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Introduction: Novel therapeutics are emerging to mitigate damage from perinatal brain injury (PBI). Few newborns with PBI suffer from a singular etiology. Most experience cumulative insults from prenatal inflammation, genetic and epigenetic vulnerability, toxins (opioids, other drug exposures, environmental exposure), hypoxia-ischemia, and postnatal stressors such as sepsis and seizures. Accordingly, tailoring of emerging therapeutic regimens with endogenous repair or neuro-immunomodulatory agents for individuals requires a more precise understanding of ligand, receptor-, and non-receptor-mediated regulation of essential developmental hormones. Given the recent clinical focus on neurorepair for PBI, we hypothesized that there would be injury induced changes in erythropoietin (EPO), erythropoietin receptor (EPOR), melatonin receptor (MLTR), NAD-dependent deacetylase sirtuin-1 (SIRT1) signaling, and hypoxia inducible factors (HIF1α, HIF2α). Specifically, we predicted that EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α alterations after chorioamnionitis (CHORIO) would reflect relative changes observed in human preterm infants. Similarly, we expected unique developmental regulation after injury that would reveal potential clues to mechanisms and timing of inflammatory and oxidative injury after CHORIO that could inform future therapeutic development to treat PBI. Methods: To induce CHORIO, a laparotomy was performed on embryonic day 18 (E18) in rats with transient uterine artery occlusion plus intra-amniotic injection of lipopolysaccharide (LPS). Placentae and fetal brains were collected at 24 h. Brains were also collected on postnatal day 2 (P2), P7, and P21. EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α levels were quantified using a clinical electrochemiluminescent biomarker platform, qPCR, and/or RNAscope. MLT levels were quantified with liquid chromatography mass spectrometry Results: Examination of EPO, EPOR, and MLTR1 at 24 h showed that while placental levels of EPO and MLTR1 mRNA were decreased acutely after CHORIO, cerebral levels of EPO, EPOR and MLTR1 mRNA were increased compared to control. Notably, CHORIO brains at P2 were SIRT1 mRNA deficient with increased HIF1α and HIF2α despite normalized levels of EPO, EPOR and MLTR1, and in the presence of elevated serum EPO levels. Uniquely, brain levels of EPO, EPOR and MLTR1 shifted at P7 and P21, with prominent CHORIO-induced changes in mRNA expression. Reductions at P21 were concomitant with increased serum EPO levels in CHORIO rats compared to controls and variable MLT levels. Discussion: These data reveal that commensurate with robust inflammation through the maternal placental-fetal axis, CHORIO impacts EPO, MLT, SIRT1, and HIF signal transduction defined by dynamic changes in EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α mRNA, and EPO protein. Notably, ligand-receptor mismatch, tissue compartment differential regulation, and non-receptormediated signaling highlight the importance, complexity and nuance of neural and immune cell development and provide essential clues to mechanisms of injury in PBI. As the placenta, immune cells, and neural cells share many common, developmentally regulated signal transduction pathways, further studies are needed to clarify the perinatal dynamics of EPO and MLT signaling and to capitalize on therapies that target endogenous neurorepair mechanisms. [ABSTRACT FROM AUTHOR]

Background Lemierre's syndrome is an infectious phenomenon characterized by oropharyngeal infection with bacteraemia, thrombophlebitis, and distant septic emboli. Septic emboli are a recognized cause of a Type 2 myocardial infarction, with a left ventricular pseudoaneurysm being a rare but important complication of this. Case summary A 19-year-old male presented with acute confusion, fevers, and a cough. Blood cultures were positive for Fusobacterium necrophorum and initial imaging showed a cavitating pneumonia. Further evaluation revealed septic emboli in the distal digits and brain. The patient initially responded to antibiotic therapy but developed chest pain with increased troponin levels. An electrocardiogram showed inferolateral ST elevation. A transthoracic echocardiogram (TTE) showed hypokinaesia of the mid to apical lateral wall, and a computed tomography (CT) scan showed a pericardial effusion with a possible purulent effusion or abscess. The patient underwent surgical drainage of a sterile effusion. A post-operative TTE and CT demonstrated a left ventricular pseudoaneurysm that was surgically repaired. The venous thrombus was encountered intra-operatively confirming a diagnosis of Lemierre's syndrome. The patient completed the regimen of antibiotics and showed a good post-operative recovery. Discussion This is the first case described of left ventricular pseudoaneurysm as a complication of Lemierre's syndrome. It highlights not only the importance of serial, multimodality imaging in both diagnostic workup and identification of complications, but also the importance of a multidisciplinary team in the management of patients with complex and rare presentations. [ABSTRACT FROM AUTHOR]

