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Abstract Background The S. mutans LrgA/B holin-like proteins have been shown to affect biofilm formation and oxidative stress tolerance, and are regulated by oxygenation, glucose levels, and by the LytST two-component system. In this study, we sought to determine if LytST was involved in regulating lrgAB expression in response to glucose and oxygenation in S. mutans. Results Real-time PCR revealed that growth phase-dependent regulation of lrgAB expression in response to glucose metabolism is mediated by LytST under low-oxygen conditions. However, the effect of LytST on lrgAB expression was less pronounced when cells were grown with aeration. RNA expression profiles in the wild-type and lytS mutant strains were compared using microarrays in early exponential and late exponential phase cells. The expression of 40 and 136 genes in early-exponential and late exponential phase, respectively, was altered in the lytS mutant. Although expression of comYB, encoding a DNA binding-uptake protein, was substantially increased in the lytS mutant, this did not translate to an effect on competence. However, a lrgA mutant displayed a substantial decrease in transformation efficiency, suggestive of a previously-unknown link between LrgA and S. mutans competence development. Finally, increased expression of genes encoding antioxidant and DNA recombination/repair enzymes was observed in the lytS mutant, suggesting that the mutant may be subjected to increased oxidative stress during normal growth. Although the intracellular levels of reaction oxygen species (ROS) appeared similar between wild-type and lytS mutant strains after overnight growth, challenge of these strains with hydrogen peroxide (H2O2) resulted in increased intracellular ROS in the lytS mutant. Conclusions Overall, these results: (1) Reinforce the importance of LytST in governing lrgAB expression in response to glucose and oxygen, (2) Define a new role for LytST in global gene regulation and resistance to H2O2, and (3) Uncover a potential link between LrgAB and competence development in S. mutans.

Abstract Background The universal ribosomal protein S4 is essential for the initiation of small subunit ribosomal assembly and translational accuracy. Being part of the information processing machinery of the cell, the gene for S4 is generally thought of as being inherited vertically and has been used in concatenated gene phylogenies. Here we report the evolution of ribosomal protein S4 in relation to a broad sharing of zinc/non-zinc forms of the gene and study the scope of horizontal gene transfer (HGT) of S4 during bacterial evolution. Results In this study we present the complex evolutionary history of ribosomal protein S4 using 660 bacterial genomes from 16 major bacterial phyla. According to conserved characteristics in the sequences, S4 can be classified into C+ (zinc-binding) and C- (zinc-free) variants, with 26 genomes (mainly from the class Clostridia) containing genes for both. A maximum likelihood phylogenetic tree of the S4 sequences was incongruent with the standard bacterial phylogeny, indicating a departure from strict vertical inheritance. Further analysis using the genome content near the S4 genes, which are usually located in a conserved gene cluster, showed not only that HGT of the C- gene had occurred at various stages of bacterial evolution, but also that both the C- and C+ genes were present before the individual phyla diverged. To explain the latter, we theorize that a gene pool existed early in bacterial evolution from which bacteria could sample S4 gene variants, according to environmental conditions. The distribution of the C+/- variants for seven other zinc-binding ribosomal proteins in these 660 bacterial genomes is consistent with that seen for S4 and may shed light on the evolutionary pressures involved. Conclusion The complex history presented for "core" protein S4 suggests the existence of a gene pool before the emergence of bacterial lineages and reflects the pervasive nature of HGT in subsequent bacterial evolution. This has implications for both theoretical models of evolution and practical applications of phylogenetic reconstruction as well as the control of zinc economy in bacterial cells.

Abstract Background Since the publication of the first draft of the human genome in 2000, bioinformatic data have been accumulating at an overwhelming pace. Currently, more than 3 million sequences and 35 thousand structures of proteins and nucleic acids are available in public databases. Finding correlations in and between these data to answer critical research questions is extremely challenging. This problem needs to be approached from several directions: information science to organize and search the data; information visualization to assist in recognizing correlations; mathematics to formulate statistical inferences; and biology to analyze chemical and physical properties in terms of sequence and structure changes. Results Here we present MultiSeq, a unified bioinformatics analysis environment that allows one to organize, display, align and analyze both sequence and structure data for proteins and nucleic acids. While special emphasis is placed on analyzing the data within the framework of evolutionary biology, the environment is also flexible enough to accommodate other usage patterns. The evolutionary approach is supported by the use of predefined metadata, adherence to standard ontological mappings, and the ability for the user to adjust these classifications using an electronic notebook. MultiSeq contains a new algorithm to generate complete evolutionary profiles that represent the topology of the molecular phylogenetic tree of a homologous group of distantly related proteins. The method, based on the multidimensional QR factorization of multiple sequence and structure alignments, removes redundancy from the alignments and orders the protein sequences by increasing linear dependence, resulting in the identification of a minimal basis set of sequences that spans the evolutionary space of the homologous group of proteins. Conclusion MultiSeq is a major extension of the Multiple Alignment tool that is provided as part of VMD, a structural visualization program for analyzing molecular dynamics simulations. Both are freely distributed by the NIH Resource for Macromolecular Modeling and Bioinformatics and MultiSeq is included with VMD starting with version 1.8.5. The MultiSeq website has details on how to download and use the software: http://www.scs.uiuc.edu/~schulten/multiseq/

