Unraveling the relationship between structure and stabilization of triarylpyridines as G-quadruplex binding ligands.

A series of novel 2,4,6-triarylpyridines have been synthesized and their interactions with intramolecular G-quadruplexes have been measured by Förster Resonance Energy Transfer (FRET) melting and Fluorescent Intercalator Displacement (FID) assays. A few of these compounds exhibit stabilization of G4-DNA that is comparable to other benchmark G4-DNA ligands with fair to excellent G4-DNA vs. duplex selectivity and significant cytotoxicity towards HeLa cells. The nature of the 4-aryl substituents along with side chain length governs the G4-DNA stabilization ability of the compounds. In addition, we demonstrate that there is a strong correlation between the ability of the compounds to stabilize the same G4-DNA sequence in K(+) and Na(+) conditions and a strong correlation between the ability of the compounds to stabilize different G4-DNA sequences in K(+) or Na(+) buffer.

In pursuit of the triple crown: mechanism-based pharmacodynamic modelling for the optimization of three-drug combinations against KPC-producing Klebsiella pneumoniae.

OBJECTIVES: Optimal combination therapy for Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) is unknown. The present study sought to characterize the pharmacodynamics (PD) of polymyxin B (PMB), meropenem (MEM) and rifampin (RIF) alone and in combination using a hollow fibre infection model (HFIM) coupled with mechanism-based modelling (MBM). METHODS: A 10-day HFIM was utilized to simulate human pharmacokinetics (PK) of various PMB, MEM and RIF dosing regimens against a clinical KPC-Kp isolate, with total and resistant subpopulations quantified to capture PD response. A MBM was developed to characterize bacterial subpopulations and synergy between agents. Simulations using the MBM and published population PK models were employed to forecast the bacterial time course and the extent of its variability in infected patients for three-drug regimens. RESULTS: In the HFIM, a PMB single-dose ('burst') regimen of 5.53 mg/kg combined with MEM 8 g using a 3-hr prolonged infusion every 8 hr and RIF 600 mg every 24 hr resulted in bacterial counts below the quantitative limit within 24 hr and remained undetectable throughout the 10-day experiment. The final MBM consisted of two bacterial subpopulations of differing PMB and MEM joint susceptibility and the ability to form a non-replicating, tolerant subpopulation. Synergistic interactions between PMB, MEM and RIF were well quantified, with the MBM providing adequate capture of the observed data. DISCUSSION: An in vitro-in silico approach answers questions related to PD optimization as well as overall feasibility of combination therapy against KPC-Kp, offering crucial insights in the absence of clinical trials.

Using machine learning to optimize antibiotic combinations: dosing strategies for meropenem and polymyxin B against carbapenem-resistant Acinetobacter baumannii.

OBJECTIVES: Increased rates of carbapenem-resistant strains of Acinetobacter baumannii have forced clinicians to rely upon last-line agents, such as the polymyxins, or empirical, unoptimized combination therapy. Therefore, the objectives of this study were: (a) to evaluate the in vitro pharmacodynamics of meropenem and polymyxin B (PMB) combinations against A. baumannii; (b) to utilize a mechanism-based mathematical model to quantify bacterial killing; and (c) to develop a genetic algorithm (GA) to define optimal dosing strategies for meropenem and PMB. METHODS: A. baumannii (N16870; MICmeropenem = 16 mg/L, MICPMB = 0.5 mg/L) was studied in the hollow-fibre infection model (initial inoculum 108 cfu/mL) over 14 days against meropenem and PMB combinations. A mechanism-based model of the data and population pharmacokinetics of each drug were used to develop a GA to define the optimal regimen parameters. RESULTS: Monotherapies resulted in regrowth to ~1010 cfu/mL by 24 h, while combination regimens employing high-intensity PMB exposure achieved complete bacterial eradication (0 cfu/mL) by 336 h. The mechanism-based model demonstrated an SC50 (PMB concentration for 50% of maximum synergy on meropenem killing) of 0.0927 mg/L for PMB-susceptible subpopulations versus 3.40 mg/L for PMB-resistant subpopulations. The GA had a preference for meropenem regimens that improved the %T > MIC via longer infusion times and shorter dosing intervals. The GA predicted that treating 90% of simulated subjects harbouring a 108 cfu/mL starting inoculum to a point of 100 cfu/mL would require a regimen of meropenem 19.6 g/day 2 h prolonged infusion (2 hPI) q5h + PMB 5.17 mg/kg/day 2 hPI q6h (where the 0 h meropenem and PMB doses should be 'loaded' with 80.5% and 42.2% of the daily dose, respectively). CONCLUSION: This study provides a methodology leveraging in vitro experimental data, a mathematical pharmacodynamic model, and population pharmacokinetics provide a possible avenue to optimize treatment regimens beyond the use of the 'traditional' indices of antibiotic action.

