Multi-omics analysis from archival neonatal dried blood spots: limitations and opportunities.

Newborn screening (NBS) programs operate in many countries, processing millions of dried bloodspot (DBS) samples annually. In addition to early identification of various adverse health outcomes, these samples have considerable potential as a resource for population-based research that could address key questions related to child health. The feasibility of archival DBS samples for emerging targeted and untargeted multi-omics analysis has not been previously explored in the literature. This review aims to critically evaluate the latest advances to identify opportunities and challenges of applying omics analyses to NBS cards in a research setting. Medline, Embase and PubMed databases were searched to identify studies utilizing DBS for genomic, proteomic and metabolomic assays. A total of 800 records were identified after removing duplicates, of which 23 records were included in this review. These papers consisted of one combined genomic/metabolomic, four genomic, three epigenomic, four proteomic and 11 metabolomic studies. Together they demonstrate that the increasing sensitivity of multi-omic analytical techniques makes the broad use of NBS samples achievable for large cohort studies. Maintaining the pre-analytical integrity of the DBS sample through storage at temperatures below -20 °C will enable this important resource to be fully realized in a research capacity.

Assessing multiparametric drug response in tissue engineered tumor microenvironment models.

The tumor microenvironment is important in promoting treatment resistance of tumor cells via multiple mechanisms. However, studying this interaction often proves difficult. In vivo animal models are costly, time-consuming, and often fail to adequately predict human response to treatment. Conversely, testing drug response on human tumor cells in vitro in 2D cell culture excludes the important contribution of stromal cells and biophysical forces seen in the in vivo tumor microenvironment. Here, we present tissue-engineered models of both human brain and breast tumor microenvironments incorporating key stromal cell populations for assessing multiple mechanisms of therapeutic response using flow cytometry. We show our physiologically-relevant systems used to interrogate a variety of parameters associated with chemotherapeutic efficacy, including cell death, proliferation, drug uptake, and invasion of cancer and stromal cell populations. The use of flow cytometry allows for single cell, quantitative, and fast assessments of multiple outcomes affecting anti-tumor therapy failure. Our system can be modified to add and remove cellular components with ease, thereby enabling the study of individual cellular contributions in the tumor microenvironment. Together, our models and analysis methods illustrate the importance of developing fast, cost-effective, and reproducible methods to model complex human systems in a physiologically-relevant manner that may prove useful for drug screening efforts in the future.

Synergistic interactions of vancomycin with different antibiotics against Escherichia coli: trimethoprim and nitrofurantoin display strong synergies with vancomycin against wild-type E. coli.

Gram-negative bacteria are normally resistant to the antibiotic vancomycin (VAN), which cannot significantly penetrate the outer membrane. We used Escherichia coli mutants that are partially sensitive to VAN to study synergies between VAN and 10 other antibiotics representing six different functional categories. We detected strong synergies with VAN and nitrofurantoin (NTR) and with VAN and trimethoprim (TMP) and moderate synergies with other drugs, such as aminoglycosides. These synergies are powerful enough to show the activity of VAN against wild-type E. coli at concentrations of VAN as low as 6.25 µg/ml. This suggests that a very small percentage of exogenous VAN does enter E. coli but normally has insignificant effects on growth inhibition or cell killing. We used the results of pairwise interactions with VAN and the other 10 antibiotics tested to place VAN into a functional category of its own, as previously defined by Yeh et al. (P. Yeh, A. I. Tschumi, and R. Kishony, Nat Genet 28:489-494, 2006, http://dx.doi.org/10.1038/ng1755).

Deoxycytidine deaminase-deficient Escherichia coli strains display acute sensitivity to cytidine, adenosine, and guanosine and increased sensitivity to a range of antibiotics, including vancomycin.

We show here that deoxycytidine deaminase (DCD)-deficient mutants of Escherichia coli are hypersensitive to killing by exogenous cytidine, adenosine, or guanosine, whereas wild-type cells are not. This hypersensitivity is reversed by exogenous thymidine. The mechanism likely involves the allosteric regulation of ribonucleotide reductase and severe limitations of the dTTP pools, resulting in thymineless death, the phenomenon of cell death due to thymidine starvation. We also report here that DCD-deficient mutants of E. coli are more sensitive to a series of different antibiotics, including vancomycin, and we show synergistic killing with the combination of vancomycin and cytidine. One possibility is that a very low, subinhibitory concentration of vancomycin enters Gram-negative cells and that this concentration is potentiated by chromosomal lesions resulting from the thymineless state. A second possibility is that the metabolic imbalance resulting from DCD deficiency affects the assembly of the outer membrane, which normally presents a barrier to drugs such as vancomycin. We consider these findings with regard to ideas of rendering Gram-negative bacteria sensitive to drugs such as vancomycin.

