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Her2 overexpression is associated with an aggressive form of breast cancer and malignant transformation. We demonstrate in this work that nanolipoprotein particles (NLPs) synthesized in a cell-free manner can be used to transfer Her2 protein into the membrane of nonmalignant cells in 3D culture in a nontoxic and facile manner. With NLP-mediated Her2 protein delivery, we observed an increased probability of nonmalignant cells forming apolar nongrowth-arrested tumor-like structures. The NLP delivery system alone or Her2-NLPs plus the Her2 inhibitor trastuzumab showed no effect on the acinar organization rate, indicating that Her2 signaling is key to this process. Transcriptomics revealed essentially no effect of empty NLPs compared to untreated cells, whereas Her2-NLPs versus either untreated or empty-NLP-treated cells revealed upregulation of several factors associated with breast cancer. Pathway analysis also suggested that known nodes downstream of Her2 were activated in response to Her2-NLP treatment. This demonstrates that Her2 protein delivery with NLPs is sufficient for the malignant transformation of nonmalignant cells. Thus, this system offers a new model for studying cell surface receptor signaling without genomic modification or transformation techniques.

(131)I-metaiodobenzylguanidine ((131)I-mIBG) is a targeted radiation therapy developed for the treatment of advanced neuroblastoma. We have previously shown that this patient cohort can be used to predict absorbed dose associated with early (131)I exposure, 72 h after treatment. We now expand these studies to identify gene expression differences associated with (131)I-mIBG exposure 15 days after treatment. Total RNA from peripheral blood lymphocytes was isolated from 288 whole blood samples representing 59 relapsed or refractory neuroblastoma patients before and after (131)I-mIBG treatment. We found that several transcripts predictive of early exposure returned to baseline levels by day 15, however, selected transcripts did not return to baseline. At 72 h, all 17 selected pathway-specific transcripts were differentially expressed. Transcripts CDKN1A (P < 0.000001), FDXR (P < 0.000001), DDB2 (P < 0.000001), and BBC3 (P < 0.000001) showed the highest up-regulation at 72 h after (131)I-mIBG exposure, with mean log(2) fold changes of 2.55, 2.93, 1.86 and 1.85, respectively. At day 15 after (131)I-mIBG, 11 of the 17 selected transcripts were differentially expressed, with XPC, STAT5B, PRKDC, MDM2, POLH, IGF1R, and SGK1 displaying significant up-regulation at 72 h and significant down-regulation at day 15. Interestingly, transcripts FDXR (P = 0.01), DDB2 (P = 0.03), BCL2 (P = 0.003), and SESN1 (P < 0.0003) maintained differential expression 15 days after (131)I-mIBG treatment. These results suggest that transcript levels for DNA repair, apoptosis, and ionizing radiation-induced cellular stress are still changing by 15 days after (131)I-mIBG treatment. Our studies showcase the use of biodosimetry gene expression panels as predictive biomarkers following early (72 h) and late (15 days) internal (131)I exposure. Our findings also demonstrate the utility of our transcript panel to differentiate exposed from non-exposed individuals up to 15 days after exposure from internal (131)I.

Introduction: Respiratory syncytial virus (RSV) is a common cause of infant hospitalization and mortality. With multiple vaccines in development, we aimed to determine: (1) the awareness of RSV among pregnant women and healthcare professionals (HCPs), and (2) attitudes toward clinical trials and routine implementation of antenatal RSV vaccination. Methods: Separate questionnaires for pregnant women and HCPs were distributed within 4 hospitals in South England (July 2017–January 2018). Results: Responses from 314 pregnant women and 204 HCPs (18% obstetricians, 75% midwives, 7% unknown) were analyzed. Most pregnant women (88%) and midwives (66%) had no/very little awareness of RSV, unlike obstetricians (14%). Among pregnant women, 29% and 75% would likely accept RSV vaccination as part of a trial, or if routinely recommended, respectively. Younger women (16–24 years), those of 21–30 weeks’ gestation, and with experience of RSV were significantly more likely to participate in trials [odds ratio (OR): 1.42 (1.72–9.86); OR: 2.29 (1.22–4.31); OR: 9.07 (1.62–50.86), respectively]. White-British women and those of 21–30 weeks’ gestation were more likely to accept routinely recommended vaccination [OR: 2.16 (1.07–4.13); OR: 2.10 (1.07–4.13)]. Obstetricians were more likely than midwives to support clinical trials [92% vs. 68%, OR: 2.50 (1.01–6.16)] and routine RSV vaccination [89% vs. 79%, OR: 4.08 (1.53–9.81)], as were those with prior knowledge of RSV, and who deemed it serious. Conclusions: RSV awareness is low among pregnant women and midwives. Education will be required to support successful implementation of routine antenatal vaccination. Research is needed to understand reasons for vaccine hesitancy among pregnant women and HCPs, particularly midwives.

A worldwide estimate of over one million STIs are acquired daily and there is a desperate need for effective preventive as well as therapeutic measures to curtail this global health burden. Vaccines have been the most effective means for the control and potential eradication of infectious diseases; however, the development of vaccines against STIs has been a daunting task requiring extensive research for the development of safe and efficacious formulations. Nanoparticle-based vaccines represent a promising platform as they offer benefits such as targeted antigen presentation and delivery, co-localized antigen-adjuvant combinations for enhanced immunogenicity, and can be designed to be biologically inert. Here we discuss promising types of nanoparticles along with outcomes from nanoparticle-based vaccine preclinical studies against non-viral STIs including chlamydia, syphilis, gonorrhea, and recommendations for future nanoparticle-based vaccines against STIs.

Her2 overexpression is associated with an aggressive form of breast cancer and malignant transformation. We sought to determine if a nano-carrier system could introduce Her2 protein to non-malignant cells and determine the effects on cell phenotype and transcriptome. With stimulation with Her2-NLPs, we observed uptake of Her2 protein and a decreased probability of individual non-malignant cells forming polar growth arrested acinar-like structures. The NLP delivery system alone or Her2-NLPs plus trastuzumab showed no effect on acinar organization rate. Transcriptomics revealed essentially no effect of empty NLPs versus untreated cells whereas Her2-NLPs versus either untreated or empty NLP treated cells revealed upregulation of several factors associated with breast cancer. Pathway analysis also suggested that known nodes downstream of Her2 were activated in response to Her2-NLP treatment. This suggests that Her2-NLPs are sufficient for malignant transformation of non-malignant cells and that this system offers a new model for studying cell surface receptor signaling without genomic modification or transformation techniques.

