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Green leafy kale (Brassica oleracea var. sabellica) has huge scientific attention because of its health-promoting functionality. In the present study the impact of NPK, energen, biostim and humate on flavonoid, phenolic compounds, vitamin C, carotenoids, malondialdehyde (MDA), protein, proline and soluble sugar in kale was investigated. The mineral nutrients mostly increased but in some cases maintained the studied metabolites. The stimulatory effect of applied mineral nutrients on the phytochemicals analyzed varied with the different combinations of macro and microelements. Lipid peroxidation was minimized in leaves treated with mineral nutrients hence a reduction in MDA levels. Contrary to the correlation between nitrogen deficiency and increase in polyphenol and vitamin C content in plants, NPK and biostim did not reduce phenolic compound levels. The results of this study showed that NPK maximized the synthesis of vitamin C and proline; energen - phenolic compounds, carotenoids and sugar; biostim – phenolic compounds, proteins and sugar; humate – flavonoids and sugar in curly kale. Therefore, the type of macronutrient and micronutrients combination increases phytochemicals in differently. To enhance the synthesis of phenolic compounds and vitamins, the most promising additives are those containing humic acids (humate and energen), and biostim proved to be more effective for the synthesis of proteins. Background. The understanding of how diet affects the incidence or treatment of disease has led to a rise in consumer’s demand for functional foods as well as created the market for natural sources of health benefitting compounds rather than the synthetic sources. Curly kale has gained scientific attention as a functional food because it contains higher levels of phytochemicals than most vegetables. These phytochemicals have shown antioxidant, antimutagenic, cytotoxic, antifungal, and antiviral activities. However, the content levels of these metabolites are influenced by not only genetic but environmental factors. It was of interest to evaluate how various mineral nutrients can elicit the accumulation of these compounds that minimize the risk of chronic diseases or aid in their treatment. Purpose. Evaluate how the mineral nutrients, NPK, energen, biostim and humate affect the content of metabolites (proteins, sugars, flavonoids, phenolic compounds, vitamin C, carotenoids, MDA and proline) in curly kale (Brassica oleracea var. sabellica). Materials and methods. Sprouts from kale seed kept wet in a Petri dish for 7 days were transferred to the field. At 6 weeks old four mineral nutrients (NPK, energen, humate and biostim) were added to the soil. Control variants were treated with water. A week later, the leaves were harvested after which, the phenolic compound, flavonoid, protein, sugar, vitamin C, carotenoid, MDA and proline contents were determined using spectrophotometric methods. Results. It was shown that humate fertilizer elicited the highest accumulation of flavonoids. Kale plants fertilized with energen were observed to have the highest phenolic compound content. NPK, energen and humate caused a similarly positive effect on vitamin C content in leaves, unlike biostim whose effect did not significantly differ from control plants. Energen treated kale had the highest increment of carotenoids. A varied reduction of MDA levels in plants treated with all four mineral nutrients was observed in kale leaves. Plants fertilized with biostim accrued the highest protein content in leaves. Proline content increased under the influence of all fertilizers studied. Sugar levels for all kale plants treated with the studied mineral nutrients were enhanced equally Conclusion. Macro and microelements supplied by mineral nutrients differentially boost the biosynthesis of health-promoting metabolites in curly kale, thereby enhancing its quality.

BACKGROUND: Myclobutanil is one of the most widely used demethylation inhibitor (DMI) fungicides for the management of apple scab, caused by Venturia inaequalis. Strains of V. inaequalis resistant to myclobutanil have been reported across the world. Tebuconazole, another DMI fungicide, has been proposed as an alternative to myclobutanil, and the extent of cross-resistance with myclobutanil therefore needs to be evaluated. The sensitivity to tebuconazole and myclobutanil of a total of 40 isolates was determined. Half the isolates came from an isolated orchard which had never been sprayed with fungicides and half from orchards sprayed regularly with myclobutanil, but still with disease control problems. The progeny of a tebuconazole resistant (R) × sensitive (S) V. inaequalis cross were analysed in order to improve understanding of the genetic control of tebuconazole sensitivity. RESULTS: There is cross-resistance between myclobutanil and tebuconazole (r=0.91; P < 0.001). Sensitivity to tebuconazole of the progeny of a R×S cross varied quantitatively in a pattern which implied at least two gene loci differing between the parental strains. In addition, the asymmetric distribution of the sensitivity in the progeny implied possible epistatic effects. CONCLUSION: Resistance to myclobutanil and tebuconazole is strongly correlated. At least two genes are involved in the control of tebuconazole resistance in V. inaequalis.

Background B-cell proliferative disorders, such as post-transplant lymphoproliferative disease (PTLD), are increased among persons afflicted by T-cell compromise. Most are Epstein–Barr virus (EBV) + and can first present with a focal lesion. Direct introduction of oncolytic viruses into localized tumors provides theoretical advantages over chemotherapy, immunotherapy and radiation therapy by reducing systemic toxicity. Despite extensive study as a vehicle for gene therapy, adeno-associated viruses (AAV) have rarely been applied to human cancer research due to technical and theoretical obstacles. Moreover, human B-cells have historically been described as resistant to AAV infection. Nonetheless, advances using different recombinant (r)AAV serotypes with unique tropisms to deliver cytotoxic therapy suggested a localized anti-tumor approach was feasible. Methods As a prelude to the development of a therapeutic vehicle, the ability of fifteen distinct EGFP-bearing rAAV serotypes to transduce human B-cells, including primary, immortalized, and B-cell tumor lines ± EBV was assessed by confocal microscopy, flow cytometry and subsequently cell viability assay. Results Rank order analysis revealed augmented transduction by rAAV6.2 and closely related virions. EBV infection of EBV-negative B-cell tumor lines and EBV immortalization of primary B-cells increased susceptibility to rAAV6.2 transduction. As a proof of concept, transduction by rAAV6.2 encoding herpes simplex virus type 1 (HSV1)-thymidine kinase (TK) eliminated TK-negative rhabdomyosarcoma cells and diminished viability of transduced B-cell lines upon incubation with ganciclovir. Conclusions rAAV serotypes differentially transduce human B-cell lines reversing the dogma that human B-cells are refractory to AAV infection. EBV + B-cells display increased susceptibility to rAAV6.2 infection, uncovering a new method for improved nucleic acid transfer into transfection-resistant B-cell lines. The introduction of a functional suicide gene into the rAAV6.2 genome identifies a candidate vector for the development of rAAV-based oncolytic therapy targeting focal EBV-bearing B-lymphoproliferative disorders.

Table tennis student-athletes should be mentally tough as they train and perform under extreme pressure. It is essential to identify other mental skills that will aid mental toughness development. Accordingly, the study focused on assessing athletic identity, self-compassion, and intra-team communication. The relationship between the constructs was also explored. A multi-part questionnaire was fielded to 230 college student-athletes participating in table tennis singles event. Findings indicate high levels of athletic identity and intra-team communication among the participants; while moderate levels for self-compassion and mental toughness. Structural equation modeling revealed the dynamic relationship between the constructs. On one hand, self-compassionate participants who highly recognize their role as athletes, and communicate with the team are more mentally tough. On the other hand, uncompassionate self-responding lead student-athletes to become mentally weak. The study provides useful insights for practitioners in designing mental skills training geared towards optimal functioning and psychological wellness of young athletes.

Key Points Question What are the typical clinical, radiographic, and bronchoscopic findings and clinical outcomes of e-cigarette, or vaping, product use–associated lung injury (EVALI)? Findings This case series of 31 patients found that EVALI typically presented as a flu-like illness with elevated inflammatory markers and an organizing pneumonia pattern on computed tomography imaging. Bronchoscopy showed lipid-laden macrophages and had a high rate of false-positive results for infection. Meaning The findings of this study suggest that EVALI has a characteristic clinical and radiographic presentation and that bronchoscopy has limited utility in its evaluation., This case series describes the clinical features, bronchoscopic findings, imaging patterns, and outcomes of e-cigarette, or vaping, product use–associated lung injury (EVALI)., Importance e-Cigarette, or vaping, product use–associated lung injury (EVALI) has caused more than 2800 illnesses and 68 deaths in the United States. Better characterization of this novel illness is needed to inform diagnosis and management. Objective To describe the clinical features, bronchoscopic findings, imaging patterns, and outcomes of EVALI. Design, Setting, and Participants This case series of 31 adult patients diagnosed with EVALI between June 24 and December 10, 2019, took place at an academic medical center in Salt Lake City, Utah. Exposures e-Cigarette use, also known as vaping. Main Outcomes and Measures Symptoms, laboratory findings, bronchoscopic results, imaging patterns, and clinical outcomes. Results Data from 31 patients (median [interquartile range] age, 24 [21-31] years) were included in the study. Patients were primarily men (24 [77%]) and White individuals (27 [87%]) who used e-cigarette products containing tetrahydrocannabinol (THC) (29 [94%]). Patients presented with respiratory (30 [97%]), constitutional (28 [90%]), and gastrointestinal (28 [90%]) symptoms. Serum inflammatory markers were elevated in all patients. Bronchoscopy was performed in 23 of 28 inpatients (82%) and bronchoalveolar lavage (BAL) revealed the presence of lipid-laden macrophages (LLMs) in 22 of 24 cases (91%). BAL samples tested positive for Pneumocystis jirovecii (3 patients [13%]), rhinovirus (2 patients [8%]), human metapneumovirus and Aspergillus (1 patient each [4%]); all except human metapneumovirus were determined to be false-positives or clinically inconsequential. The exclusive or dominant computed tomography (CT) pattern was organizing pneumonia in 23 of 26 cases (89%). Patients received antibiotics (26 [84%]) and corticosteroids (24 [77%]), and all survived; 20 patients (65%) seen in follow-up showed marked improvement, but residual symptoms (13 [65%]), radiographic opacities (8 [40%]), and abnormal pulmonary function tests (8 of 18 [44%]) were common. Conclusions and Relevance In this case series, patients with EVALI characteristically presented with a flu-like illness with elevated inflammatory markers, LLMs on BAL samples, and an organizing pneumonia pattern on CT imaging. Bronchoscopic testing for infection had a high incidence of false-positive results. Patients had substantial residual abnormal results at early follow-up. These data suggest a limited role for bronchoscopy in typical presentations of EVALI without risk factors for alternative diagnoses and the need for careful longitudinal follow-up.

