Bioconversion of citrus waste into mucic acid by xylose-fermenting Saccharomyces cerevisiae.

Mucic acid holds promise as a platform chemical for bio-based nylon synthesis; however, its biological production encounters challenges including low yield and productivity. In this study, an efficient and high-yield method for mucic acid production was developed by employing genetically engineered Saccharomyces cerevisiae expressing the NAD+-dependent uronate dehydrogenase (udh) gene. To overcome the NAD+ dependency for the conversion of pectin to mucic acid, xylose was utilized as a co-substrate. Through optimization of the udh expression system, the engineered strain achieved a notable output, producing 20 g/L mucic acid with a highest reported productivity of 0.83 g/L-h and a theoretical yield of 0.18 g/g when processing pectin-containing citrus peel waste. These results suggest promising industrial applications for the biological production of mucic acid. Additionally, there is potential to establish a viable bioprocess by harnessing pectin-rich fruit waste alongside xylose-rich cellulosic biomass as raw materials.

Glutamine increases stability of TPH1 mRNA via p38 mitogen-activated kinase in mouse mastocytoma cells.

Expression changes for tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in serotonin synthesis, by environmental glutamine (GLN) were examined in mouse mastocytoma-derived P815-HTR cells. GLN-treated cells exhibited a robust increase in TPH1 mRNA after a 6 h exposure to GLN. 6-Diazo-5-oxo-L-norleucine (DON), a glutamine-utilizing glutaminase inhibitor, significantly inhibited the GLN-induction of TPH1 mRNA. Nuclear run-on assays and mRNA decay experiments demonstrated that the primary mechanism leading to increased TPH1 mRNA levels was not due to transcriptional changes, but rather due to increased TPH1 RNA stability induced by GLN. Treatment with GLN also led to activation of p38 MAP kinase, but not p42/44 MAPK. In addition, SB203580, a p38 MAP kinase specific inhibitor, completely abolished the GLN-mediated increase of TPH1 mRNA levels, suggesting the pathway stabilizing TPH1 mRNA might be mediated by the activated p38 MAP kinase pathway. Additionally, SB203580 significantly reduced the stability of TPH1 mRNA, and this reduction of the stability was not affected by GLN in the culture medium, implying a sequential signaling from GLN being mediated by p38 MAP kinase, resulting in alteration of TPH1 mRNA stability. TPH1 mRNA stability loss was also dependent on de novo protein synthesis as shown by treatment of cells with a transcriptional/translational blocker. We provide evidence that TPH1 mRNA levels are increased in response to increased exogenous GLN in mouse mastocytoma cells via a stabilization of TPH1 mRNA due to the activity of the p38 MAP kinase.

Metastable hexagonal close-packed palladium hydride in liquid cell TEM.

Metastable phases-kinetically favoured structures-are ubiquitous in nature1,2. Rather than forming thermodynamically stable ground-state structures, crystals grown from high-energy precursors often initially adopt metastable structures depending on the initial conditions, such as temperature, pressure or crystal size1,3,4. As the crystals grow further, they typically undergo a series of transformations from metastable phases to lower-energy and ultimately energetically stable phases1,3,4. Metastable phases sometimes exhibit superior physicochemical properties and, hence, the discovery and synthesis of new metastable phases are promising avenues for innovations in materials science1,5. However, the search for metastable materials has mainly been heuristic, performed on the basis of experiences, intuition or even speculative predictions, namely 'rules of thumb'. This limitation necessitates the advent of a new paradigm to discover new metastable phases based on rational design. Such a design rule is embodied in the discovery of a metastable hexagonal close-packed (hcp) palladium hydride (PdHx) synthesized in a liquid cell transmission electron microscope. The metastable hcp structure is stabilized through a unique interplay between the precursor concentrations in the solution: a sufficient supply of hydrogen (H) favours the hcp structure on the subnanometre scale, and an insufficient supply of Pd inhibits further growth and subsequent transition towards the thermodynamically stable face-centred cubic structure. These findings provide thermodynamic insights into metastability engineering strategies that can be deployed to discover new metastable phases.

