Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction.

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.

A dual modality 99mTc/Re(i)-labelled T140 analogue for imaging of CXCR4 expression.

The C-X-C chemokine receptor 4 (CXCR4) has been shown to be overexpressed in at least 23 types of cancer, including prostate cancer which has been shown to have a significant distinction of expression rates between cancerous compared to healthy or benign tissue. In an attempt to exploit the difference in expression, we have synthesized a derivative of T140, a peptide antagonist for CXCR4, containing a fluorescent 4-amino-1,8-naphthalimide appended with a di-(2-picolyl)amine binding unit to chelate rhenium or technetium-99m for fluorescence or SPECT imaging. The rhenium-coordinated variant was shown to have similar binding affinity for the receptor as T140 and showed specific uptake by fluorescence microscopy in CXCR4 expressing cells. The peptide was radiolabelled with technetium-99m in decay corrected radiochemical yields ranging from 60-85%, radiochemical purities >95%, and molar activities of 36-44 GBq µmol-1. The technetium-99m labelled peptide showed two-fold higher uptake in U87 cells expressing CXCR4 compared to non-transfected cells. Ex vivo biodistribution studies were performed using the technetium-99m labelled peptide in NOD/SCID mice bearing tumors derived from U87 cells with CXCR4. Tumor uptake of 0.51 ± 0.09% ID g-1 was observed two-hours post-injection. Our novel T140 derivative is suitable for imaging of CXCR4 expression by confocal microscopy. Further structural modifications to the peptide or metal complex may result in improved biodistribution for use in SPECT imaging of CXCR4 expressing tumors.

Amino-Substituted 2,2'-Bipyridine Ligands as Fluorescent Indicators for ZnII and Applications for Fluorescence Imaging of Prostate Cells.

ZnII concentrations in malignant prostate tissues are much lower than in benign or healthy, suggesting that ZnII levels are a potential biomarker for prostate cancer (PCa). Five 2,2'-bipyridine ligands were synthesized containing amino substituents with varying electron-donating ability for investigation as fluorescent ZnII indicators. The excited state characteristics of the ligands were explored by UV/Vis and fluorescence spectroscopy. 3,3'-Diamino-2,2'-bipyridine (1) was previously shown to be weakly fluorescent as a result of π→π* transitions. The other four ligands have properties consistent with an n→π* intraligand charge transfer excited state. Strongly donating amino and aminophenyl (2 and 4) substituents gave low quantum yields, while weaker donating benzimidazole substituents (6 and 7) gave high quantum yields. Absorption and fluorescence wavelengths underwent bathochromic shifts upon ZnII binding in a majority of cases. Quantum yields drastically increased upon ZnII binding for 1 and 2, but decreased for 4, 6, and 7. Compounds 6 and 7 were incubated with PC-3, DU 145 and BPH-1 cells to determine their ZnII sensing abilities in a biological system. Weak fluorescence was observed in BPH-1 cells and subsequent incubation with ZnII caused fluorescence intensity to increase. No fluorescence was observed in PCa cell lines. Further investigation of these ligands may allow for quantitative determination of ZnII concentrations in ex vivo tissue samples.

Establishing a multidisciplinary initiative for interoperable electronic health record innovations at an academic medical center.

OBJECTIVE: To establish an enterprise initiative for improving health and health care through interoperable electronic health record (EHR) innovations. MATERIALS AND METHODS: We developed a unifying mission and vision, established multidisciplinary governance, and formulated a strategic plan. Key elements of our strategy include establishing a world-class team; creating shared infrastructure to support individual innovations; developing and implementing innovations with high anticipated impact and a clear path to adoption; incorporating best practices such as the use of Fast Healthcare Interoperability Resources (FHIR) and related interoperability standards; and maximizing synergies across research and operations and with partner organizations. RESULTS: University of Utah Health launched the ReImagine EHR initiative in 2016. Supportive infrastructure developed by the initiative include various FHIR-related tooling and a systematic evaluation framework. More than 10 EHR-integrated digital innovations have been implemented to support preventive care, shared decision-making, chronic disease management, and acute clinical care. Initial evaluations of these innovations have demonstrated positive impact on user satisfaction, provider efficiency, and compliance with evidence-based guidelines. Return on investment has included improvements in care; over $35 million in external grant funding; commercial opportunities; and increased ability to adapt to a changing healthcare landscape. DISCUSSION: Key lessons learned include the value of investing in digital innovation initiatives leveraging FHIR; the importance of supportive infrastructure for accelerating innovation; and the critical role of user-centered design, implementation science, and evaluation. CONCLUSION: EHR-integrated digital innovation initiatives can be key assets for enhancing the EHR user experience, improving patient care, and reducing provider burnout.

A study of 99mTc/Re-tricarbonyl complexes of 4-amino-1,8-naphthalimides.