A microfluidic platform to synthesize a G-quadruplex binding ligand.

An aromatic triarylpyridine chromophore promotes pi-stacking interactions with the terminal G-tetrad in quadruplex DNA, stabilizing the structure and presenting a pathway towards cancer treatment by inhibition of telomerase. An interesting parent compound in this class is the dimethylamino functionalised 4'-aryl-2,6-bis(4-aminophenyl)pyridine. However, access to this compound using traditional batch synthetic methodology is limited, due to thermodynamic and kinetic constraints. A novel approach to the synthesis of this compound has been developed, involving dynamic thin films, overcoming a series of competing reactions, effectively controlling chemical reactivity and selectivity.

Phosphate uptake in chick kidney cells: effects of 1,25(OH)2D3 and 24,25(OH)2D3.

Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.

Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein.

Protein misfolding is the basis of a number of human diseases and presents an obstacle to the production of soluble recombinant proteins. We present a general method to assess the solubility and folding of proteins in vivo. The basis of this assay is structural complementation between the alpha- and omega- fragments of beta-galactosidase (beta-gal). Fusions of the alpha-fragment to the C terminus of target proteins with widely varying in vivo folding yield and/or solubility levels, including the Alzheimer's amyloid beta (A beta) peptide and a non-amyloidogenic mutant thereof, reveal an unambiguous correlation between beta-gal activity and the solubility/folding of the target. Thus, structural complementation provides a means of monitoring protein solubility/misfolding in vivo, and should find utility in the screening for compounds that influence the pathological consequences of these processes.

Enabling dual cellular destinations of polymeric nanoparticles for treatment following partial injury to the central nervous system.

Following neurotrauma, oxidative stress is spread via the astrocytic syncytium and is associated with increased aquaporin 4 (AQP4), inflammatory cell infiltration, loss of neurons and glia and functional deficits. Herein we evaluate multimodal polymeric nanoparticles functionalized with an antibody to an extracellular epitope of AQP4, for targeted delivery of an anti-oxidant as a therapeutic strategy following partial optic nerve transection. Using fluorescence microscopy, spectrophotometry, correlative nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy, in vitro and in vivo, we demonstrate that functionalized nanoparticles are coated with serum proteins such as albumin and enter both macrophages and astrocytes when administered to the site of a partial optic nerve transection in rat. Antibody functionalized nanoparticles synthesized to deliver the antioxidant resveratrol are effective in reducing oxidative damage to DNA, AQP4 immunoreactivity and preserving visual function. Non-functionalized nanoparticles evade macrophages more effectively and are found more diffusely, including in astrocytes, however they do not preserve the optic nerve from oxidative damage or functional loss following injury. Our study highlights the need to comprehensively investigate nanoparticle location, interactions and effects, both in vitro and in vivo, in order to fully understand functional outcomes.

Pattern of malformations in the axial skeleton in human triploid fetuses.

We examined the axial skeleton in 15 human triploid fetuses (10 with XXX and 5 with XXY sex chromosomes). All fetuses 14-29 weeks of gestational age (GA), underwent whole-body radiography, permitting analysis of the nasal bone and the spine. From 9 of these, detailed radiographs were taken of midsagittal blocks of the cranial base and the spine, permitting detailed analysis of the cranial base. NASAL BONE: Of 14 fetuses, where the nasal bone was seen on lateral projection, it appeared short in 10 cases. SPINE: The spine was normal in 7 of 15 fetuses; malformations occurred in 8. These were osseous fusions between 2 or more vertebral bodies, most frequently in the cervical and thoracic regions, and disproportions in the sizes of the cervical bodies. Fusions occurred in 5 cases alone, and in one case in combination with disproportions of vertebral size. Disproportions alone occurred in 2 cases. CRANIAL BASE: Malformation of the basilar part of the occipital bone was found in 5 of the 9 fetuses investigated. Of 9 fetuses, bilateral ossification centers of the postsphenoid bone occurred in 7, and shell-like ossification centers in 2. There was no difference in the type of malformations in the different axial fields related to genotype (XXX and XXY). CONCLUSION: The most remarkable findings in the axial skeleton of triploid fetuses are vertebral fusions in 6 of 15 cases; clefts of vertebral bodies, previously reported as common findings in trisomy fetuses, are not demonstrated.

Mosaic partial trisomy 17 due to a ring chromosome identified by fluorescence in situ hybridisation.