Skin cancer risk is increased by exposure to ultraviolet radiation (UVR). Because UVR exposure accumulates over time and lighter skin is more susceptible to UVR, age and skin tone are risk factors for skin cancer. However, measurements of somatic mutations in healthy-appearing skin have not been used to calculate skin cancer risk. In this study, we developed a noninvasive test that quantifies somatic mutations in healthy-appearing sun-exposed skin and applied it to a 1038-subject cohort. Somatic mutations were combined with other known skin cancer risk factors to train a model to calculate risk. The final model (DNA-Skin Cancer Assessment of Risk) was trained to predict personal history of skin cancer from age, family history, skin tone, and mutation count. The addition of mutation count significantly improved model performance (OR = 1.3, 95% confidence interval = 1.14-1.48; P = 5.3 × 10 -6 ) and made a more significant contribution than skin tone. Calculations of skin cancer risk matched the known United States population prevalence, indicating that DNA-Skin Cancer Assessment of Risk was well-calibrated. In conclusion, somatic mutations in healthy-appearing sun-exposed skin increase skin cancer risk, and mutations capture risk information that is not accounted for by other risk factors. Clinical utility is supported by the noninvasive nature of skin sample collection through adhesive patches., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In human cells and yeast, an intact "hydrophobic patch" substrate docking site is needed for mitotic cyclin centrosomal localization. A hydrophobic patch mutant (HPM) of the fission yeast mitotic cyclin Cdc13 cannot enter mitosis, but whether this is due to defective centrosomal localization or defective cyclin-substrate docking more widely is unknown. Here, we show that artificially restoring Cdc13-HPM centrosomal localization promotes mitotic entry and increases CDK (cyclin-dependent kinase) substrate phosphorylation at the centrosome and in the cytoplasm. We also show that the S-phase B-cyclin hydrophobic patch is required for centrosomal localization but not for S phase. We propose that the hydrophobic patch is essential for mitosis due to its requirement for the local concentration of cyclin-CDK with CDK substrates and regulators at the centrosome. Our findings emphasize the central importance of the centrosome as a hub coordinating cell-cycle control and explain why the cyclin hydrophobic patch is essential for mitosis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic highlighted the importance of vaccine innovation in public health. Hundreds of vaccines built on numerous technology platforms have been rapidly developed against SARS-CoV-2 since 2020. Like all vaccine platforms, an important bottleneck to viral-vectored vaccine development is manufacturing. Here, we describe a scalable manufacturing protocol for replication-competent SARS-CoV-2 Spike-pseudotyped vesicular stomatitis virus (S-VSV)-vectored vaccines using Vero cells grown on microcarriers in a stirred-tank bioreactor. Using Cytodex 1 microcarriers over 6 days of fed-batch culture, Vero cells grew to a density of 3.95 ± 0.42 ×10 6 cells/mL in 1-L stirred-tank bioreactors. Ancestral strain S-VSV reached a peak titer of 2.05 ± 0.58 ×10 8 plaque-forming units (PFUs)/mL at 3 days postinfection. When compared to growth in plate-based cultures, this was a 29-fold increase in virus production, meaning a 1-L bioreactor produces the same amount of virus as 1,284 plates of 15 cm. In addition, the omicron BA.1 S-VSV reached a peak titer of 5.58 ± 0.35 × 10 6 PFU/mL. Quality control testing showed plate- and bioreactor-produced S-VSV had similar particle-to-PFU ratios and elicited comparable levels of neutralizing antibodies in immunized hamsters. This method should enhance preclinical and clinical development of pseudotyped VSV-vectored vaccines in future pandemics., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Equine mesenchymal stromal cells (MSCs) have been found to be beneficial for the treatment of many ailments, including orthopedic injuries, due to their superior differentiation potential and immunomodulating properties. Cell therapies require large cell numbers, which are not efficiently generated using conventional static expansion methods. Expansion of equine cord blood-derived MSCs (eCB-MSCs) in bioreactors, using microcarriers as an attachment surface, has the potential to generate large numbers of cells with increased reproducibility and homogeneity compared with static T-flask expansion. This study investigated the development of an expansion process using Vertical-Wheel (VW) bioreactors, a single-use bioreactor technology that incorporates a wheel instead of an impeller. Initially, microcarriers were screened at small scale to assess eCB-MSC attachment and growth and then in bioreactors to assess cell expansion and harvesting. The effect of different donors, serial passaging, and batch versus fed batch were all examined in 0.1 L VW bioreactors. The use of VW bioreactors with an appropriate microcarrier was shown to be able to produce cell densities of up to 1E6 cells/mL, while maintaining cell phenotype and functionality, thus demonstrating great potential for the use of these bioreactors to produce large cell numbers for cell therapies., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Mesenchymal stromal cells (MSCs) are an ideal cell source for allogenic cell therapy due to their immunomodulatory and differentiation properties. Equine MSCs (eMSCs) have been found to be a promising treatment for equine joint injuries including meniscal injuries, cartilage degradation, and osteoarthritis. Although the use of eMSCs has shown efficacy in preliminary studies, challenges associated with biomanufacturing remain. To achieve the required cell numbers for clinical application, bioreactor-based processes are required. Initial studies have shown that eMSCs can be cultivated in microcarrier-based, stirred suspension bioreactor culture at the laboratory 0.1 L scale using a Vertical-Wheel ® (VW) bioreactor. However, investigations regarding scale up of these processes to the required biomanufacturing scales are required. This study investigated the scale-up of a equine cord blood MSC (eCB-MSC) bioprocess in VW bioreactors at three scales. This included scale-up from the 0.1-0.5 L bioreactor, scale-up from static culture to the 3 L computer-controlled bioreactor, and scale-up into the 3 L computer-controlled bioreactor using a mock clinical trial process. Results from the various scale-up experiments demonstrated similar cell expansion at the various tested scales. The 3 L computer-controlled system resulted in a final cell densities of 1.5 × 10 5 cells/cm 2 on average, achieving 1.5 × 10 9 harvested cells. Biological testing of the cells showed that cell phenotype and functionality were maintained after scale-up. These findings demonstrate the scalability of an eCB-MSC bioprocess using microcarriers in VW bioreactors to achieve clinically relevant cell numbers, a critical step to translate MSC treatments from research to clinical applications. This study also represents the first known published study expanding any cell type in the 3 L VW bioreactor., Competing Interests: Author TGK is the founder, CEO and a shareholder in eQcell. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Roberts, Abraham, Dang, Gysel, Mehrpouyan, Alizadeh, Koch and Kallos.)

Understanding the interaction between biomaterials and blood is critical in the design of novel biomaterials for use in biomedical applications. Depending on the application, biomaterials can be designed to promote hemostasis, slow or stop bleeding in an internal or external wound, or prevent thrombosis for use in permanent or temporary medical implants. Bacterial nanocellulose (BNC) is a natural, biocompatible biopolymer that has recently gained interest for its potential use in blood-contacting biomedical applications (e.g., artificial vascular grafts), due to its high porosity, shapeability, and tissue-like properties. To promote hemostasis, BNC has been modified through oxidation or functionalization with various peptides, proteins, polysaccharides, and minerals that interact with the coagulation cascade. For use as an artificial vascular graft or to promote vascularization, BNC has been extensively researched, with studies investigating different modification techniques to enhance endothelialization such as functionalizing with adhesion peptides or extracellular matrix (ECM) proteins as well as tuning the structural properties of BNC such as surface roughness, pore size, and fiber size. While BNC inherently exhibits comparable mechanical characteristics to endogenous blood vessels, these mechanical properties can be enhanced through chemical functionalization or through altering the fabrication method. In this review, we provide a comprehensive overview of the various modification techniques that have been implemented to enhance the suitability of BNC for blood-contacting biomedical applications and different testing techniques that can be applied to evaluate their performance. Initially, we focused on the modification techniques that have been applied to BNC for hemostatic applications. Subsequently, we outline the different methods used for the production of BNC-based artificial vascular grafts and to generate vasculature in tissue engineered constructs. This sequential organization enables a clear and concise discussion of the various modifications of BNC for different blood-contacting biomedical applications and highlights the diverse and versatile nature of BNC as a natural biomaterial., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Mesenchymal stromal cells (MSC) are a promising platform for regenerative medicine applications because of their multilineage differentiation abilities and ease of collection, isolation, and growth ex vivo. To meet the demand for clinical applications, large-scale manufacturing will be required using three-dimensional culture platforms in vessels such as stirred suspension bioreactors. As MSCs are an adherent cell type, microcarriers are added to the culture to increase the available surface area for attachment and growth. Although extensive research has been performed on efficiently culturing MSCs using microcarriers, challenges persist in downstream processing, including harvesting, filtration, and volume reduction, which all play a critical role in the translation of cell therapies to the clinic. The objective of this review is to assess the current state of downstream technologies available for microcarrier-based MSC cultures. This includes a review of current research within the three stages: harvesting, filtration, and volume reduction. Using this information, a downstream process for MSCs is proposed, which can be applied to a wide range of applications., (© 2022 Wiley Periodicals LLC.)

