced mid-infrared (Mid-IR) spectroscopy techniques to evaluate faba bean structural and molecular properties.

Differential item functioning to validate setting of delivery compatibility in PROMIS-global health.

PURPOSE: Patient-reported outcomes measures (PROMs) such as PROMIS are increasingly utilized in healthcare to assess patient perception and functional status, but the effect of delivery setting remains to be fully investigated. To our knowledge, no current study establishes the absence of differential item functioning (DIF) across delivery setting for these PROMIS- Global Health (PROMIS-GH) measures among orthopedic patients. We sought to investigate the correlation of PROMIS-GH scores across in-clinic versus remote delivery by evaluating DIF within the Global Physical Health (GPH) and Global Mental Health (GMH) items. We hypothesize that the setting of delivery of the GPH and GMH domains of PROMIS-GH will not impact the results of the measure, allowing direct comparison between the two delivery settings. METHODS: Five thousand and seven hundred and eighty-five complete PROMIS-Global Health measures were analyzed retrospectively using the 'Lordif' package on the R platform. DIF was measured for GPH and GMH domains across setting of response (in-clinic vs remote) during the pre-operative period, immediate post-operative period, and 1-year post-operative period using Monte Carlo estimation. McFadden pseudo-R2 thresholds (> 0.02) were used to assess the magnitude of DIF for individual PROMIS items. RESULTS: No GPH or GMH items contained in the PROMIS-GH instrument yielded DIF across in-clinic vs remote delivery setting during the pre-operative, immediate post-operative, or 1-year post-operative window. CONCLUSION: The GPH and GMH domains within the PROMIS-GH instrument may be delivered in the clinic or remotely with comparable accuracy. This cross-delivery setting validation analysis may aid to improve the quality of patient care by allowing mixed platform PROMIS-GH data tailored to individual patient circumstance.

LapG mediates biofilm dispersal in Vibrio fischeri by controlling maintenance of the VCBS-containing adhesin LapV.

Efficient symbiotic colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri depends on bacterial biofilm formation on the surface of the squid's light organ. Subsequently, the bacteria disperse from the biofilm via an unknown mechanism and enter through pores to reach the interior colonization sites. Here, we identify a homolog of Pseudomonas fluorescens LapG as a dispersal factor that promotes cleavage of a biofilm-promoting adhesin, LapV. Overproduction of LapG inhibited biofilm formation and, unlike the wild-type parent, a ΔlapG mutant formed biofilms in vitro. Although V. fischeri encodes two putative large adhesins, LapI (near lapG on chromosome II) and LapV (on chromosome I), only the latter contributed to biofilm formation. Consistent with the Pseudomonas Lap system model, our data support a role for the predicted c-di-GMP-binding protein LapD in inhibiting LapG-dependent dispersal. Furthermore, we identified a phosphodiesterase, PdeV, whose loss promotes biofilm formation similar to that of the ΔlapG mutant and dependent on both LapD and LapV. Finally, we found a minor defect for a ΔlapD mutant in initiating squid colonization, indicating a role for the Lap system in a relevant environmental niche. Together, these data reveal new factors and provide important insights into biofilm dispersal by V. fischeri.

In vitro stem cell modelling demonstrates a proof-of-concept for excess functional mutant TIMP3 as the cause of Sorsby fundus dystrophy.

Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disease of the macula that leads to bilateral loss of central vision and is caused by mutations in the TIMP3 gene. However, the mechanisms by which TIMP3 mutations cause SFD are poorly understood. Here, we generated human induced pluripotent stem cell-derived retinal pigmented epithelial (hiPSC-RPE) cells from three SFD patients carrying TIMP3 p.(Ser204Cys) and three non-affected controls to study disease-related structural and functional differences in the RPE. SFD-hiPSC-RPE exhibited characteristic RPE structure and physiology but showed significantly reduced transepithelial electrical resistance associated with enriched expression of cytoskeletal remodelling proteins. SFD-hiPSC-RPE exhibited basolateral accumulation of TIMP3 monomers, despite no change in TIMP3 gene expression. TIMP3 dimers were observed in both SFD and control hiPSC-RPE, suggesting that mutant TIMP3 dimerisation does not drive SFD pathology. Furthermore, mutant TIMP3 retained matrix metalloproteinase activity. Proteomic profiling showed increased expression of ECM proteins, endothelial cell interactions and angiogenesis-related pathways in SFD-hiPSC-RPE. By contrast, there were no changes in VEGF secretion. However, SFD-hiPSC-RPE secreted higher levels of monocyte chemoattractant protein 1, PDGF and angiogenin. Our findings provide a proof-of-concept that SFD patient-derived hiPSC-RPE mimic mature RPE cells and support the hypothesis that excess accumulation of mutant TIMP3, rather than an absence or deficiency of functional TIMP3, drives ECM and angiogenesis-related changes in SFD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Perioperative Antibiotic Prophylaxis: Single and 24-Hour Antibiotic Dosages are Equally Effective at Preventing Periprosthetic Joint Infection in Total Joint Arthroplasty.