Simulation Model for Prediction of Gas Separation in Membrane Contactor Process.

The purpose of this study is to establish a practical simulation model based on mass balance, mass transport equations and equilibrium equation between gas and liquid phases across a porous membrane in membrane contactor process in order to predict the separation behavior by the gassing process of gas mixture in membrane contactor. The established simulation model was verified by comparison between the simulated values and real process values in the separation of CH4/CO2 mixture, showing an excellent agreement between them. The parameter R-value in the model, which is a kind of the permeability of permeant across porous membrane, has been determined by fitting a numerical solution of the model equation to the experimental data to obtain a practical value of the parameter. A parametric study on the gassing process of N2/CO2 mixture in membrane contactor was made with the help of the practical simulation model to investigate the effects of operation parameters on separation performance and to characterize the separation behavior of membrane contactor process. A series of simulations of the separation of N2/CO2 mixture in membrane contactor were conducted, and the optimization on the membrane process was discussed to maximize the separation performance in terms of N2 recovery percent in retentate and CO2 permeation rate. It was observed from the analysis of the result of the simulation that liquid flow rate has a negative effect on N2 recovery percent in retentate but a positive effect on the separation of CO2, while R-value affects the separation performance in the other way. It is confirmed in this study that the developed simulation can be used as a tool to optimize the parameters, i.e., feed gas pressure, liquid flow rate and R-value to maximize the separation performance.

Perfusion-Decellularized Larynx as a Natural 3D Scaffold in a Rabbit Model.

INTRODUCTION: Decellularized larynges could be used as scaffolds to regenerate the larynx. The purpose of this study was to establish a perfusion decellularization protocol to produce a 3-dimensional whole laryngeal extracellular matrix (ECM) scaffold in a rabbit model. METHODS: The larynges of 20 rabbits assigned to the study group were harvested and decellularized using a perfusion decellularization protocol, while the larynges of 10 rabbits in the control group were harvested and untreated. Macroscopic and microscopic morphological analyses, a molecular analysis, a cellular content analysis, and scanning electron microscopy were performed. RESULTS: A histological analysis showed the absence of cellular components, the presence of the ECM, and an intact cartilage structure filled with chondrocytes. The mean total DNA amounts of the native larynx, decellularized larynx, and decellularized cartilage-free larynx were 1,826.40, 434.70, and 41.40 µg/µL, respectively; those for the decellularized larynx and decellularized cartilage-free larynx were significantly lower (p < 0.001 and p < 0.001, respectively). The total amount of DNA in the decellularized sample was significantly lower compared to that in the native sample, at 57.2% in cartilage (p < 0.001), 2.4% in the thyroid gland (p < 0.001), 2.7% in muscle (p < 0.001), 1.6% in vessels (p < 0.001), and 4.8% in the vocal cords (p < 0.001). CONCLUSION: Our perfusion decellularization protocol is feasible and reproducible to produce a 3-dimensional whole laryngeal ECM scaffold in a rabbit.

Synthesis of Sulfonated Poly(Arylene Ether Sulfone)s Containing Aliphatic Moieties for Effective Membrane Electrode Assembly Fabrication by Low-Temperature Decal Transfer Methods.

The purpose of this study was to investigate the effect of the aliphatic moiety in the sulfonated poly(arylene ether sulfone) (SPAES) backbone. A new monomer (4,4'-dihydroxy-1,6-diphenoxyhexane) was synthesized and polymerized with other monomers to obtain partially alkylated SPAESs. According to differential scanning calorimetry analysis, the glass transition temperature (Tg) of these polymers ranged from 85 to 90 °C, which is 100 °C lower than that of the fully aromatic SPAES. Due to the low Tg values obtained for the partially alkylated SPAESs, it was possible to prepare a hydrocarbon electrolyte membrane-based membrane electrode assembly (MEA) with Nafion® binder in the electrode through the use of a decal transfer method, which is the most commercially suitable system to obtain an MEA of proton exchange membrane fuel cells (PEMFCs). A single cell prepared using this partially alkylated SPAES as an electrolyte membrane exhibited a peak power density of 539 mW cm-2.

Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae.

Being a microbial host for lignocellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway; however, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (Δpho13) has been reported to be a crucial genetic perturbation in improving xylose fermentation. A confirmed mechanism of the Δpho13 effect on xylose fermentation is that the Δpho13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the current study, we found a couple of engineered strains, of which phenotypes were not affected by Δpho13 (Δpho13-negative), among many others we examined. Genome resequencing of the Δpho13-negative strains revealed that a loss-of-function mutation in GCR2 was responsible for the phenotype. Gcr2 is a global transcriptional factor involved in glucose metabolism. The results of RNA-seq confirmed that the deletion of GCR2 (Δgcr2) led to the upregulation of PPP genes as well as downregulation of glycolytic genes, and changes were more significant under xylose conditions than those under glucose conditions. Although there was no synergistic effect between Δpho13 and Δgcr2 in improving xylose fermentation, these results suggested that GCR2 is a novel knockout target in improving lignocellulosic ethanol production.

Polystyrene-Based Hydroxide-Ion-Conducting Ionomer: Binder Characteristics and Performance in Anion-Exchange Membrane Fuel Cells.

Polystyrene-based polymers with variable molecular weights are prepared by radical polymerization of styrene. Polystyrene is grafted with bromo-alkyl chains of different lengths through Friedel-Crafts acylation and quaternized to afford a series of hydroxide-ion-conducting ionomers for the catalyst binder for the membrane electrode assembly in anion-exchange membrane fuel cells (AEMFCs). Structural analyses reveal that the molecular weight of the polystyrene backbone ranges from 10,000 to 63,000 g mol-1, while the ion exchange capacity of quaternary-ammonium-group-bearing ionomers ranges from 1.44 to 1.74 mmol g-1. The performance of AEMFCs constructed using the prepared electrode ionomers is affected by several ionomer properties, and a maximal power density of 407 mW cm-2 and a durability exceeding that of a reference cell with a commercially available ionomer are achieved under optimal conditions. Thus, the developed approach is concluded to be well suited for the fabrication of next-generation electrode ionomers for high-performance AEMFCs.

Recent advances in the biological valorization of citrus peel waste into fuels and chemicals.

In the quest to reduce global food loss and waste, fruit processing wastes, particularly citrus peel waste (CPW), have emerged as a promising and sustainable option for biorefinery without competing with human foods and animal feeds. CPW is largely produced and, as recent studies suggest, has the industrial potential of biological valorization into fuels and chemicals. In this review, the promising aspects of CPW as an alternative biomass were highlighted, focusing on its low lignin content. In addition, specific technical difficulties in fermenting CPW are described, highlighting that citrus peel is high in pectin that consist of non-fermentable sugars, mainly galacturonic acid. Last, recent advances in the metabolic engineering of yeast and other microbial strains that ferment CPW-derived sugars to produce value-added products, such as ethanol and mucic acid, are summarized. For industrially viable CPW-based biorefinery, more studies are needed to improve fermentation efficiency and to diversify product profiles.

High-yield electrochemical hydrogen peroxide production from an enhanced two-electron oxygen reduction pathway by mesoporous nitrogen-doped carbon and manganese hybrid electrocatalysts.

Electrochemical hydrogen peroxide (H2O2) production by the direct two-electron (2e-) oxygen reduction reaction (ORR) has received much attention as a promising alternative to the industrially developed anthraquinone fabrication process. Transition metal (M) and nitrogen doped carbon (M-N-C, M = Fe or Co) catalysts are known to be active for four electron ORR pathways via two + two electron transfer, where the former is for the ORR and the latter for the peroxide reduction reaction (PRR). Here, we report mesoporous N-doped carbon/manganese hybrid electrocatalysts composed of MnO and Mn-Nx coupled with N-doped carbons (Mn-O/N@NCs), which have led to the development of electrocatalysis towards the 2e- ORR route. Based on the structural and electrochemical characterization, the number of transferred electrons during the ORR on the Mn-O/N@NCs was found to be close to the theoretical value of the 2e- process, indicating their high activity toward H2O2. The favored ORR process arose due to the increased number of Mn-Nx sites within the mesoporous N-doped carbon materials. Furthermore, there was a strong indication that the PRR is significantly suppressed by adjacent MnO species, demonstrating its highly selective production of H2O2 (>80%) from the oxygen electrochemical process. The results of a real fuel cell device test demonstrated that an Mn-O/N@NC catalyst sustains a very stable current, and we attributed its outstanding activity to a combination of site-dependent facilitation of 2e- transfer and a favorable porosity for mass transport.

