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A longstanding challenge in human microbiome research is achieving the taxonomic and functional resolution needed to generate testable hypotheses about the gut microbiome’s impact on health and disease. More recently, this challenge has extended to a need for in-depth understanding of the pharmacokinetics and pharmacodynamics of clinical microbiome-based interventions. Whole genome metagenomic sequencing provides high taxonomic resolution and information on metagenome functional capacity, but the required deep sequencing is costly. For this reason, short-read sequencing of the bacterial 16S ribosomal RNA (rRNA) gene is the standard for microbiota profiling, despite its poor taxonomic resolution. The recent falling costs and improved fidelity of long-read sequencing warrant an evaluation of this approach for clinical microbiome analysis. We used samples from participants enrolled in a Phase 1b clinical trial of a novel live biotherapeutic product to perform a comparative analysis of short-read and long-read amplicon and metagenomic sequencing approaches to assess their value for generating informative and actionable clinical microbiome data. Comparison of ubiquitous short-read 16S rRNA amplicon profiling to long-read profiling of the 16S-ITS-23S rRNA amplicon showed that only the latter provided strain-level community resolution and insight into novel taxa. Across all methods, overall community taxonomic profiles were comparable and relationships between samples were conserved, highlighting the accuracy of modern microbiome analysis pipelines. All methods identified an active ingredient strain in treated study participants, though detection confidence was higher for long-read methods. Read coverage from both metagenomic methods provided evidence of active ingredient strain replication in some treated participants. Compared to short-read metagenomics, approximately twice the proportion of long reads were assigned functional annotations (63% vs. 34%). Finally, similar bacterial metagenome-assembled genomes (MAGs) were recovered across short-read and long-read metagenomic methods, although MAGs recovered from long reads were more complete. Overall, despite higher costs, long-read microbiome characterization provides added scientific value for clinical microbiome research in the form of higher taxonomic and functional resolution and improved recovery of microbial genomes compared to traditional short-read methodologies.Data SummaryAll supporting data, code and protocols have been provided within the article or as supplementary data files. Two supplementary figures and four supplementary tables are available with the online version of this article. Sequencing data are accessible in the National Center for Biotechnology Information (NCBI) database under BioProject accession number PRJNA754443. The R code and additional data files used for analysis and figure generation are accessible in a GitHub repository (https://github.com/jeanette-gehrig/Gehrig_et_al_sequencing_comparison).Impact StatementAccurate sequencing and analysis are essential for informative microbiome profiling, which is critical for the development of novel microbiome-targeted therapeutics. Recent improvements in long-read sequencing technology provide a promising, but more costly, alternative to ubiquitous short-read sequencing. To our knowledge, a direct comparison of the informational value of short-read and HiFi long-read sequencing approaches has not been reported for clinical microbiome samples. Using samples from participants in a Phase 1b trial of a live biotherapeutic product, we compare microbiome profiles generated from short-read and long-read sequencing for both amplicon-based 16S ribosomal RNA profiling and metagenomic sequencing. Though overall taxonomic profiles were similar across methods, only long-read amplicon sequencing provided strain-level resolution, and long-read metagenomic sequencing resulted in a significantly greater proportion of functionally annotated genes. Detection of a live biotherapeutic active ingredient strain in treated participants was achieved with all methods, and both metagenomic methods provided evidence of active replication of this strain in some participants. Similar taxonomies were recovered through metagenomic assemblies of short and long reads, although assemblies were more complete with long reads. Overall, we show the utility of long-read microbiome sequencing in direct comparison to commonly used short-read methods for clinically relevant microbiome profiling.

Identifying and tracking microbial strains as microbiomes evolve are major challenges in the field of microbiome research. We utilized a new sequencing kit that combines DNA extraction with PCR amplification of a large region of the rRNA operon and downstream bioinformatic data analysis. Longitudinal microbiome samples of coadmitted twins from two different neonatal intensive care units (NICUs) were analyzed using an ∼2,500-base amplicon that spans the 16S and 23S rRNA genes and mapped to a new, custom 16S-23S rRNA database. Amplicon sequence variants (ASVs) inferred using DADA2 provided sufficient resolution for the differentiation of rRNA variants from closely related but not previously sequenced Klebsiella, Escherichia coli, and Enterobacter strains, among the first bacteria colonizing the gut of these infants after admission to the NICU. Distinct ASV groups (fingerprints) were monitored between coadmitted twins over time, demonstrating the potential to track the source and spread of both commensals and pathogens. The high-resolution taxonomy obtained from long amplicon sequencing enables the tracking of strains temporally and spatially as microbiomes are established in infants in the hospital environment.IMPORTANCE Achieving strain-level resolution is a major obstacle for source tracking and temporal studies of microbiomes. In this study, we describe a novel deep-sequencing approach that provides species- and strain-level resolution of the neonatal microbiome. Using Klebsiella, E. coli, and Enterobacter as examples, we could monitor their temporal dynamics after antibiotic treatment and in pairs of twins. The strain-level resolution, combined with the greater sequencing depth and decreased cost per read of PacBio Sequel 2, enables this advantageous source- and strain-tracking analysis method to be implemented widely across more complex microbiomes.