BACKGROUND: Perioperative antibiotic prophylaxis is used to prevent surgical site infection and periprosthetic joint infection (PJI) after total joint arthroplasty (TJA). Secondary to a national shortage of cefazolin, patients at our institution began receiving a single preoperative prophylactic antibiotic dose for primary TJA and no 24-hour postoperative antibiotic prophylaxis. The purpose of the study was to compare the efficacy of single-dose antibiotic use versus 24-hour dosing of prophylactic antibiotics in the prevention of acute PJI and short-term complications after primary TJA. METHODS: A retrospective review of 3317 patients undergoing primary TJA performed from January 2015 to December 2019 identified 554 patients who received a single dose of preoperative antibiotic prophylaxis during the antibiotic shortage and 2763 patients who received post-TJA 24-hour antibiotic prophylaxis before the shortage. Patient records were evaluated for acute PJI, superficial infection, 90-day reoperation, and 90-day complications. RESULTS: There were no significant differences in patient characteristics between single-dose and 24-hour antibiotic groups. Similarly, there were no significant differences in rates of acute PJI (0.7% vs 0.2%; P = .301), superficial infection (2.4% vs 1.4%; P = .221), 90-day reoperation (2.1% vs 1.1%; P = .155), and 90-day complications (9.9% vs 7.9%; P = .169) between single and 24-hour antibiotic dose. Post hoc power analysis demonstrated adequate sample size, beta = 93%. CONCLUSION: Single-dose prophylactic antibiotics did not lead to an increased risk of acute PJI or short-term complications after TJA. Our study suggests that administration of a single antibiotic dose may be safely considered in patients undergoing routine primary TJA.

Comparison of Tip- Versus Hub-Oscillating Saw Blade Control in a Total Knee Arthroplasty Model.

BACKGROUND: Oscillating saws are commonly used for bone preparation in total knee arthroplasty but can cause injury to the posterior neurovascular bundle during tibial resection. Tip-oscillating saw blades are a recent innovation that could improve saw control due to decreased excursion; however, the tactile feedback to the surgeon is different. METHODS: To compare traditional hub and new tip-oscillating saw blades, 16 participants of varying levels of experience were video-recorded during composite tibial bone model resections to measure posterior saw blade plunge. Subjective perceptions of saw control and preference were also surveyed. RESULTS: Saw blade design and level of surgical experience did not produce a significant difference in posterior saw blade plunge (P > .05). Independent of saw blade design, subjects with no previous saw experience had significantly decreased posterior tibial plunge over subsequent resections. Tip-oscillating saw blades were perceived to be easier to use and control by less experienced participants (P = .0163). CONCLUSION: Tip-oscillating saw blades do not alter the risk of posterior tibial saw plunge compared with traditional saw blades.

Extracellular Acidic pH Inhibits Acetate Consumption by Decreasing Gene Transcription of the Tricarboxylic Acid Cycle and the Glyoxylate Shunt.