Postpartum gestational diabetes insipidus related to preeclampsia: A case report.

Gestational diabetes insipidus (GDI) is a rare complication of pregnancy thought to be due to increased vasopressinase produced by the placenta. It typically occurs at the end of the second or in the third trimester. This report describes a case of GDI diagnosed postpartum in the setting of newly diagnosed superimposed preeclampsia. A 39-year-old Hispanic woman (gravida 2 para 2) presented ten days postpartum with a persistent headache and elevated blood pressures in the setting of a history of chronic hypertension, meeting criteria for superimposed preeclampsia. Repeat lab work was notable for mild elevation of liver function enzymes. Despite normalization of blood pressures, her headache persisted and further workup revealed polyuria, suspected to be vasopressinase-induced diabetes insipidus. The patient was started on oral desmopressin with improvement of polyuria and symptoms.

The aminoglycoside antibiotic kanamycin damages DNA bases in Escherichia coli: caffeine potentiates the DNA-damaging effects of kanamycin while suppressing cell killing by ciprofloxacin in Escherichia coli and Bacillus anthracis.

The distribution of mutants in the Keio collection of Escherichia coli gene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ, holC, holD, and priA knockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded by recA, recB, and recC, among others. Additionally, caffeine partially protects cells of both Escherichia coli and Bacillus anthracis from killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

Designing Patient-Driven, Tissue-Engineered Models of Primary and Metastatic Breast Cancer.

The rising survival rate for early-stage breast cancer in the United States has created an expanding population of women in remission at risk for distant recurrence, with metastatic spread to the brain demonstrating an especially poor prognosis. The current standard of care for breast cancer brain metastases is not well defined or differentiated from the treatment of brain metastases from other primary sites. Here, we present tissue-engineered models of the primary and brain metastatic breast cancer microenvironments informed by analysis of patient tumor resections. We find that metastatic resections demonstrate distinct cellular and matrix components compared with primary resections or non-cancerous controls. Using our model systems, we find that the observed deposition of collagen I after metastasis to the brain may enhance breast cancer invasion. Future optimization of these models will present a novel platform to examine tumor-stroma interactions and screen therapeutics for the management of metastatic breast cancer.

Selective advantage of resistant strains at trace levels of antibiotics: a simple and ultrasensitive color test for detection of antibiotics and genotoxic agents.

Many studies have examined the evolution of bacterial mutants that are resistant to specific antibiotics, and many of these focus on concentrations at and above the MIC. Here we ask for the minimum concentration at which existing resistant mutants can outgrow sensitive wild-type strains in competition experiments at antibiotic levels significantly below the MIC, and we define a minimum selective concentration (MSC) in Escherichia coli for two antibiotics, which is near 1/5 of the MIC for ciprofloxacin and 1/20 of the MIC for tetracycline. Because of the prevalence of resistant mutants already in the human microbiome, allowable levels of antibiotics to which we are exposed should be below the MSC. Since this concentration often corresponds to low or trace levels of antibiotics, it is helpful to have simple tests to detect such trace levels. We describe a simple ultrasensitive test for detecting the presence of antibiotics and genotoxic agents. The test is based on the use of chromogenic proteins as color markers and the use of single and multiple mutants of Escherichia coli that have greatly increased sensitivity to either a wide range of antibiotics or specific antibiotics, antibiotic families, and genotoxic agents. This test can detect ciprofloxacin at 1/75 of the MIC.

Linker-based lecithin microemulsions for transdermal delivery of lidocaine.

In this work, we introduce alcohol-free lecithin microemulsions formulated with linkers to produce transdermal delivery vehicles. The food-grade linker system consists of a combination of sodium caprylate and caprylic acid (hydrophilic linkers), and sorbitan monooleate (lipophilic linker). A "carrier" oil (isopropyl myristate) was used to predissolve a model lipophilic drug, lidocaine. The first part of the article describes the phase behavior and physical properties of these linker-based lecithin microemulsions. In the second part of the article, we evaluate the transdermal permeation and cytotoxicity of lidocaine formulated in oil-in-water (Type I), water-in-oil (Type II), and bicontinuous (Type IV) linker microemulsions. The transdermal permeation studies show that compared to a conventional Type II alcohol-based lecithin microemulsion, Type II linker-based microemulsions provide twice the absorption and penetration of lidocaine through skin. The larger flux obtained with linker systems is due to the presence of sodium caprylate and caprylic acid. These hydrophilic linkers accelerate the microemulsion-skin mass transfer by reducing the interfacial rigidity of the systems. Furthermore, the cytotoxicity studies show that these linker microemulsions are significantly less toxic than the alcohol-based system. The Type II linker microemulsion (containing approximately 4% lidocaine) has a comparable cytotoxicity to water saturated with lidocaine (0.4% lidocaine).

