Predicting Experimental B22 Values and the Effects of Histidine Charge States for Monoclonal Antibodies Using Coarse-Grained Molecular Simulations.

Static light scattering (SLS) was used to characterize five monoclonal antibodies (MAbs) as a function of total ionic strength (TIS) at pH values between 5.5 and 7.0. Second osmotic virial coefficient (B22) values were determined experimentally for each MAb as a function of TIS using low protein concentration SLS data. Coarse-grained molecular simulations were performed to predict the B22 values for each MAb at a given pH and TIS. To include the effect of charge fluctuations of titratable residues in the B22 calculations, a statistical approach was introduced in the Monte Carlo algorithm based on the protonation probability based on a given pH value and the Henderson-Hasselbalch equation. The charged residues were allowed to fluctuate individually, based on the sampled microstates and the influence of electrostatic interactions on net protein-protein interactions during the simulations. Compared to static charge simulations, the new approach provided improved results compared to experimental B22 values at pH conditions near the pKa of titratable residues.

Why do silanes reduce electron-rich phosphine oxides faster than electron-poor phosphine oxides?

Organophosphine-mediated reactions that generate P[double bond, length as m-dash]O-bonded byproducts can be transformed into catalytic processes by reducing the R3P[double bond, length as m-dash]O byproduct back to PR3in situ with a silane. DFT calculations explain why the most readily reduced phosphine oxides are those incorporating electron-rich (e.g. alkyl) substituents rather than electron-deficient (e.g. aryl) substituents.

Physiological effects of high-flow nasal cannula therapy in preterm infants.

OBJECTIVE: High-flow nasal cannula (HFNC) therapy is increasingly used in preterm infants despite a paucity of physiological studies. We aimed to investigate the effects of HFNC on respiratory physiology. STUDY DESIGN: A prospective randomised crossover study was performed enrolling clinically stable preterm infants receiving either HFNC or nasal continuous positive airway pressure (nCPAP). Infants in three current weight groups were studied: <1000 g, 1000-1500 g and >1500 g. Infants were randomised to either first receive HFNC flows 8-2 L/min and then nCPAP 6 cm H2O or nCPAP first and then HFNC flows 8-2 L/min. Nasopharyngeal end-expiratory airway pressure (pEEP), tidal volume, dead space washout by nasopharyngeal end-expiratory CO2 (pEECO2), oxygen saturation and vital signs were measured. RESULTS: A total of 44 preterm infants, birth weights 500-1900 g, were studied. Increasing flows from 2 to 8 L/min significantly increased pEEP (mean 2.3-6.1 cm H2O) and reduced pEECO2 (mean 2.3%-0.9%). Tidal volume and transcutaneous CO2 were unchanged. Significant differences were seen between pEEP generated in open and closed mouth states across all HFNC flows (difference 0.6-2.3 cm H2O). Infants weighing <1000 g received higher pEEP at the same HFNC flow than infants weighing >1000 g. Variability of pEEP generated at HFNC flows of 6-8 L/min was greater than nCPAP (2.4-13.5 vs 3.5-9.9 cm H2O). CONCLUSIONS: HFNC therapy produces clinically significant pEEP with large variability at higher flow rates. Highest pressures were observed in infants weighing <1000 g. Flow, weight and mouth position are all important determinants of pressures generated. Reductions in pEECO2 support HFNC's role in dead space washout.

Reduction Rate of 1-Phenyl Phospholane 1-Oxide Enhanced by Silanol Byproducts: Comprehensive DFT Study and Kinetic Modeling Linked to Reagent Design.

Important stoichiometric transformations like Wittig and Appel reactions have been implemented in a catalytic fashion in the past decade. The phosphine oxide generated in situ can be reintroduced as phosphine into the catalytic cycle using mild and selective silane reagents (redox-driven catalysis). While the field of experimental investigation has been fully expanding in the past decade, theoretical studies are still sparse. In this present work, density functional theory (DFT) has been used to characterize the free energy surfaces of the reduction of 1-phenyl phospholane 1-oxide with four different silanes. Found stationary points have been studied in-depth to highlight mechanistic peculiarities, like the effect of substituents at the silicon center and the parallel and competitive reactivity between the precursor silanes and their semioxidized byproducts. Calculated thermodynamic parameters in combination with "real" values for concentrations have been used in the formulation of rate equations for simple bimolecular and monomolecular steps of the mechanism. The deterministic integration concentrations versus time of such rate equations led to a realistic description of the systems under study and paved the way to strategic and rational design of new silanes with increased reactivity.

