Influence of surface roughness on silicone rubber voice prostheses on in vitro biofilm formation and clinical lifetime in laryngectomised patients.

OBJECTIVES: Evaluation of the influence of a smooth surface moulding technique of silicone rubber indwelling voice prostheses on in vitro biofilm formation and analysis of the clinical in situ lifetime. DESIGN: Biofilm formation on smooth and Groningen ultra low resistance (URL) prostheses was studied in an artificial throat model. The clinical lifetime of smooth voice prostheses was compared to the previous lifetime of URL by counting the number of replacements in a consecutive 6-month period in the same patient. PARTICIPANTS: Eleven laryngectomised patients in follow-up who required frequent replacement of their voice prostheses. SETTINGS: Tertiary University Medical Center. RESULTS: Use of a smoother mould and less viscous silicone rubber yielded a decrease in surface roughness from 46 to 8 nm and was accompanied by a 40% reduction in the prevalence of bacteria and yeast in in vitro formed biofilms. Clinically, the lifetime was significantly (P<.005) increased by a factor of 2.1. CONCLUSIONS: This combined in vitro and clinical study suggests that the choice of material and in particular its surface finishing may be determining factors with respect to the clinical lifetime of silicone rubber implants and devices failing due to biofilm formation.

Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase.

The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.

Managing a nosocomial outbreak of carbapenem-resistant Klebsiella pneumoniae: an early Australian hospital experience.

BACKGROUND: Carbapenems are traditionally reserved as the last line of defence for treatment of serious infections with multiresistant Gram-negative bacilli. Reports of Klebsiella pneumoniae carbapenemase (KPC)-producing organisms have been emerging globally, but rare in Australasia to date. We describe an outbreak of KPC-2 producing K. pneumoniae at an Australian hospital. METHODS: After initial detection in October 2012, a retrospective review of patients with meropenem-resistant K. pneumoniae to June 2012, and ongoing prospective surveillance, was undertaken. Included patients were admitted to the hospital after June 2012 and had meropenem-resistant K. pneumoniae isolated from any site. Available isolates underwent detection of the KPC-2 gene by polymerase chain reaction and molecular typing was performed to determine genetic relatedness between isolates. Point-prevalence screening was performed on selected wards to detect asymptomatic carriage. Infection control procedures were implemented to contain the outbreak. RESULTS: Ten cases were identified in the initial cluster. Eight were localised to a single inpatient ward. Point-prevalence screening revealed one extra case. After temporary containment, re-emergence of KPC-producing isolates was observed post October 2013 with 18 further cases identified. Four K. pneumoniae isolates in the 2012 cluster and 16 from the 2013-2014 cluster were referred for further testing. All carried the KPC-2 beta-lactamase gene. The 2012 isolates were genetically similar to the 2014 isolates. CONCLUSION: KPC-2 mediated resistance is an emerging threat in Australia. The re-emergence of KPC despite initial containment emphasises the need for constant vigilance in the microbiology laboratory and ongoing maintenance of infection control and antimicrobial stewardship activity.

Rothia aeria mitral valve endocarditis complicated by multiple mycotic aneurysms: laboratory identification expedited using MALDI-TOF MS.

Rothia aeria has only rarely been described as a human pathogen. We describe a case of Rothia aeria causing mitral valve endocarditis and multiple mycotic aneurysms, including cerebral mycotic aneurysms. In the case described, early identification of Rothia aeria was achieved using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Cross-strand histidine-aromatic interactions enhance acyl-transfer rates in beta-hairpin peptide catalysts.

A reactive tagging methodology was used to select the species most reactive to an acylation reagent from a solid phase library of beta hairpin peptides. Hits bearing an electron-rich aromatic residue across strand from a reactive histidine were found to competitively become N-acylated. In addition to displaying rapid N-acylation rates the hit peptide was additionally deacylated in the presence of a nucleophile, thus closing a putative catalytic cycle. Variants of the hit peptide were studied to elucidate both the magnitude (up to 18,000-fold over background, kcat/kuncat = 94,000,000, or 45-fold over Boc-histidine methyl ester) and mechanism of acyl transfer catalysis. A combination of CH-π, cation-π and HisH(+)-O interactions in the cationic imidazole transition state is implicated in the rate acceleration, in addition to the fidelity of the beta hairpin fold. Moreover, NMR structural data on key intermediates or models thereof suggest that a key feature of this catalyst is the ability to access several different stabilizing conformations along the catalysis reaction coordinate.

