Selectivity and Cooperativity of Modulatory Ions in a Neurotransmitter Receptor

Ions play a modulatory role in many proteins. Kainate receptors, members of the ionotropic glutamate receptor family, require both monovalent anions and cations in the extracellular milieu for normal channel activity. Molecular dynamics simulations and extensive relative binding free energy calculations using thermodynamic integration were performed to elucidate the rank order of binding of monovalent cations, using x-ray crystal structures of the GluR5 kainate receptor dimers with bound cations from the alkali metal family. The simulations show good agreement with experiments and reveal that the underlying backbone structure of the binding site is one of the most rigid regions of the protein. A simplified model where the partial charge of coordinating oxygens was varied suggests that selectivity arises from the presence of two carboxylate groups. Furthermore, using a potential of mean force derived from umbrella sampling, we show that the presence of cations lower the energy barrier for anion approach and binding in the buried anion binding cavity.     

Identifying Conservation Successes,Failures and Future Opportunities; Assessing Recovery Potential of Wild Ungulates and Tigers in Eastern Cambodia

Conservation investment, particularly for charismatic and wide-ranging large mammal species, needs to be evidence-based. Despite the prevalence of this theme within the literature, examples of robust data being generated to guide conservation policy and funding decisions are rare. We present the first published case-study of tiger conservation in Indochina, from a site where an evidence-based approach has been implemented for this iconic predator and its prey. Despite the persistence of extensive areas of habitat, Indochina''s tiger and ungulate prey populations are widely supposed to have precipitously declined in recent decades. The Seima Protection Forest (SPF), and broader Eastern Plains Landscape, was identified in 2000 as representing Cambodia''s best hope for tiger recovery; reflected in its designation as a Global Priority Tiger Conservation Landscape. Since 2005 distance sampling, camera-trapping and detection-dog surveys have been employed to assess the recovery potential of ungulate and tiger populations in SPF. Our results show that while conservation efforts have ensured that small but regionally significant populations of larger ungulates persist, and density trends in smaller ungulates are stable, overall ungulate populations remain well below theoretical carrying capacity. Extensive field surveys failed to yield any evidence of tiger, and we contend that there is no longer a resident population within the SPF. This local extirpation is believed to be primarily attributable to two decades of intensive hunting; but importantly, prey densities are also currently below the level necessary to support a viable tiger population. Based on these results and similar findings from neighbouring sites, Eastern Cambodia does not currently constitute a Tiger Source Site nor meet the criteria of a Global Priority Tiger Landscape. However, SPF retains global importance for many other elements of biodiversity. It retains high regional importance for ungulate populations and potentially in the future for Indochinese tigers, given adequate prey and protection.     

Bortezomib Is Cytotoxic to the Human Growth Plate and Permanently Impairs Bone Growth in Young Mice

Bortezomib, a novel proteasome inhibitor approved for the treatment of cancer in adults, has recently been introduced in pediatric clinical trials. Any tissue-specific side effects on bone development have to our knowledge not yet been explored. To address this, we experimentally studied the effects of bortezomib in vivo in young mice and in vitro in organ cultures of rat metatarsal bones and human growth plate cartilage, as well as in a rat chondrocytic cell line. We found that bortezomib while efficiently blocking the ubiquitin/proteasome system (UPS) caused significant growth impairment in mice, by increasing resting/stem-like chondrocyte apoptosis. Our data support a local action of bortezomib, directly targeting growth plate chondrocytes leading to decreased bone growth since no suppression of serum levels of insulin-like growth factor-I (IGF-I) was observed. A local effect of bortezomib was confirmed in cultured rat metatarsal bones where bortezomib efficiently caused growth retardation in a dose dependent and irreversible manner, an effect linked to increased chondrocyte apoptosis, mainly of resting/stem-like chondrocytes. The cytotoxicity of bortezomib was also evaluated in a unique model of cultured human growth plate cartilage, which was found to be highly sensitive to bortezomib. Mechanistic studies of apoptotic pathways indicated that bortezomib induced activation of p53 and Bax, as well as cleavage of caspases and poly-ADP-ribose polymerase (PARP) in exposed chondrocytes. Our observations, confirmed in vivo and in vitro, suggest that bone growth could potentially be suppressed in children treated with bortezomib. We therefore propose that longitudinal bone growth should be closely monitored in ongoing clinical pediatric trials of this promising anti-cancer drug.     

hSSB1 (NABP2/ OBFC2B) is required for the repair of 8-oxo-guanine by the hOGG1-mediated base excision repair pathway

The maintenance of genome stability is essential to prevent loss of genetic information and the development of diseases such as cancer. One of the most common forms of damage to the genetic code is the oxidation of DNA by reactive oxygen species (ROS), of which 8-oxo-7,8-dihydro-guanine (8-oxoG) is the most frequent modification. Previous studies have established that human single-stranded DNA-binding protein 1 (hSSB1) is essential for the repair of double-stranded DNA breaks by the process of homologous recombination. Here we show that hSSB1 is also required following oxidative damage. Cells lacking hSSB1 are sensitive to oxidizing agents, have deficient ATM and p53 activation and cannot effectively repair 8-oxoGs. Furthermore, we demonstrate that hSSB1 forms a complex with the human oxo-guanine glycosylase 1 (hOGG1) and is important for hOGG1 localization to the damaged chromatin. In vitro, hSSB1 binds directly to DNA containing 8-oxoguanines and enhances hOGG1 activity. These results underpin the crucial role hSSB1 plays as a guardian of the genome.     