Starvation ketoacidosis (SKA) represents one of three metabolic acidoses caused by the accumulation of ketone bodies within the bloodstream. While easily treated, it is a diagnosis that can be easily missed in patients with an unexplained metabolic acidosis. In this case report, we discuss two patients presenting with a starvation ketoacidosis and psychiatric illness. This link between SKA and psychiatric disease is especially pertinent as this is a cohort of patients who may not be able to give an accurate history, which can potentially lead to a delay in diagnosis and treatment. Furthermore, the patient cohort described here have higher rates of alcohol dependence and are therefore at risk of alcoholic ketoacidosis. It is important to recognise that these conditions may coexist and should be managed as such, with thiamine prior to carbohydrate replacement in all at-risk patients. [ABSTRACT FROM AUTHOR]

Soluble amyloid-beta (Aβ) oligomers are the prime causative agents of cognitive deficits during early stages of Alzheimer's disease (AD). The transient nature of the oligomers makes them difficult to characterize by traditional techniques, suggesting that advanced approaches are necessary. Previously developed fluorescence-based tethered approach for probing intermolecular interactions (TAPIN) and AFM-based single-molecule force spectroscopy are capable of probing dimers of Aβ peptides. In this paper, a novel polymer nanoarray approach to probe trimers and tetramers formed by the Aβ(14–23) segment of Aβ protein at the single-molecule level is applied. By using this approach combined with TAPIN and AFM force spectroscopy, the impact of pH on the assembly of these oligomers was characterized. Experimental results reveal that pH affects the oligomer assembly process. At neutral pH, trimers and tetramers assemble into structures with a similar stability, while at acidic conditions (pH 3.7), the oligomers adopt a set of structures with different lifetimes and strengths. Models for the assembly of Aβ(14–23) trimers and tetramers based on the results obtained is proposed. [ABSTRACT FROM AUTHOR]

Triple-helix-forming oligodeoxynucleotides (ODNs) altered at the 3'- and 5'-ends with the clinically used nitrogen mustard chlorambucil, can very efficiently, and with very good specificity, alkylate both Watson and Crick strands of a DNA target. ODNs provide a route to examine whether sequence-targeted interstrand cross-linkage of the genomic DNA can achieve selective and irrevocable gene inactivation.

Pregnancy is an inflammatory process that is carefully regulated by the placenta via immunomodulation and cell-to-cell communication of maternal and fetal tissues. Exosomes, types of extracellular vesicles, facilitate the intercellular communication and traffic biologically modifying cargo within the maternal-placental-fetal axis in normal and pathologic pregnancies. Chorioamnionitis is characterized by inflammation of chorioamniotic membranes that produces systemic maternal and fetal inflammatory responses of cytokine dysregulation and has been associated with brain injury and neurodevelopmental disorders. This review focuses on how pathologic placental exosomes propagate acute and chronic inflammation leading to brain injury. The evidence reviewed here highlights the need to investigate exosomes from pathologic pregnancies and those with known brain injury to identify new diagnostics, biomarkers, and potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Targeting DNA repair proteins with small-molecule inhibitors became a proven anti-cancer strategy. Previously, we identified an inhibitor of a major protein of homologous recombination (HR) RAD51, named B02. B02 inhibited HR in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo. Here, using a medicinal chemistry approach, we aimed to improve the potency of B02. We identified the B02 analog, B02-isomer, which inhibits HR in human cells with significantly higher efficiency. We also show that B02-iso sensitizes triple-negative breast cancer MDA-MB-231 cells to the PARP inhibitor (PARPi) olaparib. [ABSTRACT FROM AUTHOR]