Escherichia coli produces acetate during aerobic growth on various carbon sources. After consuming the carbon substrate, E. coli can further grow on the acetate. This phenomenon is known as the acetate switch, where cells transition from producing acetate to consuming it. In this study, we investigated how pH governs the acetate switch. When E. coli was grown on a glucose-supplemented medium initially buffered to pH 7, the cells produced and then consumed the acetate. However, when the initial pH was dropped to 6, the cells still produced acetate but were only able to consume it when little (<10 mM) acetate was produced. When significant acetate was produced in acidic medium, which occurs when the growth medium contains magnesium, amino acids, and sugar, the cells were unable to consume the acetate. To determine the mechanism, we characterized a set of metabolic mutants and found that those defective in the tricarboxylic acid (TCA) cycle or glyoxylate shunt exhibited reduced rates of acetate consumption. We further found that the expression of the genes in these pathways was reduced during growth in acidic medium. The expression of the genes involved in the AckA-Pta pathway, which provides the principal route for both acetate production and consumption, was also inhibited in acidic medium but only after glucose was depleted, which correlates with the acetate consumption phase. On the basis of these results, we conclude that growth in acidic environments inhibits the expression of the acetate catabolism genes, which in turn prevents acetate consumption.IMPORTANCE Many microorganisms produce fermentation products during aerobic growth on sugars. One of the best-known examples is the production of acetate by Escherichia coli during aerobic growth on sugars. In E. coli, acetate production is reversible: once the cells consume the available sugar, they can consume the acetate previously produced during aerobic fermentation. We found that pH affects the reversibility of acetate production. When the cells produce significant acetate during growth in acidic environments, they are unable to consume it. Unconsumed acetate may accumulate in the cell and inhibit the expression of pathways required for acetate catabolism. These findings demonstrate how acetate alters cell metabolism; they also may be useful for the design of aerobic fermentation processes.

Global Lysine Acetylation in Escherichia coli Results from Growth Conditions That Favor Acetate Fermentation.

Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regard to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of Escherichia coli on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets accessible lysines, which may or may not alter enzyme activity. They further suggest that lysine acetylation principally results from conditions that favor accumulation of acetyl phosphate, the principal acetate donor in E. coliIMPORTANCE Bacteria alter their metabolism in response to nutrient availability, growth conditions, and environmental stresses using a number of different mechanisms. One is lysine acetylation, a posttranslational modification known to target many metabolic enzymes. However, little is known about this regulatory mode. We investigated the factors inducing changes in lysine acetylation by comparing growth on glucose and xylose. We found that the specific sugar used for growth did not alter the pattern of acetylation; rather, the amount of sugar did, with more sugar causing more acetylation. These results imply that lysine acetylation is a global regulatory mechanism that is responsive not to the specific carbon source per se but rather to the accumulation of downstream metabolites.

Effects of processing methods (rolling vs. pelleting vs. steam-flaking) of cool-season adapted oats on dairy cattle production performance and metabolic characteristics compared with barley.

Several processing techniques can be used to slow the degradation rate in the rumen and thus provide more bypass crude protein (CP) and starch to the small intestine. The aim of this study was to evaluate the effects of processing methods on cool-season adapted oat grain compared with dry-rolled barley grain, when fed as total mixed ration (TMR) for lactating dairy cows. Eight lactating Holstein cows were used in a replicated 4 × 4 Latin square design with 21-d periods and fed TMR with 1 of 4 treatments: dry-rolled oats, steam-flaked oats, pelleted oats, or dry-rolled barley. Dry matter intake (DMI) ranged from 28.19 to 31.61 kg/d and was lower for rolled oats compared with pelleted oats. Despite the nutrient intake being higher for cows fed pelleted oats, those fed rolled oats had the highest milk production and milk fat percentage (49.23 kg/d and 4%, respectively). Ruminal fermentation characteristics were similar across treatments, with only significant differences in concentrations of acetate (lowest for pelleted oats) and total short-chain fatty acids (highest value for rolled barley) and a lower pH for flaked oats at the 9-h and 12-h points. Dietary treatments did not affect total-tract digestibility of dry matter, organic matter, or CP; digestibility of starch was the lowest for rolled barley (89.04%). Measured blood metabolites, urea, glucose, and ß-hydroxybutyrate, were not affected by dietary treatment. Purine derivatives and microbial N supply were also unaffected by dietary treatments. Cows fed flaked oat-based TMR showed the lowest N excretion in milk; however, the lack of difference between diets with regard to urinary N and fecal N excretion resulted in no significant changes in N balance between diets. Therefore, rolled oats allow cows to have higher milk production with lower DMI compared with all other treatments in this study.

Effects of silencing TT8 and HB12 on in vitro nutrients degradation and VFA production in relation to molecular structures of alfalfa (Medicago sativa).