Nicotinic acid and DP1 blockade: studies in mouse models of atherosclerosis.

The use of nicotinic acid to treat dyslipidemia is limited by induction of a "flushing" response, mediated in part by the interaction of prostaglandin D(2) (PGD(2)) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr(-/-)ApoE(-/-) mice versus ApoE(-/-) mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr(-/-)ApoE(-/-) mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE(-/-) mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr(-/-) mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.

Trophic state resilience to hurricane disturbance of Lake Yojoa, Honduras.

Cyclones are a poorly described disturbance in tropical lakes, with the potential to alter ecosystems and compromise the services they provide. In November 2020, Hurricanes Eta and Iota made landfall near the Nicaragua-Honduras border, inundating the region with a large amount of late-season precipitation. To understand the impact of these storms on Lake Yojoa, Honduras, we compared 2020 and 2021 conditions using continuous (every 16 days) data collected from five pelagic locations. The storms resulted in increased Secchi depth and decreased algal abundance in December 2020, and January and February 2021, and lower-than-average accumulation of hypolimnetic nutrients from the onset of stratification (April 2021) until mixus in November 2021. Despite the reduced hypolimnetic nutrient concentrations, epilimnetic nutrient concentrations returned to (and in some cases exceeded) pre-hurricane levels following annual water column turnover in 2021. This response suggests that Lake Yojoa's trophic state had only an ephemeral response to the disturbance imposed by the two hurricanes, likely due to internal input of sediment derived nutrients. These aseasonal storms acted as a large-scale experiment that resulted in nutrient dilution and demonstrated the resilience of Lake Yojoa's trophic state to temporary nutrient reductions.

Isotonitazene, a novel psychoactive substance opioid, detected in two cases following a local surge in opioid overdoses.

BACKGROUND: Isotonitazene is a novel opioid that was first reported in Europe in 2019. There have been no reports of the detection of isotonitazene in patients presenting to the emergency department with acute drug toxicity. AIM: There was an increase in presentations to our emergency department with acute opioid toxicity in August 2021. We aim to describe this outbreak and provide detail on two cases in which isotonitazene was quantified in serum samples. METHODS: Serum samples were available for comprehensive toxicological analysis for two presentations. Written consent was obtained and the samples were analysed through a Thermo XRS ultrahigh-performance liquid chromatography system, interfaced to a Thermo Q Exactive high-resolution accurate mass spectrometer, operating in heated positive ion electrospray mode. Acquired data were processed using Toxfinder software (Thermo) against a regularly updated in-house database. RESULTS: There was an increase in acute opioid presentations to our emergency department from a median of 10 per month to 36 in August 2021. Twenty were treated with naloxone, and 23 were admitted to the hospital for observation and treatment. Serum sample analysis from two patients with acute opioid toxicity responsive to naloxone detected the presence of isotonitazene (0.18 and 0.81 ng/ml). CONCLUSION: We report a cluster of acute opioid toxicity presentations to our Emergency Department with detection of isotonitazene in two cases. Analytical screening is important in determining the presence of novel psychoactive substances (NPS) and to help inform the public health of the implications of NPS use, particularly during clusters of acute recreational drug toxicity presentations.

A retrospective cohort study of infection risk in hospitalized patients evaluated for Sweet syndrome.

Sweet syndrome (SS) is divided into malignancy-associated, classical, and drug-induced subtypes. Features associated with SS, such as fevers, neutropenia, and cancer, are also high risk for serious infection. We aimed to describe hospitalized patients with a documented concern for SS on initial dermatologic evaluation, their risk of infection, and the impact of SS subtype on treatment and outcomes. We descriptively analyzed hospitalizations at The Ohio State University evaluated for SS by dermatology and performed a retrospective cohort analysis of malignancy-associated and non-malignancy-associated SS patients. Eighty-seven patient hospitalizations were evaluated for SS from 2012 to 2019. Thirty-one hospitalizations were complicated by neutropenia. Lesions in 12.9% (n = 4/31) of neutropenic hospitalizations were infected with Fusarium species (n = 2) or methicillin-resistant staphylococcus aureus (n = 2). One patient with fungal disease died within 30 days of hospitalization. Thirty-three patients were confirmed to have a final diagnosis of SS. In the confirmed SS cohort, malignancy was associated with greater overall dapsone use (p = .021), less initial (p = .046) and overall (p = .013) corticosteroid use, and fewer SS-related readmissions within one year (p = .020) and overall (p = .004). Corticosteroid treatment delay should be considered for a short period in neutropenic patients while excluding infection. Malignancy-associated SS patients were more frequently treated with dapsone and favorable outcomes were seen in cancer patients.