Introduction: Novel therapeutics are emerging to mitigate damage from perinatal brain injury (PBI). Few newborns with PBI suffer from a singular etiology. Most experience cumulative insults from prenatal inflammation, genetic and epigenetic vulnerability, toxins (opioids, other drug exposures, environmental exposure), hypoxia-ischemia, and postnatal stressors such as sepsis and seizures. Accordingly, tailoring of emerging therapeutic regimens with endogenous repair or neuro-immunomodulatory agents for individuals requires a more precise understanding of ligand, receptor-, and non-receptor-mediated regulation of essential developmental hormones. Given the recent clinical focus on neurorepair for PBI, we hypothesized that there would be injury-induced changes in erythropoietin (EPO), erythropoietin receptor (EPOR), melatonin receptor (MLTR), NAD-dependent deacetylase sirtuin-1 (SIRT1) signaling, and hypoxia inducible factors (HIF1α, HIF2α). Specifically, we predicted that EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α alterations after chorioamnionitis (CHORIO) would reflect relative changes observed in human preterm infants. Similarly, we expected unique developmental regulation after injury that would reveal potential clues to mechanisms and timing of inflammatory and oxidative injury after CHORIO that could inform future therapeutic development to treat PBI. Methods: To induce CHORIO, a laparotomy was performed on embryonic day 18 (E18) in rats with transient uterine artery occlusion plus intra-amniotic injection of lipopolysaccharide (LPS). Placentae and fetal brains were collected at 24 h. Brains were also collected on postnatal day 2 (P2), P7, and P21. EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α levels were quantified using a clinical electrochemiluminescent biomarker platform, qPCR, and/or RNAscope. MLT levels were quantified with liquid chromatography mass spectrometry. Results: Examination of EPO, EPOR, and MLTR1 at 24 h showed that while placental levels of EPO and MLTR1 mRNA were decreased acutely after CHORIO, cerebral levels of EPO, EPOR and MLTR1 mRNA were increased compared to control. Notably, CHORIO brains at P2 were SIRT1 mRNA deficient with increased HIF1α and HIF2α despite normalized levels of EPO, EPOR and MLTR1, and in the presence of elevated serum EPO levels. Uniquely, brain levels of EPO, EPOR and MLTR1 shifted at P7 and P21, with prominent CHORIO-induced changes in mRNA expression. Reductions at P21 were concomitant with increased serum EPO levels in CHORIO rats compared to controls and variable MLT levels. Discussion: These data reveal that commensurate with robust inflammation through the maternal placental-fetal axis, CHORIO impacts EPO, MLT, SIRT1, and HIF signal transduction defined by dynamic changes in EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α mRNA, and EPO protein. Notably, ligand-receptor mismatch, tissue compartment differential regulation, and non-receptor-mediated signaling highlight the importance, complexity and nuance of neural and immune cell development and provide essential clues to mechanisms of injury in PBI. As the placenta, immune cells, and neural cells share many common, developmentally regulated signal transduction pathways, further studies are needed to clarify the perinatal dynamics of EPO and MLT signaling and to capitalize on therapies that target endogenous neurorepair mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kitase, Madurai, Hamimi, Hellinger, Odukoya, Ramachandra, Muthukumar, Vasan, Sevensky, Kirk, Gall, Heck, Ozen, Orsburn, Robinson and Jantzie.)