Data for simultaneous fermentation of galacturonic acid and five-carbon sugars by engineered Saccharomyces cerevisiae.

Saccharomyces cerevisiae expressing heterologous pathways for xylose, arabinose, and galacturonic acid metabolism has been constructed by a Cas9-based genome editing technology [1]. The fermentation performance of the final strain (YE9) was tested under various substrate conditions, and the fermentation parameters were calculated. The dataset can be used for designing bioprocesses for pectin-rich biomass.

Xylose utilization in Saccharomyces cerevisiae during conversion of hydrothermally pretreated lignocellulosic biomass to ethanol.

With growing interest in alternative fuels to minimize carbon and particle emissions, research continues on the production of lignocellulosic ethanol and on the development of suitable yeast strains. However, great diversities and continued technical advances in pretreatment methods for lignocellulosic biomass complicate the evaluation of developed yeast strains, and strain development often lags industrial applicability. In this review, recent studies demonstrating developed yeast strains with lignocellulosic biomass hydrolysates are compared. For the pretreatment methods, we highlight hydrothermal pretreatments (dilute acid treatment and autohydrolysis), which are the most commonly used and effective methods for lignocellulosic biomass pretreatment. Rather than pretreatment conditions, the type of biomass most strongly influences the composition of the hydrolysates. Metabolic engineering strategies for yeast strain development, the choice of xylose-metabolic pathway, adaptive evolution, and strain background are highlighted as important factors affecting ethanol yield and productivity from lignocellulosic biomass hydrolysates. A comparison of the parameters from recent studies demonstrating lignocellulosic ethanol production provides useful information for future strain development.

Simultaneous fermentation of galacturonic acid and five-carbon sugars by engineered Saccharomyces cerevisiae.

Pectin-rich biomass has garnered attention as an alternative biomass source. However, some monomers derived from pectin-rich biomass, namely d-galacturonic acid, l-arabinose, and d-xylose, are not fermentable by industrial microorganisms such as Saccharomyces cerevisiae. The purpose of this study is to develop a S. cerevisiae strain capable of fermenting the pectin monomers. Expressions of eight heterologous genes and deletion of two endogenous genes, all of which were successfully completed by Cas9-based in vivo assembly and integration strategy, allowed the consumption of pectin monomers as sole carbon sources. To facilitate the consumption of galacturonic acid, which had the most limitations, the use of a co-substrate was tested using various sugars. As a result, we found that arabinose and xylose allowed simultaneous consumption of galacturonic acid. Based on intracellular metabolite profiling, it was concluded that the five-carbon sugars partially resolve the metabolic bottleneck of galacturonic acid.

Preoperative serum VEGF-C but not VEGF-A level is correlated with lateral neck metastasis in papillary thyroid carcinoma.

BACKGROUND: This study aimed to investigate the relationships between serum vascular endothelial growth factor (VEGF)-A or VEGF-C levels and lymph node metastasis (LNM) status in patients with papillary thyroid carcinoma (PTC). METHODS: The study enrolled 150 patients with pathologically proven PTC who underwent surgery: PTC without LNM, PTC with central neck metastasis, and PTC with lateral neck metastasis. RESULTS: Preoperative serum VEGF-A levels were 300.12 ± 80.80 pg/mL overall and were not correlated with the presence of LNM. Preoperative serum VEGF-C levels were 132.41 ± 48.48 pg/mL overall and were significantly correlated with the presence of LNM. Serum VEGF-C levels were further increased in patients with lateral neck metastasis and positively correlated with the number of metastatic LNs (rho = 0.252, P = 0.002). Serum VEGF-C, but not VEGF-A, was identified as a significant predictor of lateral neck metastasis. CONCLUSION: Serum VEGF-C might be a clinically relevant biomarker of lateral neck metastasis in patients with PTC.

