Reply to Crawford et al.: Why Trap-Neuter-Return (TNR) Is an Ethical Solution for Stray Cat Management

The recently published article, ‘A Case of Letting the Cat out of the Bag—Why Trap-Neuter-Return Is Not an Ethical Solution for Stray Cat (Felis catus) Management,’ by Crawford et al. warrants rebuttal. The case presented in the paper, opposing the initiation of TNR trials in Australia, ignores peer-reviewed evidence which substantiates the effectiveness of TNR at reducing unowned urban cat numbers. In addition, the paper’s authors offer a number of unrealistic recommendations, which are little more than a rebranding of the failed status quo. Urban stray cats have long been considered a problem across Australia. Current practice calls for the trapping and killing of thousands of healthy urban stray cats and kittens each year with no apparent effect on the total population. In contrast, the literature offers numerous examples, including two recent studies in Australia, of reductions in urban stray cat numbers where TNR has been implemented. TNR has also been associated with reduced feline intake and euthanasia at shelters, which improves both animal welfare and the well-being of shelter staff. A large-scale trial of TNR in an urban Australian context is scientifically justified and long overdue

Integral membrane protein structure determination using pseudocontact shifts.

Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.This work was supported by the Biotechnology and Biological Sciences Research Council BBSRC [BB/K01983X/1].This paper was originally published in the Journal of Bimolecular NMR (Crick DJ, Wang JX, Graham B, Swarbrick JD, Mott HR, Nietlispach D, Journal of Biomolecular NMR 2015, doi:10.1007/s10858-015-9899-6)

RIDME distance measurements using Gd(iii) tags with a narrow central transition

Methods based on pulse electron paramagnetic resonance allow measurement of the electron-electron dipolar coupling between two spin labels. Here we compare the most popular technique, Double Electron-Electron Resonance (DEER or PELDOR), with the dead-time free 5-pulse Relaxation-Induced Dipolar Modulation Enhancement (RIDME) method for Gd(iii)-Gd(iii) distance measurements at W-band (94.9 GHz, ≈3.5 T) using Gd(iii) tags with a small zero field splitting (ZFS). Such tags are important because of their high EPR sensitivity arising from their narrow central transition. Two systems were investigated: (i) a rigid model compound with an inter-spin distance of 2.35 nm, and (ii) two mutants of a homodimeric protein, both labeled with a DOTA-based Gd(iii) chelate and characterized by an inter-spin distance of around 6 nm, one having a narrow distance distribution and the other a broad distribution. Measurements on the model compound show that RIDME is less sensitive to the complications arising from the failure of the weak coupling approximation which affect DEER measurements on systems characterized by short inter-spin distances between Gd(iii) tags having a narrow central transition. Measurements on the protein samples, which are characterized by a long inter-spin distance, emphasize the complications due to the appearance of harmonics of the dipolar interaction frequency in the RIDME traces for S > 1/2 spin systems, as well as enhanced uncertainties in the background subtraction. In both cases the sensitivity of RIDME was found to be significantly better than DEER. The effects of the experimental parameters on the RIDME trace are discussed.This research was supported by the Israeli Science Foundation (grant 334/14) and was made possible in part by the historic generosity of the Harold Perlman Family (D. G.). We also acknowledge the Australian Research Council for a Discovery grant to G. O. and B. G. (DP150100383) and a Future Fellowship to B. G. (FT130100838). D. G. holds the Erich Klieger professorial chair in Chemical Physics

A New Gd³⁺ Spin Label for Gd³⁺-Gd³⁺ Distance Measurements in Proteins Produces Narrow Distance Distributions

Gd(3+) tags have been shown to be useful for performing distance measurements in biomolecules via the double electron-electron resonance (DEER) technique at Q- and W-band frequencies. We introduce a new cyclen-based Gd(3+) tag that exhibits a relatively narrow electron paramagnetic resonance (EPR) spectrum, affording high sensitivity, and which yields exceptionally narrow Gd(3+)-Gd(3+) distance distributions in doubly tagged proteins owing to a very short tether. Both the maxima and widths of distance distributions measured for tagged mutants of the proteins ERp29 and T4 lysozyme, featuring Gd(3+)-Gd(3+) distances of ca. 6 and 4 nm, respectively, were well reproduced by simulated distance distributions based on available crystal structures and sterically allowed rotamers of the tag. The precision of the position of the Gd(3+) ion is comparable to that of the nitroxide radical in an MTSL-tagged protein and thus the new tag represents an attractive tool for performing accurate distance measurements and potentially probing protein conformational equilibria.This work was supported by the Israel Science Foundation, the Australian Research Council (ARC), and an AustraliaWeizmann Making Connections grant. B.G. thanks the ARC for a Future Fellowship (FT130100838). D.G. holds the Erich Klieger Professorial Chair in Physical Chemistry