Pair-wise interactions at the single-molecule level can be done with nanoprobing techniques, such as AFM force spectroscopy, optical tweezers, and magnetic tweezers. These techniques can be used to probe interactions between well-characterized assemblies of biomolecules, such as monomer-dimer, dimer-dimer, and trimer-monomer. An important step of these techniques is the proper assembly of dimers, trimers, and higher oligomers to enable the interactions to be probed. We have developed a novel approach in which a defined number of peptides are assembled along a flexible polymeric molecule that serves as a linear matrix, termed as flexible nanoarray (FNA). The construct is synthesized with the use of phosphoramidite chemistry (PA), in which non-nucleoside PA spacers and standard oligonucleotide synthesis are used to grow the polymeric chain with the desired length. The reactive sites are incorporated during FNA synthesis. As a result, the FNA polymer contains a set of predesigned reactive sites to which the peptides are covalently conjugated. We describe the protocol for the synthesis of FNA and the application of this methodology to measure the molecular interactions between amyloid peptides of monomer-monomer, monomer-dimer, and dimer-dimer.

Bacillus anthracis is a tier 1 select agent with the potential to quickly cause severe disease. Rapid identification of this pathogen may accelerate treatment and reduce mortality in the event of a bioterrorism attack. We developed a rapid and sensitive assay to detect B. anthracis bacteremia using a system that is suitable for point-of-care testing. A filter-based cartridge that included both sample processing and PCR amplification functions was loaded with all reagents needed for sample processing and multiplex nested PCR. The assay limit of detection (LOD) and dynamic range were determined by spiking B. anthracis DNA into individual PCR mixtures and B. anthracis CFU into human blood. One-milliliter blood samples were added to the filter-based detection cartridge and tested for B. anthracis on a GeneXpert instrument. Assay specificity was determined by testing blood spiked with non-anthrax bacterial isolates or by testing blood samples drawn from patients with concurrent non- B. anthracis bacteremia or nonbacteremic controls. The assay LODs were 5 genome equivalents per reaction and 10 CFU/ml blood for both the B. anthracis Sterne and V1B strains. There was a 6-log 10 dynamic range. Assay specificity was 100% for tests of non- B. anthracis bacterial isolates and patient blood samples. Assay time was less than 90 min. This automated system suitable for point-of-care detection rapidly identifies B. anthracis directly from blood with high sensitivity. This assay might lead to early detection and more rapid therapy in the event of a bioterrorism attack., (Copyright © 2017 American Society for Microbiology.)

The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection. IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R., (Copyright © 2017 Chakravorty et al.)

Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrs genes and the promoters of inhA and eis genes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of Mycobacterium tuberculosis in 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods., (Copyright © 2016 American Society for Microbiology.)

Probing of biomolecular complexes by single-molecule force spectroscopy (SMFS) methods including AFM requires proper and suitable coupling methods for immobilization of biomolecules onto the AFM tip and the surface. The use of flexible tethers for the coupling process has dual advantages. First, they allow the specific immobilization of interacting molecules, and second, their flexibility facilitates the proper orientation of the interacting partners. Recently, we developed an approach termed Flexible Nano Array (FNA) in which interacting partners are located on the same polymeric FNA molecule separated by a flexible segment with a defined length. In this paper, we modified the FNA tether approach by incorporating click chemistry with non-metal modification. FNA was synthesized using DNA synthesis chemistry, in which phosphoramidite (PA) spacers containing six ethylene glycol units were used instead of nucleoside triphosphates. During the synthesis, two T modifiers conjugated to two dibenzocyclooctyl (DBCO) residues were incorporated at selected positions within the FNA. The DBCO functionality allows for coupling azide labeled biomolecules via click chemistry. Amyloid peptide Aβ(14-23) terminated with azide was incorporated into the FNA and the reaction was controlled with mass-spectrometry. Assembly of tethered Aβ(14-23) peptides into dimers was characterized by AFM force spectroscopy experiments in which the AFM tip functionalized with FNA terminated with biotin probed a streptavidin-coated mica surface. The formation of the peptide dimer was verified with force spectroscopy that showed the appearance of a specific fingerprint for dimer dissociation followed by a rupture event for the biotin-streptavidin link. The developed approach is capable of multiple probing events to allow the collection of a large set of data for a quantitative analysis of the force spectroscopy events., Competing Interests: Conflict of Interests The authors declare no competing interests.