The genetic basis and mechanisms of disparate antitumor immune response was investigated in Diversity Outbred (DO) F1 mice that express human HER2. DO mouse stock samples nearly the entire genetic repertoire of the species. We crossed DO mice with syngeneic HER2 transgenic mice to study the genetics of an anti-self HER2 response in a healthy outbred population. Anti-HER2 IgG was induced by Ad/E2TM or naked pE2TM, both encoding HER2 extracellular and transmembrane domains. The response of DO F1 HER2 transgenic mice was remarkably variable. Still, immune sera inhibited HER2+ SKBR3 cell survival in a dose-dependent fashion. Using DO quantitative trait locus (QTL) analysis, we mapped the QTL that influences both total IgG and IgG2(a/b/c) Ab response to either Ad/E2TM or pE2TM. QTL from these four datasets identified a region in chromosome 17 that was responsible for regulating the response. A/J and NOD segments of genes in this region drove elevated HER2 Ig levels. This region is rich in MHC-IB genes, several of which interact with inhibitory receptors of NK cells. (B6xA/J)F1 and (B6xNOD)F1 HER2 transgenic mice received Ad/E2TM after NK cell depletion, and they produced less HER2 IgG, demonstrating positive regulatory function of NK cells. Depletion of regulatory T cells enhanced response. Using DO QTL analysis, we show that MHC-IB reactive NK cells exert positive influence on the immunity, countering negative regulation by regulatory T cells. This new, to our knowledge, DO F1 platform is a powerful tool for revealing novel immune regulatory mechanisms and for testing new interventional strategies.

Introduction. Pregnant women are at a high risk of anaemia, with iron-folate deficiency being the most common cause of anaemia among pregnant women. Despite the well-known importance of iron and folic acid supplementation (IFAS) during pregnancy, adherence to these supplements is relatively low and associated factors were not well identified in the study area. This study is aimed at investigating adherence to IFAS and associated factors among pregnant women in Kasulu district, north-western Tanzania. Methods. A health facility cross-sectional survey with a mixed-method approach was conducted in Kasulu district from March to April 2019. A structured questionnaire was given to 320 women with children aged 0-6 months to assess factors associated with adherence to IFAS among pregnant women. Data were entered into SPSS version 22.0 for analysis. Binary logistic regression was further employed to determine the factors associated with adherence to IFAS. Focus group discussions were done with 19 pregnant women and 15 mothers of children aged 0-6 months to obtain more clarifications on the factors associated with adherence to IFAS. Furthermore, in-depth interviews were done with six health care providers to explore their perceptions of IFAS. Results. Out of the 320 respondents of the survey, 20.3% (n=65) adhered to IFAS. Factors associated with adherence to IFAS among pregnant women included time to start ANC (AOR=3.72, 95% CI: 1.42, 9.79), knowledge of anaemia (AOR=3.84, 95% CI: 1.335, 10.66), counseling on the importance of the iron-folic acid (AOR=3.86, 95% CI: 1.42, 10.50), IFAS given during clinical visit (AOR=15.72, 95% CI: 5.34, 46.31), number of meals consumed (AOR=3.44, 95% CI: 1.28, 9.21), number of children (AOR=3.462, 95% CI: 1.035, 11.58), and distance to health facility (AOR=0.34, 95% CI: 0.131, 0.886). Qualitative findings revealed that delayed first ANC visit, lack of remainder for pregnant women to take IFAS, low awareness about the negative effects of anaemia, low of knowledge of IFAS and management of side effects, negative beliefs about the use of IFAS, and follow-up mechanism were major reasons for poor adherence. Conclusion. Adherence to iron-folic acid supplementation during pregnancy was low. Strengthening systems for creating reminding mechanism, raising community awareness through educational programs to pregnant women and health providers could improve adherence to IFAS.

Although humans are heterogeneous, evaluation of tumorigenesis and immunotherapy has relied on the use of inbred mouse strains, which may lead to biased results. In order to investigate the effect(s) of genetic background on tumor incidence and immune response, we have taken a unique new approach to identify genes that are associated with tumor onset time & tumor growth rate using Diversity Outbred (DO) mice. Specifically, we crossed DO female mice (J:DO, Jackson Labs) with BALB/NeuT (NeuT) mice to introduce a heterogeneous background in (BALBxDO) F1 NeuT mice. NeuT express transforming rat neu, the homologue of HER2, and develop spontaneous tumors in all 10 mammary glands between 14 and 19 weeks of age (woa). DO mice are generated by non-sibling crosses of 8 founder strains: A/J, C57BL/6J, 129S1/SvlmJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, WSB/EiJ to encompass greater than 90% of murine polymorphic alleles. The genetic makeup of each F1 mouse is defined using 143,600 chromosomal markers (mostly SNP) in the Giga Mouse Universal Genotyping Array. SNP analysis identifies the origin of quantitative trait loci (QTL), using the R/QTL package. The program reads the GigaMUGA genotyping results to perform haplotype reconstruction and determine the founder strain haplotype of each marker locus. DO F1 mice have been successfully used to identify the critical role of MHC-IB, and NK cells, in generation of HER2 Ab response following vaccination of HER2 tolerant mice (Wei et al., 2020). In (BALBxDO) F1 NeuT female mice most spontaneous mammary tumors develop over a wider range of time from 9 to 23 woa. QTL analysis of tumor onset time revealed QTLs in chromosomes (Chr) 1 and X with peak LOD scores of 7.7 (p = 0.01) and 8.6 (p < 0.05), respectively. Tumor growth rate was associated with a QTL in Chr 10. More specifically, PWK and CAST haplotypes in these QTLs were associated with early and faster growing tumors. About 10-15 genes in the 3 QTLs contain missense SNP alterations unique to PWK and CAST, and the genes have been associated with human breast cancer prognosis. As an example, Mid2 identified in ChrX QTL interacts with BRCA1, and is negatively associated with patient survival using the TCGA BRCA database. Single cell RNA sequencing revealed that Mid2 is expressed in NeuT mammary tumor tissue. Studies are ongoing to determine the functional significance of candidate genes in HER2/neu mammary tumorigenesis. DO F1 QTL combined with scRNA transcriptomic analysis opens important new opportunities to define critical genes in cancer biology and immunotherapy, with the potential for novel intervention and disease prevention. Citation Format: Jennifer Jacob, Kuang Wei, Joyce D. Reyes, Cong-Cong Yin, Elizabeth Rondini, Qing-Sheng Mi, James Granneman, Prahlad Parajuli, K Wagner, Wei-Zen Wei. A Diversity outbred (DO) mouse model reveals regulators of HER2-driven tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2920.

Objectives In the past years, there have been increases in S. aureus infections, intensifying the demand for new and effective drugs. Apparently, old drugs such as statins have shown unexpected pleiotropic effects, and may be exploited or repurposed to meet new demands on antimicrobial therapy. In this study, the antibacterial activity of atorvastatin and possible interactions with ampicillin were evaluated by determining the minimum inhibitory concentration (MIC), synergism, and biofilm inhibition of these drugs against S. aureus ATCC 29213. Methods In this study, atorvastatin and ampicillin, alone or in combination, were tested for antibacterial property against S. aureus using the MIC assay, and tested for synergism via the checkerboard microdilution assay. Both drugs were then tested against biofilm formation to check its relevance to sessile bacterial states. Results Atorvastatin did not display anti-bacterial activity consistent with previous reports, but synergistically inhibited planktonic S. aureus growth by lowering the MIC of ampicillin by 8-fold. Further, atorvastatin abrogated biofilm formation in 48h-grown S. aureus − implicative of its role in chronic infection, but lost this property when co-administered with ampicillin in a ratio of 1:1. Conclusion These suggest that atorvastatin and ampicillin may in fact be targeting a common bacterial pathway integral to biofilm formation, and this warrants further verification on the mechanisms involved as well as on the identification of novel enzyme off-targets that could potentially impact the treatment of S. aureus infections.

Novel, tumor-selective therapies are needed to increase the survival rate of pancreatic cancer patients. K-Ras-mutant-driven NAD(P)H:quinone oxidoreductase 1 (NQO1) is over-expressed in pancreatic tumor versus associated normal tissue, while catalase expression is lowered compared to levels in associated normal pancreas tissue. ARQ761 undergoes a robust, futile redox cycle in NQO1+ cancer cells, producing massive hydrogen peroxide (H2 O2 ) levels; normal tissues are spared by low NQO1 and high catalase expression. DNA damage created by ARQ761 in pancreatic cancer cells "hyperactivates" PARP1, causing metabolic catastrophe and NAD ± keresis cell death. NQO1: catalase levels (high in tumor, low in normal tissue) are an attractive therapeutic window to treat pancreatic cancer. Based on a growing body of literature, we are leading a clinical trial to evaluate the combination of ARQ761 and chemotherapy in patients with pancreatic cancer.