Micronutrients applied as nanoparticles of metal oxides have shown efficacy in vegetable and other crops for improving yield and reducing Fusarium diseases, but their role in ornamental crop management has not been investigated. In 2017, 2018, and 2020, nanoparticles of CuO, Mn 2 O 3 , or ZnO were foliarly applied at 500 μg/mL (0.6 mg/plant) to chrysanthemum transplants and planted in potting soil noninfested or infested with Fusarium oxysporum f. sp. chrysanthemi . An untreated control and a commercial fungicide, Fludioxonil, was also included. Chrysanthemums treated with nanoscale CuO had a 55, 30, and 32% reduction in disease severity ratings compared to untreated plants in 2017, 2018, and 2020, respectively. Specifically, the average dry biomass for the three years was reduced 22% by disease, but treatment with nanoscale CuO led to a 23% increase when compared to controls. Similar trends with plant height were observed. Horticultural quality was improved 28% with nano CuO and was equal to the fungicide. Nanoscale Mn 2 O 3 and the fungicide did not consistently reduce disease ratings or increase dry biomass each year. Nanoscale ZnO was ineffective. Nanoscale CuO-treated plants had 24 to 48% more Cu/g tissue than controls ( P < 0.001). These findings agree with past reports on food crops where single applications of nanoscale CuO improved plant health, growth, and yield and could offer significant impacts for managing plant diseases on ornamentals.

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19., (© 2021 The Author(s).)

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir., (© 2021 The Author(s).)

Human induced pluripotent stem cells (hiPSCs) have generated a great deal of attention owing to their capacity for self-renewal and differentiation into the three germ layers of the body. Their discovery has facilitated a new era in biomedicine for understanding human development, drug screening, disease modeling, and cell therapy while reducing ethical issues and risks of immune rejection associated with traditional embryonic stem cells. Bioreactor-based processes have been the method of choice for the efficient expansion and differentiation of stem cells in controlled environments. Current protocols for the expansion of hiPSCs use horizontal impeller, paddle, or rocking wave mixing method bioreactors which require large static cell culture starting populations and achieve only moderate cell fold increases. This study focused on optimizing inoculation, agitation, oxygen, and nutrient availability for the culture of hiPSCs as aggregates in single-use, low-shear, vertical-wheel bioreactors. Under optimized conditions, we achieved an expansion of more than 30-fold in 6 days using a small starting population of cells and minimal media resources throughout. Importantly, we showed that that this optimized bioreactor expansion protocol could be replicated over four serial passages resulting in a cumulative cell expansion of 1.06E6-fold in 28 days. Cells from the final day of the serial passage were of high quality, maintaining a normal karyotype, pluripotent marker staining, and the ability to form teratomas in vivo. These findings demonstrate that a vertical-wheel bioreactor-based bioprocess can provide optimal conditions for efficient, rapid generation of high-quality hiPSCs to meet the demands for clinical manufacturing of therapeutic cell products., (© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Escherichia coli carrying prophage with genes that encode for Shiga toxins are categorized as Shiga toxin-producing E. coli (STEC) pathotype. Illnesses caused by STEC in humans, which are often foodborne, range from mild to bloody diarrhea with life-threatening complications of renal failure and hemolytic uremic syndrome and even death, particularly in children. As many as 158 of the total 187 serogroups of E. coli are known to carry Shiga toxin genes, which makes STEC a major pathotype of E. coli . Seven STEC serogroups, called top-7, which include O26, O45, O103, O111, O121, O145, and O157, are responsible for the majority of the STEC-associated human illnesses. The STEC serogroups, other than the top-7, called "non-top-7" have also been associated with human illnesses, more often as sporadic infections. Ruminants, particularly cattle, are principal reservoirs of STEC and harbor the organisms in the hindgut and shed in the feces, which serves as a major source of food and water contaminations. A number of studies have reported on the fecal prevalence of top-7 STEC in cattle feces. However, there is paucity of data on the prevalence of non-top-7 STEC serogroups in cattle feces, generally because of lack of validated detection methods. The objective of our study was to develop and validate 14 sets of multiplex PCR (mPCR) assays targeting serogroup-specific genes to detect 137 non-top-7 STEC serogroups previously reported to be present in cattle feces. Each assay included 7-12 serogroups and primers were designed to amplify the target genes with distinct amplicon sizes for each serogroup that can be readily identified within each assay. The assays were validated with 460 strains of known serogroups. The multiplex PCR assays designed in our study can be readily adapted by most laboratories for rapid identification of strains belonging to the non-top-7 STEC serogroups associated with cattle., (Copyright © 2020 Ludwig, Shi, Shridhar, Roberts, DebRoy, Phebus, Bai and Nagaraja.)

Embryonic stem cells (ESCs) have almost unlimited proliferation capacity in vitro and can retain the ability to contribute to all cell lineages, making them an ideal platform material for cell-based therapies. ESCs are traditionally cultured in static flasks on a feeder layer of murine embryonic fibroblast cells. Although sufficient to generate cells for research purposes, this approach is impractical to achieve large quantities for clinical applications. In this study, we have developed protocols that address a variety of challenges that currently bottleneck clinical translation of ESCs expanded in stirred suspension bioreactors. We demonstrated that mouse ESCs (mESCs) cryopreserved in the absence of feeder cells could be thawed directly into stirred suspension bioreactors at extremely low inoculation densities (100 cells/ml). These cells sustained proliferative capacity through multiple passages and various reactor sizes and geometries, producing clinically relevant numbers (10 9 cells) and maintaining pluripotency phenotypic and functional properties. Passages were completed in stirred suspension bioreactors of increasing scale, under defined batch conditions which greatly improved resource efficiency. Output mESCs were analyzed for pluripotency marker expression (SSEA-1, SOX-2, and Nanog) through flow cytometry, and spontaneous differentiation and teratoma analysis was used to demonstrate functional maintenance of pluripotency., (© 2020 Wiley Periodicals, Inc.)