BACKGROUND: Transparent Testa8 (TT8) and Homeobox12 (HB12) are two transcriptional factors in plant phenylpropanoid pathways and were reported to be positively related to lignin content. Alfalfa with silenced TT8 (TT8i) and HB12 (HB12i) was therefore generated using the RNA interference (RNAi) technique. Although lignin was found to be high in HB12i, such gene-silencing of alfalfa resulted in nutrient profiles that might be suitable for grazing. To extend the nutritional evaluation of transformed alfalfa, ground samples of 11 HB12i, 5 TT8i and 4 wild type (WT) were incubated in rumen fluid : buffer solution for 0, 2, 4, 8, 12, 24 and 48 h at 39 °C. Dry matter (DM) and neutral detergent fiber (NDF) degradations at each time point, and production of volatile fatty acids (VFA) at 4, 12, 24 and 48 h were analyzed, as well as degradation and production kinetics. The correlations and regressions between nutritive profiles and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectral parameters were determined. RESULTS: Both transformed genotypes had lower DM degradation and HB12i had lower VFA production compared with WT. Structural carbohydrate (STC) parameters were found to be negatively correlated with DM degradation and VFA production. The kinetics of DM degradation and VFA production were predicted from spectral parameters with good estimation power. CONCLUSION: Silencing of HB12 and TT8 affected fermentation characteristics of alfalfa and some fermentation characteristics were predictable from spectral parameters using ATR-FTIR spectroscopy. © 2019 Society of Chemical Industry.

Effect of fibrolytic enzymes on lactational performance, feeding behavior, and digestibility in high-producing dairy cows fed a barley silage-based diet.

The objectives of this study were to evaluate the effects of pretreating dairy cow rations with a fibrolytic enzyme derived from Trichoderma reesei (FETR; mixture of xylanase and cellulase; AB Vista, Wiltshire, UK) on lactation performance, digestibility, and feeding behavior in response to feeding a barley silage-based diet. Before starting the dairy trial, in vitro incubations were conducted to determine whether the addition of FETR would have an effect on these animal performance characteristics when applied to a barley silage-based diet for dairy cows. The dairy trial was performed using 8 Holstein dairy cows. The cows were blocked by parity and assigned randomly to 1 of 4 treatments: 0, 0.5, 0.75, and 1 mL of FETR/kg of dry matter (DM) diet in a replicated Latin square design. The pretreatment was applied to the complete diet during the mixing process. The experimental period continued for 22 d, with each experimental period consisting of a 16-d adaptation period and a 6-d sampling period. The daily feed intake of each individual cow was monitored using Insentec feed bins (RIC system, Insentec, Marknesse, the Netherlands). Feeding behavior characteristics were measured during the entire sampling period using the feed bin attendance data. Milk samples were collected in the last 3 d of each experimental period. The addition of FETR linearly increased the in vitro DM digestibility and tended to improve the in vitro digestibility of barley silage. There was a cubic effect of the enzyme levels on the total-tract DM and neutral detergent fiber digestibility. Maximal digestibility was reached at 0.75 mL of FETR/kg of TMR. The milk fat yield, fat-corrected milk, and energy-corrected milk quadratically responded to the incremental levels of FETR. The milk protein percentage linearly improved in response to FETR. Increasing FETR levels resulted in a quadratic effect on feed efficiency. There was no effect of FETR level on feeding behavior. In conclusion, pretreating dairy cow barley silage-based diet with 0.75 mL of FETR/kg of TMR increased the milk production efficiency of dairy cows fed diet containing 34% barley silage (DM basis). The positive effect of adding FETR could benefit the dairy industry in western Canada, where barley silage-based diets are common.

Molecular Structural Changes in Alfalfa Detected by ATR-FTIR Spectroscopy in Response to Silencing of TT8 and HB12 Genes.

This study investigated the spectral changes in alfalfa molecular structures induced by silencing of Transparent Testa 8 (TT8) and Homeobox 12 (HB12) genes with univariate and multivariate analyses. TT8-silenced (TT8i), HB12-silenced (HB12i) and wild type (WT) alfalfa were grown in a greenhouse under normal conditions and were harvested at early-to-mid vegetative stage. Samples were free-dried and grounded through 0.02 mm sieve for spectra collections with attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Afterwards, both univariate and multivariate analyses were conducted on amide, carbohydrate and lipid regions. Univariate results showed that silencing of TT8 and HB12 genes affected peak heights of most total carbohydrate (TC) and structural carbohydrate (STC), and structural carbohydrate area (STCA) in carbohydrate regions; and ß-sheet height, amide areas, and ratios of amide I/II and α-helix/ß-sheet in amide region; and symmetric CH2 (SyCH2), asymmetric CH2 (AsCH2) and (a)symmetric CH2 and CH3 area (ASCCA) in the lipid region. Multivariate analysis showed that both hierarchy cluster analysis (HCA) and principal component analysis (PCA) clearly separated WT from transgenic plants in all carbohydrate regions and (a)symmetric CH2 and CH3 (ASCC) lipid region. In the amide region, PCA separated WT, TT8i and HB12i into different groups, while HCA clustered WT into a separate group. In conclusion, silencing of TT8 and HB12 affected intrinsic molecular structures of both amide and carbohydrate profiles in alfalfa, and multivariate analyses successfully distinguished gene-silenced alfalfa from its parental WT control.