Cancer-associated fibroblasts (CAFs) are a prominent stromal cell type in solid tumors and molecules secreted by CAFs play an important role in tumor progression and metastasis. CAFs coexist as heterogeneous populations with potentially different biological functions. Although CAFs are a major component of the breast cancer stroma, molecular and phenotypic heterogeneity of CAFs in breast cancer is poorly understood. In this study, we investigated CAF heterogeneity in triple-negative breast cancer (TNBC) using a syngeneic mouse model, BALB/c-derived 4T1 mammary tumors. Using single-cell RNA sequencing (scRNA-seq), we identified six CAF subpopulations in 4T1 tumors including: 1) myofibroblastic CAFs, enriched for &alpha, smooth muscle actin and several other contractile proteins, 2) &lsquo, inflammatory&rsquo, CAFs with elevated expression of inflammatory cytokines, and 3) a CAF subpopulation expressing major histocompatibility complex (MHC) class II proteins that are generally expressed in antigen-presenting cells. Comparison of 4T1-derived CAFs to CAFs from pancreatic cancer revealed that these three CAF subpopulations exist in both tumor types. Interestingly, cells with inflammatory and MHC class II-expressing CAF profiles were also detected in normal breast/pancreas tissue, suggesting that these phenotypes are not tumor microenvironment-induced. This work enhances our understanding of CAF heterogeneity, and specifically targeting these CAF subpopulations could be an effective therapeutic approach for treating highly aggressive TNBCs.

Purpose Few tools exist to predict clinical outcomes after radiopharmaceutical therapy. Our goal was to identify associations between blood-based biomarkers of radiation effect and clinical outcomes after 131 I-metaiodobenzylguanidine ( 131 I-MIBG) therapy in patients with neuroblastoma. Methods and Materials We conducted a prospective, single-center cohort study in children with advanced neuroblastoma treated with 131 I-MIBG as monotherapy or in combination with systemic putative radiation sensitizers. We collected serial peripheral blood samples after 131 I-MIBG infusions and quantified a panel of protein and messenger RNA markers. We plotted relative change from baseline to assess degree of modulation over time and then evaluated association of marker modulation with toxicity and response endpoints. Results The cohort included 40 patients (30 male/10 female; median age 7 years). We observed significant modulation of the majority of markers between baseline and hour 72 after 131 I-MIBG. Greater fold increase of plasma FLT3 ligand was associated with subsequent grade 4 neutropenia ( P =.039). Modulation of peripheral blood BCLXL and DDB2 was associated with grade 3+ nonhematologic toxicity ( P =.043 and .048, respectively). No markers were associated with tumor response. Greater plasma FLT3 ligand, BCLXL , and BCL2 modulation was observed in patients receiving 131 I-MIBG in combination with radiation sensitizers. Among 9 patients who received 2 courses, the degree of modulation in serum amylase was significantly lower after the second course ( P =.012). Conclusions Peripheral blood biomarkers relevant to radiation exposure are significantly modulated during the acute period after 131 I-MIBG. The degree of modulation of a subset of these markers is associated with toxicity and receipt of concomitant radiation sensitizers.

Background Recombinant expression of toxic proteins remains a challenging problem. One potential method to shield toxicity and thus improve expression of these proteins is to encapsulate them within protein compartments to sequester them away from their targets. Many bacteria naturally produce so-called bacterial microcompartments (BMCs) in which enzymes comprising a biosynthetic pathway are encapsulated in a proteinaeous shell, which is in part thought to shield the cells from the toxicity of reaction intermediates. As a proof-of-concept, we attempted to encapsulate toxic, lysis protein E (E) from bacteriophage ϕX174 inside recombinant BMCs to enhance its expression and achieve higher yields during downstream purification. Results E was fused with various N-terminal BMC targeting tags (PduP-, PduD-, and EutC-tags, 18–20 amino acids) and co-expressed with appropriate BMC shell proteins that associate with the tags and are required to form BMCs. Only BMC targeted E fusions, but not non-tagged E, could be successfully cloned, suggesting that the BMC tags reduce the toxicity of E. A PduP-tagged E system appeared to achieve the highest expression of E. Co-expression of Pdu BMC shell proteins with PduP-E increased its expression by 20–50%. Affinity purification of PduP-E via Ni–NTA in the presence of Empigen BB detergent yielded 270 µg of PduP-E per L of induced culture. Removal of the PduP-tag via proteolysis resulted in a final yield of 200 µg of E per L of induced culture, a nearly order of magnitude (~sevenfold) improvement compared to prior reports. Conclusions These results demonstrate improved expression of ϕX174 lysis protein E via re-directed BMC systems and ultimately higher E purification yields. Similar strategies can be used to enhance expression of other toxic proteins in recombinant Escherichia coli systems. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0685-x) contains supplementary material, which is available to authorized users.

Tumor cells, including cancer stem cells (CSCs) resistant to radio- and chemotherapy, must enhance metabolism to meet the extra energy demands to repair and survive such genotoxic conditions. However, such stress-induced adaptive metabolic alterations, especially in cancer cells that survive radiotherapy, remain unresolved. In this study, we found that CPT1 (Carnitine palmitoyl transferase I) and CPT2 (Carnitine palmitoyl transferase II), a pair of rate-limiting enzymes for mitochondrial fatty acid transportation, play a critical role in increasing fatty acid oxidation (FAO) required for the cellular fuel demands in radioresistant breast cancer cells (RBCs) and radiation-derived breast cancer stem cells (RD-BCSCs). Enhanced CPT1A/CPT2 expression was detected in the recurrent human breast cancers and associated with a worse prognosis in breast cancer patients. Blocking FAO via a FAO inhibitor or by CRISPR-mediated CPT1A/CPT2 gene deficiency inhibited radiation-induced ERK activation and aggressive growth and radioresistance of RBCs and RD-BCSCs. These results revealed that switching to FAO contributes to radiation-induced mitochondrial energy metabolism, and CPT1A/CPT2 is a potential metabolic target in cancer radiotherapy.