New nanoscale toughening mechanisms mitigate embrittlement in binary nanocrystalline alloys.

Nanocrystalline metals offer significant improvements in structural performance over conventional alloys. However, their performance is limited by grain boundary instability and limited ductility. Solute segregation has been proposed as a stabilization mechanism, however the solute atoms can embrittle grain boundaries and further degrade the toughness. In the present study, we confirm the embrittling effect of solute segregation in Pt-Au alloys. However, more importantly, we show that inhomogeneous chemical segregation to the grain boundary can lead to a new toughening mechanism termed compositional crack arrest. Energy dissipation is facilitated by the formation of nanocrack networks formed when cracks arrested at regions of the grain boundaries that were starved in the embrittling element. This mechanism, in concert with triple junction crack arrest, provides pathways to optimize both thermal stability and energy dissipation. A combination of in situ tensile deformation experiments and molecular dynamics simulations elucidate both the embrittling and toughening processes that can occur as a function of solute content.

Biophysical characterization and molecular simulation of electrostatically driven self-association of a single-chain antibody.

Colloidal protein-protein interactions (PPI) are often expected to impact key behaviors of proteins in solution, such as aggregation rates and mechanisms, aggregate structure, protein solubility, and solution viscosity. PPI of an anti-fluorescein single chain antibody variable fragment (scFv) were characterized experimentally at low to intermediate ionic strength using a combination of static light scattering and sedimentation equilibrium ultracentrifugation. Surprisingly, the results indicated that interactions were strongly net-attractive and electrostatics promoted self-association. Only repulsive interactions were expected based on prior work and calculations based a homology model of a related scFv crystal structure. However, the crystal structure lacks the charged, net-neutral linker sequence. PyRosetta was used to generate a set of scFv structures with different linker conformations, and coarse-grained Monte Carlo simulations were used to evaluate the effect of different linker configurations via second osmotic virial coefficient (B22 ) simulations. The results show that the configuration of the linker has a significant effect on the calculated B22 values, and can result in strong electrostatic attractions between oppositely charged residues on the protein surface. This is particularly relevant for development of non-natural antibody products, where charged linkers and other loop regions may be prevalent. The results also provide a preliminary computational framework to evaluate the effect of unstructured linkers on experimental protein-protein interaction parameters such as B22 .

A novel culture medium for isolation of rapidly-growing mycobacteria from the sputum of patients with cystic fibrosis.

BACKGROUND: Isolation of mycobacteria from the sputum of patients with cystic fibrosis (CF) is challenging due to the overgrowth of cultures by other bacteria and fungi. In this setting, Burkholderia cepacia selective agar (BCSA) has been recommended as a convenient and effective culture medium for the isolation of rapidly-growing, non-tuberculous mycobacteria (NTM). A novel selective culture medium (RGM medium) was evaluated for the isolation of rapidly-growing NTM from the sputum of children and adults with CF. METHODS: A total of 118 isolates of rapidly-growing mycobacteria and 98 other bacteria and fungi were inoculated onto RGM medium. These were assessed for growth at 30°C over a seven day period. A total of 502 consecutive sputum samples were collected from 210 patients with CF. Each sample was homogenized and cultured onto RGM medium and also onto BCSA. Cultures were incubated for 10days at 30°C. RESULTS: Of 118 isolates of mycobacteria all but one grew well on RGM medium, whereas 94% of other bacteria and fungi were inhibited. A total of 55 sputum samples (from 33 distinct patients) yielded NTM using a combination of both RGM and BCSA (prevalence: 15.7%). NTM were recovered from 54 sputum samples using RGM medium compared with only 17 samples using BCSA (sensitivity 98% vs. 31%; P≤0.0001). A total of 419 isolates of non-mycobacteria were recovered from sputum samples on BCSA compared with 46 on RGM medium. CONCLUSIONS: RGM medium offers a simple and effective culture method for the isolation of rapidly-growing mycobacteria from sputum samples from patients with CF without decontamination of samples. RGM medium allows for the systematic screening of all sputum samples routinely referred for culture from patients with CF.

Catalytic wittig reactions of semi- and nonstabilized ylides enabled by ylide tuning.

The first examples of catalytic Wittig reactions with semistabilized and nonstabilized ylides are reported. These reactions were enabled by utilization of a masked base, sodium tert-butyl carbonate, and/or ylide tuning. The acidity of the ylide-forming proton was tuned by varying the electron density at the phosphorus center in the precatalyst, thus facilitating the use of relatively mild bases. Steric modification of the precatalyst structure resulted in significant enhancement of E selectivity up to >95:5, E/Z.