Immobilization is a key step involved in probing molecular interactions using single-molecule force spectroscopy methods, including atomic force microscopy (AFM). To our knowledge, we describe a novel approach termed flexible nanoarray (FNA) in which the interaction between the two internally immobilized amyloid β peptides is measured by pulling of the tether. The FNA tether was synthesized with nonnucleotide phosphoramidite monomers using the DNA synthesis chemistry. The two anchoring points for immobilization of the peptides inside the tether were incorporated at defined distances between them and from the ends of the polymer. Decamers of amyloid β peptide capable of dimer formation were selected as a test system. The formation of the peptide dimers was verified by AFM force spectroscopy by pulling the tether at the ends. In these experiments, the thiolated end of the FNA tether was covalently immobilized on the AFM substrate functionalized with maleimide. The other end of the FNA tether was functionalized with biotin to form a noncovalent link with the streptavidin functionalized AFM tip during the approach stage. The dimers' rupture fingerprint was unambiguously identified on the force curves by its position and the force value. The FNA design allowed reversible experiments in which the monomers were allowed to associate after the rupture of the dimers by performing the approach stage before the rupture of the biotin-streptavidin link. This suggests that the FNA technique is capable of analyzing multiple intermolecular interactions in the same molecular complex. The computational analysis showed that the tethered peptides assemble into the same dimer structure as that formed by nontethered peptides, suggesting that the FNA tether has the necessary flexibility to enable assembly of the dimer even during the course of the force spectroscopy experiment., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

This article describes sample preparation techniques for AFM imaging of DNA and protein-DNA complexes. The approach is based on chemical functionalization of the mica surface with aminopropyl silatrane (APS) to yield an APS-mica surface. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations, and in a broad range of pH. The chapter describes the methodologies for the preparation of APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purification of APS is also provided. The AFM applications are illustrated with examples of images of DNA and protein-DNA complexes.

Surface preparation is a key step for reliable and reproducible imaging of DNA and protein-DNA complexes with atomic force microscopy (AFM). This article describes the approaches for chemical functionalization of the mica surface. One approach utilizes 3-aminopropyl-trietoxy silane (APTES), enabling one to obtain a smooth surface termed AP-mica. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations and in a broad range of pH. Another method utilizes aminopropyl silatrane (APS) to yield an APS-mica surface. The advantage of APS-mica compared with AP-mica is the ability to obtain reliable and reproducible time-lapse images in aqueous solutions. The chapter describes the methodologies for the preparation of AP-mica and APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purification of APS is also provided. The applications are illustrated with a number of examples.

Despite their rather recent invention, atomic force microscopes are widely available commercially. AFM and its special modifications (tapping mode and noncontact operation in solution) have been successfully used for topographic studies of a large number of biological objects including DNA, RNA, proteins, cell membranes, and even whole cells. AFM was also successfully applied to studies of nucleic acids and various protein DNA complexes. Part of this success is due to the development of reliable sample preparation procedures. This chapter describes one of the approaches based on chemical functionalization of mica surface with 1-(3-aminopropyl) silatrane (APS). One of the most important properties of APS-mica approach is that the sample can be deposited on the surface in a wide range of ionic strengths, in the absence of divalent cations and a broad range of pH. In addition to imaging of dried sample, APS-mica allows reliable and reproducible time lapse imaging in aqueous solutions. Finally, APS mica is terminated with reactive amino groups that can be used for covalent and ionic attachment of molecules for AFM force spectroscopy studies. The protocols for the preparation of APS, functionalization with APS mica and AFM probes, preparation of samples for imaging in air and in aqueous solutions, and force spectroscopy studies are outlined. All these applications are illustrated with a few examples.