Identifying and tracking microbial strains as microbiomes evolve is a major challenge in the field of microbiome research. Longitudinal microbiome samples of co-admitted twins from two different neonatal intensive care units (NICUs) were analyzed using a ∼2,500 base amplicon that spans the 16S and 23S rRNA genes, and mapped to a new 16S-23S rRNA database. Amplicon Sequence Variants inferred using DADA2 provided sufficient resolution for differentiation of rRNA variants from closely related, but not previously sequenced Klebsiella, E. coli, and Enterobacter, among the first bacteria colonizing the gut of these infants after admission to the NICU. Distinct ASV groups (fingerprints) were followed between co-admitted twins over time, demonstrating the potential to track the source and spread of both commensals and pathogens.The high-resolution taxonomy obtained from long amplicon sequencing enable tracking of strains temporally and spatially as microbiomes are established in infants in the hospital environment.

Background Ankle valgus is frequently encountered in skeletally immature patients in association with a variety of musculoskeletal disorders. Guided growth with temporary medial malleolar transphyseal screw (MMS) hemiepiphysiodesis is an established surgical treatment capable of correcting the angular deformity, but is often complicated by symptomatic screw head prominence and difficult hardware removal. Tension-band plate (TBP) hemiepiphysiodesis has recently been advocated as an alternative; however, the relative efficacy of these 2 techniques has not been directly investigated. Thus, the purpose of this study was to compare MMS and TBP in treatment of pediatric ankle valgus deformity. Methods Medical records and radiographs of all patients undergoing distal tibial medial hemiepiphysiodesis for ankle valgus between January 1, 2005 and November 1, 2010 at a pediatric orthopaedic specialty hospital were retrospectively reviewed. Radiographs obtained preoperatively and at 6-month intervals postoperatively were reviewed and the tibiotalar angle was measured. Patient age, sex, underlying diagnosis, concurrent surgical procedures, surgical and postoperative complications, and the presence or absence of symptomatic hardware complaints were documented. Results Sixty ankles in 42 patients met the inclusion criteria, with adequate radiographs and minimum postoperative follow-up of 12 months (mean: 34 mo). Thirty-five ankles were treated with MMS, and 25 with TBP. Good mean correction of the tibiotalar angle was achieved in both groups (MMS: pre-77.1 degrees to post-87.8 degrees over 25.2 mo; TBP: pre-81.3 to post-87.6 over 20.0 mo). The mean rate of correction was faster in ankles treated with MMS than TBP, but differences did not reach statistical significance (0.55 vs. 0.36 degrees/mo, respectively; P=0.057). Complications included 6 hardware-related surgical complications in MMS ankles (17.1%) and 1 in TBP ankles (4.0%). The incidence of symptomatic hardware complaints was low in both groups (MMS, 5.7%; TBP, 0%). Conclusions Both MMS and TBP techniques can result in successful correction of ankle valgus in the growing child. Although the rate of deformity correction may be faster with MMS, TBP seems to be associated with fewer hardware-related complications. This information may aid the clinician in selecting the surgical option most appropriate for each individual patient. Level of evidence Level II-retrospective study.

Fluorescent dyes that bind DNA have been demonstrated as a useful alternative to radionucleotides for the quantification of DNA and the in vitro measurement of the activity of DNA polymerases and nucleases. However, this approach is generally used in a semi-quantitative way to determine relative rates of reaction. In this report, we demonstrate a method for the simultaneous quantification of DNA in both its single-strand and double-strand forms using the dye PicoGreen. This approach is used in a steady-state assay of DNA polymerase Klenow fragment exo(−), where we determine kcat and Km values for the DNA polymerase that are in excellent agreement with literature values.

Extracting kinetic parameters from DNA polymerase-catalyzed processive polymerization data using traditional initial-rate analysis has proven to be problematic for multiple reasons. The first substrate, DNA template, is a heterogeneous polymer and binds tightly to DNA polymerase. Further, the affinity and speed of incorporation of the second substrate, deoxynucleoside triphosphate (dNTP), vary greatly depending on the nature of the templating base and surrounding sequence. Here, we present a mathematical model consisting of the DNA template-binding step and a Michaelis–Menten-type nucleotide incorporation step acting on a DNA template with a finite length. The model was numerically integrated and globally fitted to experimental reaction time courses. The time courses were determined by monitoring the processive synthesis of oligonucleotides of lengths between 50 and 120 nucleotides by DNA polymerase I (Klenow fragment exo–) using the fluorophore PicoGreen. For processive polymerization, we were able to estimate an enzyme–template association rate k1 of 7.4 μM–1 s–1, a disassociation rate k–1 of 0.07 s–1, and a Kd of 10 nM, and the steady-state parameters for correct dNTP incorporation give kcat values of 2.5–3.3 s–1 and Km values of 0.51–0.86 μM. From the analysis of time courses measured between 5 and 25 °C, an activation energy for kcat of 82 kJ mol–1 was calculated, and it was found that up to 73% of Klenow fragment becomes inactivated or involved in unproductive binding at lower temperatures. Finally, a solvent deuterium kinetic isotope effect (KIE) of 3.0–3.2 was observed under processive synthesis conditions, which suggests that either the intrinsic KIE is unusually high, at least 30–40, or previous findings, showing that the phosphoryl transfer step occurs rapidly and is flanked by two slow conformational changes, need to be re-evaluated. We suggest that the numerical integration of rate equations provides a high level of flexibility and generally produces superior results compared to those of initial-rate analysis in the study of DNA polymerase kinetics and, by extension, other complex enzyme systems.