The cyclin-dependent kinases (CDKs) are the major cell-cycle regulators that phosphorylate hundreds of substrates, controlling the onset of S phase and M phase [1-3]. However, the patterns of substrate phosphorylation increase are not uniform, as different substrates become phosphorylated at different times as cells proceed through the cell cycle [4, 5]. In fission yeast, the correct ordering of CDK substrate phosphorylation can be established by the activity of a single mitotic cyclin-CDK complex [6, 7]. Here, we investigate the substrate-docking region, the hydrophobic patch, on the fission yeast mitotic cyclin Cdc13 as a potential mechanism to correctly order CDK substrate phosphorylation. We show that the hydrophobic patch targets Cdc13 to the yeast centrosome equivalent, the spindle pole body (SPB), and disruption of this motif prevents both centrosomal localization of Cdc13 and the onset of mitosis but does not prevent S phase. CDK phosphorylation in mitosis is compromised for approximately half of all mitotic CDK substrates, with substrates affected generally being those that require the highest levels of CDK activity to become phosphorylated and those that are located at the SPB. Our experiments suggest that the hydrophobic patch of mitotic cyclins contributes to CDK substrate selection by directing the localization of Cdc13-CDK to centrosomes and that this localization of CDK contributes to the CDK substrate phosphorylation necessary to ensure proper entry into mitosis. Finally, we show that mutation of the hydrophobic patch prevents cyclin B1 localization to centrosomes in human cells, suggesting that this mechanism of cyclin-CDK spatial regulation may be conserved across eukaryotes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Computational fluid dynamics (CFD) modeling can be applied to understand hydrodynamics in stirred suspension bioreactors, which can in turn affect cell viability, proliferation, pluripotency and differentiation. In this study, we developed a CFD model to determine the effects of average shear rates and turbulent eddies on the formation and growth of murine embryonic stem cell aggregates. We found a correlation between average eddy size and aggregate size, which depended on bioreactor agitation rates. By relating these computational and biological variables, CFD modeling can predict optimal agitation rates to grow embryonic stem cell aggregates in stirred suspension bioreactors. To examine the effect of hydrodynamics on pluripotency, mESCs cultured in bioreactors under various agitation rates were tested for SSEA-1, Sox-2, and Nanog expression. Cells maintained a minimum of 95% positive expression with no change in the intensity distribution pattern between the different bioreactor conditions. This indicates that the average level of pluripotency marker expression is independent of changes in the hydrodynamic profile and resulting aggregate size distribution. The findings here can be further extended to other cell types that grow as aggregates in stirred suspension bioreactors and offer important insights necessary to realize cell therapies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Equine mesenchymal stromal cells (MSCs) are increasingly investigated for their clinical therapeutic utility. Such cell-based treatments can require cell numbers in the millions or billions, with conventional expansion methods using static T-flasks typically inefficient in achieving these cell numbers. Equine cord blood-derived MSCs (eCB-MSCs), are promising cell candidates owing to their capacity for chondrogenic differentiation and immunomodulation. Expansion of eCB-MSCs in stirred suspension bioreactors with microcarriers as an attachment surface has the potential to generate clinically relevant numbers of cells while decreasing cost, time and labour requirements and increasing reproducibility and yield when compared to static expansion. As eCB-MSCs have not yet been expanded in stirred suspension bioreactors, a robust protocol was required to expand these cells using this method. This study outlines the development of an expansion bioprocess, detailing the inoculation phase, expansion phase, and harvesting phase, followed by phenotypic and trilineage differentiation characterization of two eCB-MSC donors. The process achieved maximum cell densities up to 75,000 cells/cm 2 corresponding to 40 million cells in a 100 mL bioreactor, with a harvesting efficiency of up to 80%, corresponding to a yield of 32 million cells from a 100 mL bioreactor. When compared to cells grown in static T-flasks, bioreactor-expanded eCB-MSC cultures did not change in surface marker expression or trilineage differentiation capacity. This indicates that the bioreactor expansion process yields large quantities of eCB-MSCs with similar characteristics to conventionally grown eCB-MSCs., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The U.S. Food and Drug Administration Escherichia coli Identification (FDA-ECID) microarray provides rapid molecular characterization of E. coli. The effectiveness of the FDA-ECID for characterizing Shiga toxin-producing E. coli (STEC) was evaluated by three federal laboratories and one reference laboratory with a panel of 54 reference E. coli strains from the External Quality Assurance program. Strains were tested by FDA-ECID for molecular serotyping (O and H antigens), Shiga toxin subtyping, and the presence of the ehxA and eae genes for enterohemolysin and intimin, respectively. The FDA-ECID O typing was 96% reproducible among the four laboratories and 94% accurate compared with the reference External Quality Assurance data. Discrepancies were due to the absence of O41 target loci on the array and to two pairs of O types with identical target sequences. H typing was 96% reproducible and 100% accurate, with discrepancies due to two strains from one laboratory that were identified as mixed by FDA-ECID. Shiga toxin (Stx) type 1 subtyping was 100% reproducible and accurate, and Stx2 subtyping was 100% reproducible but only 64% accurate. FDA-ECID identified most Stx2 subtypes but had difficulty distinguishing among stx 2a , stx 2c , and stx 2d genes because of close similarities of these sequences. FDA-ECID was 100% effective for detecting ehxA and eae and accurately subtyped the eae alleles. This interlaboratory study revealed that FDA-ECID for STEC characterization was highly reproducible for molecular serotyping, stx and eae subtyping, and ehxA detection. However, the array was less useful for distinguishing among the highly homologous O antigen genes and the stx 2a , stx 2c , and stx 2d subtypes.

Background: In previous reports, the antibacterial properties of certain tetrazole derivatives have been described. We have previously reported the antibacterial properties of aryl 1Htetrazole compounds., Objective: To study the antibacterial activity of 5-substituted heteroaryl, vinyl, benzyl, and alkyl 1H-tetrazole derivatives., Methods: The antibacterial properties of heteroaryl, vinyl, benzylic, and aliphatic tetrazole derivatives were investigated against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The activity was assessed by determining the minimum inhibitory concentration of these tetrazole derivatives and comparing them to the known antibiotics amoxicillin, trimethoprim and sulfamethoxazole., Results: The tetrazole compounds were prepared utilizing cerium(III) chloride heptahydrate catalysis at 160 °C for 1-4 h in a microwave reactor using an aqueous solvent mixture. The most active derivatives exhibited minimum inhibitory concentration values between 125-250 µg/mL against Escherichia coli. More importantly, these compounds were considerably more active when used in combination with trimethoprim and a significant synergistic effect was observed (MIC = 0.98-7.81 µg/mL) against E. coli and S. aureus., Conclusion: The tetrazole derivatives were synthesized in high yield and short reaction times in water. Several of the tetrazole compounds showed a significant synergistic antibacterial effect when used with trimethoprim., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The reduction of synovial tissue macrophages is a reliable biomarker for clinical improvement in patients with rheumatoid arthritis (RA), and macrophages are reduced in synovial tissue shortly after initiation of TNF inhibitors. The mechanism for this initial response is unclear. These studies were performed to identify the mechanisms responsible for the initial reduction of macrophages following TNF inhibition, positing that efflux to draining lymph nodes was involved. RA synovial tissue and synovial fluid macrophages expressed CCR7, which was increased in control macrophages following incubation with TNF-α. Human TNF transgenic (hTNF-Tg) mice were treated with infliximab after development of arthritis. Ankles were harvested and examined by histology, immunohistochemistry, quantitative RT-PCR, ELISA, and flow cytometry. hTNF-Tg mice treated with infliximab demonstrated significant clinical and histologic improvement 3 d after the initiation of therapy, at which time Ly6C + macrophages were significantly reduced in the ankles. However, no evidence was identified to support a role of macrophage efflux to draining lymph nodes following treatment with infliximab. In contrast, apoptosis of Ly6C + macrophages in the ankles and popliteal lymph nodes, decreased migration of monocytes into the ankles, and a reduction of CCL2 were identified following the initiation of infliximab. These observations demonstrate that Ly6C + macrophage apoptosis and decreased ingress of circulating monocytes into the joint are responsible for the initial reduction of macrophages following infliximab treatment in hTNF-Tg mice., (Copyright © 2017 by The American Association of Immunologists, Inc.)

