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Deborah Gorth,1 Sabrina Puckett,1 Batur Ercan,1 Thomas J Webster,1 Mohamed Rahaman,2 B Sonny Bal31School of Engineering and Department of Orthopaedics, Brown University, Providence, RI, 2Department of Materials Science and Engineering, Missouri University of Science and Technology, Rolla, MO, 3Department of Orthopaedic Surgery, School of Medicine, University of Missouri, Columbia, MO, USAAbstract: A significant need exists for orthopedic implants that can intrinsically resist bacterial colonization. In this study, three biomaterials that are used in spinal implants – titanium (Ti), poly-ether-ether-ketone (PEEK), and silicon nitride (Si3N4) – were tested to understand their respective susceptibility to bacterial infection with Staphylococcus epidermidis, Staphlococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus. Specifically, the surface chemistry, wettability, and nanostructured topography of respective biomaterials, and the effects on bacterial biofilm formation, colonization, and growth were investigated. Ti and PEEK were received with as-machined surfaces; both materials are hydrophobic, with net negative surface charges. Two surface finishes of Si3N4 were examined: as-fired and polished. In contrast to Ti and PEEK, the surface of Si3N4 is hydrophilic, with a net positive charge. A decreased biofilm formation was found, as well as fewer live bacteria on both the as-fired and polished Si3N4. These differences may reflect differential surface chemistry and surface nanostructure properties between the biomaterials tested. Because protein adsorption on material surfaces affects bacterial adhesion, the adsorption of fibronectin, vitronectin, and laminin on Ti, PEEK, and Si3N4 were also examined. Significantly greater amounts of these proteins adhered to Si3N4 than to Ti or PEEK. The findings of this study suggest that surface properties of biomaterials lead to differential adsorption of physiologic proteins, and that this phenomenon could explain the observed in-vitro differences in bacterial affinity for the respective biomaterials. Intrinsic biomaterial properties as they relate to resistance to bacterial colonization may reflect a novel strategy toward designing future orthopedic implants.Keywords: silicon nitride, nanostructure, anti-infective, biofilm, protein adsorption

A recently developed variational Bayesian analysis using pattern recognition and machine learning of single viral ribonucleoprotein (RNP) particle tracks in the cytoplasm of living cells provides a quantitative molecular explanation for active diffusion, a concept previously "explained" largely by hypothetical models based on indirect analyses such as continuum microrheology. Machine learning shows that vesicular stomatitis virus (VSV) RNP particles are temporarily confined to dynamic traps or pores made up of cytoskeletal elements. Active diffusion occurs when the particles escape from one trap to a nearby trap. In this paper, we demonstrate that actin filament disruption increased RNP mobility by increasing trap size. Inhibition of nonmuscle myosin II ATPase decreased mobility by decreasing trap size. Trap sizes were observed to fluctuate with time, dependent on nonmuscle myosin II activity. This model for active diffusion is likely to account for the dominant motion of other viral and cellular elements. IMPORTANCE RNA virus ribonucleoproteins (RNPs) are too large to freely diffuse in the host cytoplasm, yet their dominant motions consist of movements in random directions that resemble diffusion. We show that vesicular stomatitis virus (VSV) RNPs overcome limitations on diffusion in the host cytoplasm by hopping between traps formed in part by actin filaments and that these traps expand and contract by nonmuscle myosin II ATPase activity. ATP-dependent random motion of cellular particles has been termed "active diffusion." Thus, these mechanisms are applicable to active diffusion of other cellular and viral elements.

The difficulty of glioblastoma treatment makes it a good candidate for novel therapies, such as oncolytic viruses. Vesicular stomatitis virus expressing Lassa virus glycoprotein (Lassa-VSV) showed significant promise in animal models using established glioblastoma cell lines. These experiments were to determine the susceptibility of low-passage, patient-derived cell lines to Lassa-VSV oncolysis. Four patient-derived glioblastoma cell lines were infected with Lassa-VSV that expresses green fluorescent protein (GFP) and analyzed by fluorescence microscopy, flow cytometry, and cell viability assays. Cells were also analyzed as tumorspheres containing primarily glioma stem-like cells. Three low-passage, patient-derived cells were further analyzed with RNA sequencing (RNA-seq). Individual cell lines varied somewhat in their levels of viral gene expression and time course of Lassa-VSV-induced cell death, but each was susceptible to Lassa-VSV. Brain Tumor Center of Excellence (BTCOE) 4765 cells had the highest level of expression of interferon-stimulated genes but were most susceptible to Lassa-VSV-induced cell death, indicating that more susceptible cells do not necessarily have lower interferon pathway activation. Cells cultured as tumorspheres and infected with Lassa-VSV also showed variable susceptibility to Lassa-VSV, but BTCOE 4765 cells were least susceptible. Thus, patient-derived brain tumor cells show variable responses to Lassa-VSV infection, but each of the lines was susceptible to VSV oncolysis., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