CYP144 from Mycobacterium tuberculosis was expressed and purified. CYP144 demonstrates heme thiolate coordination in its ferric form, but the cysteinate is protonated to thiol in both the carbon monoxide-bound and ligand-free ferrous forms (forming P420 in the former). Tight binding of various azole drugs was shown, with affinity for miconazole ( K d = 0.98 μM), clotrimazole (0.37 μM) and econazole (0.78 μM) being highest. These azoles are also the trio with the highest affinity for the essential CYP121 and for the cholesterol oxidase CYP125 (essential for host infection), and have high potency as anti-mycobacterial drugs. Construction of a Mtb gene knockout strain demonstrated that CYP144 is not essential for growth in vitro . However the deletion strain was more sensitive to azole inhibition in culture suggesting an important role for CYP144 in cell physiology and/or in mediating azole resistance. The biophysical and genetic features of CYP144 are compared to those of other characterized Mtb P450s, identifying both commonality in properties (including thiolate protonation in ferrous P450s) and intriguing differences in thermodynamic and spectroscopic features. Our developing knowledge of the Mtb P450s has revealed unusual biochemistry and gene essentiality, highlighting their potential as drug targets in this human pathogen.

Background: Chimeric antigen receptor (CAR) T-cell therapy is a rapidly emerging form of treatment for hematologic malignancies including lymphoma, multiple myeloma, and leukemia. Hospitalizations and readmissions after CAR T-cell therapy have not been systematically studied. A better understanding of hospital utilization patterns could inform design of clinical trials, advanced planning of hospitalizations, timing of discharge, and frequency and type of outpatient follow-up. Methods: We conducted a retrospective analysis of all patients admitted to the Massachusetts General Hospital for CAR T-cell therapy between 3/2016 and 3/2018. The primary outcome was hospital readmission within 30 days following discharge from CAR-T treatment visit. Secondary outcomes were ICU admission and inpatient mortality. Exploratory analyses were also conducted to determine whether patient age, length of initial hospitalization, laboratory measurements including ferritin and C-reactive protein (CRP), and tocilizumab exposure were associated with 30-day readmission. Summary statistics were reported by disease types, CAR-T products, and the primary and secondary outcomes using appropriate statistical functions. Comparison of laboratory values (ferritin and CRP) between the index admission (CAR T infusion) and first-readmission visits were analyzed using random intercept linear mixed effects models. Pairwise comparisons regarding mean difference of laboratory measurements during index admission and 30-day readmission/non-readmission were conducted and 95% confidence intervals reported. Bonferroni method was used for p-value adjustment (number of comparison groups = 4). This study was approved by the Partners Healthcare Institutional Review Board. Results: Forty-two patients were treated with CAR T-cells between 3/2016 and 3/2018. Thirty-six patients had non-Hodgkins lymphoma (NHL), 1 acute lymphoblastic leukemia, 1 chronic lymphocytic leukemia, and 4 multiple myeloma. Eight (19%) received the standard of care product, axicabtagene ciloleucel and all others were treated on a clinical trial with an investigational product. Median age at first treatment was 62.2 years old. Twenty-nine patients were male and 13 female. Fourteen patients (33.3%) experienced readmission within 30 days following discharge from CAR T infusion hospitalization. Twelve patients (29%) were readmitted within the first 14 days and 20 patients (47.6%) were readmitted to the hospital within 90 days of discharge (Figure 1). Patient age was not associated with 30-day readmission (P=0.642). Thirty-four (81%) patients received one CAR-T treatment and 8 (19%) received 2 treatments. Among those who received a second CAR-T treatment, 4 (50%) experienced 30-day readmission. Overall, 3 (7%) patients required transfer to the ICU (1 patient after both CAR T infusions) and two patients with NHL died during their first CAR T treatment admission. One patient died of progressive disease 44 days post-CAR T and the other died of disseminated candidemia 18 day post-CAR T. Our mixed effects model showed that median ferritin level was significantly elevated during the 30-day readmission visit as compared to initial CAR T infusion visit (1526.5 vs 1005.5 ug/L, difference in mean = 1088.5, 95% CI: 437.4 to 1739.6, adjusted P = 0.009). CAR T infusion visit ferritin levels did not differ among patients who were readmitted within 30 days and those who were not (1143.0 vs 1005.5 ug/L, P = 1.0). For readmissions which occurred after 30 days, we did not find a statistically significant increase in ferritin levels (P = 0.809). We did not find an association between initial length of hospitalization, CRP levels, and tocilizumab usage and early readmissions (all P-values > 0.05). Conclusions: CAR T-cell therapy is a promising evolving therapy for the treatment of relapsed, refractory hematologic malignancies. Further evaluation of pooled data may allow for early identification of patterns of deterioration which may limit premature hospital discharge, early readmission, and therapy-associated mortality. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

The Mycobacterium tuberculosis cytochrome P450 enzyme CYP142 is encoded in a large gene cluster involved in metabolism of host cholesterol. CYP142 was expressed and purified as a soluble, low spin P450 hemoprotein. CYP142 binds tightly to cholesterol and its oxidized derivative cholest-4-en-3-one, with extensive shift of the heme iron to the high spin state. High affinity for azole antibiotics was demonstrated, highlighting their therapeutic potential. CYP142 catalyzes either 27-hydroxylation of cholesterol/cholest-4-en-3-one or generates 5-cholestenoic acid/cholest-4-en-3-one-27-oic acid from these substrates by successive sterol oxidations, with the catalytic outcome dependent on the redox partner system used. The CYP142 crystal structure was solved to 1.6 Å, revealing a similar active site organization to the cholesterol-metabolizing M. tuberculosis CYP125, but having a near-identical organization of distal pocket residues to the branched fatty acid oxidizing M. tuberculosis CYP124. The cholesterol oxidizing activity of CYP142 provides an explanation for previous findings that ΔCYP125 strains of Mycobacterium bovis and M. bovis BCG cannot grow on cholesterol, because these strains have a defective CYP142 gene. CYP142 is revealed as a cholesterol 27-oxidase with likely roles in host response modulation and cholesterol metabolism.