Structure, function, and regulation of thioesterases

Thioesterases are present in all living cells and perform a wide range of important biological functions by catalysing the cleavage of thioester bonds present in a diverse array of cellular substrates. Thioesterases are organised into 25 families based on their sequence conservation, tertiary and quaternary structure, active site configuration, and substrate specificity. Recent structural and functional characterisation of thioesterases has led to significant changes in our understanding of the regulatory mechanisms that govern enzyme activity and their respective cellular roles. The resulting dogma changes in thioesterase regulation include mechanistic insights into ATP and GDP-mediated regulation by oligomerisation, the role of new key regulatory regions, and new insights into a conserved quaternary structure within TE4 family members. Here we provide a current and comparative snapshot of our understanding of thioesterase structure, function, and regulation across the different thioesterase families

Redefining the Expression and Function of the Inhibitor of Differentiation 1 in Mammary Gland Development

The accumulation of poorly differentiated cells is a hallmark of breast neoplasia and progression. Thus an understanding of the factors controlling mammary differentiation is critical to a proper understanding of breast tumourigenesis. The Inhibitor of Differentiation 1 (Id1) protein has well documented roles in the control of mammary epithelial differentiation and proliferation in vitro and breast cancer progression in vivo. However, it has not been determined whether Id1 expression is sufficient for the inhibition of mammary epithelial differentiation or the promotion of neoplastic transformation in vivo. We now show that Id1 is not commonly expressed by the luminal mammary epithelia, as previously reported. Generation and analysis of a transgenic mouse model of Id1 overexpression in the mammary gland reveals that Id1 is insufficient for neoplastic progression in virgin animals or to prevent terminal differentiation of the luminal epithelia during pregnancy and lactation. Together, these data demonstrate that there is no luminal cell-autonomous role for Id1 in mammary epithelial cell fate determination, ductal morphogenesis and terminal differentiation

Overcoming artificial broadening in Gd³⁺–Gd³⁺ distance distributions arising from dipolar pseudo-secular terms in DEER experiments

By providing accurate distance measurements between spin labels site-specifically attached to bio-macromolecules, double electron–electron resonance (DEER) spectroscopy provides a unique tool to probe the structural and conformational changes in these molecules. Gd3+-tags present an important family of spin-labels for such purposes, as they feature high chemical stability and high sensitivity in high-field DEER measurements. The high sensitivity of the Gd3+ ion is associated with its high spin (S = 7/2) and small zero field splitting (ZFS), resulting in a narrow spectral width of its central transition at high fields. However, under the conditions of short distances and exceptionally small ZFS, the weak coupling approximation, which is essential for straightforward DEER data analysis, becomes invalid and the pseudo-secular terms of the dipolar Hamiltonian can no longer be ignored. This work further explores the effects of pseudo-secular terms on Gd3+–Gd3+ DEER measurements using a specifically designed ruler molecule; a rigid bis-Gd3+-DOTA model compound with an expected Gd3+–Gd3+ distance of 2.35 nm and a very narrow central transition at the W-band (95 GHz). We show that the DEER dipolar modulations are damped under the standard W-band DEER measurement conditions with a frequency separation, Δν, of 100 MHz between the pump and observe pulses. Consequently, the DEER spectrum deviates considerably from the expected Pake pattern. We show that the Pake pattern and the associated dipolar modulations can be restored with the aid of a dual mode cavity by increasing Δν from 100 MHz to 1.09 GHz, allowing for a straightforward measurement of a Gd3+–Gd3+ distance of 2.35 nm. The increase in Δν increases the contribution of the |−5/2〉 → |−3/2〉 and |−7/2〉 → |−5/2〉 transitions to the signal at the expense of the |−3/2 〉 → |−1/2〉 transition, thus minimizing the effect of dipolar pseudo-secular terms and restoring the validity of the weak coupling approximation. We apply this approach to the A93C/N140C mutant of T4 lysozyme labeled with two different Gd3+ tags that have narrow central transitions and show that even for a distance of 4 nm there is still a significant (about two-fold) broadening that is removed by increasing Δν to 636 MHz and 898 MHz.This research was supported by the Israeli Science Foundation (grant 334/14) and made possible in part by the historic generosity of the Harold Perlman Family. D. G. holds the Erich Klieger professorial chair in Chemical Physic