A key principle of oncolytic viral therapy is that many cancers develop defects in their antiviral responses, making them more susceptible to virus infection. However, some cancers display resistance to viral infection. Many of these resistant cancers constitutively express interferon-stimulated genes (ISGs). The goal of these experiments was to determine the role of two tumor suppressor genes, MAP3K7 and CHD1 , in viral resistance and ISG expression in PC3 prostate cancer cells resistant to oncolytic vesicular stomatitis virus (VSV). MAP3K7 and CHD1 are often co-deleted in aggressive prostate cancers. Silencing expression of MAP3K7 and CHD1 in PC3 cells increased susceptibility to the matrix (M) gene mutant M51R-VSV, as shown by increased expression of viral genes, increased yield of progeny virus, and reduction of tumor growth in nude mice. Silencing MAP3K7 alone had a greater effect on virus susceptibility than did silencing CHD1 . Silencing MAP3K7 and CHD1 decreased constitutive expression of ISG mRNAs and proteins, whereas silencing MAP3K7 alone decreased expression of ISG proteins, but actually increased expression of ISG mRNAs. These results suggest a role for the protein product of MAP3K7 , transforming growth factor β-activated kinase 1 (TAK1), in regulating translation of ISG mRNAs and a role of CHD1 in maintaining the transcription of ISGs., (© 2020 The Authors.)

Updated clinical practice guidelines for screening blood pressures in pediatric patients were published in 2017. They differ from the previous guideline, known as the Fourth Report, providing updated population normal values and blood pressure categorization. We hypothesized that the prevalence of abnormal blood pressure in children and adolescents would be higher using the new clinical practice guidelines. We present a cross-sectional study of screening blood pressure values for children 3 to 18 years of age obtained during well-child visits at a primary care clinic. All blood pressure values were categorized using both the Fourth Report and the Clinical Practice Guideline. A total of 2635 blood pressure measurements were extracted, and 2600 were eligible for analysis. Using the clinical practice guideline, the prevalence of hypertension increased to 17.85% compared to 9.5% per the Fourth Report (P < 0.0001). Of those patients classified as having a normal blood pressure by the Fourth Report, 12% changed to abnormal when applying the Clinical Practice Guideline. All subgroups had a significant increase in the prevalence of abnormal blood pressure. The most dramatic increase in the prevalence of stage 1 and stage 2 hypertension was seen in six patient subgroups: males, 3-12 years of age, Hispanic ethnicity, race designated as other, normal weight, and overweight. Applying the new Clinical Practice Guideline increased the prevalence of elevated blood pressure and stage 1 and stage 2 hypertension in children and adolescents, requiring more follow-up and intervention than previously expected for this patient population., (©2019 Wiley Periodicals, Inc.)

Recombinant vesicular stomatitis virus (VSV) is a promising platform for vaccine development. M51R VSV, an attenuated, M protein mutant strain, is an effective inducer of Type I interferon and dendritic cell (DC) maturation, which are desirable properties to exploit for vaccine design. We have previously evaluated M51R VSV (M51R) and M51R VSV that produces flagellin (M51R-F) as vaccine vectors using murine models, and found that flagellin enhanced DC activation and VSV-specific antibody production after low-dose vaccination. In this report, the immunogenicity of M51R vectors and the adjuvant effect of virus-produced flagellin were evaluated in nonhuman primates following high-dose (10⁸ pfu) and low-dose (10⁵ pfu) vaccination. A single intramuscular vaccination of African green monkeys with M51R or M51R-F induced VSV-specific, dose-dependent humoral immune responses. Flagellin induced a significant increase in antibody production (IgM, IgG and neutralizing antibody) at the low vaccination dose. A VSV-specific cellular response was detected at 6 weeks post-vaccination, but was neither dose-dependent nor enhanced by flagellin; similar numbers of VSV-specific, IFNγ-producing cells were detected in lymph node and spleen of all animals. These results indicate that virus-directed, intracellular flagellin production may improve VSV-based vaccines encoding heterologous antigens by lowering the dose required to achieve humoral immunity., Competing Interests: The authors declare no conflicts of interest.