Conformational stability as a design target to control protein aggregation.

Non-native protein aggregation is a prevalent problem occurring in many biotechnological manufacturing processes and can compromise the biological activity of the target molecule or induce an undesired immune response. Additionally, some non-native aggregation mechanisms lead to amyloid fibril formation, which can be associated with debilitating diseases. For natively folded proteins, partial or complete unfolding is often required to populate aggregation-prone conformational states, and therefore one proposed strategy to mitigate aggregation is to increase the free energy for unfolding (ΔGunf) prior to aggregation. A computational design approach was tested using human γD crystallin (γD-crys) as a model multi-domain protein. Two mutational strategies were tested for their ability to reduce/increase aggregation rates by increasing/decreasing ΔGunf: stabilizing the less stable domain and stabilizing the domain-domain interface. The computational protein design algorithm, RosettaDesign, was implemented to identify point variants. The results showed that although the predicted free energies were only weakly correlated with the experimental ΔGunf values, increased/decreased aggregation rates for γD-crys correlated reasonably well with decreases/increases in experimental ΔGunf, illustrating improved conformational stability as a possible design target to mitigate aggregation. However, the results also illustrate that conformational stability is not the sole design factor controlling aggregation rates of natively folded proteins.

Part I: the development of the catalytic Wittig reaction.

We have developed the first catalytic (in phosphane) Wittig reaction (CWR). The utilization of an organosilane was pivotal for success as it allowed for the chemoselective reduction of a phosphane oxide. Protocol optimization evaluated the phosphane oxide precatalyst structure, loading, organosilane, temperature, solvent, and base. These studies demonstrated that to maintain viable catalytic performance it was necessary to employ cyclic phosphane oxide precatalysts of type 1. Initial substrate studies utilized sodium carbonate as a base, and further experimentation identified N,N-diisopropylethylamine (DIPEA) as a soluble alternative. The use of DIPEA improved the ease of use, broadened the substrate scope, and decreased the precatalyst loading. The optimized protocols were compatible with alkyl, aryl, and heterocyclic (furyl, indolyl, pyridyl, pyrrolyl, and thienyl) aldehydes to produce both di- and trisubstituted olefins in moderate-to-high yields (60-96%) by using a precatalyst loading of 4-10 mol%. Kinetic E/Z selectivity was generally 66:34; complete E selectivity for disubstituted α,ß-unsaturated products was achieved through a phosphane-mediated isomerization event. The CWR was applied to the synthesis of 54, a known precursor to the anti-Alzheimer drug donepezil hydrochloride, on a multigram scale (12.2 g, 74% yield). In addition, to our knowledge, the described CWR is the only transition-/heavy-metal-free catalytic olefination process, excluding proton-catalyzed elimination reactions.

Breaking the ring through a room temperature catalytic Wittig reaction.

One ring no longer rules them all: Employment of 2.5-10 mol % of 4-nitrobenzoic acid with phenylsilane led to the development of a room temperature catalytic Wittig reaction (see scheme). Moreover, these enhanced reduction conditions also facilitated the use of acyclic phosphine oxides as catalysts for the first time. A series of alkenes were produced in moderate to high yield and selectivity.

Vitamin D in children with cystic fibrosis.

PURPOSE: The Cystic Fibrosis Trust in 2007 published a recommended target of 75-150 nmol/L for 25-hydroxyvitamin D (25-OHD). In 2008 we found that only 10% of pancreatic insufficient (PI) children met this target. An increase in supplementation was implemented and a repeat audit performed in 2010. METHODS: PI children ≥1 year under sole-care in our regional centre were included. Vitamin D3 supplementation increased by >450% to either 3800 IU/day liquid or 800 IU daily plus 20,000 IU weekly tablets. In 2010 pancreatic sufficient (PS) children were also audited separately. RESULTS: The median 25-OHD level increased from 51.5 nmol/L in 2008 (n=78, 10% >75 nmol/L) to 72 nmol/L in 2010 (n=72, 51% >75 nmol/L), p<0.0001. In PS children (n=15 in 2010) 87% had 25-OHD levels <75 nmol/L. CONCLUSIONS: A substantial increase in supplementation led to a significant increase in 25-OHD levels but around half still failed to reach the recommended target.

Use of cyclin D1 in conjunction with human papillomavirus status to predict outcome in oropharyngeal cancer.