Enhancement of endogenous immunity to tumor-associated self-antigens and neoantigens is the goal of preventive vaccination. Toward this goal, we compared the efficacy of the following HER2 DNA vaccine constructs: vaccines encoding wild-type HER2, hybrid HER2 vaccines consisting of human HER2 and rat Neu, HER2 vaccines with single residue substitutions and a novel human HER2 DNA vaccine, ph(es)E2TM. ph(es)E2TM was designed to contain five evolution-selected substitutions: M198V, Q398R, F425L, H473R and A622T that occur frequently in 12 primate HER2 sequences. These ph(es)E2TM substitutions score 0 to 1 in blocks substitutions matrix (BLOSUM), indicating minimal biochemical alterations. h(es)E2TM recombinant protein is recognized by a panel of anti-HER2 mAbs, demonstrating the preservation of HER2 protein structure. Compared to native human HER2, electrovaccination of HER2 transgenic mice with ph(es)E2TM induced a threefold increase in HER2-binding antibody (Ab) and elevated levels of IFNγ-producing T cells. ph(es)E2TM, but not pE2TM immune serum, recognized HER2 peptide p95 355LPESFDGDPASNTAP369, suggesting a broadening of epitope recognition induced by the minimally modified HER2 vaccine. ph(es)E2TM vaccination reduced tumor growth more effectively than wild-type HER2 or HER2 vaccines with more extensive modifications. The elevation of tumor immunity by ph(es)E2TM vaccination would create a favorable tumor microenvironment for neoantigen priming, further enhancing the protective immunity. The fundamental principle of exploiting evolution-selected amino acid substitutions is novel, effective and applicable to vaccine development in general.

Whether the human tumor virus, Epstein–Barr Virus (EBV), promotes breast cancer remains controversial and a potential mechanism has remained elusive. Here we show that EBV can infect primary mammary epithelial cells (MECs) that express the receptor CD21. EBV infection leads to the expansion of early MEC progenitor cells with a stem cell phenotype, activates MET signaling and enforces a differentiation block. When MECs were implanted as xenografts, EBV infection cooperated with activated Ras and accelerated the formation of breast cancer. Infection in EBV-related tumors was of a latency type II pattern, similar to nasopharyngeal carcinoma (NPC). A human gene expression signature for MECs infected with EBV, termed EBVness, was associated with high grade, estrogen-receptor-negative status, p53 mutation and poor survival. In 11/33 EBVness-positive tumors, EBV-DNA was detected by fluorescent in situ hybridization for the viral LMP1 and BXLF2 genes. In an analysis of the TCGA breast cancer data EBVness correlated with the presence of the APOBEC mutational signature. We conclude that a contribution of EBV to breast cancer etiology is plausible, through a mechanism in which EBV infection predisposes mammary epithelial cells to malignant transformation, but is no longer required once malignant transformation has occurred., Highlights • Mammary epithelial cells, but not breast cancer cells, express CD21 and can be infected by EBV. • EBV infection of MECs rewires mitogenic signaling with activation of the MET pathway and gene expression changes (EBVness). • EBV infection of MECs lowers the threshold for malignant transformation in xenografts. • Human tumors with ‘EBVness’ are associated with poor prognosis and presence of remnant EBV DNA (but not RNA). Research in context: This study shows that Epstein–Barr Virus (EBV) infection occurs in breast epithelial cells (MECs), but not in breast cancer cells. EBV infection of MECs leads to changes in gene expression, activation of oncogenic signaling via c-MET and lowers the threshold for cancerous transformation. In human breast cancer, these gene expression changes, termed ‘EBVness’, are associated with high grade and the finding of remnant EBV DNA. EBV infection of breast epithelial cells may increase the odds of later breast cancer formation in predisposed individuals, which could potentially be avoided by a childhood vaccine against EBV.

Obesity results from the accumulation of excess white adipose tissue, and its increasing rates worldwide pose a significant risk to overall health due to its multiple comorbidities, like type 2 diabetes. A novel treatment strategy is to increase energy expenditure through activation of brown and beige adipocytes, which can use uncoupling protein 1 (UCP1) for thermogenesis. In addition to having different developmental lineages, brown and beige adipocytes differ functionally regarding the additional mechanisms by which they generate heat. Most human fat depots are not easily accessible, so little is known about their developmental and functional characteristics. In this study, we collected adipose tissue from 11 patients (age 16-84, 4 female/7 male) who had undergone autopsy. We sampled from 7 anatomically distinct fat depots: subcutaneous (sc), omental (om), retroperitoneal (rp), pericardial (pc), periadrenal (pa), paraspinal (ps), and supraclavicular (sv). We measured mRNA levels of lineage and functional genes associated with brown, beige, and white adipocytes. Energy storing white adipocytes were defined by high leptin expression, while energy expending brown and beige cells were defined by high UCP1; these were further distinguished as having a brown lineage using Zic1 and beige via Tbx1. Principal component analysis showed three different clusters of genes consistent with the brown, beige, and white lineages. Functionally, based on expression of leptin and UCP1 for white and brown fat, respectively, the sc and om were white (P In summary, human adipose tissue is not merely white or brown but instead is composed of many depots with distinct developmental lineages and functional capacities. These differences will be important when trying to use brown and beige adipocyte thermogenesis to treat obesity and diabetes. Disclosure J.W. Johnson: None. C. Cero: None. A. O'Mara: None. J.D. Linderman: None. A.S. Baskin: None. A. Cypess: None.

IFNγ is an attractive target for imaging active antitumor immunity due to its function in the T-cell signaling axis. Here, we test an IFNγ immuno-PET (immunoPET) probe for its capacity to identify adaptive immunotherapy response after HER2/neu vaccination in both spontaneous salivary and orthotopic neu+ mouse mammary tumors. IFNγ immunoPET detected elevated cytokine levels in situ after vaccination, which inversely correlated with tumor growth rate, an indicator of response to therapy. In a model of induced T-cell anergy where CD8 T cells infiltrate the tumor, but upregulate PD-1, IFNγ tracer uptake was equivalent to isotype control, illustrating a lack of antitumor T-cell activity. The IFNγ immunoPET tracer detected IFNγ protein sequestered on the surface of tumor cells, likely in complex with the IFNγ receptor, which may explain imaging localization of this soluble factor in vivo. Collectively, we find that the activation status of cytotoxic T cells is annotated by IFNγ immunoPET, with reduced off-target binding to secondary lymphoid tissues compared with imaging total CD3+ tumor-infiltrating lymphocytes. Targeting of soluble cytokines such as IFNγ by PET imaging may provide valuable noninvasive insight into the function of immune cells in situ. Significance: This study presents a novel approach to monitor therapeutic outcomes via IFNγ-targeted positron emission tomography. Cancer Res; 78(19); 5706–17. ©2018 AACR.

Glycosaminoglycans (GAGs) such as chondroitin are ubiquitous disaccharide carbohydrate chains that contribute to the formation and function of proteoglycans at the cell membrane and in the extracellular matrix. Although GAG-modifying enzymes are required for diverse cellular functions, the role of these proteins in human development and disease is less well understood. Here, we describe two sisters out of seven siblings affected by congenital limb malformation and malignant lymphoproliferative disease. Using Whole-Genome Sequencing (WGS), we identified in the proband deletion of a 55 kb region within chromosome 12q23 that encompasses part of CHST11 (encoding chondroitin-4-sulfotransferase 1) and an embedded microRNA (MIR3922). The deletion was homozygous in the proband but not in each of three unaffected siblings. Genotyping data from the 1000 Genomes Project suggest that deletions inclusive of both CHST11 and MIR3922 are rare events. Given that CHST11 deficiency causes severe chondrodysplasia in mice that is similar to human limb malformation, these results underscore the importance of chondroitin modification in normal skeletal development. Our findings also potentially reveal an unexpected role for CHST11 and/or MIR3922 as tumor suppressors whose disruption may contribute to malignant lymphoproliferative disease.

In rodents, brown adipose tissue (BAT) regulates cold- and diet-induced thermogenesis (CIT; DIT). Whether BAT recruitment is reversible and how it impacts on energy metabolism have not been investigated in humans. We examined the effects of temperature acclimation on BAT, energy balance, and substrate metabolism in a prospective crossover study of 4-month duration, consisting of four consecutive blocks of 1-month overnight temperature acclimation (24°C [month 1] → 19°C [month 2] → 24°C [month 3] → 27°C [month 4]) of five healthy men in a temperature-controlled research facility. Sequential monthly acclimation modulated BAT reversibly, boosting and suppressing its abundance and activity in mild cold and warm conditions (P < 0.05), respectively, independent of seasonal fluctuations (P < 0.01). BAT acclimation did not alter CIT but was accompanied by DIT (P < 0.05) and postprandial insulin sensitivity enhancement (P < 0.05), evident only after cold acclimation. Circulating and adipose tissue, but not skeletal muscle, expression levels of leptin and adiponectin displayed reciprocal changes concordant with cold-acclimated insulin sensitization. These results suggest regulatory links between BAT thermal plasticity and glucose metabolism in humans, opening avenues to harnessing BAT for metabolic benefits.