High-affinity chromodomains engineered for improved detection of histone methylation and enhanced CRISPR-based gene repression.

Histone methylation is an important post-translational modification that plays a crucial role in regulating cellular functions, and its dysregulation is implicated in cancer and developmental defects. Therefore, systematic characterization of histone methylation is necessary to elucidate complex biological processes, identify biomarkers, and ultimately, enable drug discovery. Studying histone methylation relies on the use of antibodies, but these suffer from lot-to-lot variation, are costly, and cannot be used in live cells. Chromatin-modification reader domains are potential affinity reagents for methylated histones, but their application is limited by their modest affinities. We used phage display to identify key residues that greatly enhance the affinities of Cbx chromodomains for methylated histone marks and develop a general strategy for enhancing the affinity of chromodomains of the human Cbx protein family. Our strategy allows us to develop powerful probes for genome-wide binding analysis and live-cell imaging. Furthermore, we use optimized chromodomains to develop extremely potent CRISPR-based repressors for tailored gene silencing. Our results highlight the power of engineered chromodomains for analyzing protein interaction networks involving chromatin and represent a modular platform for efficient gene silencing.

Lack of authentic atrial fibrillation in commonly used murine atrial fibrillation models.

The mouse is a useful preclinical species for evaluating disease etiology due to the availability of a wide variety of genetically modified strains and the ability to perform disease-modifying manipulations. In order to establish an atrial filtration (AF) model in our laboratory, we profiled several commonly used murine AF models. We initially evaluated a pharmacological model of acute carbachol (CCh) treatment plus atrial burst pacing in C57BL/6 mice. In an effort to observe micro-reentrant circuits indicative of authentic AF, we employed optical mapping imaging in isolated mouse hearts. While CCh reduced atrial refractoriness and increased atrial tachyarrhythmia vulnerability, the left atrial (LA) excitation patterns were rather regular without reentrant circuits or wavelets. Therefore, the atrial tachyarrhythmia resembled high frequency atrial flutter, not typical AF per se. We next examined both a chronic angiotensin II (Ang II) infusion model and the surgical model of transverse aortic constriction (TAC), which have both been reported to induce atrial and ventricular structural changes that serve as a substrates for micro-reentrant AF. Although we observed some extent of atrial remodeling such as fibrosis or enlarged LA diameter, burst pacing-induced atrial tachyarrhythmia vulnerability did not differ from control mice in either model. This again suggested that an AF-like pathophysiology is difficult to demonstrate in the mouse. To continue searching for a valid murine AF model, we studied mice with a cardiac-specific deficiency (KO) in liver kinase B1 (Cardiac-LKB1), which has been reported to exhibit spontaneous AF. Indeed, the electrocardiograms (ECG) of conscious Cardiac-LKB1 KO mice exhibited no P waves and had irregular RR intervals, which are characteristics of AF. Histological evaluation of Cardiac-LKB1 KO mice revealed dilated and fibrotic atria, again consistent with AF. However, atrial electrograms and optical mapping revealed that electrical activity was limited to the sino-atrial node area with no electrical conduction into the atrial myocardium beyond. Thus, Cardiac-LKB1 KO mice have severe atrial myopathy or atrial standstill, but not AF. In summary, the atrial tachyarrhythmias we observed in the four murine models were distinct from typical human AF, which often exhibits micro- or macro-reentrant atrial circuits. Our results suggest that the four murine AF models we examined may not reflect human AF well, and raise a cautionary note for use of those murine models to study AF.

Membrane tethering in intracellular transport.

Studies of various membrane trafficking steps over the past year indicate that membranes are tethered together prior to the interaction of v-SNAREs and t-SNAREs across the membrane junction. The tethering proteins identified to date are quite large, being either fibrous proteins or multimeric protein complexes. The tethering factors employed at different steps are evolutionarily unrelated, yet their function seems to be closely tied to the more highly conserved Rab GTPases. Tethering factors may collaborate with Rabs and SNAREs to generate targeting specificity in the secretory pathway.