BackgroundUptake rates of influenza and pertussis vaccination in pregnancy remain suboptimal.AimTo determine the acceptability of routine vaccination among pregnant women; the confidence of maternity healthcare professionals (HCPs) discussing vaccination; and HCP opinion with regards to the optimum healthcare site for vaccine administration.MethodSeparate questionnaires for pregnant women and maternity HCPs were distributed within four NHS trusts in South England from July 2017–January 2018.ResultsResponses from 314 pregnant women and 204 HCPs (18% obstetricians, 75% midwives [both hospital and community], 7% unidentified) were analysed. Actual/intended uptake of influenza and pertussis vaccination was 78% and 92%, respectively. The commonest reason for declining vaccination was feared side effects for their child. White British women (79%) were significantly more accepting of influenza (odds ratio [OR] 3.25, 95% confidence interval [CI] = 1.67 to 6.32) and pertussis vaccination (OR 4.83, 95% CI = 1.77 to 13.19) compared with non-white British women. Among HCPs, 25% were not-at-all or slightly confident discussing vaccination. Obstetricians felt significantly more confident discussing pertussis vaccination than midwives (OR 2.05, 95% CI = 1.02 to 4.12). Among HCPs, 53%, 25%, and 16% thought vaccines should be administered in primary care (general practice), community midwifery, and the hospital setting, respectively.ConclusionMisconceptions exist regarding safety and efficacy of maternal vaccination, and framing information towards safety for the child may increase uptake. Education of HCPs is essential, and vaccine promotion should be incorporated into routine antenatal care, with an emphasis on women from ethnic minorities. Administration of vaccines in primary care may present a logistical barrier to women, however support for alternative sites appears low among HCPs.

Chlamydia trachomatis is a sexually transmitted bacterium that infects over 130 million individuals worldwide annually. To implement a vaccine, we developed a cell-free co-translational system to express the Chlamydia muridarum major outer membrane protein (MOMP). This approach uses a nanolipoprotein particles (tNLP) made from ApoA1 protein, amphiphilic telodendrimer and lipids that self-assemble to form 10–25 nm discs. These tNLP provide a protein-encapsulated lipid support to solubilize and fold membrane proteins. The cell-free system co-translated MOMP and ApoA1 in the presence of telodendrimer mixed with lipids. The MOMP-tNLP complex was amenable to CpG and FSL-1 adjuvant addition. To investigate the ability of MOMP-tNLP+CpG+FSL-1 to induce protection against an intranasal (i.n.) C. muridarum challenge, female mice were vaccinated intramuscularly (i.m.) or i.n. and i.m. simultaneously 4 weeks apart. Following vaccination with MOMP-tNLP+CpG+FSL-1, mice mounted significant humoral and cell-mediated immune responses. Following the i.n. challenge, mice vaccinated with MOMP-tNLP+CpG+FSL-1 i.n. + i.m. group were protected as determined by the percentage change in body weight and by the number of C. muridarum inclusion forming units (IFU) recovered from the lungs. To our knowledge, this is the first time a MOMP-based vaccine formulated in tNLP has been shown to protect against C. muridarum.

The advances in the development of preclinical models for various cancers have tremendously helped study of the biology and genetics of human cancers as well as for development of therapeutics. Recently, patient-derived organoids (PDOs) have emerged as a robust preclinical platform that can provide insight into the patient specific genetic mechanisms, cancer progression and drug susceptibility. We have developed a novel PDO platform by embedding patient-derived organoids in a 3D hydrogel suspension culture. Our previous studies have shown that, when cultured in hydrogel, spheroids derived from cancer cell lines demonstrated upregulated gene expression signatures in relation to migration and angiogenesis compared to cultures in media, suggesting a more hypoxic, nutrient deficient environment. Given that hypoxia and nutrient deficiency are important feathers of solid tumors, the hydrogel environment could provide a good platform for in vitro study of tumor development and resistance to treatment. In the current study, we embedded PDOs derived from liver metastasis of colorectal cancer into the 3D suspension hydrogel culture. The PDOs adapted well in the hydrogel gel environment. We assessed the response of PDOs in hydrogel to anticancer agents, and compared that to those cultured in media. The PDOs cultured in hydrogel showed significantly elevated resistance to the treatments. The IC50s of chemotherapeutic drugs 5-fluorouracil and oxaliplatin are increased by ~5-10 fold when cultured in hydrogel compared to those in media. Further studies through RNAseq analysis revealed differentially activated signaling pathways. In particular, the levels of the CXC ligand family chemokines are significantly up-regulated in the PDOs cultured in the hydrogel. The CXC ligand family chemokines are important immune stimulants that are known to play key role in the chemotaxis and tumor recruitment of immune cells including neutrophil, T-cell and B-cells. Interestingly, the elevated levels of CXC-ligands have been shown to relate to resistance of colorectal cancer against drug treatment such as 5-fluorouracil. Our hydrogel model could help elucidate the mechanisms behind such resistance. Future study will focus on evaluating how well the hydrogel PDO platform can recapitulate tumor microenvironment and its potential in screening for personalized medicine. Citation Format: He Wei, Eveliina Karelehto, Karen Dubbin, Aimy Sebastian, Robert Warren, Monica Moya, Gaby Loots, Elizabeth Wheeler, Matthew Coleman. Developing 3d hydrogel model for patient-derived organoids of metastatic colorectal cancer [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 2638.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most incurable types of cancer. Cancer that develops in the acinar cells of the pancreas is typically not diagnosed until later stages, such as stage 3 or 4. As such, this form of cancer is particularly lethal with only 9% of patients reaching 5-year survival. PDAC is known to have a particularly dense extracellular matrix composed of fibroblasts, which have been previously shown to play an important role in promoting resistance to drug therapy. Characterization of the stromal networks involved in PDAC tumor development as well as protein markers of fibroblast subpopulations within the tumor stroma are critical to developing new fibroblast-targeted therapeutic approaches as well as understanding key signaling molecules that ultimately promote tumor progression and drug resistance. Single-cell RNA sequencing (scRNAseq) was utilized to analyze cancer-associated fibroblasts (CAFs) from KPC mouse-derived, MT3 subcutaneous murine allografts along with two fibroblast lines derived from human PDAC tumors: CRC-811 and IA-1340. Single cell gene expression profiling and subsequent analysis of MT3-derived CAFs resulted in the identification of three CAF subpopulations including a myofibroblast subpopulation that expressed high levels of smooth muscle actin (Acta2), which was also observed in both human samples. Fibroblast subpopulations enriched in Acta2 expression, expressed high levels of Wnt5a along with several other secreted factors including Tgfb1, Tgfb2 and Ctgf. Wnt5a is a secreted protein that activates non-canonical Wnt signaling pathways and is known to regulate normal developmental processes, including proliferation, differentiation, migration, adhesion and polarity. However Wnt5a is not natively expressed in MT3 cancer cells derived from syngeneic tumors, but potentially in the stroma. We hypothesize it is exclusively derived from fibroblasts. Previously it has been shown that Wnt5a inhibition suppressed gastric cancer metastasis, therefore further validation of the role of myofibroblast-derived Wnt5a on PDAC disease progression is warranted. This study received funding by LDRD 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM Release Number: LLNL-ABS-798442. Citation Format: Kelly A. Martin, Aimy Sebastian, Nicholas R. Hum, Stephen Byers, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Analysis of stromal myofibroblasts identifies secreted proteins that promote pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2757.