2517 Background: NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron oxidoreductase expressed in multiple tumor types at levels 5- to 200-fold above normal tissue. ARQ761 is a β-lapachone hydroquinone analog that exploits the unique elevation of NQO1 found in solid tumors to cause tumor-specific cell death by eliciting a futile redox cycle generating high levels of reactive oxygen species and ultimately PARP1 hyperactivation-dependent cell death. Methods: 3+3 dose escalation study of 3 schedules (weekly, every other week, 2/3 weeks) of ARQ 761 as a 1-hr or 2-hr infusion. Eligible patients had refractory advanced solid tumors, ECOG 0-1, adequate organ function, and central venous access. Blood samples were analyzed for ARQ761 levels and NQO1 polymorphisms. Archival tumor tissue was analyzed for NQO1 staining intensity and prevalence. After 18 patients were analyzed, enrollment was restricted to patients with NQO1-positive tumors (defined as Histo-score ≥200). Results: A total of 42 patients were treated. Median number of prior lines of therapy was 4. For all schedules, the maximum tolerated dose (MTD) was 390 mg/m2 as a 2-hr infusion. DLT was hemolytic anemia. The most common treatment-related adverse events were anemia (79%), fatigue (45%), hypoxia (33%), hemolysis (17%), nausea (17%) and vomiting (17%). Transient grade 3 hypoxia, due to methemoglobinemia, occurred in 26% of patients. Among 31 evaluable patients, the best response was stable disease (n = 11) and progressive disease (n = 19). For the 18 analyzed cases analyzed prior to NQO1 enrollment biomarker, clinical benefit appeared associated with tissue NQO1 expression: disease control rate was 65% in NQO1-positive tumors and 18% in NQO1-negative tumors ( P=0.06). Analysis of all 31 evaluable patients did not show a significant difference in progression-free survival (PFS) according to NQO1 status ( P=0.26), but 3-mo PFS rate was numerically greater among NQO1-positive cases (40% versus 20%). Conclusions: ARQ 761 has clinical activity in NQO1-positive tumors. Principal toxicities include hemolytic anemia and methemoglobinemia. Combination studies are underway.

Percutaneous cryoablation is a minimally invasive procedure for tumor destruction, which can potentially initiate or amplify antitumor immunity through the release of tumor-associated antigens. However, clinically efficacious immunity is lacking and regional recurrences are a limiting factor relative to surgical excision. To understand the mechanism of immune activation by cryoablation, comprehensive analyses of innate immunity and HER2/neu humoral and cellular immunity following cryoablation with or without peritumoral CpG injection were conducted using two HER2/neu+ tumor systems in wild-type (WT), neu-tolerant, and SCID mice. Cryoablation of neu+ TUBO tumor in BALB/c mice resulted in systemic immune priming, but not in neu-tolerant BALB NeuT mice. Cryoablation of human HER2+ D2F2/E2 tumor enabled the functionality of tumor-induced immunity, but secondary tumors were refractory to antitumor immunity if rechallenge occurred during the resolution phase of the cryoablated tumor. A step-wise increase in local recurrence was observed in WT, neu-tolerant, and SCID mice, indicating a role of adaptive immunity in controlling residual tumor foci. Importantly, local recurrences were eliminated or greatly reduced in WT, neu tolerant, and SCID mice when CpG was incorporated in the cryoablation regimen, showing significant local control by innate immunity. For long-term protection, however, adaptive immunity was required because most SCID mice eventually succumbed to local tumor recurrence even with combined cryoablation and CpG treatment. This improved understanding of the mechanisms by which cryoablation affects innate and adaptive immunity will help guide appropriate combination of therapeutic interventions to improve treatment outcomes. Cancer Res; 74(19); 5409–20. ©2014 AACR.

SummaryRediscovery of cold-activated brown adipose tissue (BAT) in humans has boosted research interest in identifying BAT activators for metabolic benefits. Of particular interest are cytokines capable of fat browning. Irisin, derived from FNDC5, is an exercise-induced myokine that drives brown-fat-like thermogenesis in murine white fat. Here we explored whether cold exposure is an afferent signal for irisin secretion in humans and compared it with FGF21, a brown adipokine in rodents. Cold exposure increased circulating irisin and FGF21. We found an induction of irisin secretion proportional to shivering intensity, in magnitude similar to exercise-stimulated secretion. FNDC5 and/or FGF21 treatment upregulated human adipocyte brown fat gene/protein expression and thermogenesis in a depot-specific manner. These results suggest exercise-induced irisin secretion could have evolved from shivering-related muscle contraction, serving to augment brown fat thermogenesis in concert with FGF21. Irisin-mediated muscle-adipose crosstalk may represent a thermogenic, cold-activated endocrine axis that is exploitable in obesity therapeutics development.

Rods and rings (RR) are protein assemblies composed of cytidine triphosphate synthetase type 1 (CTPS1) and inosine monophosphate dehydrogenase type 2 (IMPDH2), key enzymes in CTP and GTP biosynthesis. Small-molecule inhibitors of CTPS1 or IMPDH2 induce RR assembly in various cancer cell lines within 15 min to hours. Since glutamine is an essential amide nitrogen donor in these nucleotide biosynthetic pathways, glutamine deprivation was examined to determine whether it leads to RR formation. HeLa cells cultured in normal conditions did not show RR, but after culturing in media lacking glutamine, short rods (2 μm) assembled after 24 h, and longer rods (5 μm) formed after 48 h. Upon supplementation with glutamine or guanosine, these RR underwent almost complete disassembly within 15 min. Inhibition of glutamine synthetase with methionine sulfoximine also increased RR assembly in cells deprived of glutamine. Taken together, our data support the hypothesis that CTP/GTP biosynthetic enzymes polymerize to form RR in response to a decreased intracellular level of glutamine. We speculate that rod and ring formation is an adaptive metabolic response linked to disruption of glutamine homeostasis.

In the century preceding the discovery of Epstein-Barr virus (EBV), physicians speculated that a common clinical syndrome characterized by fever, tonsillar adenopathy, splenomegaly, and mononuclear leukocytosis termed glandular fever was caused by a pathogen (1). In 1920, the name infectious mononucleosis (IM) was introduced by Thomas P. Sprunt and Frank A. Evans to characterize this syndrome (2). Three years later, Hal Downey and C.A. McKinlay described the now-classic atypical lymphocyte as a common feature of this disease (3), and in 1932 John Rodman Paul and Walls Willard Bunnell demonstrated high titers of spontaneously occurring heterophile antibodies in the sera of patients with IM (4), ensuring more accurate diagnosis. In 1961, the British surgeon Denis P. Burkitt gave the first account outside of Africa of “The Commonest Children's Cancer in Tropical Africa” at Middlesex Hospital London, detailing the geographic relationship between Burkitt's lymphoma (BL) and conditions of temperature, altitude, and rainfall associated with development of Plasmodium falciparum malaria (5, 6). M. Anthony Epstein, who was in the audience, became intrigued by the idea that a biological agent might be involved in the etiology of BL, and in 1964, the Epstein laboratory analyzed BL biopsy samples by thin-section electron microscopy and discovered a new, large, icosahedral herpesvirus that could be directly reactivated from in vitro–grown BL cells (7). These initial findings were reported in The Lancet, and the virus was named after Epstein and his graduate student Yvonne Barr (8). Shortly thereafter, two independent groups (9, 10) demonstrated the ability of EBV to transform primary human B lymphocytes into permanently growing lymphoblastoid cell lines (LCLs), providing the first concrete evidence of the ability of EBV to promote human cancer. In 1968, Gertrude Henle and Werner Henle made two further critical observations: (1) that EBV seroconversion occurred during the course of acute IM (AIM) in a laboratory technician (9) and (2) that EBV-carrying LCLs spontaneously formed from a peripheral blood leukocyte culture obtained during the acute phase of the technician's illness. The Henles confirmed their observations through study of sera provided by James Corson Niederman and Robert W. McCollum, who collected blood from incoming Yale freshmen and later from individuals who developed AIM. This study and others demonstrated EBV-specific antibodies in the sera of the students who developed AIM, confirming the etiologic association between EBV and AIM (11).

Rods and Rings (RR) are conserved, non-membrane-bound intracellular polymeric structures composed in part of inosine monophosphate dehydrogenase, a key enzyme leading to GMP and GTP biosynthesis. RR formation is induced by IMPDH inhibitors as well as glutamine deprivation. They also form upon treatment of cells with glutamine synthetase inhibitors. We now report that depriving cells of serine and glycine promotes RR formation, and we have traced these effects to dihydrofolate reductase (DHFR) and serine hydroxymethyltransferase-2 (SHMT2), pivotal enzymes in one-carbon metabolism and nucleotide biosynthesis. RR assembly is likewise induced upon DHFR inhibition by methotrexate or aminopterin as well as siRNA-mediated knockdown of DHFR or SHMT2. Because RR assembly occurs when guanine nucleotide biosynthesis is inhibited, and because RR rapidly disassemble after the addition of guanine nucleotide precursors, RR formation may be an adaptive homeostatic mechanism, allowing IMPDH to sense changes in the one-carbon folate pathway.