Development of field test kits for determination of microencapsulated iron in double-fortified salt.

BACKGROUND: Efficacy studies have shown that salt double-fortified with iodine and iron can significantly reduce the incidence rates of iron-deficiency anemia and iodine-deficiency disorders. Double-fortified salt can be prepared by mixing microencapsulated iron compounds into conventionally iodated salt. Effective implementation of a double fortification program requires field-based analytical methods to ensure iron levels in double-fortified salt. OBJECTIVE: To develop semiquantitative and qualitative field test kits by adopting standard analytical methods for iron determination to the analysis of iron in double-fortified salt. METHODS: Thermal, mechanical, and chemical strategies were assessed to enable contact between analytical reagents and the encapsulated iron compounds during the analysis. A chemical approach using nonpolar solvents was adopted in semiquantitative and qualitative field tests. The fat coating of the iron premix was removed by solvents, releasing the iron for subsequent colorimetric determination. RESULTS: Both semiquantitative and qualitative field tests were based on initial removal of the microencapsulant, followed by iron quantitation. Solvent dissolution of the coating layer was most useful for rapid release of iron. A semiquantitative field test kit was developed using a mixture of 5% heptane and 95% tetrachloroethylene to free the iron, which was then determined by the 1,10-phenanthroline method. The field test had a useful detection range of 0 to 2,000 ppm of iron. Statistical analyses revealed that the results obtained with the kit correlated well with those obtained by standard laboratory methods (p < .001). A qualitative field test kit was developed to identify the presence of iron. Microencapsulated iron was freed with the use of tetrachloroethylene and then reacted with phenanthroline to form a visually observable coloration on the salt sample. CONCLUSION: Semiquantitative and qualitative field test kits for iron determination in double-fortified salt have been developed and tested. These kits could be useful in quality control of double fortification of salt in small salt-production facilities and in the field, particularly in developing countries.

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections.

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within patient samples of glioblastoma, the deadliest and most invasive brain cancer. Not only are quantitative methods beneficial for accurately describing diseased tissues, they can also potentially contribute to more accurate prognosis, diagnosis, and the development of tissue-engineered systems and replacements. In glioblastoma, glial cells, such as microglia and astrocytes, have been independently correlated with poor prognosis based on pathologist grading. However, the state of these cells and other glial cell components has not been well-described quantitatively. This can be difficult due to the large processes that mark these glial cells. Furthermore, most histological analyses focus on the overall tissue sample or only within the bulk of the tumor, as opposed to delineating quantifications based on regions within the highly heterogeneous tissue. Here, we describe a method for identifying and quantitatively analyzing the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations in patient tumor resections and ImageJ to analyze percent coverage of staining for each glial population. With these techniques we are able to better describe the glial cells throughout regions of the glioma tumor microenvironment.

Quantitative Analysis of the Cellular Microenvironment of Glioblastoma to Develop Predictive Statistical Models of Overall Survival.

Glioblastomas, the most common primary malignant brain tumors, have a distinct tissue microenvironment. Although non-neoplastic cells contribute to glioblastoma progression, very few quantitative studies have shown the effect of tumor microenvironmental influences on patient survival. We examined relationships of the cellular microenvironment, including astrocytes, microglia, oligodendrocytes, and blood vessels, to survival in glioblastoma patients. Using histological staining and quantitative image analyses, we examined the tumor-associated parenchyma of 33 patients and developed statistical models to predict patient outcomes based on the cellular picture of the tumor parenchyma. We found that blood vessel density correlated with poorer prognosis. To examine the role of adjacent parenchymal versus higher tumor cell density bulk parenchymal tissue, we examined the glial components in these highly variable regions. Comparison of bulk and adjacent astrocytes and microglia in tissue yielded the strongest prediction of survival, with high levels of adjacent astrocytes predicted poor prognosis and high levels of microglia correlated with a better prognosis. These results indicate that parenchymal components predict survival in glioblastoma patients and in particular that the balance between reactive glial populations is important for patient prognosis.

Extreme dNTP pool changes and hypermutability in dcd ndk strains.

Cells lacking deoxycytidine deaminase (DCD) have been shown to have imbalances in the normal dNTP pools that lead to multiple phenotypes, including increased mutagenesis, increased sensitivity to oxidizing agents, and to a number of antibiotics. In particular, there is an increased dCTP pool, often accompanied by a decreased dTTP pool. In the work presented here, we show that double mutants of Escherichia coli lacking both DCD and NDK (nucleoside diphosphate kinase) have even more extreme imbalances of dNTPs than mutants lacking only one or the other of these enzymes. In particular, the dCTP pool rises to very high levels, exceeding even the cellular ATP level by several-fold. This increased level of dCTP, coupled with more modest changes in other dNTPs, results in exceptionally high mutation levels. The high mutation levels are attenuated by the addition of thymidine. The results corroborate the critical importance of controlling DNA precursor levels for promoting genome stability. We also show that the addition of certain exogenous nucleosides can influence replication errors in DCD-proficient strains that are deficient in mismatch repair.