Enzymes of central carbon metabolism are essential mediators of Mycobacterium tuberculosis (Mtb) physiology and pathogenicity, but are often perceived to lack sufficient species selectivity to be pursued as potential drug targets. Fumarase (Fum) is an enzyme of the canonical tricarboxylic acid cycle and is dispensable in many organisms. Transposon mutagenesis studies in Mtb, however, indicate that Fum is required for optimal growth. Here, we report the generation and characterization of a genetically engineered Mtb strain in which Fum expression is conditionally regulated. This revealed that Fum deficiency is bactericidal in vitro and during both the acute and chronic phases of mouse infection. This essentiality is linked to marked accumulations of fumarate resulting in protein and metabolite succination, a covalent modification of cysteine thiol residues. These results identify Mtb Fum as a potentially species-specific drug target whose inactivation may kill Mtb through a covalently irreversible form of metabolic toxicity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The glyoxylate shunt is a metabolic pathway of bacteria, fungi, and plants used to assimilate even-chain fatty acids (FAs) and has been implicated in persistence of Mycobacterium tuberculosis ( Mtb ). Recent work, however, showed that the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), may mediate survival of Mtb during the acute and chronic phases of infection in mice through physiologic functions apart from fatty acid metabolism. Here, we report that malate synthase (MS), the second enzyme of the glyoxylate shunt, is essential for in vitro growth and survival of Mtb on even-chain fatty acids, in part, for a previously unrecognized activity: mitigating the toxicity of glyoxylate excess arising from metabolism of even-chain fatty acids. Metabolomic profiling revealed that MS-deficient Mtb cultured on fatty acids accumulated high levels of the ICL aldehyde endproduct, glyoxylate, and increased levels of acetyl phosphate, acetoacetyl coenzyme A (acetoacetyl-CoA), butyryl CoA, acetoacetate, and β-hydroxybutyrate. These changes were indicative of a glyoxylate-induced state of oxaloacetate deficiency, acetate overload, and ketoacidosis. Reduction of intrabacterial glyoxylate levels using a chemical inhibitor of ICL restored growth of MS-deficient Mtb , despite inhibiting entry of carbon into the glyoxylate shunt. In vivo depletion of MS resulted in sterilization of Mtb in both the acute and chronic phases of mouse infection. This work thus identifies glyoxylate detoxification as an essential physiologic function of Mtb malate synthase and advances its validation as a target for drug development.