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media.

Complex media are routinely used to cultivate diverse bacteria. However, this complexity can obscure the factors that govern cell growth. While studying protein acetylation in buffered tryptone broth supplemented with glucose (TB7-glucose), we observed that Escherichia coli did not fully consume glucose prior to stationary phase. However, when we supplemented this medium with magnesium, the glucose was completely consumed during exponential growth, with concomitant increases in cell number and biomass but reduced cell size. Similar results were observed with other sugars and other peptide-based media, including lysogeny broth. Magnesium also limited cell growth for Vibrio fischeri and Bacillus subtilis in TB7-glucose. Finally, magnesium supplementation reduced protein acetylation. Based on these results, we conclude that growth in peptide-based media is magnesium limited. We further conclude that magnesium supplementation can be used to tune protein acetylation without genetic manipulation. These results have the potential to reduce potentially deleterious acetylated isoforms of recombinant proteins without negatively affecting cell growth.IMPORTANCE Bacteria are often grown in complex media. These media are thought to provide the nutrients necessary to grow bacteria to high cell densities. In this work, we found that peptide-based media containing a sugar are magnesium limited for bacterial growth. In particular, magnesium supplementation is necessary for the bacteria to use the sugar for cell growth. Interestingly, in the absence of magnesium supplementation, the bacteria still consume the sugar. However, rather than use it for cell growth, the bacteria instead use the sugar to acetylate lysines on proteins. As lysine acetylation may alter the activity of proteins, this work demonstrates how lysine acetylation can be tuned through magnesium supplementation. These findings may be useful for recombinant protein production, when acetylated isoforms are to be avoided. They also demonstrate how to increase bacterial growth in complex media.

Sorsby fundus dystrophy - A review of pathology and disease mechanisms.

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular dystrophy with an estimated prevalence of 1 in 220,000 and an onset of disease around the 4th to 6th decade of life. Similar to age-related macular degeneration (AMD), ophthalmoscopy reveals accumulation of protein/lipid deposits under the retinal pigment epithelium (RPE), referred to as drusen, in the eyes of patients with SFD. SFD is caused by variants in the gene for tissue inhibitor of metalloproteinases-3 (TIMP3), which has been found in drusen-like deposits of SFD patients. TIMP3 is constitutively expressed by RPE cells and, in healthy eyes, resides in Bruch's membrane. Most SFD-associated TIMP3 variants involve the gain or loss of a cysteine residue. This suggests the protein aberrantly forms intermolecular disulphide bonds, resulting in the formation of TIMP3 dimers. It has been demonstrated that SFD-associated TIMP3 variants are more resistant to turnover, which is thought to be a result of dimerisation and thought to explain the accumulation of TIMP3 in drusen-like deposits at the level of Bruch's membrane. An important function of TIMP3 within the outer retina is to regulate the thickness of Bruch's membrane. TIMP3 performs this function by inhibiting the activity of matrix metalloproteinases (MMPs), which have the function of catalysing breakdown of the extracellular matrix. TIMP3 has an additional function to inhibit vascular endothelial growth factor (VEGF) signalling and thereby to inhibit angiogenesis. However, it is unclear whether SFD-associated TIMP3 variant proteins retain these functions. In this review, we discuss the current understanding of the potential mechanisms underlying development of SFD and summarise all known SFD-associated TIMP3 variants. Cell culture models provide an invaluable way to study disease and identify potential treatments. These allow a greater understanding of RPE physiology and pathophysiology, including the ability to study the blood-retinal barrier as well as other RPE functions such as phagocytosis of photoreceptor outer segments. This review describes some examples of such recent in vitro studies and how they might provide new insights into degenerative diseases like SFD. Thus far, most studies on SFD have been performed using ARPE-19 cells or other, less suitable, cell-types. Now, induced pluripotent stem cell (iPSC) technologies allow the possibility to non-invasively collect somatic cells, such as dermal fibroblast cells and reprogram those to produce iPSCs. Subsequent differentiation of iPSCs can generate patient-derived RPE cells that carry the same disease-associated variant as RPE cells in the eyes of the patient. Use of these patient-derived RPE cells in novel cell culture systems should increase our understanding of how SFD and similar macular dystrophies develop.