Asplenic individuals are compromised not only in their ability to destroy infectious agents, but are at increased risk for death from autoimmune disease, certain tumors, and ischemic heart disease. Enhanced mortality is attributed to lack of phagocytes sequestered in spleen that efficiently engulf and destroy appropriate targets, although related cells are found elsewhere. To determine whether a unique population regulates RBC-pathogen clearance and filtration of altered self, we reviewed the anatomic literature and analyzed in situ by immunohistochemistry and immunofluorescence the expression patterns of a little-characterized cell that dominates the splenic red pulp of humans and closely related primates: the venous sinus-lining or littoral cell (LC). High expression of the formin homology domain protein 1 outlines the LC population. Although LCs are endothelial-like in distribution, they express several macrophage-directed proteins, the RBC Duffy Ag receptor for chemokines and T cell coreceptor CD8α/α, yet they lack lineage-associated markers CD34 and CD45. Strikingly, SIRPα (CD172a) expression in human spleen concentrates on LCs, consistent with recent demonstration of a key role in RBC turnover and elimination versus release of infected or altered self. Our results indicate human LCs (SIRPα+, formin homology domain protein 1+, CD8α/α+, CD34−, CD45−) comprise a highly plastic barrier cell population that emerged late in primate evolution coordinate with CD8 expression. Unique to Hominidae, LCs may be the ultimate determinant of which cells recirculate after passage through human spleen.

Ganciclovir (GCV) and acyclovir (ACV) are guanine nucleoside analogues that inhibit lytic herpesvirus replication. GCV and ACV must be monophosphorylated by virally encoded enzymes to be converted into nucleotides and incorporated into viral DNA. However, whether GCV and/or ACV phosphorylation in Epstein-Barr virus (EBV)-infected cells is mediated primarily by the EBV-encoded protein kinase (EBV-PK), the EBV-encoded thymidine kinase (EBV-TK), or both is controversial. To examine this question, we constructed EBV mutants containing stop codons in either the EBV-PK or EBV-TK open reading frame and selected for stable 293T clones latently infected with wild-type EBV or each of the mutant viruses. Cells were induced to the lytic form of viral replication with a BZLF1 expression vector in the presence and absence of various doses of GCV and ACV, and infectious viral titers were determined by a green Raji cell assay. As expected, virus production in wild-type EBV-infected 293T cells was inhibited by both GCV (50% inhibitory concentration [IC 50 ] = 1.5 μM) and ACV (IC 50 = 4.1 μM). However, the EBV-PK mutant (which replicates as well as the wild-type (WT) virus in 293T cells) was resistant to both GCV (IC 50 = 19.6 μM) and ACV (IC 50 = 36.4 μM). Expression of the EBV-PK protein in trans restored GCV and ACV sensitivity in cells infected with the PK mutant virus. In contrast, in 293T cells infected with the TK mutant virus, viral replication remained sensitive to both GCV (IC 50 = 1.2 μM) and ACV (IC 50 = 2.8 μM), although susceptibility to the thymine nucleoside analogue, bromodeoxyuridine, was reduced. Thus, EBV-PK but not EBV-TK mediates ACV and GCV susceptibilities.

Background & objectives: Culex quinquefasciatus is a peridomestic mosquito known for its ability to transmit pathogenic diseases such as filariasis and Japanese encephalitis. The development and use of novel and innovative vector control measures such as the utilization of Wolbachia, along with the existing ones, are necessary to prevent the transmission of these diseases. Studies exploring the diversity of Wolbachia, particularly in Cx. quinquefasciatus are very limited in the Philippines. Thus, the aim of the study was to detect the presence, distribution, and phylogenetic relationship of Wolbachia infections in Cx. quinquefasciatus in Metropolitan Manila, Philippines. Methods: Adult Cx. quinquefasciatus mosquitoes were collected using a commercially available light-trap from May 2014–January 2015. Based on their sampling grids (n = 51), the adult mosquito abdomens were pooled and subjected to Wolbachia surface protein (wsp) gene amplification assay. Five selected wsp-positive samples were then sequenced and further analyzed to infer their phylogenetic relationship with known Wolbachia strains. Results: A total of 1090 adult Cx. quinquefasciatus mosquitoes were collected. Pooled abdomens (n = 53) were then sorted based on their sampling grids for subsequent screening of wsp gene. Wolbachia infection rate was 59% (31/53). These infections were located at 29 (57%) sampling grids, and were observed to be widely distributed in the study area. Phylogenetic analysis indicated that the sample sequences were Wolbachia pipientis isolated from known hosts, Cx. pipiens and Cx. quinquefasciatus belonging to supergroup B clade. Interpretation & conclusion: The study was able to demonstrate the prevalence and distribution of Wolbachia in Cx. quinquefasciatus in Metropolitan Manila, Philippines. The findings of this study are geared towards proposing a vector control program that utilizes the potential of Wolbachia as a biological control agent in preventing the transmission of Culex-borne diseases.

Immune tolerance to tumor-associated self-antigens poses a major challenge in the ability to mount an effective cancer vaccine response. To overcome immune tolerance to HER-2, we formulated DNA vaccines that express both human HER-2 and heterologous rat Neu sequences in separate plasmids or as single hybrid constructs that encode HER-2/Neu fusion proteins. Candidate vaccines were tested in Her-2 transgenic (Tg) mice of BALB/c (BALB), BALB/c × C57BL/6 F1 (F1), or C57BL/6 (B6) background, which exhibit decreasing immune responsiveness to HER-2. Analysis of various cocktails or hybrid vaccines defined a requirement for particular combination of HER/2/Neu sequences to effectively prime immune effector cells in HER-2 Tg mice. In B6 HER-2 Tg mice, rejection of HER-2–positive tumors protected mice from HER-2–negative tumors, providing evidence of epitope spreading. Our findings show that a strategy of combining heterologous antigen with self-antigens could produce a potent DNA vaccine that may be applicable to other tumor-associated antigens. Cancer Res; 70(1); 119–28

Domestic cats share human living environments and genetic traits. They develop spontaneous feline mammary carcinoma (FMC) with similar histopathology to human breast cancer. HER2 and AKT phosphorylation was demonstrated in primary FMC by immunoblot analysis, indicating HER2 as a therapeutic target. FMC lines K12 and K248 expressing HER1, HER2, and HER3 were sensitive to receptor tyrosine kinase (RTK) inhibitors gefitinib and lapatinib. To test HER2 vaccine response in cats, purpose-bred, healthy cats were electrovaccinated with heterologous (xenogeneic) or point-mutated feline HER2 DNA. T-cell reactivity to feline self-HER2 was detected in 4 of 10 cats that received bear HER2, human–rat fusion HER2 (E2Neu) or mutant feline HER2 (feHER2-K), which contains a single amino acid substitution. The variable T-cell responses may resemble that in the genetically heterogeneous human population. All immune sera to heterologous HER2 recognized feline HER2 expressed in 3T3 cells (3T3/HER2), but not that in FMC K12 or K248. Immune sera to mutant pfeHER2-K bound 3T3/HER2 cells weakly, but they showed better recognition of K12 and K248 cells that also express HER1 and HER3, suggesting distinct HER2 epitopes displayed by FMC that may be simulated by feHER2-K. In summary, HER2 DNA electroporation overcomes T-cell immune tolerance in approximately 40% of healthy cats and induces antibodies with distinct specificity. Vaccination studies in domestic cats can expedite vaccine iteration to guide human vaccine design and better predict outcome, with the added benefit of helping feline mammary tumor patients. Cancer Immunol Res; 3(7); 777–86. ©2015 AACR.

Malignancies associated with latent Epstein-Barr virus (EBV) are resistant to nucleoside-type antiviral agents because the viral enzyme target of these antiviral drugs, thymidine kinase (TK), is not expressed. Short-chain fatty acids, such as butyrate, induce EBV-TK expression in latently infected B cells. As butyrate has been shown to sensitize EBV+ lymphoma cells in vitro to apoptosis induced by ganciclovir, arginine butyrate in combination with ganciclovir was administered in 15 patients with refractory EBV+ lymphoid malignancies to evaluate the drug combination for toxicity, pharmacokinetics, and clinical responses. Ganciclovir was administered twice daily at standard doses, and arginine butyrate was administered by continuous infusion in an intrapatient dose escalation, from 500 mg/(kg/day) escalating to 2000 mg/(kg/day), as tolerated, for a 21-day cycle. The MTD for arginine butyrate in combination with ganciclovir was established as 1000 mg/(kg/day). Ten of 15 patients showed significant antitumor responses, with 4 CRs and 6 PRs within one treatment cycle. Complications from rapid tumor lysis occurred in 3 patients. Reversible somnolence or stupor occurred in 3 patients at arginine butyrate doses of greater than 1000 mg/(kg/day). The combination of arginine butyrate and ganciclovir was reasonably well-tolerated and appears to have significant biologic activity in vivo in EBV+ lymphoid malignancies which are refractory to other regimens.

An 81-year-old male patient suffered from recurrent acute hemolytic transfusion reactions after transfusion with phenotyped cross-match-negative red blood cells (RBCs). Extensive posttransfusion workup eventually revealed Dombrock (a) (Do(a)) antibodies. Because commercially available cell panels do not allow for identification of anti-Do(a) and owing to the lack of Do(a) typing serum samples, selection of matched units of RBCs is dependent on negative cross-match results. In this case, selection of Do(a-) units by cross-matching failed, indicating that serologic methods were not reliable. A polymerase chain reaction with sequence-specific priming assay was used to detect DOA and DOB alleles, which encode Do(a) and Do(b) antigens, respectively. The patient was confirmed to be DOB/DOB by DNA sequencing. Furthermore, the involved mismatched units in each of the three hemolytic episodes were shown to be Do(a+). In the presenting case, DNA typing appeared to be superior to serologic methods in selecting matched RBC units in the presence of anti-Do(a).