Anatomic pathologists are familiar with stains used in light microscopy to identify cells, storage products, tissue deposits, and pathogens. Assessment of the surrounding tissue with special stains may reveal aspects of interest for the tissue or the species. We illustrate the expected staining characteristics of normal rainbow trout gill tissue with routine hematoxylin and eosin and 18 other histochemical stains.

Modification of membrane receptor makeup is one of the most efficient ways to control input-output signals but is usually achieved by expressing DNA or RNA-encoded proteins or by using other genome-editing methods, which can be technically challenging and produce unwanted side effects. Here we develop and validate a nanodelivery approach to transfer in vitro synthesized, functional membrane receptors into the plasma membrane of living cells. Using β2-adrenergic receptor (β2AR), a prototypical G-protein coupled receptor, as an example, we demonstrated efficient incorporation of a full-length β2AR into a variety of mammalian cells, which imparts pharmacologic control over cellular signaling and affects cellular phenotype in an ex-vivo wound-healing model. Our approach for nanodelivery of functional membrane receptors expands the current toolkit for DNA and RNA-free manipulation of cellular function. We expect this approach to be readily applicable to the synthesis and nanodelivery of other types of GPCRs and membrane receptors, opening new doors for therapeutic development at the intersection between synthetic biology and nanomedicine.

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP–tNLP). The cell-free expressed mMOMP–tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP–tNLP complex in a 1-ml cell-free reaction. The mMOMP–tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP–tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.

Curcumin is a phytochemical with potent anti-neoplastic properties. The antitumoral effects of curcumin in cells derived from chronic or acute myeloid leukemia have been already described. However, a comparative study of the cytostatic and cytotoxic effects of curcumin on chronic and acute myeloid leukemia cells has not yet been performed. In the present study, the cellular effects of curcumin on cell lines derived from chronic or acute myeloid leukemia were examined. Dose and time-response assays were performed with curcumin on HL-60 and K562 cells. Cell viability was evaluated with trypan blue exclusion test and cell death by flow cytometry using a fluorescent molecular probe. A cell cycle profile was analyzed, and protein markers of cell cycle progression and cell death were investigated. In the present study, the K562 cells showed a higher sensitivity to the cytostatic and cytotoxic effects of curcumin compared with HL-60. In addition, curcumin induced G1 phase arrest in HL-60 cells and G2/M phase arrest in K562 cells. Furthermore, curcumin-related cell death in HL-60 was associated with the processed forms of caspases-9 and -3 proteins, whereas in K562 cells, both the processed and the unprocessed forms were present. Accordingly, activity of these caspases was significantly higher in HL-60 cells compared with that in K562. In conclusion, curcumin elicits different cellular mechanisms in chronic or acute myeloid leukemia cells and the powerful antitumoral effect was more potent in K562 compared with HL-60 cells.

Preclinical cancer models have been vital contributors in minimizing this burden as well as understanding basic cancer cell biology, however conventional and modern cancer models do not accurately or reliably recapitulate the complex in vivo tumor environment. Clinical significance of discoveries made using in vitromodels requires an understanding of the limitations imparted from cancer cells in a non-native environment. An ideal pre-clinical cancer platform that mimicks in vivo molecular phenotypes is essential for achieving effective drug screening and personalized treatments. This study aims to elucidate biological processes deficient in conventional in vitro methods from in vivo grown allograft cancer cells via transcriptome analysis. The effects of culturing conditions on cancer cells were analyzed via whole transcriptome RNA sequencing on a mouse mammary carcinoma (4T1) cell line grown in multiple culture conditions: 2D (monolayer) or 3D (spheroid) constructs under static or dynamic flow in addition to 4T1 cells isolated from subcutaneous or orthotopically grown tumors into the mammary fat pad of immune-competent, BALB/c mice. Comparative analysis of whole transcriptomic profiles of 4T1 cells in differing culturing conditions reveals distinct biological processes fostered by their environment. Monolayer culture shows enrichment in gene ontologies promoting proliferation, cell cycle progression, and protein synthesis. Compared to monolayer culture all 3-dimensional culturing methods encouraged the expression of proteins known to be critical to tumor progression such as extracellular matrix remodeling, adhesion, and differentiation. Furthermore, spheroid culture introduced heterogeneity as evidenced by upregulation of hypoxic induced genes and regulation of multicellular organism development processes. As expected, 4T1 cells expanded in vivo upregulated genes associated with processes difficult to recapitulate in vitro such as cell migration, inflammatory response, and angiogenesis. 3D culturing methods are able to recapitulate aspects of tumor heterogeneity yet fail to incorporate the complex heterogeneous cell composition and transient fluxes in nutrients and drugs found in vivo. Findings from this study demonstrate the behavioral and transcriptional alterations imparted from environmental factors. Additionally, clinically relevant in vitro testing can be improved by the incorporation of factors found in the native tumor microenvironment to existing 3D culturing approaches. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas Hum, Aimy Sebastian, Wei He, Monica L. Moya, William F. Hynes, Jonathan J. Adorno, Aubree Hinckley, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. RNA-seq comparisons of in vitro and in vivo cancer model platforms: Monolayer, spheroids, immunodeficient, and syngeneic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 37.