Protein acetylation dynamics in response to carbon overflow in Escherichia coli.

In Escherichia coli, acetylation of proteins at lysines depends largely on a non-enzymatic acetyl phosphate-dependent mechanism. To assess the functional significance of this post-translational modification, we first grew wild-type cells in buffered tryptone broth with glucose and monitored acetylation over time by immunochemistry. Most acetylation occurred in stationary phase and paralleled glucose consumption and acetate excretion, which began upon entry into stationary phase. Transcription of rprA, a stationary phase regulator, exhibited similar behavior. To identify sites and substrates with significant acetylation changes, we used label-free, quantitative proteomics to monitor changes in protein acetylation. During growth, both the number of identified sites and the extent of acetylation increased with considerable variation among lysines from the same protein. As glucose-regulated lysine acetylation was predominant in central metabolic pathways and overlapped with acetyl phosphate-regulated acetylation sites, we deleted the major carbon regulator CRP and observed a dramatic loss of acetylation that could be restored by deleting the enzyme that degrades acetyl phosphate. We propose that acetyl phosphate-dependent acetylation is a response to carbon flux that could regulate central metabolism.

Molecular basis of bacterial protein Hen1 activating the ligase activity of bacterial protein Pnkp for RNA repair.

Ribotoxins cleave essential RNAs for cell killing in vivo, and the bacterial polynucleotide kinase-phosphatase (Pnkp)/hua enhancer 1 (Hen1) complex has been shown to repair ribotoxin-cleaved RNAs in vitro. Bacterial Pnkp/Hen1 is distinguished from other RNA repair systems by performing 3'-terminal 2'-O-methylation during RNA repair, which prevents the repaired RNA from repeated cleavage at the same site. To ensure the opportunity of 2'-O-methylation by bacterial Hen1 during RNA repair and, therefore, maintain the quality of the repaired RNA, Pnkp/Hen1 has evolved to require the participation of Hen1 in RNA ligation, because Pnkp alone is unable to carry out the reaction despite possessing all signature motifs of an RNA ligase. However, the precise role of Hen1 in RNA ligation is unknown. Here, we present the crystal structure of an active RNA ligase consisting of the C-terminal half of Pnkp (Pnkp-C) and the N-terminal half of Hen1 (Hen1-N) from Clostridium thermocellum. The structure reveals that the N-terminal domain of Clostridium thermocellum (Cth) Hen1, shaped like a left hand, grabs the flexible insertion module of CthPnkp and locks its conformation via further interaction with the C-terminal addition module of CthPnkp. Formation of the CthPnkp-C/Hen1-N heterodimer creates a ligation pocket with a width for two strands of RNA, depth for two nucleotides, and the adenosine monophosphate (AMP)-binding pocket at the bottom. The structure, combined with functional analyses, provides insight into the mechanism of how Hen1 activates the RNA ligase activity of Pnkp for RNA repair.

Effect of oxygen tension on the amino acid utilisation of human embryonic stem cells.

BACKGROUND/AIMS: Human embryonic stem cells (hESCs) are a potential source of cells for treatment of many degenerative diseases, but in culture have a propensity to spontaneously differentiate, possibly due to suboptimal conditions. Culture at low oxygen tensions improves hESC maintenance and regulates carbohydrate metabolism. Hence, a greater understanding of the nutrient requirements of hESCs will allow production of more appropriate culture media. This study aims to investigate the effect of environmental oxygen tension on the amino acid metabolism of hESCs. METHODS: The production or depletion of amino acids by hESCs cultured at 5% or 20% oxygen in the presence or absence of FGF2 was measured by reversephase HPLC. RESULTS: Atmospheric oxygen, or removal of FGF2 from hESCs cultured at 5% oxygen, perturbed the uptake or release of individual amino acids and the total amino acid turnover compared to hESCs cultured at 5% oxygen. In particular, serine uptake was reduced at 20% oxygen and by removal of FGF2. CONCLUSIONS: Highly pluripotent hESCs, cultured at 5% oxygen, demonstrate a greater amino acid turnover than hESCs cultured at 20% oxygen, or without FGF2. These data suggest that amino acid turnover could be used as a measure of the self-renewal capacity of hESCs.