We report on a 3-year-old-girl with mosaic partial trisomy 17 due to an additional ring chromosome 17 in 13% of cells analysed. This was identified by fluorescence in situ hybridisation (FISH) using a whole chromosome 17 specific paint as well as probes specific for the Smith-Magenis and Miller-Dieker regions of chromosome 17p. This girl showed mild developmental delay with subtle facial and other minor abnormalities including single palmar creases, generalised joint laxity, and a scoliosis.

Atypical mycobacterial lymphadenitis in childhood--a clinicopathological study of 17 cases.

AIMS: To assess the clinical and pathological features of atypical mycobacterial lymphadenitis in childhood to define the salient clinical and histological features. METHODS: 17 cases were included on the basis of positive culture or demonstration of bacilli of appropriate morphology and staining characteristics. RESULTS: The mean age at diagnosis was 4.86 years. All children were systemically well, with clear chest x rays. Unilateral cervical lymphadenopathy was the commonest mode of presentation. Differential Mantoux testing played no part in diagnosis. Clinical diagnosis improved with awareness. Treatment varied with surgeons opting for excision and paediatricians adding six months antituberculous chemotherapy. Acid- and alcohol-fast bacilli were identified in nine cases. Bacterial cultures were conducted in 16 cases and were positive for atypical or nontuberculous mycobacteria in 14, the main organism being M avium-intracellulare complex (11 cases). Histologically, 12 cases had bright eosinophilic serpiginous necrosis with nuclear debris scattered throughout the necrotic foci. Langhans type giant cells featured in the majority of cases but infiltration by plasma cells and neutrophils was not consistent. CONCLUSIONS: Atypical mycobacterial lymphadenitis of childhood represents a rare but significant disease with characteristic clinical and histological features.

Inversion (14)(q11q32) in a patient with childhood T-cell acute lymphoblastic leukemia.

We report a rare example of inversion (14)(q11q32) in childhood T-ALL and its further involvement in a subsequent translocation. We discuss the possible clinical significance of inv(14) in childhood T-ALL.

Synthetic oligonucleotide cocktails as probes for detection of human parvovirus B19.

A cocktail of 10 oligonucleotides selected at intervals along the length of the genome of human parvovirus B19 was labelled enzymically with digoxigenin and chemically with either digoxigenin (DIG) or dinitrophenyl (DNP). Chemical labelling was easier and more practical for the production of large quantities of probe. Pools labelled with either digoxigenin or DNP could detect 10 fg of B19 DNA in a dot blot reaction using an alkaline phosphatase antibody conjugate and colorimetric detection. Formalin fixed tissue from 11 consecutive cases of fetal hydrops were examined by in situ hybridisation (ISH). Both probe cocktails detected human parvovirus B19 DNA in 3 cases, with positive cells in all tissues examined and with equal sensitivity. The DNP pool is significantly cheaper and simpler to produce and could provide an inexpensive reagent suitable for diagnostic detection of viral nucleic acid in histopathological material.

Three-year follow-up of bibliotherapy for depression.

This study examined the durability of cognitive bibliotherapy for mild to moderately depressed adults by conducting a 3-year follow-up of participants from a previous study (C. Jamison & F. Scogin, 1995). The Hamilton Rating Scale for Depression, Beck Depression Inventory, and questions relating to participants' perceptions of the program were administered. Results indicated that treatment gains were maintained over the 3-year follow-up period and support the usefulness of cognitive bibliotherapy as an adjunct to traditional treatment modalities in a general adult population.

Chronic periphlebitis retinae in multiple sclerosis. A histopathological study.

Retinal periphlebitis in multiple sclerosis is of particular interest in relation to our understanding of the pathogenesis of the demyelinating central nervous system plaques. Previous studies have largely been clinical, and there is little detailed histopathological information relating to this condition. We present the first detailed report in the neurological literature on the histological findings in chronic periphlebitis retinae associated with multiple sclerosis. The most significant abnormalities of the affected retinal veins were the presence of thick laminated collagen in the wall, associated with a scanty infiltration of plasma cells.

Broadsheet number 56: Mechanisms of fetal loss.

The most significant task of the pathologist examining a fetal death is, if possible, the provision of an explanation for the event, which will allow the clinical attendants to counsel the family in an informed and relevant manner. In some cases, no adequate explanation will be possible, though many conditions will be excluded and the importance of this exercise is worth emphasising. Often, however, a combination of fetal, maternal or placental conditions can be found and a full or partial explanation offered, and it is the purpose of this broadsheet to highlight some of these conditions and provide suggestions as to sources of further practical help.