The vast unexplored virus biodiversity makes the application of virus templates to nanomaterial synthesis especially promising. Here, a new biotemplate, Barley stripe mosaic virus (BSMV) was successfully used to synthesize organic-metal nanorods of similarly high quality to those produced with Tobacco mosaic virus (TMV). The mineralization behavior was characterized in terms of the reduction and adsorption of precursor and nanocrystal formation processes. The BSMV surface-mediated reduction of Pd (2+) proceeded via first-order kinetics in both Pd (2+) and BSMV. The adsorption equilibrium relationship of PdCl 3 H 2 O - on the BSMV surface was described by a multistep Langmuir isotherm suggesting alternative adsorbate-adsorbent interactions when compared to those on TMV. It was deduced that the first local isotherm is governed by electrostatically driven adsorption, which is then followed by sorption driven by covalent affinity of metal precursor molecules for amino acid residues. Furthermore, the total adsorption capacity of palladium species on BSMV is more than double of that on TMV. Finally, study of the BSMV-Pd particles by combining USAXS and SAXS enabled the characterization of all length scales in the synthesized nanomaterials. Results confirm the presence of core-shell cylindrical particles with 1-2 nm grains. The nanorods were uniform and monodisperse, with controllable diameters and therefore, of similar quality to those synthesized with TMV. Overall, BSMV has been confirmed as a viable alternate biotemplate with unique biomineralization behavior. With these results, the biotemplate toolbox has been expanded for the synthesis of new materials and comparative study of biomineralization processes.

Background: Tetrazole derivatives such as 1-substituted dinitrobenzyl tetrazoles and their oxa and selanyl analogs have previously been studied against drug-susceptible and multidrug-resistant mycobacteria. In addition, other tetrazole derivatives have been shown to inhibit CTX-M class A b-lactamases., Objective: To study the antibacterial activity of 5-substituted aryl 1H-tetrazole derivatives., Methods: The antibacterial activity of several known 5-substituted aryl 1H-tetrazole derivatives was evaluated against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The activity was assessed by determining the minimum inhibitory concentration of these tetrazole derivatives and comparing them to the known antibiotics amoxicillin, trimethoprim and sulfamethoxazole., Results: Some derivatives showed significant antibacterial activity with the most active derivatives exhibiting a minimum inhibitory concentration (MIC) of 125-250 mg/mL against Staphylococcus aureus and Escherichia coli. Using some of these tetrazole compounds in combination with trimethoprim led to a synergistic effect that gave MIC values ranging from 0.24-1.95 mg/mL against Escherichia coli and 3.91-31.3 mg/mL against Staphylococcus aureus. The tetrazole derivatives were prepared in an isopropanol/water mixture using microwave heating at 160 oC for 1 h. The cycloaddition between organonitriles and sodium azide was catalyzed by indium chloride., Conclusion: This study shows a significant synergistic effect between the tetrazole compounds tested and trimethoprim which could be used to potentially develop new antibacterial agents.

There is a lack of fundamental information about the molecular processes governing biomineralization of inorganic materials to produce nanostructures on biological templates. This information is essential for the directed synthesis of high quality nanomaterials via biotemplating. We characterized palladium (Pd) mineralization via the individual adsorption, reduction, and nanocrystal growth processes, which simultaneously occur during the hydrothermal synthesis on the Tobacco mosaic virus (TMV). The adsorption of precursor and reduction of palladium were decoupled through UV-vis Spectroscopy and in situ X-ray absorption spectroscopy studies. The role of additional cysteine (Cys) residues, ionic strength, and coating density on the fundamental parameters describing these processes were quantitatively evaluated. Primary nanocrystal growth and structural orientation of Pd nanoparticles was characterized using in situ small angle X-ray scattering. The adsorption, reduction of Pd species, and nanocrystal sizes were significantly changed on addition of Cys residues to the amino terminus of the TMV coat protein. Reduction of Pd on an already coated virion was dependent on the Pd surface area, and was hindered by the presence of residual salt. Furthermore, trends in Pd adsorption intensity and capacity suggested that chloride ions affected the adsorption equilibrium. Application of this fundamental approach with further optimization of parameters dictating biomineralization would facilitate directed synthesis and scale up of bioinorganic systems., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Shiga toxin-producing Escherichia coli O157:H7 (STEC) cause food-borne illness that may be fatal. STEC strains enumerate two types of potent Shiga toxins (Stx1 and Stx2) that are responsible for causing diseases. It is important to detect the E. coli O157 and Shiga toxins in food to prevent outbreak of diseases. We describe the development of two multi-analyte antibody-based lateral flow immunoassays (LFIA); one for the detection of Stx1 and Stx2 and one for the detection of E. coli O157 that may be used simultaneously to detect pathogenic E. coli O157:H7. The LFIA strips were developed by conjugating nano colloidal gold particles with monoclonal antibodies against Stx1 and Stx2 and anti-lipid A antibodies to capture Shiga toxins and O157 antigen, respectively. Our results indicate that the LFIA for Stx is highly specific and detected Stx1 and Stx2 within three hours of induction of STEC with ciprofloxacin at 37 °C. The limit of detection for E. coli O157 LFIA was found to be 10⁵ CFU/mL in ground beef spiked with the pathogen. The LFIAs are rapid, accurate and easy to use and do not require sophisticated equipment or trained personnel. Following the assay, colored bands on the membrane develop for end-point detection. The LFIAs may be used for screening STEC in food and the environment.

Escherichia coli strains are classified based on O-antigens that are components of the lipopolysaccharide (LPS) in the cell envelope. O-antigens are important virulence factors, targets of both the innate and adaptive immune system, and play a role in host-pathogen interactions. Because they are highly immunogenic and display antigenic specificity unique for each strain, O-antigens are the biomarkers for designating O-types. Immunologically, 185 O-serogroups and 11 OX-groups exist for classification. Conventional serotyping for O-typing entails agglutination reactions between the O-antigen and antisera generated against each O-group. The procedure is labor intensive, not always accurate, and exhibits equivocal results. In this report, we present the sequences of 71 O-antigen gene clusters (O-AGC) and a comparison of all 196 O- and OX-groups. Many of the designated O-types, applied for classification over several decades, exhibited similar nucleotide sequences of the O-AGCs and cross-reacted serologically. Some O-AGCs carried insertion sequences and others had only a few nucleotide differences between them. Thus, based on these findings, it is proposed that several of the E. coli O-groups may be merged. Knowledge of the O-AGC sequences facilitates the development of molecular diagnostic platforms that are rapid, accurate, and reliable that can replace conventional serotyping. Additionally, with the scientific knowledge presented, new frontiers in the discovery of biomarkers, understanding the roles of O-antigens in the innate and adaptive immune system and pathogenesis, the development of glycoconjugate vaccines, and other investigations, can be explored.