Predictors of size for gestational age in St. Louis City and County.

OBJECTIVE: To identify social, behavioral, and physiological risk factors associated with small for gestational age (SGA) by gestational age category in St. Louis City and County. METHODS: A retrospective cohort study was conducted using birth certificate and fetal death records from 2000 to 2009 (n = 142,017). Adjusted associations of risk factors with SGA were explored using bivariate logistic regression. Four separate multivariable logistic regression analyses, stratified by gestational age, were conducted to estimate adjusted odds ratios. RESULTS: Preeclampsia and inadequate weight gain contributed significantly to increased odds for SGA across all gestational age categories. The point estimates ranged from a 3.41 increased odds among women with preeclampsia and 1.76 for women with inadequate weight gain at 24-28 weeks' gestational age to 2.19 and 2.11 for full-term infants, respectively. Among full-term infants, smoking (aOR = 2.08), chronic hypertension (aOR = 1.46), and inadequate prenatal care (aOR = 1.25) had the next most robust and significant impact on SGA. CONCLUSION: Preeclampsia and inadequate weight gain are significant risk factors for SGA, regardless of gestational age. Education on the importance of nutrition and adequate weight gain during pregnancy is vital. In this community, disparities in SGA and smoking rates are important considerations for interventions designed to improve birth outcomes.

Effect of surfactant concentration on transdermal lidocaine delivery with linker microemulsions.

A limited number of studies have been conducted to investigate the effect of surfactant concentration on microemulsion-mediated transdermal transport. Some studies suggest that increasing surfactant concentration reduces the partition of the active in the skin, and the overall transport. Other studies suggest that increasing surfactant concentration improves mass transport across membranes by increasing the number of "carriers" available for transport. To decouple these partition and mass transport effects, a three-compartment (donor, skin, receiver) mass balance model was introduced. The model has three permeation parameters, the skin-donor partition coefficient (K(sd)), the donor-skin mass transfer coefficient (k(ds)) and the skin-receiver mass transfer coefficient (k(sr)), also known as skin permeability. The model was used to fit the permeation profile of lidocaine formulated in oil-in-water (Type I) and water-in-oil (Type II) lecithin-linker microemulsions. The results show that surfactant concentration has a relatively minor effect on the mass transfer coefficients, suggesting that permeation enhancement via disruption of the structure of the skin is not a relevant mechanism in these lecithin-linker microemulsions. The most significant effect was the increase in the concentration of lidocaine in the skin with increasing surfactant concentration. For Type I systems such increase in lidocaine concentration in the skin was linked to the increase in lidocaine solubilization in the microemulsion with increasing surfactant concentration. For Type II systems, the increase in lidocaine concentration in the skin was linked to the increase in skin-donor partition. A surfactant-mediated absorption/permeation mechanism was proposed to explain the increase in lidocaine concentration in skin with increasing surfactant concentration. The penetration profiles of hydrophobic and amphiphilic fluorescence probes are consistent with the proposed mechanism.

Extended release of lidocaine from linker-based lecithin microemulsions.

In a previous article we reported on the use of linker-based lecithin microemulsions as effective transdermal delivery vehicles for lidocaine [Yuan, J.S., Ansari, M., Samaan, M., Acosta, E., 2008. Linker-based lecithin microemulsions for transdermal delivery of lidocaine. Int. J. Pharm. 349, 130-143]. It was determined at that time that the performance of these vehicles was in part due to a permeability enhancement effect, but also due to the amount of lidocaine absorbed in the skin. In the present article we take advantage of this drug absorbed in the skin to produce an extended release profile where the lidocaine-loaded skin is used as an in situ patch. The release of lidocaine from the skin is modeled using a differential mass balance that yields a first order release profile. This profile depends on the mass of drug initially loaded in the skin and a mass transfer coefficient. When the release profile of lidocaine was evaluated as a function of the concentration of lidocaine in the microemulsion, application time, and microemulsion dosage; we observed that all these different conditions only change the mass of lidocaine initially loaded in the skin. However, these parameters do not change the mass transfer coefficient. When the release profile of Types I and II microemulsions was compared, it was determined that the mass transfer coefficient of Type II systems was larger than that of Type I. This suggests that the morphology of the microemulsion plays an important role on the release kinetics. These linker microemulsions were able to release 90% of their content over a 24-h period which rivals the performance of some polymer-based patches. Fluorescence micrographs of transversal cuts of skin loaded with Nile red are consistent with the observed release profiles.