Bilateral Wilms' tumor: a clinicopathologic review.

Bilateral Wilms' tumor occurs at a younger age than unilateral disease. While it generally has a good prognosis, it presents a therapeutic dilemma to balance curative surgical resection with preservation of renal tissue. A 15 year review of bilateral Wilms' tumors diagnosed at Princess Margaret Hospital was undertaken. Of 46 Wilms' tumor cases, eight were designated bilateral by diagnostic imaging (median age 1.1 years compared with 3.5 years for unilateral tumors). The surgical management entailed primary nephrectomy with contralateral biopsy in two patients, and bilateral biopsy and delayed resection in all remaining surviving patients (one patient died of perioperative complications). Seven patients had localized disease (stage I/II) and the six surviving patients received chemotherapy with vincristine and actinomycin; no patient received radiotherapy. All are alive and well (median follow-up 5.1 years). The remaining patient presented with pulmonary metastases and died of disease progression. Pathologic review revealed that four patients had truly bilateral disease demonstrable by histology, three had unilateral Wilms' tumor with contralateral nephrogenic rests, and in one patient the biopsies of the contralateral kidney showed neither tumor nor nephrogenic rests. In most cases pathological material was subject to external review. Follow-up demonstrates excellent renal function with compensatory hypertrophy in the remaining renal tissue. Conservative surgery and simple out-patient based, low toxicity chemotherapy is curative in most patients.

The spectrum of presentation at autopsy of myocarditis in infancy and childhood.

To characterize the clinicopathological presentation of patients with myocarditis coming to autopsy in childhood, 32 cases of histologically-proven myocarditis were obtained from the files of the Adelaide Children's Hospital. In 16 of the cases (Group A), myocarditis was the only significant finding and death was ascribed to this condition. In the remaining 16 (Group B) myocarditis was found in association with other severe disease processes. Clinical histories of the 2 groups showed sudden death to be a feature in 5 out of 16 cases in Group A, 3 of whom had no prodromal symptoms. Five patients in Group B also suffered sudden death, but this was associated with a variety of causes, including bronchopneumonia, and asphyxia. These cases demonstrate the variability in clinicopathological presentation of myocarditis in infancy and childhood and suggest that myocarditis should always be considered a possible diagnosis at autopsy in the pediatric age group, even in the presence of coincident lethal disease.

The use of complementary medicines by those with asthma.

Complementary therapies attract considerable media attention and previous surveys of members of an asthma patient organisation suggested that their use by those with asthma was commonplace. This report concerns a study of a stratified cross section of the asthma population designed to give a more representative insight into current usage of complementary therapies. A sift questionnaire was used to identify those with asthma and 785 of those so identified undertook a semi-structured face-to-face interview. Only 6% of the study population were current users of complementary therapies with use being more common amongst those who expressed most concern regarding their current medication. Low use of complementary therapies may well reflect satisfaction with current management and suggests that previous surveys may have been unrepresentative of a more balanced population of those with asthma.

Do plant secondary compounds determine feeding preferences of snowshoe hares?

We investigated the food preferences of captive snowshoc hares (Lepus americanus) in winter to test three hypotheses proposed to explain food choices by hares: (1) that food choice is related to the protein content of twigs; 92) that defensive chemicals present in twigs are negatively correlated with hare food preferences; and (3) that hares eat less-preferred but protein-rich twigs when their diet is buffered by large amounts of palatable food. Hares exhibited striking and consistent preferences for different species and, in general, preferred mature twigs to juvenile growth stages. Preferences across species among mature twigs were not, however, the same as preferences for juvenile growth stages across species. None of the three hypotheses adequately explained food choice by hares. Hares did not (1) select twigs that were high in protein content. They also did not (2) consistently select twigs that were low in resins or phenols. Finally (3), hares generally ate less, not more of non-preferred twigs in the presence of a protein and energy rich alternative food, commercial rabbit chow. Food preferences of hares must presumably have some chemical basis, but no simple theory has yet explained what this is. We suggest that hares may not be under severe dietary constraints imposed by chemical defenses in winter.

Evaluation of a lactate dehydrogenase-nitro blue tetrazolium method of detecting unapparent myocardial infarction using a standardised sampling technique.

Using a standardised block technique, the microscopic lactate dehydrogenase-nitro blue tetrazolium (LDH-nitro BT) and conventional haematoxylin and eosin staining methods were used to investigate the presence of myocardial infarction in a series of fifty forensic autopsies. The microscopic LDH-nitro BT method was found to be reliable; however, it did not reveal the presence of myocardial infarction in any of the cases where this had not been suspected.