Background and Purpose: There is a paucity of literature that supports the Consortium of Multiple Sclerosis Centers guideline that proton density MR imaging is a core spinal cord sequence. We hypothesized that proton density fast spin-echo imaging is superior to T2 fast spin-echo MR imaging for the detection of cervical cord MS lesions. This study compared the detection rate and conspicuity of cervical cord MS lesions on sagittal 1.5T proton density fast spin-echo and T2 fast spin-echo MR imaging., Materials and Methods: One hundred consecutive patients with MS imaged with 1.5T sagittal proton density fast spin-echo and T2 fast spin-echo cervical cord MR imaging between September 2012 and October 2013 were retrospectively included. The number of MS lesions detected on each sequence was recorded; conspicuity was assessed quantitatively with the lesion-to-cord contrast ratio and lesion-contrast-to-noise ratio. Statistical analysis was performed by using the Wilcoxon signed rank test., Results: Seventy-eight patients had MS cord lesions detected. Proton density fast spin-echo imaging detected a greater number of lesions (n = 181) compared with T2 fast spin-echo imaging (n = 137, P < .001). Fifteen patients (19%) with abnormal findings on proton density fast spin-echo imaging had normal findings on T2 fast spin-echo imaging; no patient with abnormal T2 fast spin-echo imaging findings had normal proton density fast spin-echo imaging findings. Although proton density fast spin-echo and T2 fast spin-echo imaging had similar lesion-to-cord contrast ratios (proton density fast spin-echo, 0.32 ± 0.01, versus T2 fast spin-echo, 0.33 ± 0.01; P = .43), proton density fast spin-echo had greater lesion-contrast-to-noise ratio (proton density fast spin-echo, 82 ± 3.0, versus T2 fast spin-echo, 64 ± 2.6; P < .001)., Conclusions: Proton density fast spin-echo imaging is superior to T2 fast spin-echo MR imaging for the detection of cervical cord MS lesions. Proton density fast spin-echo detects cord lesions in patients in whom T2 fast spin-echo findings appear normal. This study forms the evidentiary base for the current Consortium of Multiple Sclerosis Centers guideline that proton density imaging is a core spinal cord sequence., (© 2016 by American Journal of Neuroradiology.)

The current study describes isolation of Extraintestinal Pathogenic Escherichia coli (ExPEC) from a juvenile male cat that died after being rescued from an animal hoarding incident. Grossly, there was evidence of pneumonia and renal abscessation. Histologically, there was diffuse interstitial pneumonia with necrosis and necrotizing and suppurative nephritis with colonies of coccobacilli. Within the lung, kidney, and mesentery there was necrotizing and suppurative vasculitis with thrombosis and coccobacilli. E. coli strain belonging to serotype O6:H1 that carried many of the virulence genes associated with ExPEC was isolated from the lung and kidney. The cat was part of a community of approximately 60 cats that lived in a house in a residential neighborhood, in which multiple cats had died. The case was of major significance to public health, as first responders, animal health professionals, and other community members were likely exposed to ExPEC, which is known to have zoonotic potential. It is important that pet owners, animal health and public health professionals, and first responders be made aware of the potential for zoonotic diseases., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Prophages make up 12% of the enterohemorrhagic Escherichia coli genome and play prominent roles in the evolution and virulence of this food-borne pathogen. Acquisition and loss of and rearrangements within prophage regions are the primary causes of differences in pulsed-field gel electrophoresis (PFGE) patterns among strains of E. coli O157:H7. Sp11 and Sp12 are two tandemly integrated and putatively defective prophages carried by E. coli O157:H7 strain Sakai. In this study, we identified 3 classes of deletions that occur within the Sp11-Sp12 region, at a frequency of ca. 7.74 × 10(-4). One deletion resulted in a precise excision of Sp11, and the other two spanned the junction of Sp11 and Sp12. All deletions resulted in shifts in the XbaI fragment pattern observed by PFGE. We sequenced the inducible prophage pool of Sakai but did not identify any mature phage particles corresponding to either Sp11 or Sp12. Deletions containing pchB and psrC, which are Sp11-carried genes encoding proteins known or suspected to regulate type III secretion, did not affect the secretion levels of the EspA or EspB effector. Alignment of the Sp11-Sp12 DNA sequence with its corresponding regions in other E. coli O157:H7 and O55:H7 strains suggested that homologous recombination rather than integrase-mediated excision is the mechanism behind these deletions. Therefore, this study provides a mechanism behind the previously observed genetic instability of this genomic region of E. coli O157:H7.

The objective of the current study was to determine the incidence of contamination by the top 7 Shiga toxin-producing Escherichia coli (STEC) O-groups, responsible for the majority of E. coli infections in human beings, in retail meat from different animal species. Samples from ground beef (n = 51), ground pork (n = 16), ground chicken (n = 16), and game meat (deer, wild boar, bison, and rabbit; n = 55) were collected from retail vendors for the detection of 7 STEC O-groups (O26, O45, O103, O111, O121, O145, and O157). Meat samples were tested by using a multiplex polymerase chain reaction assay targeting the wzx gene of O antigen gene clusters of the 7 STEC O-groups. The positive samples were further tested for Shiga toxin genes (stx1 and stx2). Out of a total of 83 ground beef, pork, and chicken samples, 17 (20%) carried O121, 9 (10%) carried O45, 8 (9%) carried O157, 3 (3%) carried O103, and 1 (1%) carried O145. None of the samples were positive for O26, O111, or the stx gene. All 3 white-tailed deer samples (100%) were positive for O45, O103, or both, 2 (10%) out of 20 red deer samples exhibited the presence of O103, and all 3 bison samples were contaminated with either O121, O145, or O157. One sample from ground deer, contaminated with E. coli O45, carried the stx1 gene. This preliminary investigation illustrates the importance of microbiological testing of pathogens in meat products, as well as the recognized need for increased surveillance and research on foodborne pathogens.

Adenosine deaminase (AD), a purine salvage enzyme, exists as AD isoform 1 (AD1) and AD isoform 2 (AD2). Plasma AD has been advocated for the screening and monitoring of cancer, as AD2 activity is increased in conditions associated with tumour growth. Plasma AD2 was measured before and seven to 10 days after the first dose of chemotherapy in patients with different tumours. A 'tumour regression score' was assessed independently based on radiological changes seen in the tumour following completion of chemotherapy. Changes in plasma AD2 were then compared with the tumour regression score. Following first-dose chemotherapy, plasma AD2 decreased on average from 22.7 +/- 10.5 U/L to 15.0 +/- 4.6 U/L. The percentage decrease in plasma AD2 correlated with the tumour regression score (r=0.5, P=0.028). These data suggest plasma AD2 may have a role in determining tumour response to treatment.