Immunodominant Tuberculosis CD8 Antigens Preferentially Restricted by HLA-B

CD8+ T cells are essential for host defense to intracellular bacterial pathogens such as Mycobacterium tuberculosis (Mtb), Salmonella species, and Listeria monocytogenes, yet the repertoire and dominance pattern of human CD8 antigens for these pathogens remains poorly characterized. Tuberculosis (TB), the disease caused by Mtb infection, remains one of the leading causes of infectious morbidity and mortality worldwide and is the most frequent opportunistic infection in individuals with HIV/AIDS. Therefore, we undertook this study to define immunodominant CD8 Mtb antigens. First, using IFN-γ ELISPOT and synthetic peptide arrays as a source of antigen, we measured ex vivo frequencies of CD8+ T cells recognizing known immunodominant CD4+ T cell antigens in persons with latent tuberculosis infection. In addition, limiting dilution was used to generate panels of Mtb-specific T cell clones. Using the peptide arrays, we identified the antigenic specificity of the majority of T cell clones, defining several new epitopes. In all cases, peptide representing the minimal epitope bound to the major histocompatibility complex (MHC)-restricting allele with high affinity, and in all but one case the restricting allele was an HLA-B allele. Furthermore, individuals from whom the T cell clone was isolated harbored high ex vivo frequency CD8+ T cell responses specific for the epitope, and in individuals tested, the epitope represented the single immunodominant response within the CD8 antigen. We conclude that Mtb-specific CD8+ T cells are found in high frequency in infected individuals and are restricted predominantly by HLA-B alleles, and that synthetic peptide arrays can be used to define epitope specificities without prior bias as to MHC binding affinity. These findings provide an improved understanding of immunodominance in humans and may contribute to a development of an effective TB vaccine and improved immunodiagnostics

Čvrsta disperzija meloksikama: faktorijalno dizajnirani dozirani pripravak za gerijatrijsku populaciju

The objective of the present work was to improve the dissolution properties of the poorly water-soluble drug meloxicam by preparing solid dispersions with hydroxyethylcellulose (HEC), mannitol and polyethylene glycol (PEG) 4000 and to develop a dosage form for geriatric population. Differential scanning calorimetry, X–ray diffractometry, Fourier transform infrared spectroscopy and scanning electron microscopy were used to investigate the solid-state physical structure of the prepared solid dispersions. Higher in vitro dissolution of solid dispersions was recorded compared to their corresponding physical mixtures and the pure drug. PEG 4000 in 1:9 drug to carrier ratio exhibited the highest drug release (100.2%), followed by mannitol (98.2%) and HEC (89.5%) in the same ratio. Meloxicam-PEG 4000 solid dispersion was formulated into suspension and optimization was carried out by 23 factorial design. Formulations containing higher levels of methyl cellulose and higher levels of either sodium citrate or Tween 80 exhibited the highest drug release.Cilj rada bio je poboljšati topljivost meloksikama u vodi pripravom čvrstih disperzija s hidroksietilcelulozom (HEC), manitolom i polietilen glikolom 4000 (PEG 4000) te razviti dozirani pripravaka za gerijatrijsku populaciju. Za ispitivanje fizičke strukture pripravljenih čvrstih disperzija korištene su diferencijalna pretražna kalorimetrija, difraktometrija rentgentskim zrakama, FTIR i pretražna elektronska mikroskopija. Čvrste disperzije su u in vitro uvjetima pokazale bolju topljivost u odnosu na fizičku smjesu i čistu ljekovitu tvar. Najbolje oslobađanje lijeka (100,2%). postignuto je iz disperzija s PEG 4000 (omjer ljekovite tvari i nosača 1:9). Slijede manitol (98,2%) i HEC (89,5%) (isti omjer meloksikama i polimera). Čvrsta disperzija meloksikama s PEG 4000 prevedena je u suspenziju te optimirana 23 faktorijalnim dizajnom. Najbolje oslobađanje ljekovite tvari postignuto je iz pripravaka koji sadrže veći udio etilceluloze i natrijevog citrata, odnosno Tween 80