The nucleoside kinase encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is a relatively inefficient enzyme with substrate specificity for thymidine alone, unlike alphaherpesvirus thymidine kinases (TKs). Similar to all gammaherpesvirus TKs, KSHV TK is composed of two distinct domains, a conserved C-terminal kinase and a novel and uncharacterized N terminus. Ectopic expression of KSHV TK in adherent cells induced striking morphological changes and anchorage independence although cells survived, a property shared with the related rhadinovirus TKs of rhesus monkey rhadinovirus and herpesvirus saimiri. To determine whether KSHV TK served alternate functions relevant to the rhadinovirus life cycle and to reveal the contribution of the N terminus, an enhanced green fluorescent protein-tagged fusion protein and serial mutants were generated for investigation of intracellular localization and cell biology. Analysis of truncation mutants showed that a proline-rich region located within the N terminus cooperated with the conserved C-terminal kinase to tether KSHV TK to a reticular network in the cytoplasm and to induce morphological change. Fusion of the KSHV N terminus to herpes simplex virus type 1 TK, a nucleus-localized enzyme, similarly resulted in cytoplasmic redistribution of the chimeric protein but did not alter cell shape or adhesion. Unlike other human herpesvirus TKs, KSHV TKs and related rhadinovirus TKs are constitutively tyrosine phosphorylated; a KSHV TK mutant that was hypophosphorylated failed to detach and grow in suspension. Loss of adhesion may enhance terminal differentiation, viral replication, and egress at the cellular level and at the organism level may facilitate detachment and distant migration of KSHV-replicating cells within body fluids—promoting oropharyngeal transmission and perhaps contributing to the multifocal lesions that characterize KS.

CD21 is a multifunctional receptor for Epstein-Barr virus (EBV), for C3dg and for CD23. Upon engagement of immune complexes CD21 modulates immunoreceptor signaling, linking innate and adaptive immune responses. The mechanisms enabling CD21 to independently relay information between the exterior and interior of the cell, however, remain unresolved. We show that formin homologue overexpressed in spleen (FHOS/FHOD1) binds the cytoplasmic domain of human CD21 through its C terminus. When expressed in cells, EGFP-FHOS localizes to the cytoplasm and accumulates with actin in membrane protrusions. Plasma membrane aggregation, redistribution and co-localization of both proteins are stimulated when EBV (ligand) binds CD21. Though widely expressed, FHOS RNA is most abundant in the littoral cell, a major constituent of the red pulp of human spleen believed to function in antigen filtration. Formins are molecular scaffolds that nucleate actin by a pathway distinct from Arp2/3 complex, linking signal transduction to actin reorganization and gene transcription. Thus, ligand stimulation of FHOS-CD21 interaction may transmit signals through promotion of cytoskeletal rearrangement. Moreover, formin recruitment to sites of actin assembly initiated by immunoreceptors could be a general mechanism whereby co-receptors such as CD21 modulate intracellular signaling.

Endochondral bone formation has been fairly well characterized from a morphological perspective and yet this process remains largely undefined at molecular and biochemical levels. In vitro and in vivo studies have shown that human bone morphogenetic protein-2 (hBMP-2) is an important developmental growth and differentiation factor, capable of inducing ectopic bone formation in vivo. This study evaluated several aspects of the osteogenic effect of hBMP-2 protein injected into quadriceps of female C57B1/6J SCID mice. Mice were euthanized 1, 2, 3, 4, 7, and 14 days postinjection and muscles were collected for several methods of analysis. Hematoxylin and eosin-stained sections of muscles injected with formulation buffer showed no evidence of osteogenesis. In contrast, sections of muscles injected with hBMP-2 showed evidence of endochondral bone formation that progressed to mineralized bone by day 14. In addition, radiographs of mice injected with hBMP-2 showed that much of the quadriceps muscle had undergone mineralization by day 14. Labeled mRNA solutions were prepared and hybridized to oligonucleotide arrays designed to monitor approximately 1300 murine, full-length genes. Changes in gene expression associated with hBMP-2 were determined from time-matched comparisons between buffer and hBMP-2 samples. A gene expression profile was created for 215 genes that showed greater than 4-fold changes at one or more of the indicated time points. One hundred twenty-two of these genes have previously been associated with bone or cartilage metabolism and showed significant increases in expression, e.g., aggrecan (Agc1), runt related transcription factor 2 (Runx2), bone Gla protein 1 (Bglap1), and procollagens type II (Col2a1) and X (Col10a1). In addition, there were 93 genes that have not been explicitly associated with bone or cartilage metabolism. Two of these genes, cytokine receptor-like factor-1 (Crlf1) and matrix metalloproteinase 23 (Mmp23), showed peak changes in gene expression of 15- and 40-fold on days 4 and 7, respectively. In situ hybridizations of muscle sections showed that Mmp23 and Crlf1 mRNAs were expressed in chondrocytes and osteoblasts, suggesting a role for both proteins in some aspect of cartilage or bone formation. In conclusion, oligonucleotide arrays enabled a broader view of endochondral bone formation than has been reported to date. An increased understanding of the roles played by these gene products will improve our understanding of skeletogenesis, fracture repair, and pathological conditions such as osteoporosis.

Iogen (Canada) is a major manufacturer of industrial cellulase and hemicellulase enzymes for the textile, pulp and paper, and poultry feed industries. Iogen has recently constructed a 40 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. The integration of enzyme and ethanol plants results in significant reduction in production costs and offers an alternative use for the sugars generated during biomass conversion. Iogen has partnered with the University of Toronto to test the fermentation performance characteristics of metabolically engineered Zymomonas mobilis created at the National Renewable Energy Laboratory. This study focused on strain AX101, a xylose- and arabinose-fermenting stable genomic integrant that lacks the selection marker gene for antibiotic resistance. The "Iogen Process" for biomass depolymerization consists of a dilute-sulpfuric acid-catalyzed steam explosion, followed by enzymatic hydrolysis. This work examined two process design options for fermentation, first, continuous cofermentation of C5 and C6 sugars by Zm AX101, and second, separate continuous fermentations of prehydrolysate by Zm AX101 and cellulose hydrolysate by either wildtype Z. mobilis ZM4 or an industrial yeast commonly used in the production of fuel ethanol from corn. Iogen uses a proprietary process for conditioning the prehydrolysate to reduce the level of inhibitory acetic acid to at least 2.5 g/L. The pH was controlled at 5.5 and 5.0 for Zymomonas and yeast fermentations, respectively. Neither 2.5 g/L of acetic acid nor the presence of pentose sugars (C6:C5 = 2:1) appreciably affected the high-performance glucose fermentation of wild-type Z. mobilis ZM4. By contrast, 2.5 g/L of acetic acid significantly reduced the rate of pentose fermentation by strain AX101. For single-stage continuous fermentation of pure sugar synthetic cellulose hydrolysate (60 g/L of glucose), wild-type Zymomonas exhibited a four-fold higher volumetric productivity compared with industrial yeast. Low levels of acetic acid stimulated yeast ethanol productivity. The glucose-to-ethanol conversion efficiency for Zm and yeast was 96 and 84%, respectively.

IOGEN Corporation of Ottawa, Canada, has recently built a 40t/d biom-ass-to-ethanol demonstration plant adjacent to its enzyme production facility. It has partnered with the University of Toronto to test the C6/C5 cofermenta-tion performance characteristics of the National Renewable Energy Labora-tory’s metabolically engineered Zymomonas mobilis using various biomass hydrolysates. IOGEN’s feedstocks are primarily agricultural wastes such as corn stover and wheat straw. Integrated recombinant Z. mobilis strain AX101 grows on D-xylose and/or L-arabinose as the sole carbon/energy sources and ferments these pentose sugars to ethanol in high yield. Strain AX101 lacks the tetracycline resistance gene that was a common feature of other recombinant Zm constructs. Genomic integration provides reliable cofermentation performance in the absence of antibiotics, another characteristic making strain AX101 attractive for industrial cellulosic ethanol production. In this work, IOGEN’s biomass hydrolysate was simulated by a pure sugar medium containing 6% (w/v) glucose, 3% xylose, and 0.35% arabinose. At a level of 3 g/L (dry solids), corn steep liquor with inorganic nitrogen (0.8 g/L of ammonium chloride or 1.2 g/L of diammonium phosphate) was a cost-effective nutritional supplement. In the absence of acetic acid, the maximum volumetric ethanol productivity of a continuous fermentation at pH 5.0 was 3.54 g/L·h. During prolonged continuous fermentation, the efficiency of sugar-to-ethanol conversion (based on total sugar load) was maintained at >85%. At a level of 0.25% (w/v) acetic acid, the productivity decreased to 1.17 g/L·h at pH 5.5. Unlike integrated, xylose-utilizing rec Zm strain C25, strain AX101 produces less lactic acid as byproduct, owing to the fact that the Escherichia coli arabinose genes are inserted into a region of the host chromosome tentatively assigned to the gene for D-lactic acid dehydrogenase. In pH-controlled batch fermentations with sugar mixtures, the order of sugar exhaustion from the medium was glucose followed by xylose and arabinose. Both the total sugar load and the sugar ratio were shown to be important determinants for efficient cofermentation. Ethanol at a level of 3% (w/v) was implicated as both inhibitory to pentose fermentation and as a potentiator of acetic acid inhibition of pentose fermentation at pH 5.5. The effect of ethanol may have been underestimated in other assessments of acetic acid sensitivity. This work underscores the importance of employing similar assay conditions in making comparative assessments of biocatalyst fermentation performance.