Proteins involved in vesicular transport and membrane fusion.

In the past year, new information about proteins involved in vesicular transport has been plentiful. Particularly noteworthy are the complementary findings that Sec17p is required for vesicle consumption in endoplasmic reticulum-to-Golgi transport in yeast and that an analogous activity in mammalian cells, termed SNAP, is required for transport from the cis to the medial cisternae of the Golgi apparatus.

The discovery of high affinity agonists of GPR109a with reduced serum shift and improved ADME properties.

Amino-anthranilic acid derivatives have been identified as a new class of low serum shifted, high affinity full agonists of the human orphan G-protein-coupled receptor GPR109a with improved ADME properties.

Growth hormone (GH), brain development and neural stem cells.

A range of observations support a role for GH in development and function of the brain. These include altered brain structure in GH receptor null mice, and impaired cognition in GH deficient rodents and in a subgroup of GH receptor defective patients (Laron dwarfs). GH has been shown to alter neurogenesis, myelin synthesis and dendritic branching, and both the GH receptor and GH itself are expressed widely in the brain. We have found a population of neural stem cells which are activated by GH infusion, and which give rise to neurons in mice. These stem cells are activated by voluntary exercise in a GH-dependent manner. Given the findings that local synthesis of GH occurs in the hippocampus in response to a memory task, and that GH replacement improves memory and cognition in rodents and humans, these new observations warrant a reappraisal of the clinical importance of GH replacement in GH deficient states.

Alterations to sediment nutrient deposition and transport along a six reservoir sequence.

Reservoir presence and construction has become commonplace along rivers due to the multitude of ecosystem services they provide. Many services are well recognized, including the effectiveness of sequestering both sediments and sediment-bound nutrients such as silts and phosphorus (P). Reservoirs are also capable of transforming or sequestering significant quantities of nutrients with more complex biogeochemical pathways, like nitrogen (N). Reservoir assessments, independent of inflow-outflow models, have primarily focused on a small number of systems creating a growing need to understand how reservoirs function both individually and as reservoir sequences within large rivers and their watersheds. Models have simulated the overall efficiency and drivers of reservoir nutrient deposition, but few have considered how a sequence of reservoirs alters deposition as an interdependent watershed-sediment-transport-system. In this study, we collected sediment cores from a six-reservoir sequence along a 5th - 6th order stream receiving treated waters from a large metropolitan area in the subtropical southeastern United States. Paleolimnological studies of subtropical reservoirs are underrepresented and are needed to understand the history of reservoir development. Using paleolimnological techniques and a known 30 year flux of riverine nutrient loading from waste water treatment facilities, we compared nutrient deposition to reservoir morphological qualities and primary producer community structure during the past ~50 years. Our findings suggest phosphorus deposition is associated with reservoir order downstream of the primary nutrient source, nitrogen deposition is linked to reservoir water retention time, and N:P is most strongly linked to reservoir surface area and watershed population density. Our results were strongly influenced by a large upstream and metropolitan nutrient source, common in large rivers, but under different conditions of nutrient loading (i.e. nonpoint source), reservoirs may express other nutrient depositional patterns.

Discovery of pyrazolyl propionyl cyclohexenamide derivatives as full agonists for the high affinity niacin receptor GPR109A.

A series of pyrazolyl propionyl cyclohexenamides were discovered as full agonists for the high affinity niacin receptor GPR109A. The structure-activity relationship (SAR) studies were aimed to improve activity on GPR109A, reduce Cytochrome P450 2C8 (CYP2C8) and Cytochrome P450 2C9 (CYP2C9) inhibition, reduce serum shift and improve pharmacokinetic (PK) profiles.

GPR109a agonists. Part 2: pyrazole-acids as agonists of the human orphan G-protein coupled receptor GPR109a.

5-Alkyl and aryl-pyrazole-acids have been identified as a new class of selective, small-molecule, agonists of the human orphan G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug nicotinic acid.

Anthranilic acid replacements in a niacin receptor agonist.

Niacin is an effective drug for raising HDL cholesterol. However, niacin must be taken in large doses and significant side effects are often observed, including facial flushing, loss of glucose tolerance, and liver toxicity. An anthranilic acid was identified as an agonist of the niacin receptor. In order to improve efficacy and provide structural diversity, replacements for the anthranilic acid were investigated and several compounds with improved properties were identified.