It is currently unclear how stromal components affect drug response and the emergence of drug resistance, in primary tumors. To determine how individual cells within the stroma of triple negative breast cancer (TNBC) allografts respond to chemotherapy, we used single cell sequencing to profile individual cells present in murine tumors with or without exposure to doxorubicin (Dox). Dox is a common chemotherapeutic agent used to treat breast cancer which inhibits breast cancer proliferation by intercalating into DNA and preventing topoisomerase II activity. Several autonomous processes have been implicated in the development of chemoresistance yet the impact of stromal and immune cells on tumor progression is still poorly understood. In this study, TNBC 4T1 cell line were utilized to generate murine allograft tumors in immunocompetent BALB/c mice. Tumor composition was analyzed via single cell RNA sequencing after 3 and 7 days of doxorubicin chemotherapeutic regiment mimicking clinical treatment. Using Cell Ranger single cell software suite and Seurat R toolkit, single cell transcriptomic analysis identified the cellular composition of tumors through expression of cell-type specific genes. Stromal cell types such as endothelial, fibroblast and epithelial cells were assessed and quantified in the tumor microenvironment. Immune cell types including neutrophils, monocytes, macrophages, T-cells and B-cells were also identified in the stroma and the responses to doxorubicin treatment was determined based on the gene expression changes. In this study, cancer-associated fibroblasts and non-canonical tumor associated macrophage subpopulations are of particular interest. As expected, we found both qualitative and quantitative changes in specific subpopulations of stromal cells in response to Dox exposure. Identification of stromal and immune cell sub-types could also lead to improved diagnostic capabilities and tumor susceptibilities. Future studies modulating non-cancerous cells in the tumor microenvironment may increase efficacy of chemotherapeutics. Further elucidating the specific cellular subpopulations within the tumor microenvironment that shift in response to drug exposure may provide new therapeutic avenues. Understanding changes in cell populations within the drug exposed tumor microenvironment can aid in future drug development to specifically target cells least sensitive to chemotherapy exposure. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas Hum, Aimy Sebastian, Sean Gilmore, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Characterization of the tumor microenvironment using single cell transcriptomics of triple negative breast cancer allografts treated with doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 130.

ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) receptor tyrosine kinases are critical for tissue development and maintenance, and frequently become oncogenic when mutated or overexpressed. In vitro analysis of ErbB receptor kinases can be difficult because of their large size and poor water solubility. Here we report improved production and assembly of the correctly folded full-length EGF receptor (EGFR) into nanolipoprotein particles (NLPs). NLPs are ~10 nm in diameter discoidal cell membrane mimics composed of apolipoproteins surrounding a lipid bilayer. NLPs containing EGFR were synthesized via incubation of baculovirus-produced recombinant EGFR with apolipoprotein and phosphoplipids under conditions that favor self-assembly. The resulting EGFR-NLPs were the correct size, formed dimers and multimers, had intrinsic autophosphorylation activity, and retained the ability to interact with EGFR-targeted ligands and inhibitors consistent with previously-published in vitro binding affinities. We anticipate rapid adoption of EGFR-NLPs for structural studies of full-length receptors and drug screening, as well as for the in vitro characterization of ErbB heterodimers and disease-relevant mutants.

Calculating internal dose from therapeutic radionuclides currently relies on estimates made from multiple radiation exposure measurements, converted to absorbed dose in specific organs using the Medical Internal Radiation Dose (MIRD) schema. As an alternative biodosimetric approach, we utilized gene expression analysis of whole blood from patients receiving targeted radiotherapy. Collected blood from patients with relapsed or refractory neuroblastoma who received (131)I-labeled metaiodobenzylguanidine ((131)I-mIBG) at the University of California San Francisco (UCSF) was used to compare calculated internal dose with the modulation of chosen gene expression. A total of 40 patients, median age 9 years, had blood drawn at baseline, 72 and 96 h after (131)I-mIBG infusion. Whole-body absorbed dose was calculated for each patient based on the cumulated activity determined from injected mIBG activity and patient-specific time-activity curves combined with (131)I whole-body S factors. We then assessed transcripts that were the most significant for describing the mixed therapeutic treatments over time using real-time polymerase chain reaction (RT-PCR). Modulation was evaluated statistically using multiple regression analysis for data at 0, 72 and 96 h. A total of 10 genes were analyzed across 40 patients: CDKN1A; FDXR; GADD45A; BCLXL; STAT5B; BAX; BCL2; DDB2; XPC; and MDM2. Six genes were significantly modulated upon exposure to (131)I-mIBG at 72 h, as well as at 96 h. Four genes varied significantly with absorbed dose when controlling for time. A gene expression biodosimetry model was developed to predict absorbed dose based on modulation of gene transcripts within whole blood. Three transcripts explained over 98% of the variance in the modulation of gene expression over the 96 h (CDKN1A, BAX and DDB2). To our knowledge, this is a novel study, which uses whole blood collected from patients treated with a radiopharmaceutical, to characterize biomarkers that may be useful for biodosimetry. Our data indicate that transcripts, which have been previously identified as biomarkers of external exposures in ex vivo whole blood and in vivo radiotherapy patients, are also good early indicators of internal exposure. However, for internal sources of radiation, the biokinetics and physical decay of the radionuclide strongly influence the gene expression.

Fanconi anemia (FA) is a rare, heritable chromosomal instability disease characterized by several congenital defects and cancer predisposition. Functional interactions between specific FA proteins and DNA damage response and repair activities have been reported, but the interplay between these mechanisms for maintaining genomic stability are not well understood. Many DNA damage response proteins are transcriptionally regulated by the tumor suppressor protein p53 (TP53), suggesting an important regulatory role for the DNA damage and stress response pathway. To better understand the association between FA and the DNA damage stress response we analyzed the levels of chromosomal damage and damage mediated gene transcription responses in lymphoblastoid cells derived from normal individuals and patients carrying the most common FA complementation group (FA-A). Chromosomal aberrations were first measured after exposure to mitomicyn C (MMC) or hydroxyurea (HU). Aliquots of the same cell were than assayed for the transcriptional response of 21 DNA damage and stress response genes using quantitative real-time PCR. The FA-A lymphoblastoid cells showed significant increases in the frequency of chromosome aberrations relative to non-FA-A lymphoblastoid lines after MMC treatment. The MMC induced damage was correlated with a general increase in expression of TP53-modulated DNA damage stress response genes involved in processes such as DNA repair, cell cycle progression, and apoptosis. Following HU treatment FA cells showed a decreased induction of CAs with much less transcriptional differences between targeted genes. Overall, the differences between the normal and FA-A cells after genotoxic treatments imply an increased activation and reliance of FA cells on the down-stream activities of TP53 for prevention of cell killing and chromosome damage from interstrand crosslinks but not for general replication arrest and double strand breaks. Furthermore, these results imply a regulatory connection between the FA pathway and activation of TP53 for responding to DNA damage. Alterations in the regulation of the DNA damage response may be related to the complex phenotypes seen in FA patients.