Rapid laboratory methods provide optimal support for active surveillance efforts to screen for methicillin-resistant Staphylococcus aureus (MRSA). Most laboratories struggle to determine the optimal use of resources, considering options to balance cost, speed, and diagnostic accuracy. To assess the performance of common methods, the first comparison of MRSA Select agar (MS) and CHROMagar MRSA (CA), with and without broth enrichment followed by a 24-h subculture to MS, was performed. Results were compared to those of the Xpert MRSA assay. For direct culture methods, the agreement between MS and CA was 98.8%. At 18 h, direct MS identified 93% of all positive samples from direct culture and 84% of those identified by the Xpert MRSA. For Trypticase soy broth-enriched MS culture, incubated overnight and then subcultured for an additional 24 h, the agreement with Xpert MRSA was 96%. The agreement between direct MS and Xpert MRSA was 100% when semiquantitative culture revealed a bacterial density of 2+ or greater; however, discrepancies between culture and Xpert MRSA arose for MRSA bacterial densities of 1+ or less, indicating low density as a common cause of false-negative culture results. Since 1+ or less was established as the most common MRSA carrier state, broth enrichment or PCR may be critical for the identification of all MRSA carriers who may be reservoirs for transmission. In this active-surveillance convenience sample, the use of broth enrichment followed by subculture to MS offered a low-cost but sensitive method for MRSA screening, with performance similar to that of Xpert MRSA PCR.

The human pathogen Mycobacterium tuberculosis has made a dramatic resurgence in recent years. Drug resistant and multidrug resistant strains are prevalent, and novel antibiotic strategies are desperately needed to counter Mtb's global spread. The M. tuberculosis genome sequence revealed an unexpectedly high number of cytochrome P450 (P450) enzymes (20), and parallel studies indicated that P450-inhibiting azole drugs had potent anti-mycobacterial activity. This article reviews current knowledge of structure/function of P450s and redox partner systems in M. tuberculosis. Recent research has highlighted potential drug target Mtb P450s and provided evidence for roles of selected P450 isoforms in host lipid and sterol/steroid transformations. Structural analysis of key Mtb P450s has provided fundamental information on the nature of the heme binding site, P450 interactions with azole drugs, the biochemical nature of cytochrome P420, and novel mutational adaptations by which azole binding to P450s may be diminished to facilitate azole resistance.

Genomic DNA was amplified about 5 billion-fold from single, flow-sorted bacterial cells by the multiple displacement amplification (MDA) reaction, using φ 29 DNA polymerase. A 662-bp segment of the 16S rRNA gene could be accurately sequenced from the amplified DNA. MDA methods enable new strategies for studying nonculturable microorganisms.

Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size. We describe a method, termed multiple displacement amplification (MDA), which provides a highly uniform representation across the genome. Amplification bias among eight chromosomal loci was less than 3-fold in contrast to 4–6 orders of magnitude for PCR-based WGA methods. Average product length was >10 kb. MDA is an isothermal, strand-displacing amplification yielding about 20–30 μg product from as few as 1–10 copies of human genomic DNA. Amplification can be carried out directly from biological samples including crude whole blood and tissue culture cells. MDA-amplified human DNA is useful for several common methods of genetic analysis, including genotyping of single nucleotide polymorphisms, chromosome painting, Southern blotting and restriction fragment length polymorphism analysis, subcloning, and DNA sequencing. MDA-based WGA is a simple and reliable method that could have significant implications for genetic studies, forensics, diagnostics, and long-term sample storage.

Estrogen signaling is mediated by ERα and -β. ERs are converted from an inactive form to a transcriptionally active state through conformational changes induced by ligand and estrogen-responsive element (ERE) sequences. We show here that ERα and ERβ bind to an ERE independently from ER ligands. We found that although the binding affinity of ERβ for an ERE is 2-fold lower than that of ERα, both ERs use the same nucleotides for DNA contacts. We show that both EREs and ligands are independent modulators of ER conformation. Specifically, the ERE primarily determines the receptor-DNA affinity, whereas the structure of the ER ligand dictates the affinity of ER for particular cofactors. We found that the ligand-dependent cofactor transcriptional intermediary factor-2, through a distinct surface, also interacts with ERα preferentially and independently of ligand. The extent of interaction, however, is dependent upon the ER-ERE affinity. In transfected cells, ERα is more transcriptionally active than ERβ. The ERE sequence, however, determines the potency of gene induction when either ER subtype binds to an agonist. Antagonists prevent ERs from inducing transcription independently from ERE sequences. Thus, ERE- and ligand-induced structural changes are independent determinants for the recruitment of cofactors and transcriptional responses. The ability of ERα to differentially recruit a cofactor could contribute to ER subtype-specific gene responses.