HeLa E-Box Binding Protein, HEB, Inhibits Promoter Activity of the Lysophosphatidic Acid Receptor Gene Lpar1 in Neocortical Neuroblast Cells.

Lysophosphatidic acid (LPA) is an endogenous lysophospholipid with signaling properties outside of the cell and it signals through specific G protein-coupled receptors, known as LPA1-6. For one of its receptors, LPA1 (gene name Lpar1), details on the cis-acting elements for transcriptional control have not been defined. Using 5'RACE analysis, we report the identification of an alternative transcription start site of mouse Lpar1 and characterize approximately 3,500 bp of non-coding flanking sequence 5' of mouse Lpar1 gene for promoter activity. Transient transfection of cells derived from mouse neocortical neuroblasts with constructs from the 5' regions of mouse Lpar1 gene revealed the region between -248 to +225 serving as the basal promoter for Lpar1. This region also lacks a TATA box. For the region between -761 to -248, a negative regulatory element affected the basal expression of Lpar1. This region has three E-box sequences and mutagenesis of these E-boxes, followed by transient expression, demonstrated that two of the E-boxes act as negative modulators of Lpar1. One of these E-box sequences bound the HeLa E-box binding protein (HEB), and modulation of HEB levels in the transfected cells regulated the transcription of the reporter gene. Based on our data, we propose that HEB may be required for a proper regulation of Lpar1 expression in the embryonic neocortical neuroblast cells and to affect its function in both normal brain development and disease settings.

Tensin-3 Regulates Integrin-Mediated Proliferation and Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

Human palatine tonsils are potential tissue source of multipotent mesenchymal stem cells (MSCs). The proliferation rate of palatine tonsil-derived MSCs (TMSCs) is far higher than that of bone marrow-derived MSCs (BMSCs) or adipose tissue-derived MSCs (ADSCs). In our previous study, we had found through DNA microarray analysis that tensin-3 (TNS3), a type of focal adhesion protein, was more highly expressed in TMSCs than in both BMSCs and ADSCs. Here, the role of TNS3 in TMSCs and its relationship with integrin were investigated. TNS3 expression was significantly elevated in TMSCs than in other cell types. Cell growth curves revealed a significant decrease in the proliferation and migration of TMSCs treated with siRNA for TNS3 (siTNS3). siTNS3 treatment upregulated p16 and p21 levels and downregulated SOX2 expression and focal adhesion kinase, protein kinase B, and c-Jun N-terminal kinase phosphorylation. siTNS3 transfection significantly reduced adipogenic differentiation of TMSCs and slightly decreased osteogenic and chondrogenic differentiation. Furthermore, TNS3 inhibition reduced active integrin beta-1 (ITGß1) expression, while total ITGß1 expression was not affected. Inhibition of ITGß1 expression in TMSCs by siRNA showed similar results observed in TNS3 inhibition. Thus, TNS3 may play an important role in TMSC proliferation and differentiation by regulating active ITGß1 expression.

Auricular Cartilage Regeneration with Adipose-Derived Stem Cells in Rabbits.

Tissue engineering cell-based therapy using induced pluripotent stem cells and adipose-derived stem cells (ASCs) may be promising tools for therapeutic applications in tissue engineering because of their abundance, relatively easy harvesting, and high proliferation potential. The purpose of this study was to investigate whether ASCs can promote the auricular cartilage regeneration in the rabbit. In order to assess their differentiation ability, ASCs were injected into the midportion of a surgically created auricular cartilage defect in the rabbit. Control group was injected with normal saline. After 1 month, the resected auricles were examined histopathologically and immunohistochemically. The expression of collagen type II and transforming growth factor-ß1 (TGF-ß1) were analyzed by quantitative polymerase chain reaction. Histopathology showed islands of new cartilage formation at the site of the surgically induced defect in the ASC group. Furthermore, Masson's trichrome staining and immunohistochemistry for S-100 showed numerous positive chondroblasts. The expression of collagen type II and TGF-ß1 were significantly higher in the ASCs than in the control group. In conclusion, ASCs have regenerative effects on the auricular cartilage defect of the rabbit. These effects would be expected to contribute significantly to the regeneration of damaged cartilage tissue in vivo.