There is increasing use of multiple molecular markers to predict prognosis in human cancer. Our aim was to examine the prognostic significance of cyclin D1 and retinoblastoma (pRb) expression in association with human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma. Clinical records and specimens of 226 patients with follow-up from 1 to 235 months postdiagnosis were retrieved. Tumor HPV status was determined by HPV E6-targeted multiplex real-time PCR/p16 semiquantitative immunohistochemistry and cyclin D1 and pRb expression by semiquantitative immunohistochemistry. Determinants of recurrence and mortality hazards were modeled using Cox regression with censoring at dates of last follow-up. The HPV-positivity rate was 37% (91% type 16). HPV was a predictor of recurrence, an event (recurrence or death) and death after adjustment for clinicopathological variables. There were inverse relationships between HPV status and cyclin D1 and pRb. On univariate analysis, cyclin D1 predicted locoregional recurrence, event and death and pRb predicted event and death. Within the HPV-positive group, after adjusting for clinicopathological factors, patients with cyclin D1-positive cancers had up to a eightfold increased risk of poor outcome relative to those with cyclin D1-negative tumors. However, within the HPV-negative group, there was only a very small adjusted increased risk. A combination of pRb and HPV did not provide additional prognostic information. Our data provide the first evidence that a combination of HPV and cyclin D1 provides more prognostic information in oropharyngeal cancer than HPV alone. If findings are confirmed, treatment based on HPV and cyclin D1 may improve outcomes.

Structure-activity relationship analysis of Pd-PEPPSI complexes in cross-couplings: a close inspection of the catalytic cycle and the precatalyst activation model.

A series of Pd-N-heterocyclic carbene (Pd-NHC) complexes with various NHC, halide and pyridine ligands (PEPPSI (pyridine, enhanced, precatalyst, preparation, stabilisation and initiation) precatalysts) were prepared, and the effects of these ligands on catalyst activation and performance were studied in the Kumada-Tamao-Corriu (KTC), Negishi, and Suzuki-Miyaura cross-coupling reactions. The lowered reactivity of more hindered 2,6-dimethylpyridyl complex 4 in the Negishi and KTC reactions is consistent with slow reductive dimerisation of the organometallic reaction partner during precatalyst activation. Comparative rate studies of complexes 1, 4 and 5 in the KTC and Suzuki-Miyaura reactions verify that 4 activated more slowly than the others. A potential on/off mechanism of pyridine coordination to NHC-Pd(0) is also plausible, in which the more basic pyridine stays bound for longer.

On the role of additives in alkyl-alkyl Negishi cross-couplings.

An additives study for the alkyl-alkyl Negishi reaction using an NHC-Pd catalyst revealed that bromide salts promote coupling while the cation is mechanistically benign. A double titration revealed that the cross-coupling begins at a 1 : 1 ratio of LiBr : (n)BuZnBr, suggesting that a higher-order zincate, presumably Li(m)Zn((n)Bu)Br(3)((2-m)-), is the active transmetalating agent.

Potential role of micro-RNAs in head and neck tumorigenesis.

A new class of regulatory molecules known as microRNAs (miRNAs) is redefining our understanding of the molecular pathways associated with tumorigenesis. These miRNAs are small noncoding RNA (ncRNA) sequences with potent regulatory potential. The aberrant expression of miRNAs has been associated with the development of various tumors. It has been suggested that miRNAs can both regulate and act as tumor-suppressor genes and oncogenes. Our understanding of the role of miRNAs in head and neck tumorigenesis is in its infancy. However, several recent studies have revealed extensive dysregulation of miRNA in head and neck tumors and have highlighted the potential of certain miRNAs to act as diagnostic and prognostic markers and targets for new therapeutic agents. The intent of this review is to discuss and summarize current findings that point to a significant role for miRNAs in head and neck tumorigenesis.

Squamous cell carcinoma of the oropharynx in Australian males induced by human papillomavirus vaccine targets.

This study provides Australian data on the incidence of human papillomavirus (HPV)-related oropharyngeal cancer to aid the debate on extending the HPV vaccination programme to males. The HPV status for 302 oropharyngeal cancers diagnosed between 1987 and 2006 was determined by HPV E6-targeted multiplex real-time PCR/p16 immunohistochemistry. The overall HPV-positivity rate was 36% (94% types 16 and 18). HPV-related cancer increased from 19% (1987-1990) to 47% (2001-2005). HPV data used in conjunction with Australian cancer incidence data 2001-2005 showed that 1.56 cases of oropharyngeal cancer per 100,000 males per year were associated with HPV types targeted by the vaccine. Vaccinating males may substantially reduce the burden of oropharyngeal cancer in Australia.