This work represents a continuation of our investigation into environmental conditions that promote lactic acid synthesis by Zymomonas mobilis. The characteristic near theoretical yield of ethanol from glucose by Z. mobilis can be compromised by the synthesis of D- and L-lactic acid. The production of lactic acid is exacerbated by the following conditions: pH 6.0, yeast extract, and reduced growth rate. At a specific growth rate of 0.048/h, the average yield of DL-lactate from glucose in a yeast extract-based medium at pH 6.0 was 0.15 g/g. This represents a reduction in ethanol yield of about 10% relative to the yield at a growth rate of 0.15/h. Very little lactic acid was produced at pH 5.0 or using a defined salts medium (without yeast extract) Under permissive and comparable culture conditions, a tetracycline-resistant, D-ldh negative mutant produced about 50% less lactic acid than its parent strain Zm ATCC 39676. D-lactic acid was detected in the cell-free spent fermentation medium of the mutant, but this could be owing to the presence of a racemase enzyme. Under the steady-state growth conditions provided by the chemostat, the specific rate of glucose consumption was altered at a constant growth rate of 0.075/h. Shifting from glucose-limited to nitrogen-limited growth, or increasing the temperature, caused an increase in the specific rate of glucose catabolism. There was good correlation between an increase in glycolytic flux and a decrease in lactic acid yield from glucose. This study points to a mechanistic link between the glycolytic flux and the control of end-product glucose metabolism. Implications of reduced glycolytic flux in pentose-fermenting recombinant Z. mobilis strains, relative to increased byproduct synthesis, is discussed.

Epstein-Barr virus (EBV) attachment to human CD21 on the B-cell surface initiates infection. Whether CD21 is a simple tether or conveys vital information to the cell interior for production of host factors that promote infection of primary B cells is controversial, as the cytoplasmic fragment of CD21 is short, though highly conserved. The ubiquity of CD21 on normal B cells, the diversity of this population, and the well-known resistance of primary B cells to gene transfer technologies have all impeded resolution of this question. To uncover the role(s) of the CD21 cytoplasmic domain during infection initiation, the full-length receptor (CD21 = CR), a mutant lacking the entire cytoplasmic tail (CT), and a control vector (NEO) were stably expressed in two pre-B-cell lines that lack endogenous receptor. Genome-wide transcriptional analysis demonstrated that stable CD21 surface expression alone (either CR or CT) produced multiple independent changes in gene expression, though both dramatically decreased class I melanoma-associated antigen (MAGE) family RNAs and upregulated genes associated with B-cell differentiation (e.g., C2TA, HLA-II, IL21R, MIC2, CD48, and PTPRCAP/CD45-associated protein). Temporal analysis spanning 72 h revealed that not only CR- but also CT-expressing lines initiated latency. In spite of this, the number and spectrum of transcripts altered in CR- compared with CT-bearing lines at 1 h after infection further diverged. Differential modulation of immediate early cellular transcripts (e.g., c-Jun and multiple histones), both novel and previously linked to CD21-initiated signaling, as well as distinct results from pathway analyses support a separate role for the cytoplasmic domain in initiation of intracellular signals. IMPORTANCE Membrane proteins that mediate virus attachment tether virus particles to the cell surface, initiating infection. In addition, upon virus interaction such proteins may transmit signals to the interior of the cell that support subsequent steps in the infection process. Here we show that expression of the Epstein-Barr virus B-cell attachment receptor, CD21, in B cells that lack this receptor results in significant changes in gene expression, both before and rapidly following EBV-CD21 interaction. These changes translate into major signaling pathway alterations that are predicted to support stable infection.

In pH-controlled batch fermentations with pure sugar synthetic hardwood hemicellulose (1% [w/v] glucose and 4% xylose) and corn stover hydrolysate (8% glucose and 3.5% xylose) lacking acetic acid, the xylose-utilizing, tetracycline (Tc)-sensitive, genomically integrated variant of Zymomonas mobilis ATCC 39676 (designated strain C25) exhibited growth and fermentation performance that was inferior to National Renewable Energy Laboratory's first-generation, Tc-resistant, plasmid-bearing Zymomonas recombinants. With C25, xylose fermentation following glucose exhaustion was markedly slower, and the ethanol yield (based on sugars consumed) was lower, owing primarily to an increase in lactic acid formation. There was an apparent increased sensitivity to acetic acid inhibition with C25 compared with recombinants 39676:pZB4L, CP4:pZB5, and ZM4:pZB5. However, strain C25 performed well in continuous fermentation with nutrient-rich synthetic corn stover medium over the dilution range 0.03-0.06/h, with a maximum process ethanol yield at D = 0.03/h of 0.46 g/g and a maximum ethanol productivity of 3 g/(L x h). With 0.35% (w/v) acetic acid in the medium, the process yield at D = 0.04/h dropped to 0.32 g/g, and the maximum productivity decreased by 50% to 1.5 g/(L x h). Under the same operating conditions, rec Zm ZM4:pZB5 performed better; however, the medium contained 20 mg/L of Tc to constantly maintain selective pressure. The absence of any need for antibiotics and antibiotic resistance genes makes the chromosomal integrant C25 more compatible with current regulatory specifications for biocatalysts in large-scale commercial operations.

Lentiviral vectors transduce dividing and postmitotic cells and thus are being developed toward therapies for many diseases affecting diverse tissues. One essential requirement for efficacy will be that vector particles are resistant to inactivation by human serum complement. Most animal studies with lentiviral vectors have utilized VSV-G pseudotyped envelopes. Here we demonstrate that VSV-G pseudotyped HIV and FIV vectors produced in human cells are inactivated by human serum complement, suggesting that alternative envelopes may be required for therapeutic efficacy for many clinical applications of lentiviral vectors.

The sequenced gammaherpesviruses each contain a single viral bcl-2 homolog (v- bcl-2 ) which may encode a protein that functions in preventing the apoptotic death of virus-infected cells. Epstein-Barr virus (EBV), a gammaherpesvirus associated with several lymphoid and epithelial malignancies, encodes the v-Bcl-2 homolog BHRF1. In this report the previously uncharacterized BALF1 open reading frame in EBV is identified as having significant sequence similarity to other v- bcl-2 homologs and cellular bcl-2 . Transfection of cells with a BALF1 cDNA conferred apoptosis resistance. Furthermore, a recombinant green fluorescent protein-BALF1 fusion protein suppressed apoptosis and associated with Bax and Bak. These results indicate that EBV encodes a second functional v- bcl-2 .

Whether Kaposi’s sarcoma herpesvirus (KSHV) is associated with multiple myeloma (MM) remains controversial. We assayed for KSHV DNA sequences in long-term bone marrow stromal cells (BMSCs) from 26 patients with MM and 4 normal donors. Polymerase chain reaction (PCR) using primers which amplify a KSHV gene sequence to yield a 233-bp fragment (KS330233 within open reading frame 26) was negative in all cases. Aliquots of these PCR products were used as templates in subsequent nested PCR, with primers that amplify a 186-bp product internal to KS330233. BMSCs from 24 of 26 (92%) patients with MM and 1 of 4 normal donors were KSHV PCR+. DNA sequence analyses showed interpatient specific mutations (2 to 3 bp). Both Southern blot and sequence analyses confirmed the specificity of PCR results. The presence of the KSHV gene sequences was further confirmed by amplifying T 1.1 (open reading frame [ORF] K7) and viral cyclin D (ORF 72), two other domains within the KSHV genome. Immunohistochemical studies of KSHV PCR+ MM BMSCs demonstrate expression of dendritic cell (DC) lineage markers (CD68, CD83, and fascin). Serological studies for the presence of KSHV lytic or latent antibodies were performed using sera from 53 MM patients, 12 normal donors, and 5 human immunodeficiency virus (HIV)/KSHV+ patients. No lytic or latent antibodies were present in sera from either MM patients or normal donors. Taken together, these findings show that KSHV DNA sequences are detectable in BMSCs from the majority of MM patients, but that serologic responses to KSHV are not present. Ongoing studies are defining whether the lack of antibody response is caused by the absence of ongoing infection, the presence of a novel viral strain associated with MM, or underlying immunodeficiency in these patients.