Unlabelled: A major challenge to oncolytic virus therapy is that individual cancers vary in their sensitivity to oncolytic viruses, even when these cancers arise from the same tissue type. Variability in response may arise due to differences in the initial genetic lesions leading to cancer development. Alternatively, susceptibility to viral oncolysis may change during cancer progression. These hypotheses were tested using cells from a transgenic mouse model of prostate cancer infected with vesicular stomatitis virus (VSV). Primary cultures from murine cancers derived from prostate-specific Pten deletion contained a mixture of cells that were susceptible and resistant to VSV. Castration-resistant cancers contained a higher percentage of susceptible cells than cancers from noncastrated mice. These results indicate both susceptible and resistant cells can evolve within the same tumor. The role of Pten deletion was further investigated using clonal populations of murine prostate epithelial (MPE) progenitor cells and tumor-derived Pten(-/-) cells. Deletion of Pten in MPE progenitor cells using a lentivirus vector resulted in cells that responded poorly to interferon and were susceptible to VSV infection. In contrast, tumor-derived Pten(-/-) cells expressed higher levels of the antiviral transcription factor STAT1, activated STAT1 in response to VSV, and were resistant to VSV infection. These results suggest that early in tumor development following Pten deletion, cells are primarily sensitive to VSV, but subsequent evolution in tumors leads to development of cells that are resistant to VSV infection. Further evolution in castration-resistant tumors leads to tumors in which cells are primarily sensitive to VSV., Importance: There has been a great deal of progress in the development of replication-competent viruses that kill cancer cells (oncolytic viruses). However, a major problem is that individual cancers vary in their sensitivity to oncolytic viruses, even when these cancers arise from the same tissue type. The experiments presented here were to determine whether both sensitive and resistant cells are present in prostate cancers originating from a single genetic lesion in transgenic mice, prostate-specific deletion of the gene for the tumor suppressor Pten. The results indicate that murine prostate cancers are composed of both cells that are sensitive and cells that are resistant to oncolytic vesicular stomatitis virus (VSV). Furthermore, androgen deprivation led to castration-resistant prostate cancers that were composed primarily of cells that were sensitive to VSV. These results are encouraging for the use of VSV for the treatment of prostate cancers that are resistant to androgen deprivation therapy., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. Depletion of FBA resulted in clearance of Mtb in both the acute and chronic phases of infection in vivo, and loss of viability in vitro when cultured on single carbon sources. Consistent with prior reports of Mtb's ability to co-catabolize multiple carbon sources, this in vitro essentiality could be overcome when cultured on mixtures of glycolytic and gluconeogenic carbon sources, enabling generation of an fba knockout (Δfba). In vitro studies of Δfba however revealed that lack of FBA could only be compensated for by a specific balance of glucose and butyrate in which growth and metabolism of butyrate were determined by Mtb's ability to co-catabolize glucose. These data thus not only evaluate FBA as a potential drug target in both replicating and persistent Mtb, but also expand our understanding of the multiplicity of in vitro conditions that define the essentiality of Mtb's FBA in vivo.

The authors investigated the effects of retention in grades 1 to 5 on students' reading and math achievement, teacher-rated engagement, and student-reported school belonging in middle school. From a multiethnic sample (N=784) of children who scored below the median on a test of literacy in grade 1, an average of 75 students subsequently retained in grades 1 to 5 were matched with an average of 299 continuously promoted students on the basis of propensity to be retained in the elementary grades. A total of 20 imputed datasets were analyzed, all of which showed good balance across the 67 baseline covariates used to calculate propensity scores. The hypothesis that retained students, who are "old for grade" when they make the transition to middle school, would have a more difficult transition to middle school than promoted peers was tested with 3-level, piecewise growth modeling. Piece 1 included assessments prior to the transition to middle school, and piece 2 included assessments after the transition. Retained and continuously promoted students did not differ on any of the outcome measures during the year prior to transition, nor did they differ in their post-transition trajectories. Discrepancies between these results and results of prior research are discussed in terms of demographic and generational differences as well as differences in methodological rigor., (Copyright © 2013 Society for the Study of School Psychology. Published by Elsevier Ltd. All rights reserved.)

One in seven American households experience food insecurity at times during the year, lack of money and other resources hinder their ability to maintain consistent access to nutritious foods. Low-income, ethnic minority, and female-headed households exhibit the greatest risk for food insecurity, which often results in higher prevalence of diet-related disease. The food insecurity-obesity paradox is one that researchers have explored to understand the factors that influence food insecurity and its impact on weight change. The aim of this inquiry was to explore new evidence in associations of food insecurity and obesity in youth, adult, and elderly populations. A literature search of publication databases was conducted, using various criteria to identify relevant articles. Among 65 results, 19 studies conducted since 2005 were selected for review. Overall, the review confirmed that food insecurity and obesity continue to be strongly and positively associated in women. Growing evidence of this association was found in adolescents; but among children, results remain mixed. Few studies supported a linear relationship between food insecurity and weight outcomes, as suggested by an earlier review. New mediators were revealed (gender, marital status, stressors, and food stamp participation) that alter the association; in fact, newer studies suggest that food stamp participation may exacerbate obesity outcomes. Continued examination through longitudinal studies, development of tools to distinguish acute and chronic food insecurity, and greater inclusion of food security measurement tools in regional and local studies are warranted.