Exposure of individuals to ionizing radiation (IR), as in the case of astronauts exploring space or radiotherapy cancer patients, increases their risk of developing secondary cancers and other health-related problems. Bone marrow (BM), the site in the body where hematopoietic stem cell (HSC) self-renewal and differentiation to mature blood cells occurs, is extremely sensitive to low-dose IR, including irradiation by high-charge and high-energy particles. Low-dose IR induces DNA damage and persistent oxidative stress in the BM hematopoietic cells. Inefficient DNA repair processes in HSC and early hematopoietic progenitors can lead to an accumulation of mutations whereas long-lasting oxidative stress can impair hematopoiesis itself, thereby causing long-term damage to hematopoietic cells in the BM niche. We report here that low-dose (1)H- and (56)Fe-IR significantly decreased the hematopoietic early and late multipotent progenitor (E- and L-MPP, respectively) cell numbers in mouse BM over a period of up to 10 months after exposure. Both (1)H- and (56)Fe-IR increased the expression of pluripotent stem cell markers Sox2, Nanog, and Oct4 in L-MPPs and 10 months post-IR exposure. We postulate that low doses of (1)H- and (56)Fe-IR may induce endogenous cellular reprogramming of BM hematopoietic progenitor cells to assume a more primitive pluripotent phenotype and that IR-induced oxidative DNA damage may lead to mutations in these BM progenitors. This could then be propagated to successive cell lineages. Persistent impairment of BM progenitor cell populations can disrupt hematopoietic homeostasis and lead to hematologic disorders, and these findings warrant further mechanistic studies into the effects of low-dose IR on the functional capacity of BM-derived hematopoietic cells including their self-renewal and pluripotency.

Inhibitors of Wnt signaling have been shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts promotes PC invasion, while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts upregulated under highly invasive conditions. We found CRIM1 overexpression to also promote cell-invasion. These findings suggest that bone-derived Wnt signaling may enhance PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of PC3 cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via the tail vein in NSG mice did not readily metastasize, and those injected intrafemorally had significantly reduced osteolysis, suggesting that targeting the molecular bone environment may influence bone metastatic prognosis in clinical settings.

Immobilized antibody microarrays were compared to the Luminex flow cytometry system that utilizes suspensions of polystyrene microbeads covalently coupled with capture antibodies. The two immunoassays were performed for comparison of reproducibility, limits of detection and dynamic range. The Luminex system showed lower limits of detection and increased dynamic range among samples whereas the protein microarrays could be more amenable to miniaturization. Both technologies were capable of sensitive multiplexed detection.

Advances in Semantic Web and Ontologies have pushed the role of semantics to a new frontier: Semantic Composition of Web Services. A good example of such compositions is the querying of multiple bioinformatics data sources. Supporting effective querying over a large collection of bioinformatics data sources presents a number of unique challenges. First, queries over bioinformatics data sources are often complex associative queries over multiple Web documents. Most associations are defined by string matching of textual fragments in two documents. Second, most of the queries required by Genomics researchers involve complex data extraction, and sophisticated workflows that implement the complex associative access. Third but not the least, complex Genomics-specific queries are often reused many times by Genomics researchers, either directly or through some refinements, and are considered as a part of the research results by Genomics researchers. In this short article we present a list of challenging issues in supporting effective querying over bioinformatics data sources and illustrate them through a selection of representative search scenarios provided by biologists. We end the article with a discussion on how the state-of-art research and technological development in Semantic Web, Ontology, Internet Data Management, and Internet Computing Systems can help addressing these issues.

Objective: To compare the cervicovaginal cytokines IL-1 beta, IL-6 and IL-8 with fetal fibronectin (fFN) and cervical dilatation in the prediction of preterm delivery. Study design: Cervicovaginal cytokine concentration and fFN status were measured in 104 women with symptoms of preterm labour and intact membranes between 24(0) and 33(6) weeks and related to delivery within 2 and 7 days, Results: A group of 18% had cervical dilatation greater than or equal to 1 cm and 18% were positive for fFN. Preterm delivery within 2 and 7 days occurred in 5 and 12%, respectively. Only IL-6 demonstrated any ability to predict delivery within 2 and 7 days (area under the ROC curve = 0.63 and 0.75, respectively). Using 35 pg/ml (75th centile) as a cut-off, IL-6 had a sensitivity and specificity of 60 and 77% for predicting delivery within 2 days, and 62 and 80% for predicting delivery within 7 days. This is similar to the performance of cervical dilatation or fFN status. Conclusions: Measurement of cervicovaginal cytokines has limited ability to predict imminent delivery apart from cervicovaginal IL-6 concentrations, which, in this population, is equivalent to that of fFN status and cervical dilatation greater than or equal to 1 cm. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

The aim of this study was to determine prospectively whether serum concentrations of corticotropin-releasing hormone, corticotropin-releasing hormone-binding protein, and activin A (1) predict preterm birth within 10 days of hospital admission or at37 weeks' gestation among women with symptoms of preterm labor and (2) are affected by glucocorticoid therapy.Serum concentrations of corticotropin-releasing hormone and activin A were measured in 94 women with symptoms of preterm labor between 24 and 34 weeks' gestation, and delivery outcomes were monitored. Corticotropin-releasing hormone-binding protein concentrations were measured in 71 of these women. In a subgroup of 15 women the serum analytes were assayed in conjunction with estriol before and 12 to 24 hours after administration of dexamethasone.Forty-six percent (6/13) of the women who were delivered within 10 days of hospital admission had a raised serum corticotropin-releasing hormone level, but the predictive relationship was not significant (chi(2) = 1.7; P =.2). Among the 31 women (including the 6 previously mentioned) who were delivered at37 weeks' gestation, 39% (12/31) had a raised corticotropin-releasing hormone level. Although a raised corticotropin-releasing hormone concentration was positively associated with delivery at37 weeks' gestation (chi(2) = 9; P =.003), the predictive diagnostic value was poor, with sensitivity, specificity, and positive and negative predictive values of 39%, 90%, 67%, and 75%, respectively. The serum concentrations of corticotropin-releasing hormone-binding protein and activin A were unrelated to gestational age at delivery. Dexamethasone markedly lowered the serum estriol level (P.001) but had no effect on concentrations of corticotropinreleasing hormone, corticotropin-releasing hormone-binding protein, and activin A.Serum concentrations of corticotropin-releasing hormone, corticotropin-releasing hormone-binding protein, and activin A are not clinically useful for the prediction of preterm delivery among women with symptoms of preterm labor and are not affected by administration of glucocorticoids.