Objective: Race/ethnic differences in carotid arterial function and structure exist among those with cerebrovascular disease, but whether differences persist among healthy populations is unknown. Our objective was to investigate differences in carotid artery diameter and stiffness between race/ethnic groups, and examine whether these race/ethnic differences were age-dependent., Methods: Carotid diameters were assessed by B-mode ultrasound among 1536 participants from the Northern Manhattan Study (NOMAS), and carotid stiffness metrics were calculated. We used multivariable linear regression models to determine the relationship between race/ethnicity and both carotid arterial stiffness and carotid diastolic diameter., Results: Mean participant age was 70 ± 9 years (Hispanics = 68 ± 8, blacks = 72 ± 9, and whites = 74 ± 9, p < 0.0001). Mean DDIAM was 6.2 ± 1.0mm (Hispanics = 6.2 ± 0.9 mm, blacks = 6.3 ± 1.0 mm, and whites = 6.3 ± 1.0 mm, p < 0.005) and mean STIFF was 8.7 ± 6.3 (Hispanics = 8.5 ± 5.7, blacks = 9.2 ± 6.2 and whites = 8.9 ± 6.9, p < 0.02). In a model that adjusted for sociodemographics and vascular risk factors including hypertension, diabetes, dislipidemia, renal function, physical acticity and a history of known coronary artery diseases; age was positively associated with greater DDIAM in Hispanics (p < 0.0001) but not among blacks or whites. Older age was associated with greater stiffness among Hispanics (p < 0.0001) and blacks (p < 0.003), but not among whites., Conclusions: We found race/ethnic differences in the association between age and arterial stiffness and diameter, including age-dependent arterial dilation observed in Hispanics that was not observed among blacks or whites., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Unlike established cell lines used in the biotechnology industry, primary cells used in tissue engineering require culture media to be supplemented with serum. The most common serum is fetal bovine serum (FBS); however, FBS is expensive, negatively affecting process economics. Less-costly alternative sera are commercially available, but their efficacy has not been documented. Therefore, bovine calf serum (BCS), bovine growth serum (BGS), and newborn calf serum (NCS) were compared with FBS. Porcine aortic valve interstitial cells (VICs) were cultured as 2-dimensional (2-D) monolayers or as 3-dimensional (3-D) collagen gels using medium supplemented with 10% FBS, BGS, BCS, or NCS. No significant difference was seen in cellular activity between VICs cultured in BCS and those cultured in FBS in 2-D cultures, whereas cells cultured in BGS and NCS had significantly lower specific growth rates coupled with markedly higher metabolic activity than cells cultured in FBS. No statistically significant differences were seen in cellular activity between any of the sera when cells were cultured in 3-D constructs. In conclusion, BCS is a suitable alternative to FBS for the 2-D and 3-D culture of VICs, which may be used to develop a tissue-engineered valve.

We report our experience with 7 consecutive patients with Parkinson's disease (PD) who received rimantadine (the alpha-methyl derivative of amantadine) in substitution of amantadine due to peripheral side effects (lower limb edema, livedo reticularis). Mean age was 67.3 +/- 5.9 years, the mean disease duration was 13 +/- 6.3 years, and mean Hoehn and Yahr stage was 2.2 +/- 0.4. A total of 3 patients experienced marked improvement of edema, and 1 patient experienced marked improvement of livedo reticularis. Only 1 of the 7 patients reported significant loss of motor benefit when amantadine was replaced with rimantadine. Our results demonstrate that rimantadine may be considered as an alternative to amantadine in patients experiencing amantadine-induced peripheral side effects., (Copyright 2005 Movement Disorder Society.)

Background: Purine nucleoside phosphorylase (PNP) is the purine salvage enzyme that converts guanosine to guanine and inosine to hypoxanthine., Methods: 279 samples from patients with differing cancers were collected during treatment at both pre- and post-dose stages for plasma PNP activity and compared with a normal population., Results: Normal plasma PNP activity was found to be 3.2+/-1.4 U/l (n=55) as compared with the cancer patients (pre-dose 12.3+/-7.4 U/l [n=215] and post-dose 11.2+/-5.9 U/l [n=64]). Levels of plasma PNP did not differ greatly between the different cancer types but were on average four times greater than that found in the reference population., (Copyright 2004 Elsevier B.V.)

Plasma guanine deaminase (guanase; GD) is well established as an indicator of hepatocellular disease, recently being applied in the detection of hepatitis C in donor blood and in the diagnosis of hepatoma. No totally efficient, simple method for the estimation of plasma GD activity is routine since both guanine and 8-azaguanine, the substrates of the enzyme, are scarcely soluble in water. This difficulty in preparing stable substrates of sufficient concentration has resulted in methods that are both troublesome and inaccurate. Here we describe the development of new colorimetric and high-performance liquid chromatography (HPLC) methods utilizing guanosine as a "prosubstrate." After an initial breakdown of the guanosine to guanine using purine nucleoside phosphorylase, the ammonia formed as a result of the breakdown of the guanine by GD was estimated colorimetrically by the Berthelot reaction. As an alternative or a complementary assay, the xanthine also formed was measured using an isocratic HPLC method. These methods are suitable for routine assays for measuring plasma GD over a wide range of activities.

Glucose had long been thought to fuel oxidative metabolism in active neurons until the recently proposed astrocyte-neuron lactate shuttle hypothesis (ANLSH) challenged this view. According to the ANLSH, activity-induced uptake of glucose takes place predominantly in astrocytes, which metabolize glucose anaerobically. Lactate produced from anaerobic glycolysis in astrocytes is then released from astrocytes and provides the primary metabolic fuel for neurons. The conventional hypothesis asserts that glucose is the primary substrate for both neurons and astrocytes during neural activity and that lactate produced during activity is removed mainly after neural activity. The conventional hypothesis does not assign any particular fraction of glucose metabolism to the aerobic or anaerobic pathways. In this review, the authors discuss the theoretical background and critically review the experimental evidence regarding these two hypotheses. The authors conclude that the experimental evidence for the ANLSH is weak, and that existing evidence and theoretical considerations support the conventional hypothesis.

Of the human salvage enzymes that deaminate ribonucleosides, two--cytidine deaminase and adenosine deaminase--have been found particularly useful for diagnostic purposes. In humans, no enzymes are present that can directly deaminate the bases of these ribonucleosides. Indeed, the only enzyme present that can directly deaminate a base is guanine deaminase, and the diagnostic usefulness of this enzyme has been well documented. The aim of this study is to identify the origin of the ammonia formed when human sera and tissue extracts are incubated with buffered guanosine, and to clarify whether the ammonia comes from the deamination of guanosine by guanosine deaminase or is produced as a result of deamination of guanine formed as a breakdown product of guanosine by purine nucleoside phosphorylase (PNP). Apparent deamination of guanosine by guanosine deaminase in human sera and tissue extracts was found to be due to two enzymes acting in tandem when the products of the reaction were examined by HPLC. The ribose was first removed from guanosine by PNP to form guanine, which was then deaminated to xanthine by guanine deaminase.