A significant need exists for orthopedic implants that can intrinsically resist bacterial colonization. In this study, three biomaterials that are used in spinal implants--titanium (Ti), polyether-ether-ketone (PEEK), and silicon nitride (Si₃N₄)--were tested to understand their respective susceptibility to bacterial infection with Staphylococcus epidermidis, Staphlococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus. Specifically, the surface chemistry, wettability, and nanostructured topography of respective biomaterials, and the effects on bacterial biofilm formation, colonization, and growth were investigated. Ti and PEEK were received with as-machined surfaces; both materials are hydrophobic, with net negative surface charges. Two surface finishes of Si₃N₄ were examined: as-fired and polished. In contrast to Ti and PEEK, the surface of Si₃N₄ is hydrophilic, with a net positive charge. A decreased biofilm formation was found, as well as fewer live bacteria on both the as-fired and polished Si₃N₄. These differences may reflect differential surface chemistry and surface nanostructure properties between the biomaterials tested. Because protein adsorption on material surfaces affects bacterial adhesion, the adsorption of fibronectin, vitronectin, and laminin on Ti, PEEK, and Si₃N₄ were also examined. Significantly greater amounts of these proteins adhered to Si₃N₄ than to Ti or PEEK. The findings of this study suggest that surface properties of biomaterials lead to differential adsorption of physiologic proteins, and that this phenomenon could explain the observed in-vitro differences in bacterial affinity for the respective biomaterials. Intrinsic biomaterial properties as they relate to resistance to bacterial colonization may reflect a novel strategy toward designing future orthopedic implants.

Infection of a transcutaneous orthopedic prosthesis is undesirable and decreases the success rate of an implant. Critical to reducing this infection is to develop materials that can decrease bacteria colonization and/or increase skin growth. Since previous studies have demonstrated significantly less bacteria colonization (yet promoted osteoblast, bone forming cell, functions) on titanium (Ti) with select nanofeatures, the objective of this in vitro study was to determine keratinocyte (skin forming cells) functions on Ti modified to have nanoscale surface features. To further promote keratinocyte functions, some Ti surfaces were functionalized with fibroblast growth factor-2 (FGF-2). Nanometer-sized topographical surface features were created on traditional Ti implants by anodization (termed nanotubular) and electron beam evaporation (termed nanorough). This study found that compared to conventional (nanosmooth) Ti, the nanotubular and nanorough Ti surfaces promoted keratinocyte density. Moreover, when functionalized with FGF-2, keratinocyte density increased on all surfaces, resulting in the greatest keratinocyte density on nanorough and nanotubular Ti-functionalized with FGF-2 after 5 days of culture. Interestingly, keratinocyte densities on nanorough and nanotubular Ti were similar to that achieved on conventional Ti functionalized with FGF-2. Therefore, when considered in the context of other studies, the results of this in vitro study demonstrated that certain nanometer-sized Ti topographies may be useful for increasing keratinocyte density while reducing bacteria adhesion and promoting bone tissue formation and, thus, should be further studied for improving the efficacy of various transcutaneous Ti-based orthopedic implants., (Copyright © 2011 Wiley Periodicals, Inc.)

Vesicular stomatitis viruses (VSVs) containing wild-type (wt) or mutant matrix (M) proteins are being developed as candidate vaccine vectors due to their ability to induce innate and adaptive immunity. Viruses with wt M protein, such as recombinant wild-type (rwt) virus, stimulate maturation of dendritic cells (DC) through Toll-like receptor 7 (TLR7) and its adaptor molecule MyD88. However, M protein mutant viruses, such as rM51R-M virus, stimulate both TLR7-positive and TLR7-negative DC subsets. The goal of this study was to determine whether the ability of rwt and rM51R-M viruses to induce maturation of human DC can be enhanced by engineering these vectors to express bacterial flagellin. Flagellin expressed from the rwt virus genome partially protected human DC from VSV-induced shutoff of host protein synthesis and promoted the production of interleukin 6 (IL-6) and IL-1β. In addition, DC infected with rwt virus expressing flagellin were more effective at stimulating gamma interferon (IFN-γ) production from CD8(+) allogeneic T cells than DC infected with rwt virus. Although rM51R-M virus effectively stimulated human DC, flagellin expressed from the rM51R-M virus genome enhanced the production of cytokines. Furthermore, mice immunized with both rwt and rM51R-M viruses expressing flagellin had enhanced anti-VSV antibody responses in vivo. Therefore, rwt and rM51R-M viruses expressing flagellin may be promising vectors for the delivery of foreign antigen due to their potential to stimulate DC function.