PROBLEM: Clinically useful tests for the prediction and diagnosis of preterm labor and delivery remain to be established. We have hypothesized that soluble intercellular adhesion molecule-1 (sICAM-1) in the cervicovaginal fluid of women with preterm labor may be a useful diagnostic tool. METHOD OF STUDY: The cervicovaginal fluid of 103 women between 24° and 33 6 weeks gestation with preterm contractions and intact membranes was assayed for sICAM-1. RESULTS: Elevated sICAM-1 concentrations predicted short intervals to delivery (area under receiver-operator characteristic (ROC) curves, 0.70-0.72 for delivery within 3, 7 and 10 days), with high specificity. Characteristics for delivery within 3 days at a 3 ng/mL threshold for a positive test were sensitivity 33.3%, specificity 98.9%, and positive and negative predictive values of 75.0% and 93.9%, respectively. Predictive ability was independent of and complementary to that of fetal fibronectin (fFN). CONCLUSIONS: Measurement of sICAM-1 in cervicovaginal fluid has potential as a predictor of preterm delivery in women with symptoms of preterm labor, particularly in conjunction with fFN testing.

Objective To assess the value of uterine artery Doppler ultrasound screening, when performed in a clinical setting, to predict complications of impaired uteroplacental blood flow in high-risk women. Design A prospective audit. Subjects A total of 116 pregnancies in 114 women at high risk of pre-eclampsia and/or small-for-gestational-age (SGA) babies attending a maternal–fetal medicine clinic at National Women’s Hospital, a tertiary referral hospital, Auckland, New Zealand. Methods Uterine artery Doppler screening was performed as part of clinical practice between 22 and 24 weeks’ gestation. A resistance index (RI) was calculated from each uterine artery and the presence or absence of a notch was determined. An RI of > 0.58 was defined as abnormal and an RI of ≥ 0.7 was defined as very abnormal. The main outcome measures were: pre-eclampsia, SGA baby (birth weight 0.58 for these outcomes was 42%, 39%, 47% and 38%, respectively. The positive predictive value of any RI of > 0.58 for the same outcomes was 37%, 33%, 58% and 24%, respectively. Among women with both RI values of ≥ 0.7, 58%, 67%, 85% and 58% developed pre-eclampsia, SGA, ‘all’ and ‘severe’ outcomes, respectively. In women with bilateral notches, 47%, 53%, 76% and 65% developed the respective outcomes. Women with both RI values of ≥ 0.7 and women with bilateral notches had relative risks of 11.1 (95% CI 2.6–46.4) and 12.7 (95% CI 4.0–40.4) for developing severe outcome, respectively. Only 5% of women with both RI values of

Purpose. Transdermal extraction of clinically relevant analytes offersa potentially non-invasive method of diagnostics. However,development of such a method is limited by the low skin permeability. In thispaper, we report a potential method for non-invasive diagnostics basedon ultrasonic skin permeabilization and subsequent extraction ofinterstitial fluid (ISF) across the skin.Methods. In vivo experiments were performed using Sprague Dawleyrats to assess ultrasound-induced skin permeabilization and subsequentextraction of various analytes. Serum and ISF concentrations of variousanalytes were measured.Results. Application of low-frequency ultrasound rapidly increasedskin permeability. Skin remained in a state of high permeability for atleast three hours. During this period, application of vacuum extractedISF across rat skin in vivo at a rate of 25.7 μl/cm2/hr. We measuredconcentrations of various analytes including glucose, albumin, calcium,urea, triglycerides, lactate, and dextran in transdermally extracted fluid.The composition of the fluid extracted transdermally is similar to thatof ISF.Conclusions. Application of low-frequency ultrasound allows skin permeabilization and extraction of ISF across the skin.

To investigate labour-associated changes in production of activin and related hormones by gestational tissues we prepared extracts from amnion, choriodecidual and placental tissues delivered at term before labour (TNL; n=15), at term after spontaneous labour (TSL; n=15) or preterm (PTD; n=31) and measured concentrations of inhibin A, activin A and follistatin by ELISA. Activin concentrations in placental tissues were significantly (Mann-Whitney U-test; P

Giant magnetoresistive (GMR) nanosensors provide a novel approach for measuring protein concentrations in blood for medical diagnosis. Using an in vivo mouse radiation model, we developed protocols for measuring Flt3 ligand (Flt3lg) and serum amyloid A1 (Saa1) in small amounts of blood collected during the first week after X-ray exposures of sham, 0.1, 1, 2, 3, or 6 Gy. Flt3lg concentrations showed excellent dose discrimination at ≥ 1 Gy in the time window of 1 to 7 days after exposure except 1 Gy at day 7. Saa1 dose response was limited to the first two days after exposure. A multiplex assay with both proteins showed improved dose classification accuracy. Our magneto-nanosensor assay demonstrates the dose and time responses, low-dose sensitivity, small volume requirements, and rapid speed that have important advantages in radiation triage biodosimetry.