Glucose has long been considered the substrate for neuronal energy metabolism in the brain. Recently, an alternative explanation of energy metabolism in the active brain, the astrocyte-neuron lactate shuttle hypothesis, has received attention. It suggests that during neural activity energy needs in glia are met by anaerobic glycolysis, whereas neuronal metabolism is fueled by lactate released from glia. In this article, we critically examine the evidence supporting this hypothesis and explain, from the perspective of enzyme kinetics and substrate availability, why neurons probably use ambient glucose, and not glial-derived lactate, as the major substrate during activity.

It has been postulated that lactate released from astrocytes may be the preferred metabolic substrate for neurons, particularly during intense neuronal activity (the astrocyte-neuron lactate shuttle hypothesis). We examined this hypothesis by exposing rat hippocampal slices to artificial cerebrospinal fluid containing either glucose or lactate and either N-methyl-D-aspartate, which activates neurons without stimulating astrocytic glucose uptake, or alpha-cyano-4-hydroxycinnamate, which blocks monocarboxylate transport across plasma and mitochondrial membranes. When exposed to N-methyl-D-aspartate, slices lost synaptic transmission and K+ homeostasis more slowly in glucose-containing artificial cerebrospinal fluid than in lactate-containing artificial cerebrospinal fluid. After N-methyl-D-aspartate exposure, slices recovered synaptic transmission more completely in glucose. These results suggest that hippocampal neurons can use glucose more effectively than lactate when energy demand is high. In experiments with alpha-cyano-4-hydroxycinnamate, 500 microM alpha-cyano-4-hydroxycinnamate caused loss of K+ homeostasis and synaptic transmission in hippocampal slices during normoxia. When 200 microM alpha-cyano-4-hydroxycinnamate was used, synaptic activity and intracellular pH in slices decreased significantly during normoxia. These results suggest that alpha-cyano-4-hydroxycinnamate may have blocked mitochondrial oxidative metabolism along with lactate transport. Thus, studies using alpha-cyano-4-hydroxycinnamate to demonstrate the presence of a lactate shuttle in the brain tissue may need reevaluation. Our findings, together with observations in the literature that (1) glucose is available to neurons during activation, (2) heightened energy demand rapidly activates glycolysis in neurons, and (3) activation of glycolysis suppresses lactate utilization, suggests that glucose is the primary substrate for neurons during neuronal activation and do not support the astrocyte-neuron lactate shuttle hypothesis.

The purpose of this study was to see how the nominal removal of bicarbonate (HCO(-)(3)) from the extracellular space of brain tissue influenced recovery of brain tissue from anoxia. Removal of HCO(-)(3) in HEPES-buffered artificial cerebrospinal fluid (aCSF) inhibited almost completely recovery of synaptic transmission in hippocampal slices after anoxia. Altered pH did not contribute to this finding because adjusting intracellular (pH(i)) and extracellular (pH(o)) pH to control levels did not reduce the effect of HCO(-)(3) removal. Our results suggest that HCO(-)(3) levels are important in determining the extent of anoxic or ischemic brain injury.

Aim: To determine if a non-ophthalmologist can accurately screen for retinopathy of prematurity (ROP) by evaluating the posterior pole blood vessels of the retina. ROP is a common ocular disorder of premature infants and may require multiple screening examinations by an ophthalmologist to allow for timely intervention. Since there is a strong correlation between posterior pole vascular abnormalities and vision threatening ROP, screening examinations performed by non-ophthalmologist may yield useful clinical information in high risk infants., Methods: Infants born at the Medical University of South Carolina who met screening criteria (n = 142) were examined by a single non-ophthalmologist using a direct ophthalmoscope to evaluate the posterior pole blood vessels for abnormalities of the venules and/or arterioles. To determine the accuracy of the non-ophthalmologist's clinical observations, infants were also examined by an ophthalmologist, using an indirect ophthalmoscope, who graded the posterior pole vessels as normal, dilated venules, or dilated and tortuous venules and arterioles (including "plus disease")., Results: There was significant correlation (p <0.001) between the non-ophthalmologist's and ophthalmologist's diagnoses of posterior pole vascular abnormalities. 47 infants had normal posterior pole blood vessels by the non-ophthalmologist examination. Of these, 31 (66%) were considered to have normal vessels and 16 (34%) to have dilated venules by the ophthalmologist. The non-ophthalmologist correctly identified abnormal posterior pole vessels in all 21 infants diagnosed with abnormal arterioles and venules by the ophthalmologist. No infants with clinically important ROP ("prethreshold" or worse) would have failed detection by this screening method., Conclusion: Using a direct ophthalmoscope, a non-ophthalmologist can screen premature infants at risk for ROP by evaluating the posterior pole blood vessels of the retina. While not necessarily recommended for routine clinical practice, this technique may nevertheless be of value to those situations where ophthalmological consultation is unavailable or difficult to obtain.

Aging alters ion regulation in brain tissue. This article describes methods useful for studying such age-related changes in the rat hippocampal slice preparation. Topics considered include (a) selection of appropriate age groups of rats for aging studies, (b) a description of methods for preparing and maintaining hippocampal slices, (c) measurement of intracellular pH with the H+-sensitive dye carboxy-SNARF-1, and (d) measurement of extracellular pH and K+ with cation-selective microelectrodes., (Copyright 1999 Academic Press.)

A subgene encoding the 87 C-terminal amino acids of the biotinyl carboxy carrier protein (BCCP) from the acetyl CoA carboxylase of Escherichia coli was overexpressed and the apoprotein biotinylated in vitro. The structures of both the apo and holo forms of the biotinyl domain were determined by means of multidimensional NMR spectroscopy. That of the holo domain was well-defined, except for the 10 N-terminal residues, which form part of the flexible linker between the biotinyl and subunit-binding domains of BCCP. In agreement with X-ray crystallographic studies [Athappilly, F. K., and Hendrickson, W. A. (1995) Structure 3, 1407-1419], the structure comprises a flattened beta-barrel composed of two four-stranded beta-sheets with a 2-fold axis of quasi-symmetry and the biotinyl-lysine residue displayed in an exposed beta-turn on the side of the protein opposite from the N- and C-terminal residues. The biotin group is immobilized on the protein surface, with the ureido ring held down by interactions with a protruding polypeptide "thumb" formed by residues 94-101. However, at the site of carboxylation, no evidence could be found in solution for the predicted hydrogen bond between the main chain O of Thr94 and the ureido HN1'. The structure of the apo domain is essentially identical, although the packing of side chains is more favorable in the holo domain, and this may be reflected in differences in the dynamics of the two forms. The thumb region appears to be lacking in almost all other biotinyl domain sequences, and it may be that the immobilization of the biotinyl-lysine residue in the biotinyl domain of BCCP is an unusual requirement, needed for the catalytic reaction of acetyl CoA carboxylase.