Estrogen-induced signaling mediated by estrogen receptors (ERs) is also affected by aberrant ERs that act as constitutively active or dominant negative modulators. Variant ERs can contribute to carcinogenesis and to the loss of estrogen responsiveness, rendering antiestrogen therapy ineffective. Determining target gene response during co-synthesis of different ER species is difficult, because dimers formed in the presence of more than one ER species are a heterogenous population of homo- or heterodimers. We engineered a homofusion ERalpha as a prototype single-chain receptor by genetically conjugating two ER monomers into a covalently fused single-chain protein to obtain a homogeneous population. This permits analysis of symmetrical or asymmetrical mutations that simulate variant homo- and heterodimers. Although a monomer, the homofusion receptor exhibited similar biochemical and functional properties to the dimeric ERalpha. We used activation function-2 (AF2) defective mutants as a model in either one or both receptor domains for a dominant-negative phenotype by suppressing the reporter activity induced by the WT receptor. When co-expressed with ERalpha, the fusion variant deficient in both AF2 functions suppressed the reporter activity effectively induced by ERalpha. These results show the utility of fusion receptors as models for generation of receptor-based agonists and antagonists.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), nucleocapsid protein (NC), genomic RNA, and the growing DNA strand all influence the copying of the HIV-1 RNA genome into DNA. A detailed understanding of these activities is required to understand the process of reverse transcription. HIV-1 viral DNA is initiated from a tRNA 3 Lys primer bound to the viral genome at the primer binding site. The U3 and R regions of the RNA genome are the first sequences to be copied. The TAR hairpin, a structure found within the R region of the viral genome, is the site of increased RT pausing, RNase H activity, and RT dissociation. Template RNA was digested approximately 17 bases behind the site where polymerase paused at the base of TAR. In most template RNAs, this was the only cleavage made by the RT responsible for initiating polymerization. If the RT that initiated DNA synthesis dissociated from the base of the TAR hairpin and an RT rebound at the end of the primer, there was competition between the polymerase and RNase H activities. After the complete heteroduplex was formed, there were additional RNase H cleavages that did not involve polymerization. Levels of NC that prevented TAR DNA self-priming did not protect genomic RNA from RNase H digestion. RNase H digestion of the 100-bp heteroduplex produced a 14-base RNA from the 5′ end of the RNA that remained annealed to the 3′ end of the minus-strand strong-stop DNA only if NC was present in the reaction.

Water stress substantially alters plant metabolism, decreasing plant growth and photosynthesis1,2,3,4 and profoundly affecting ecosystems and agriculture, and thus human societies5. There is controversy over the mechanisms by which stress decreases photosynthetic assimilation of CO2. Two principal effects are invoked2,4: restricted diffusion of CO2 into the leaf, caused by stomatal closure6,7,8, and inhibition of CO2 metabolism9,10,11. Here we show, in leaves of sunflower (Helianthus annuus L.), that stress decreases CO2 assimilation more than it slows O2 evolution, and that the effects are not reversed by high concentrations of CO212,13. Stress decreases the amounts of ATP9,11 and ribulose bisphosphate found in the leaves, correlating with reduced CO2 assimilation11, but the amount and activity of ribulose bisphosphate carboxylase-oxygenase (Rubisco) do not correlate. We show that ATP-synthase (coupling factor) decreases with stress and conclude that photosynthetic assimilation of CO2 by stressed leaves is not limited by CO2 diffusion but by inhibition of ribulose biphosphate synthesis, related to lower ATP content resulting from loss of ATP synthase.

Cholic acid derivatives appended with amine groups have been prepared for use as permeabilizers of the outer membranes of Gram-negative bacteria. These compounds interact synergistically with antibiotics such as erythromycin and novobiocin to inhibit growth of Gram-negative bacteria. When a hydrophobic chain is included on the permeabilizers, they can be converted into potent antibiotics. The role of the hydrophobic chain is to facilitate self-promoted transport of the cholic acid derivatives across the outer membrane of Gram-negative bacteria. These compounds share activities found with polymyxin B and derivatives.

The estrogen receptor (ER) is a transcription factor that binds to a specific DNA sequence found in the regulatory regions of estrogen-responsive genes, called the estrogen response element (ERE). Many genes that contain EREs have been identified, and most of these EREs contain one or more changes from the core consensus sequence, a 13-nucleotide segment with 10 nucleotides forming an inverted repeat. A number of genes have multiple copies of these imperfect EREs. In order to understand why natural EREs have developed in this manner, we have attempted to define the basic sequence requirements for ER binding. To this end, we measured the binding of homodimeric ER to a variety of nonconsensus EREs. We discovered that an ERE containing even a single change from the consensus may be unable to bind ER. However, an ERE with two changes from the consensus may be capable of binding avidly to ER in the context of certain flanking sequences. We found that changes in the sequences flanking a nonconsensus ERE can greatly alter ER-ERE affinity, either positively or negatively. Careful study of sequences flanking a series of EREs made it possible to develop rules that predict whether ER binds to a given natural ERE and also to predict the relative amounts of binding when comparing two EREs.