DNA damage and repair are hallmarks of cellular responses to ionizing radiation. We hypothesized that monitoring the expression of DNA repair-associated genes would enhance the detection of individuals exposed to radiation versus other forms of physiological stress. We employed the human blood ex vivo radiation model to investigate the expression responses of DNA repair genes in repeated blood samples from healthy, non-smoking men and women exposed to 2 Gy of X-rays in the context of inflammation stress mimicked by the bacterial endotoxin lipopolysaccharide (LPS). Radiation exposure significantly modulated the transcript expression of 12 genes of 40 tested (2.2E-06

The protein family known as G-protein coupled receptors (GPCRs) comprises an important class of membrane-associated proteins, which remains a difficult family of proteins to characterize because their function requires a native-like lipid membrane environment. This paper focuses on applying a single step method leading to the formation of nanolipoprotein particles (NLPs) capable of solubilizing functional GPCRs for biophysical characterization. NLPs were used to demonstrate increased solubility for multiple GPCRs such as the Neurokinin 1 Receptor (NK1R), the Adrenergic Receptor â2 (ADRB2) and the Dopamine Receptor D1 (DRD1). All three GPCRs showed affinity for their specific ligands using a simple dot blot assay. The NK1R was characterized in greater detail to demonstrate correct folding of the ligand pocket with nanomolar specificity. Electron paramagnetic resonance (EPR) spectroscopy validated the correct folding of the NK1R binding pocket for Substance P (SP). Fluorescence correlation spectroscopy (FCS) was used to identify SP-bound NK1R-containing NLPs and measure their dissociation rate in an aqueous environment. The dissociation constant was found to be 83 nM and was consistent with dot blot assays. This study represents a unique combinational approach involving the single step de novo production of a functional GPCR combined with biophysical techniques to demonstrate receptor association with the NLPs and binding affinity to specific ligands. Such a combined approach provides a novel path forward to screen and characterize GPCRs for drug discovery as well as structural studies outside of the complex cellular environment.

Acute kidney injury (AKI) is a serious complication that significantly increases morbidity, mortality and cost of care after cardiac surgery. In this review we identify the current literature that addresses strategies for renal protection and the prevention of AKI after cardiac surgery.Even with aggressive medical care and renal replacement therapy (RRT) the morbidity, mortality and cost of postoperative AKI after cardiac surgery is substantial. An emphasis on preventive strategies would therefore appear to be the most cost-effective approach. Recent literature offers hope that as our understanding of the pathogenesis of AKI after cardiac surgery continues to improve, new directions for the prevention and amelioration of AKI will emerge. Approaches to the prevention of postoperative AKI include careful risk stratification of patients, allowing adequate recovery following a prior AKI, consideration of less extensive surgical procedures, avoidance of cardiopulmonary bypass, minimizing injury from radiocontrast dyes or other nephrotoxic agents, and optimizing cardiovascular function and oxygen delivery. Early identification of AKI and prompt, judicious application of RRT may also improve outcomes. Interest in pharmacologic renoprotection is currently directed toward statins and sodium bicarbonate.Postoperative AKI is a serious complication after cardiac surgery. Therapeutic interventions and RRT have limited influence on the outcome of AKI, and a preventive strategy remains the mainstay to attenuate its impact.

To conduct a literature review of candidate protein biomarkers for individual radiation biodosimetry of exposure to ionizing radiation.Reviewed approximately 300 publications (1973 - April 2006) that reported protein effects in mammalian systems after either in vivo or in vitro radiation exposure.We found 261 radiation-responsive proteins including 173 human proteins. Most of the studies used high doses of ionizing radiation (4 Gy) and had no information on dose- or time-responses. The majority of the proteins showed increased amounts or changes in phosphorylation states within 24 h after exposure (range: 1.5- to 10-fold). Of the 47 proteins that are responsive at doses of 1 Gy and below, 6 showed phosphorylation changes at doses below 10 cGy. Proteins were assigned to 9 groups based on consistency of response across species, dose- and time-response information and known role in the radiation damage response.ATM (Ataxia telengiectasia mutated), H2AX (histone 2AX), CDKN1A (Cyclin-dependent kinase inhibitor 1A), and TP53 (tumor protein 53) are top candidate radiation protein biomarkers. Furthermore, we recommend a panel of protein biomarkers, each with different dose and time optima, to improve individual radiation biodosimetry for discriminating between low-, moderate-, and high-dose exposures. Our findings have applications for early triage and follow-up medical assessments.

The complete genome sequence of Thiobacillus denitrificans ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, β-proteobacterium. Analysis of the 2,909,809-bp genome will facilitate our molecular and biochemical understanding of the unusual metabolic repertoire of this bacterium, including its ability to couple denitrification to sulfur-compound oxidation, to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV), and to oxidize mineral electron donors. Notable genomic features include (i) genes encoding c -type cytochromes totaling 1 to 2 percent of the genome, which is a proportion greater than for almost all bacterial and archaeal species sequenced to date, (ii) genes encoding two [NiFe]hydrogenases, which is particularly significant because no information on hydrogenases has previously been reported for T. denitrificans and hydrogen oxidation appears to be critical for anaerobic U(IV) oxidation by this species, (iii) a diverse complement of more than 50 genes associated with sulfur-compound oxidation (including sox genes, dsr genes, and genes associated with the AMP-dependent oxidation of sulfite to sulfate), some of which occur in multiple (up to eight) copies, (iv) a relatively large number of genes associated with inorganic ion transport and heavy metal resistance, and (v) a paucity of genes encoding organic-compound transporters, commensurate with obligate chemolithoautotrophy. Ultimately, the genome sequence of T. denitrificans will enable elucidation of the mechanisms of aerobic and anaerobic sulfur-compound oxidation by β-proteobacteria and will help reveal the molecular basis of this organism's role in major biogeochemical cycles (i.e., those involving sulfur, nitrogen, and carbon) and groundwater restoration.

Immunoassays have been widely used in commercial, scientific and medical research for detection and quantification of analytes in complex mixtures. There is however a need for a point-of-care, multiplex diagnostic assays capable of providing rapid and quantitative measurements of analytes present in samples that are sufficiently simple to carry out without use of a laboratory or individuals trained in chemical analysis. We are developing a fluorescent lateral flow immunoassay platform to perform simultaneous, multiplexed detection of analytes in a complex fluid mixture along with instrumentation to optically quantitate the analytes in the sample. Our prototype imaging system is based on conventional 16-bit CCD optics, which enables the development of a rugged diagnostic instrument that can be further scaled down for point-of-care applications. We have compared protein microarrays with lateral flow assays (LFAs) to determine the sensitivity of each system for the measurement of distinct proteins in complex samples. We are pursuing the LFA platform such that it can easily be scaled to meet the requirements of any given screening application, and be implemented for use in a medical or surgical setting.