Three 4-amino-1,8-naphthalimide analogues were synthesized, consisting of the tridentate chelators di-2-picolylamine, (pyridin-2-ylmethyl)glycinate, and iminodiacetate conjugated to the naphthalimide scaffold. Coordination with fac-99mTc/Re(CO)3 resulted in metal complexes with overall charges of -1, 0, or +1. Upon coordination of Re(i), the initial naphthalimide-based fluorescence is largely maintained for both negative and neutral complexes compared to their free ligand forms, while an increase in fluorescence quantum yield was observed for the positively charged complex. OVCAR-8 ovarian cancer cells were stained with each of the complexes, demonstrating that the positive complex is more lipophilic and cell membrane permeable than the neutral and negative complexes. Each of the three technetium-99m labelled naphthalimide complexes were successfully produced from fac-[99mTc(CO)3(H2O)3]+ in 15 minutes at 70 °C and isolated in radiochemical yields ranging from 60-95% with radiochemical purities greater than 95%. These fluorescent metal complexes of various charges can be used to tune pharmacokinetic and cellular uptake properties of 99mTc/Re-naphthalimide-bioconjugates, while still maintaining desirable fluorescence properties.

Comprehensive in vitro characterization of PD-L1 small molecule inhibitors.

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction has emerged as a powerful strategy in cancer immunotherapy. Recently, there have been enormous efforts to develop potent PD-1/PD-L1 inhibitors. In particular, Bristol-Myers Squibb (BMS) and Aurigene Discovery Technologies have individually disclosed several promising PD-1/PD-L1 inhibitors, whose detailed experimental data are not publicly disclosed. In this work, we report the rigorous and systematic in vitro characterization of a selected set of potent PD-1/PD-L1 macrocyclic peptide (BMSpep-57) and small-molecule inhibitors (BMS-103, BMS-142) from BMS and a peptidomimetic small-molecule inhibitor from Aurigene (Aurigene-1) using a series of biochemical and cell-based assays. Our results confirm that BMS-103 and BMS-142 are strongly active in biochemical assays; however, their acute cytotoxicity greatly compromised their immunological activity. On the other hand, Aurigene-1 did not show any activity in both biochemical and immunological assays. Furthermore, we also report the discovery of a small-molecule immune modulator, whose mode-of-action is not clear; however, it exhibits favorable drug-like properties and strong immunological activity. We hope that the results presented here will be useful in guiding the development of next-generation PD-1/PD-L1 small molecule inhibitors.

Cross-sectional and longitudinal relations between mother-child talk about conflict and children's social understanding.

We examined the relations between the ways 48 mothers and their 3- to 5-year-olds talked about a conflict depicted in a picture book and their children's current and subsequent level of social understanding. We distinguished explanatory talk, which directed attention to the actions that generated the conflict, from non-explanatory talk, which discussed the conflict in terms of, for example, making up or saying sorry. Controlling for child age and overall talk by mother, explanatory talk was positively associated with contemporaneous social understanding. Social understanding at time one was also positively associated with social understanding 30 months later. These data suggest that dialogue about conflict may be helpful for 3- to 5-year old children's understanding of the mental world, to the extent that it facilitates their understanding of particular social situations.

The ear region of the marsupial sabertooth, Thylacosmilus: Influence of the sabertooth lifestyle upon it, and convergence with placental sabertooths.

The mastoid auditory bulla of the extinct marsupial sabertooth, Thylacosmilus, has an enlarged, complex hypotympanic sinus but lacks an alisphenoid contribution. These are marked departures from the usual marsupial condition. Details of the ear region of Thylacosmilus are compared with those of the convergent, extinct placental sabertooth, Smilodon, and each is compared with less specialized related forms to define differences and similarities of the evolutionary paths that led to the striking overall convergence. Functional factors such as pressure transformer ratio (PTR), impedance transformer ratio (ITR), acoustic impedence at the eardrum, and the fraction of the sound energy theoretically transmitted to the inner ear cannot be estimated for Thylacosmilus because certain critical measures are still unknown (tympanum size, ossicle lever arm ratios). However, in both sabertooths enlarged complex hypotympanic sinuses, characterized by expansions and contractions, are greatly developed. They vastly increase middle ear space (volume) and must have influenced these factors. In both, the sinuses provide the large air volume needed to prevent excessive damping of sound energy transmission (Hunt and Korth, '80), and both are believed to have achieved a further modulation of the system from the cushioning or "pillow" effect of the confined air as it directly damps the tympanum itself. Thylacosmilus has still another feature that may have given greater control over damping of sound energy transmission: the direct opening (probably membrane covered) of one of the sinus cavities into the side of the meatal tube. In this feature, as in others noted earlier (Turnbull, '76, '78), we see a greater degree of specialization in this marsupial sabertooth than in a placental counterpart.