Diglyme (Diethylene glycol dimethyl ether, CAS No. 111-96-6) is a glycol ether which has been used in solvent formulations. To assess the potential developmental toxicity of this chemical, groups of pregnant Crl:CD BR rats were exposed to either 0 (control, room air only), 25, 100, or 400 ppm diglyme by inhalation for 6 hrs/day for Days 7 through 16 or gestation (day on which the copulation plug was detected was designation Day 1 G). All female rats were euthanized on day 21G and the fetuses were examined. An additional group of rats was treated with 25 ppm 2-methoxethanol (2ME) to serve as a positive control and for comparison of relative potencies. Maternal toxicity evident as depressed feed consumption at 400 ppm and increased liver weights at 100 ppm. There were no dams in the 400 ppm group with live fetuses (all litters consisted on resorbed conceptuses). Embryo viability was unaffected by concentrations of diglyme as high as 100 ppm. 2ME produced increased liver weights and depressed feed consumption at 25 ppm. Embryo-fetal toxicity was evident as a concentration-related decrease in fetal weight at diglyme concentrations as high as 100 ppm (and with 2ME). There were no fetuses derived from the 400 ppm diglyme-treated dams. A low incidence of structural malformations was observed in all diglyme groups (as well as with 2ME). The incidence of variations, (primarily delayed skeletal ossification and rudimentary ribs) was increased in the 25 and 100 ppm diglyme groups. The incidence and severity in the diglyme and 2ME groups exposed to 25 ppm was essentially the same suggesting similar potency for producing structural variations. In this study, diglyme was embryolethal at 400 ppm; a level that otherwise was only marginally toxic to the dam. Maternal and fetal toxicity also were demonstrated at 100 ppm. Although the fetal defects detected following diglyme exposure at 25 ppm were not significantly different from control values (with the exception of the incidence of skeletal developmental variations), the pattern, type, and incidence of variations were similar to those seen at 100 ppm, suggesting that 25 ppm was an effect level that approaches the lower end of the developmental toxicity response curve. Therefore, the no-observable-effect level (NOEL) for diglyme exposure in the dam is 25 ppm and a NOEL was not clearly demonstrated for the conceptus.

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) was identified as a low abundance protein in bovine uterus that co-purified with estrogen receptor (ER) in a ligand-independent manner and was separated from the ER by its lower retention on estrogen response element (ERE)-Sepharose. In gel mobility shift assays, COUP-TF bound as an apparent dimer to ERE and ERE half-sites. COUP-TF bound to an ERE half-site with high affinity,K d = 1.24 nm. In contrast, ER did not bind a single ERE half-site. None of the class II nuclear receptors analyzed, i.e. retinoic acid receptor, retinoid X receptor, thyroid receptor, peroxisome proliferator-activated receptor, or vitamin D receptor, were constituents of the COUP-TF·DNA binding complex detected in gel mobility shift assays. Direct interaction of COUP-TF with ER was indicated by GST “pull-down” and co-immunoprecipitation assays. The nature of the ER ligand influenced COUP-TF-ERE half-site binding. When ER was liganded by the antiestrogen 4-hydroxytamoxifen (4-OHT), COUP-TF-half-site interaction decreased. Conversely, COUP-TF transcribed and translated in vitroenhanced the ERE binding of purified estradiol (E2)-liganded ER but not 4-OHT-liganded ER. Co-transfection of ER-expressing MCF-7 human breast cancer cells with an expression vector for COUP-TFI resulted in a dose-dependent inhibition of E2-induced expression of a luciferase reporter gene under the control of three tandem copies of EREc38. The ability of COUP-TF to bind specifically to EREs and half-sites, to interact with ER, and to inhibit E2-induced gene expression suggests COUP-TF regulates ER action by both direct DNA binding competition and through protein-protein interactions.

Background: Ankle valgus is encountered in children with a variety of congenital musculoskeletal disorders, including multiple hereditary exostoses (MHE). Guided growth with temporary distal tibial medial hemiepiphysiodesis (DTMH) may correct the deformity; however, exostoses about the ankle commonly observed in MHE patients may hinder correction and increase the risk of recurrence. Thus, the purpose of this study was to review the outcomes of DTMH in treatment of ankle valgus in MHE versus other diagnosis (non-MHE). Methods: Medical records and radiographs of patients undergoing DTMH for ankle valgus between January 1, 2005, and November 1, 2010, at a single pediatric orthopedic hospital were retrospectively analyzed. Radiographs obtained preoperatively and at 6-month intervals postoperatively were reviewed and the tibiotalar angle was measured. Results: Fifty-eight ankles in 41 patients met inclusion criteria, with minimum follow-up of 12 months (mean, 34 months). Mean age was 10 years (range, 4-14 years). MHE was the most common underlying diagnosis (19 ankles, 33%). The rate of tibiotalar angle correction (mean ± standard deviation) with hardware in place was 0.37 ± 0.28 deg/mo in MHE ankles and 0.51 ± 0.42 deg/mo in non-MHE ankles ( P = .161). Following hardware removal, the rate of recurrence was faster in MHE (0.29 ± 0.25 deg/mo) compared with non-MHE ankles (0.12 ± 0.19 deg/mo) ( P = .059), and more total recurrent valgus deformity was observed in MHE (7.8 ± 8.2 degrees) than non-MHE ankles (3.4 ± 4.6 degrees) ( P = .08) over a similar follow-up period (mean 23.4 vs 23.6 months, respectively), with differences approaching statistical significance. Conclusion: MHE is a common cause of ankle valgus in children. Guided growth interventions in this population can be successful but require special consideration given the potential for relatively gradual deformity correction and rapid recurrence following hardware removal in the skeletally immature. Level of Evidence: Level III, retrospective comparative study.