The Epstein-Barr virus (EBV) thymidine kinase (TK) was expressed in mammalian 143B TK − cells to investigate its substrate specificity. The herpes simplex virus type 1 (HSV-1) TK was similarly expressed for comparison. Both viral TKs conferred a TK + phenotype on 143B TK − cells. The nucleoside analog ganciclovir (GCV) did not affect the growth of 143B EBV TK or 143B TK − cells but effectively killed 143B HSV-1 TK cells. Furthermore, lysates of 143B EBV TK cells could not phosphorylate GCV, which was confirmed by high-performance liquid chromatography. EBV TK, HSV-1 TK, and EBV TK N − , a truncated EBV TK missing 243 N-terminal amino acids, were purified as fusion proteins expressed in bacteria, and all had TK activity. In addition, EBV TK was observed to have a thymidylate kinase activity but could not phosphorylate GCV, acyclovir, or 2′-deoxycytidine. In competition assays, only nucleoside analogs of thymidine significantly inhibited thymidine phosphorylation by EBV TK, with the following rank order: 5-bromodeoxyuridine > zidovudine > stavudine > sorivudine. These results demonstrate that EBV TK substrate specificity is narrower than those of alphaherpesvirus TKs and that thymidine analogs may be the most suitable nucleoside antivirals to target the enzyme. Clinical implications for gammaherpesviruses are discussed.

In large and complex vectors a single restriction enzyme recognition site may be available for introduction of additional DNA requiring the development of linker fragments to create compatible insertion sites. This technology can be time consuming and costly. We describe the construction of a simple phagemid, pSFI, with a polylinker that contains six pairs of dual, rare-cutting, restriction enzyme recognition sites (NotI,SpeI,EcoRV,PstI,SacII,EagI) with multiple unique sites between each pair. This has permitted rapid subcloning of DNA with creation of single flanking restriction enzyme sites. pSFI was used to expedite transfer of viral genes to a LacZ-inducible expression vector and to an adenovirus expression cassette for production of replication-defective virus. The use of this phagemid has facilitated complex vector manipulations and is a valuable adjunct to the family of multifunctional cloning vectors.

The effects of nitric oxide (NO) on vascular reactivity and platelet function in the obese (cp/cp) and lean (+/?) JCR:LA-cp rats were investigated. Phenylephrine (PE; 0.1 nM–10 μM) induced contraction of isolated aortic rings in both genotypes (cp/cp and +/?) of JCR:LA-cp rats. The sensitivity to contraction with PE was enhanced in cp/cp compared with +/? rings. Rings from both genotypes showed an increased contraction upon removal of the endothelium. Acetylcholine (ACh; 0.1 nM–10 μM)-induced endothelium-dependent relaxation of rings was not significantly different in the two genotypes. Both were inhibited to a similar extent by NG-nitro-L-arginine methyl ester (L-NAME; 0.01–1 mM) when administered in vitro. The nitric oxide synthase (NOS) inhibitor (L-NAME; 0.3, 1 or 3 mg ml−1, p.o.) when administered in vivo increased blood pressure in cp/cp rats but not in +/? rats. L-NAME resulted in greater inhibition of ACh-induced relaxation in cp/cp rings compared with +/? rings. L-NAME treatment in vivo caused a decrease in cyclic GMP and NOS activity in rings from cp/cp but not +/? rats. The NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 0.1 nM–10 μM)-induced relaxation of rings from +/? rats, an effect enhanced by the treatment with L-NAME in vivo. Oral administration of L-NAME did not enhance the vasorelaxant effect of SNAP on rings of aorta from cp/cp animals. Platelet aggregation and NOS activity were similar in both genotypes and were not modified by oral administration of L-NAME. These results show that unimpaired generation of NO is crucial for maintenance of vascular tone particularly under conditions of vascular insult exemplified by insulin resistance, obesity and dyslipidemia detected in cp/cp rats.

Prevention of hepatitis B virus infection in transplant recipients can be difficult. Patients may be unresponsive to vaccination and intolerant of the intramuscular injections required to administer hepatitis B immune globulin (HBIG). A recipient of HBsAg-positive donor cells for a bone marrow transplant received multiple i.m. injections of HBIG. This mode of antibody delivery was limited by his thrombocytopenia and neutropenia and alternative forms of passive immunization were sought. Four lots of IGIV were investigated for anti-hepatitis B surface antibody (anti-HBs) content and all were found to contain significant antibody titer. Moreover, IGIV that was administered to four bone marrow transplant recipients for medical purposes unrelated to HBV transmission produced protective anti-HBs titers in all. These studies suggest IGIV may be useful for HBV prophylaxis in the appropriate setting or if HBIG is unavailable. The optimum regimen for HBV prevention in distinct transplant settings needs to be determined.

This study documents the similar pH-dependent shift in pyruvate metabolism exhibited by Zymomonas mobilis ATCC 29191 and ATCC 39676 in response to controlled changes in their steady-state growth environments. The usual high degree of ethanol selectivity associated with glucose fermentation by Z. mobilis is associated with conditions that promote rapid and robust growth, with about 95% of the substrate (5% w/v glucose) being converted to ethanol and C)2, and the remaining 5% being used for the synthesis of cell mass. Conditions that promote energetic uncoupling cause the conversion efficiency to increase to 98% as a result of the reduction in growth yield (cell mass production). Under conditions of glucose-limited growth in a chemostat, with the pH controlled at 6.0, the conversion efficiency was observed to decrease from 95% at a specific growth rate of 0.2/h to only 80% at 0.042/h. The decrease in ethanol yield was solely attributable to the pH-dependent shift in pyruvate metabolism, resulting in the production of lactic acid as a fermentation byproduct. At a dilution rate (D) of 0.042/h, decreasing from pH 6.0 to 5.5 resulted in a decrease in lactic acid from 10.8 to 7.5 g/L. Lactic acid synthesis depended on the presence of yeast extract (YE) or tryptone in the 5% (w/v) glucose-mineral salts medium. At D = 0.15/h, reduction in the level of YE from 3 to 1 g/L caused a threefold decrease in the steady-state concentration of lactic acid at pH 6. No lactic acid was produced with the same mineral salts medium, with ammonium chloride as the sole source of assimilable nitrogen. With the defined salts medium, the conversion efficiency was 98% of theoretical maximum. When chemostat cultures were used as seed for pH-stat batch fermentations, the amount of lactic acid produced correlated well with the activity of the chemostat culture; however, the mechanism of this prolonged induction effect is unknown. The levels of lactic acid produced by Z. mobilis in this study have not been previously reported. Zymomonas is Gram-negative, and at no time did microscopic inspection of lactic-acid-producing cultures indicate the presence of Gram-positive organisms. Although these observations are very preliminary in nature, they have implications for the regulation of glycolytic flux in Zymomonas, and demonstrate the possibility of an alternative fate for pyruvate previously presumed not to exits.

The infectious bronchitis virus (IBV) spike glycoprotein S1 subunit is required to initiate infection and contains virus-neutralizing and serotype-specific epitope(s). Reported are the S1 gene nucleotide and predicted amino acid sequences for the Florida 18288 strain and isolates GA-92, CV-56b, CV-9437, CV-1686, and 1013. These sequences were compared with previously published gene sequences of IBV strains, and phylogenetic relationships are reported. The S1 amino acid sequence of Florida 18288 was 94.9% similar to the Connecticut strain, and GA-92 was 92.8% similar to the Arkansas 99 strain. S1 amino acid sequences of the California variants, CV-56b, CV-9437, and CV-1686, were 97.6–99.3% similar to one another and only 76.6%–76.8% similar to the Arkansas-type strains. Isolate 1013, also from California, was 84.0% similar to Ark DPI and 77.9% similar to CV-56b. When comparing 19 viruses isolated from the United States, sequence variations were observed between amino acids 55–96, 115–149, 255–309, and 378–395. Similar regions are reported to be involved in virus-neutralizing and/or serotype-specific epitopes. These data demonstrate that variant IBV strains continue to emerge, and unique variants may circulate among poultry in geographically isolated areas.

Insulin resistance is associated with an increased risk of cardiovascular disease that is probably related to abnormalities of vascular wall function. The JCR:LA-cp rat is a unique animal model of human vascular disease that exhibits a profound insulin resistance, vasculopathy, and cardiovascular disease, and allows study of the relationships between insulin resistance and vascular function. Conductance and resistance arteries serve different functions, thus vascular disease may affect these types of artery differently. Concentration-response curves to norepinephrine, phenylephrine, and acetylcholine were studied in conductance vessels (aortic rings) and resistance vessels (mesenteric arteries) from 12-week-old male obese and lean JCR:LA-cp rats. The aortic rings and mesenteric arteries from obese rats showed increased maximal response to phenylephrine compared with those from lean rats, whereas only the mesenteric arteries from obese rats showed increased maximal response to norepinephrine. In aortic rings, relaxation to acetylcholine was similar for both genotypes, but the mesenteric arteries of obese rats showed impaired relaxation to acetylcholine. We conclude that the sensitivity to vasoconstriction is enhanced in aortic rings and mesenteric arteries of obese male JCR:LA-cp rats, but endothelial function is impaired only in the mesenteric resistance arteries of these animals. Hence functional aberrations in the obese, insulin-resistant state are more pronounced in mesenteric resistance arteries than in a major conductance artery.Key words: insulin resistance, arterial contraction and relaxation, resistance vessels, JCR:LA-cp rat, smooth muscle cells,endothelium.

Mutations in the hepatocyte nuclear factor–1α (HNF-1α) gene have recently been shown to cause maturityonset diabetes of the young (MODY). We have examined 15 U.K. MODY families for mutations in the coding region of the HNF-1α gene. Eight different mutations, three frameshift (P291fsinsC, P379fsdelCT, and A443fsdelCA) and five missense mutations (P129T, R131W, R159W, P519L, and T620I), were identified in eleven families (73%). The previously reported mutation P291fsinsC was found in four pedigrees. A screen of a further 32 probands with early onset (