The art and science of selecting graduate students in the biomedical sciences: Performance in doctoral study of the foundational sciences.

The goal of this study was to investigate associations between admissions criteria and performance in Ph.D. programs at Boston University School of Medicine. The initial phase of this project examined student performance in the classroom component of a newly established curriculum named "Foundations in Biomedical Sciences (FiBS)". Quantitative measures including undergraduate grade point average (GPA), graduate record examination (GRE; a standardized, computer-based test) scores for the verbal (assessment of test takers' ability to analyze, evaluate, and synthesize information and concepts provided in writing) and quantitative (assessment of test takers' problem-solving ability) components of the examination, previous research experience, and competitiveness of previous research institution were used in the study. These criteria were compared with competencies in the program defined as students who pass the curriculum as well as students categorized as High Performers. These data indicated that there is a significant positive correlation between FiBS performance and undergraduate GPA, GRE scores, and competitiveness of undergraduate institution. No significant correlations were found between FiBS performance and research background. By taking a data-driven approach to examine admissions and performance, we hope to refine our admissions criteria to facilitate an unbiased approach to recruitment of students in the life sciences and to share our strategy to support similar goals at other institutions.

TSH-independent release of thyroid hormones through cold exposure in aging rats.

Thyroid function decreases and cold exposure response becomes impaired with increasing age. We investigated the age-related changes in thyroid structure and function and cold-induced changes in the thyroid activity of aging rats. Thirty-two male Sprague-Dawley rats were randomly divided into four groups (8 rats per group): young (7 months) and old (22 months) groups exposed to room temperature and cold stress. The active follicle ratio and serum free T3, T4 and TSH, and TSH receptor (TSHR) concentrations in the thyroid tissues of the rats from each group were compared. At room temperature, old rats had significantly lower active follicle ratio and free T3 and T4 concentrations than young rats. Furthermore, old rats displayed higher TSH level than young. Exposure to cold temperature led to significantly increased active colloid ratio and free T3 and T4 concentrations among old rats, but no significant differences were found among young rats. Additionally, no significant changes in the TSH and TSHR levels were observed after cold exposure in both young and old rats. Old rats have lower thyroid function than young rats under normal temperature. Aging rats are more susceptible to cold stress than young rats, and cold-induced thyroid activation occurs independently of TSH. We investigated the age-related changes in the thyroid structure and function and cold-induced changes in the thyroid activity of aging rats. Aging rats have structurally less active thyroid follicles and functionally lower thyroid hormone levels than young rats. Furthermore, old rats are more susceptible to cold stress than young rats, and cold-induced thyroid activation occurs independently of TSH.

Transition metal alloying effect on the phosphoric acid adsorption strength of Pt nanoparticles: an experimental and density functional theory study.

The effect of alloying with transition metals (Ni, Co, Fe) on the adsorption strength of phosphoric acid on Pt alloy surfaces was investigated using electrochemical analysis and first-principles calculations. Cyclic voltammograms of carbon-supported Pt3M/C (M = Ni, Co, and Fe) electrocatalysts in 0.1 M HClO4 with and without 0.01 M H3PO4 revealed that the phosphoric acid adsorption charge density near the onset potential on the nanoparticle surfaces was decreased by alloying with transition metals in the order Co, Fe, Ni. First-principles calculations based on density functional theory confirmed that the adsorption strength of phosphoric acid was weakened by alloying with transition metals, in the same order as that observed in the electrochemical analysis. The simulation suggested that the weaker phosphoric acid adsorption can be attributed to a lowered density of states near the Fermi level due to alloying with transition metals.