Background: Indications for surgical meniscal repair are limited, and failure rates remain high. Thus, new ways to augment repair and stimulate meniscal regeneration are needed. Mesenchymal stem cells are multipotent cells present in mature individuals and accessible from peripheral connective tissue sites, including synovium. The purpose of this study was to quantitatively evaluate the effect of implantation of synovial tissue-derived mesenchymal stem cells on meniscal regeneration in a rabbit model of partial meniscectomy. Methods: Synovial mesenchymal stem cells were harvested from the knee of one New Zealand White rabbit, expanded in culture, and labeled with a fluorescent marker. A reproducible 1.5-mm cylindrical defect was created in the avascular portion of the anterior horn of the medial meniscus bilaterally in fifteen additional rabbits. Allogenic synovial mesenchymal stem cells suspended in phosphate-buffered saline solution were implanted into the right knees, and phosphate-buffered saline solution alone was placed in the left knees. Meniscal regeneration was evaluated histologically at four, twelve, and twenty-four weeks for (1) quantity and (2) quality (with use of an established three-component scoring system). A similar procedure was performed in four additional rabbits with use of green fluorescent protein-positive synovial mesenchymal stem cells for the purpose of tracking progeny following implantation. Results: The quantity of regenerated tissue in the group that had implantation of synovial mesenchymal stem cells was greater at all end points, reaching significance at four and twelve weeks (p < 0.05). Tissue quality scores were also superior in knees treated with mesenchymal stem cells compared with controls at all end points, achieving significance at twelve and twenty-four weeks (3.8 versus 2.8 at four weeks [p = 0.29], 5.7 versus 1.7 at twelve weeks [p = 0.008], and 6.0 versus 3.9 at twenty-four weeks [p = 0.021]). Implanted cells adhered to meniscal defects and were observed in the regenerated tissue, where they differentiated into type-I and II collagen-expressing cells, at up to twenty-four weeks. Conclusions: Synovial mesenchymal stem cells adhere to sites of meniscal injury, differentiate into cells resembling meniscal fibrochondrocytes, and enhance both quality and quantity of meniscal regeneration. Clinical Relevance: These results may stimulate further exploration into the utility of synovial mesenchymal stem cells in the treatment of meniscal injury in large animals and humans.

Purpose To evaluate knee stability after anterior cruciate ligament (ACL) reconstruction using 2 modern clinically relevant single-bundle constructs. Methods Two arthroscopic ACL reconstructions were performed on 6 fresh-frozen human cadaveric knees using bone–patellar tendon–bone autografts. The tibial tunnel was centered in the anatomic tibial footprint. The femoral tunnel was reamed through the anteromedial (AM) portal and centered alternately in either the AM portion of the femoral footprint (center-AM) or the center of the femoral footprint (center-center). Two external loading conditions were applied: (1) a 134-N anterior tibial load and (2) a 10-Nm valgus load combined with a 5-Nm internal tibial torque. Resulting kinematics were determined under 4 conditions: (1) ACL intact, (2) ACL deficient, (3) center-AM reconstruction, and (4) center-center reconstruction. Results In response to anterior tibial loading, anterior translation was similar in the ACL-intact knee and the 2 reconstructions at 0° to 60° of flexion but was greater in the reconstructed specimens at 90°. In response to the complex rotatory load, internal tibial rotation (ITR) at 30° of flexion was slightly greater in center-AM knees compared with ACL-intact knees (11.0° ± 0.6° v 10.5° ± 0.6°, P = .03). At other angles tested, ITR in both reconstructions was similar to the ACL-intact knee ( P > .05). When we compared the 2 reconstruction alternatives, however, center-center knees exhibited greater resistance to ITR at all angles ( P Conclusion Anatomic single-bundle ACL reconstruction performed with the femoral tunnel placed through the AM portal restores translational and rotational knee stability to an extent that closely approximates the ACL-intact condition. When compared with the AM femoral tunnel position, a femoral tunnel positioned in the anatomic center of the femoral origin of the ACL may further improve rotatory stability without sacrificing anterior stability. Clinical Relevance This study provides additional biomechanical evidence in support of anatomic single-bundle ACL reconstruction with tunnels positioned in the center of the femoral and tibial footprints.

The human pathogen Mycobacterium tuberculosis (Mtb) encodes 20 cytochrome P450 (P450) enzymes. Gene essentiality for viability or host infection was demonstrated for Mtb P450s CYP128, CYP121 and CYP125. Structure/function studies on Mtb P450s revealed key roles contributing to bacterial virulence and persistence in the host. Various azole-class drugs bind with high affinity to the Mtb P450 heme and are potent Mtb antibiotics. This paper reviews the current understanding of the biochemistry of Mtb P450s, their interactions with azoles and their potential as novel Mtb drug targets. Mtb multidrug resistance is widespread and novel therapeutics are desperately needed. Simultaneous drug targeting of several Mtb P450s crucial to bacterial viability/persistence could offer a new route to effective antibiotics and minimize the development of drug resistance.

Cholic acid derivatives appended with amine groups have been prepared for use as permeabilizers of the outer membranes of Gram-negative bacteria. These compounds interact synergistically with antibiotics such as erythromycin and novobiocin to inhibit growth of Gram-negative bacteria. When a hydrophobic chain is included on the permeabilizers, they can be converted into potent antibiotics. The role of the hydrophobic chain is to facilitate self-promoted transport of the cholic acid derivatives across the outer membrane of Gram-negative bacteria. These compounds share activities found with polymyxin B and derivatives.

Background Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods. Results A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point. Conclusions Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.

Background Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. Results SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. Conclusions Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.