Institutional Delivery and Satisfaction among Indigenous and Poor Women in Guatemala,Mexico, and Panama

Indigenous women in Mesoamerica experience disproportionately high maternal mortality rates and are less likely to have institutional deliveries. Identifying correlates of institutional delivery, and satisfaction with institutional deliveries, may help improve facility utilization and health outcomes in this population. We used baseline surveys from the Salud Mesoamérica Initiative to analyze data from 10,895 indigenous and non-indigenous women in Guatemala and Mexico (Chiapas State) and indigenous women in Panama. We created multivariable Poisson regression models for indigenous (Guatemala, Mexico, Panama) and non-indigenous (Guatemala, Mexico) women to identify correlates of institutional delivery and satisfaction. Compared to their non-indigenous peers, indigenous women were substantially less likely to have an institutional delivery (15.2% vs. 41.5% in Guatemala (P<0.001), 29.1% vs. 73.9% in Mexico (P<0.001), and 70.3% among indigenous Panamanian women). Indigenous women who had at least one antenatal care visit were more than 90% more likely to have an institutional delivery (adjusted risk ratio (aRR) = 1.94, 95% confidence interval (CI): 1.44–2.61), compared to those who had no visits. Indigenous women who were advised to give birth in a health facility (aRR = 1.46, 95% CI: 1.18–1.81), primiparous (aRR = 1.44, 95% CI: 1.24–1.68), informed that she should have a Caesarean section (aRR = 1.41, 95% CI: 1.21–1.63), and had a secondary or higher level of education (aRR = 1.36, 95% CI: 1.04–1.79) also had substantially higher likelihoods of institutional delivery. Satisfaction among indigenous women was associated with being able to be accompanied by a community health worker (aRR = 1.15, 95% CI: 1.05–1.26) and facility staff speaking an indigenous language (aRR = 1.10, 95% CI: 1.02–1.19). Additional effort should be exerted to increase utilization of birthing facilities by indigenous and poor women in the region. Improving access to antenatal care and opportunities for higher-level education may increase institutional delivery rates, and providing culturally adapted services may improve satisfaction.     

Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.     

Prey availability and intraguild competition regulate the spatiotemporal dynamics of a modified large carnivore guild

Effective conservation management requires an understanding of the spatiotemporal dynamics driving large carnivore density and resource partitioning. In African ecosystems, reduced prey populations and the loss of competing guild members, most notably lion (Panthera leo), are expected to increase the levels of competition between remaining carnivores. Consequently, intraguild relationships can be altered, potentially increasing the risk of further population decline. Kasungu National Park (KNP), Malawi, is an example of a conservation area that has experienced large‐scale reductions in both carnivore and prey populations, leaving a resident large carnivore guild consisting of only leopard (Panthera pardus) and spotted hyena (Crocuta crocuta). Here, we quantify the spatiotemporal dynamics of these two species and their degree of association, using a combination of co‐detection modeling, time‐to‐event analyses, and temporal activity patterns from camera trap data. The detection of leopard and spotted hyena was significantly associated with the detection of preferred prey and competing carnivores, increasing the likelihood of species interaction. Temporal analyses revealed sex‐specific differences in temporal activity, with female leopard activity patterns significantly different to those of spotted hyena and male conspecifics. Heightened risk of interaction with interspecific competitors and male conspecifics may have resulted in female leopards adopting temporal avoidance strategies to facilitate coexistence. Female leopard behavioral adaptations increased overall activity levels and diurnal activity rates, with potential consequences for overall fitness and exposure to sources of mortality. As both species are currently found at low densities in KNP, increased risk of competitive interactions, which infer a reduction in fitness, could have significant implications for large carnivore demographics. The protection of remaining prey populations is necessary to mitigate interspecific competition and avoid further alterations to the large carnivore guild.     

Pain Associated with Wound Care Treatment among Buruli Ulcer Patients from Ghana and Benin

Buruli ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. People living in remote areas in tropical Sub Saharan Africa are mostly affected. Wound care is an important component of BU management; this often needs to be extended for months after the initial antibiotic treatment. BU is reported in the literature as being painless, however clinical observations revealed that some patients experienced pain during wound care. This was the first study on pain intensity during and after wound care in BU patients and factors associated with pain. In Ghana and Benin, 52 BU patients above 5 years of age and their relatives were included between December 2012 and May 2014. Information on pain intensity during and after wound care was obtained during two consecutive weeks using the Wong-Baker Pain Scale. Median pain intensity during wound care was in the lower range (Mdn = 2, CV = 1), but severe pain (score > 6) was reported in nearly 30% of the patients. Nevertheless, only one patient received pain medication. Pain declined over time to low scores 2 hours after treatment. Factors associated with higher self-reported pain scores were; male gender, fear prior to treatment, pain during the night prior to treatment, and pain caused by cleaning the wound. The general idea that BU is painless is incorrect for the wound care procedure. This procedural pain deserves attention and appropriate intervention.