Understanding how viral components collaborate to convert the human immunodeficiency virus type 1 genome from single-stranded RNA into double-stranded DNA is critical to the understanding of viral replication. Not only must the correct reactions be carried out, but unwanted side reactions must be avoided. After minus-strand strong stop DNA (−sssDNA) synthesis, degradation of the RNA template by the RNase H domain of reverse transcriptase (RT) produces single-stranded DNA that has the potential to self-prime at the imperfectly base-paired TAR hairpin, making continued DNA synthesis impossible. Although nucleocapsid protein (NC) interferes with −sssDNA self-priming in reverse transcription reactions in vitro, NC alone did not prevent self-priming of a synthetic −sssDNA oligomer. NC did not influence DNA bending and therefore cannot inhibit self-priming at hairpins by directly blocking hairpin formation. Using DNA oligomers as a model for genomic RNA fragments, we found that a 17-base DNA fragment annealed to the 3′ end of the −sssDNA prevented self-priming in the presence of NC. This implies that to avoid self-priming, an RNA-DNA hybrid that is more thermodynamically stable than the hairpin must remain within the hairpin region. This suggests that NC prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the kinetically favored TAR hairpin. In support of this idea, sequence changes that increased base pairing in the DNA TAR hairpin resulted in an increase in self-priming in vitro. We present a model describing the role of NC-dependent inhibition of self-priming in first-strand transfer.

Estrogen-inducible genes contain an enhancer called the estrogen response element (ERE), a double-stranded inverted repeat. The estrogen receptor (ER) is generally thought to bind to the double-stranded ERE. However, some reports provide evidence that an ER homodimer can bind a single strand of the ERE and suggest that single-stranded ERE binding is the preferred binding mode for ER. Since these two models describe quite different mechanisms of receptor action, we have attempted to reconcile the observations. Analyzing DNA structure by nuclease sensitivity, we found that two identical molecules of a single strand of DNA containing the ERE sequence can partially anneal in an antiparallel manner. Bimolecular annealing produces double-stranded inverted repeats, with adjacent unannealed tails. The amount of annealing correlates exactly with the ability of ER to bind bimolecular EREs. Either strand of an ERE could anneal to itself in a way that would bind ER. We conclude that ER binds only the annealed double-stranded ERE both in vitro and in vivo.

Quantitative DNase I footprinting assays were employed to simultaneously measure the amount of estrogen receptor (ER) bound to each site in constructs containing multiple estrogen response elements (EREs). These assays revealed identical, high affinity ER-ERE binding, K d of approximately 0.25 nM, for estradiol-liganded ER (E 2 -ER), 4-hydroxytamoxifen liganded ER (4-OHT-ER), tamoxifen aziridine liganded ER (TAz-ER), and unliganded dimeric ER, for each ERE in constructs containing up to four tandem EREs. Increasing concentrations of ER resulted in the same pattern of occupancy for each ERE, whether or not the site was located near other EREs. Similarly, the presence or absence of E 2 , 4-OHT, or TAz ligand did not change ER-ERE interaction. Since activated ER-ERE binding affinity is identical, whether ER is liganded or unliganded, ligand cannot regulate ER-ERE binding affinity. These results support the hypothesis that ligand-dependent conformational changes primarily determine how ER interacts with components of the transcription initiation complex that mediate gene transactivation. In addition, footprint assays revealed that, following ER binding, an AT-rich site adjacent to the ERE becomes hypersensitive to DNase I digestion. This sequence may be easily or intrinsically bent, assisting in recruiting ER to ERE sites.

During the aggregation of Dictyostelium discoideum extracellular cAMP is known to act as a chemotractant and as an inducer of cellular differentiation. However, its intracellular role as a second messenger remains obscure. We have constructed a fusion gene consisting of the cDNA encoding the regulatory subunit (R) of the cAMP-dependent protein kinase fused to the promoter and N-terminal-proximal sequences of a Dictyostelium actin gene. Stable transformants, containing multiple copies of this gene, overproduce the R subunit which accumulates prematurely relative to the endogenous protein. These transformants fail to aggregate. Detailed analysis has shown that they are blocked at interphase, the period prior to aggregation, and that they are severely defective in most responses to cAMP including the induction of gene expression. Our observations suggest that intracellular cAMP acts, presumably by activation of the catalytic subunit of the cAMP-dependent protein kinase, to facilitate early development.

The present study examined the effects of physostigmine, a cholinesterase inhibitor, on activity in young rats whose mothers consumed isocaloric liquid diets containing 35% or 0% ethanol-derived calories on days 6-20 of pregnancy. A pair-feeding procedure was utilized and an ad libitum lab chow group was included. Physostigmine was administered to 18-day-old offspring on 3 consecutive days with activity measures recorded for 30 min each day. Injections of physostigmine produced significant reductions in activity in alcohol-exposed offspring relative to control groups. These results provide support for a functional cholinergic deficit in offspring exposed to alcohol in utero and may have relevant clinical implications in the treatment of attentional deficit disorder which can occur following prenatal alcohol exposure.

A field trial of artificial insemination in horses with transproted, chilled semen was conducted using a specially designed container which permitted a controlled, slow initial rate of cooling (-0.3 degrees C/min) and maintenance of a final temperature of 4 degrees -6 degrees C for more than 36 hrs. Forty-six mares in 23 states were inseminated with semen from three German Warmblood stallions standing at stud in Hamilton, Massachusetts. A third-cycle conception rate of 91% was obtained.