Retrospective Follow-up Assessment of Risk Variables Influencing the Outcome of Autologous Tooth Transplantation.

INTRODUCTION: This retrospective study aimed to identify which patient-, donor tooth-, recipient site-, and surgical procedure-related variables may influence the outcome of tooth autotransplantation. METHODS: The sample included 128 autotransplants performed in 122 patients. Single-visit clinical/imaging examinations were used to define the outcome as successful, survival, or failure. The association of potential indicators with the survival or failure categories was analyzed individually and adjusted for confounders through multivariate logistic regression models. RESULTS: After a follow-up period of 1 to 30.11 years, success was achieved in 71.8% of autotransplants, whereas the survival and failure groups had rates of 14.1% each, and the grouped success/survival rate reached 85.9%. An extraoral time >15 minutes and difficult handling/placement were strong/independent risk covariates for survival and failure categories (odds ratio >1, P < .05). Additionally, unerupted/partially erupted status of the donor tooth was a significant indicator for survival, whereas deficient bone level at the recipient site, surgical extraction, poor initial stability, and lack of prophylactic antibiotics were independently linked to failure (odds ratio > 1, P < .05). The root morphology and socket status acted as modifiers of the effect of the recipient site location on the survival group (P > .05). CONCLUSION: Based on the results of this study, unerupted/partially erupted status of the donor tooth, surgical extraction, total extraoral time >15 minutes, deficient recipient's bone level, difficult handling/placement of the autotransplant, poor initial stability, and lack of prophylactic antibiotics during the surgical procedure must be considered with caution when performing autotransplantation because of their deleterious influence on the outcome.

Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer.

Chimeric antigen receptor T (CAR-T) cells have demonstrated promising results against hematological malignancies, but have encountered significant challenges in translation to solid tumors. To overcome these hurdles, we have developed a switchable CAR-T cell platform in which the activity of the engineered cell is controlled by dosage of an antibody-based switch. Herein, we apply this approach to Her2-expressing breast cancers by engineering switch molecules through site-specific incorporation of FITC or grafting of a peptide neo-epitope (PNE) into the anti-Her2 antibody trastuzumab (clone 4D5). We demonstrate that both switch formats can be readily optimized to redirect CAR-T cells (specific for the corresponding FITC or PNE) to Her2-expressing tumor cells, and afford dose-titratable activation of CAR-T cells ex vivo and complete clearance of the tumor in rodent xenograft models. This strategy may facilitate the application of immunotherapy to solid tumors by affording comparable efficacy with improved safety owing to switch-based control of the CAR-T response.

Targeted Delivery of LXR Agonist Using a Site-Specific Antibody-Drug Conjugate.

Liver X receptor (LXR) agonists have been explored as potential treatments for atherosclerosis and other diseases based on their ability to induce reverse cholesterol transport and suppress inflammation. However, this therapeutic potential has been hindered by on-target adverse effects in the liver mediated by excessive lipogenesis. Herein, we report a novel site-specific antibody-drug conjugate (ADC) that selectively delivers a LXR agonist to monocytes/macrophages while sparing hepatocytes. The unnatural amino acid para-acetylphenylalanine (pAcF) was site-specifically incorporated into anti-CD11a IgG, which binds the α-chain component of the lymphocyte function-associated antigen 1 (LFA-1) expressed on nearly all monocytes and macrophages. An aminooxy-modified LXR agonist was conjugated to anti-CD11a IgG through a stable, cathepsin B cleavable oxime linkage to afford a chemically defined ADC. The anti-CD11a IgG-LXR agonist ADC induced LXR activation specifically in human THP-1 monocyte/macrophage cells in vitro (EC50-27 nM), but had no significant effect in hepatocytes, indicating that payload delivery is CD11a-mediated. Moreover, the ADC exhibited higher-fold activation compared to a conventional synthetic LXR agonist T0901317 (Tularik) (3-fold). This novel ADC represents a fundamentally different strategy that uses tissue targeting to overcome the limitations of LXR agonists for potential use in treating atherosclerosis.

Multiformat T-cell-engaging bispecific antibodies targeting human breast cancers.

Four different formats of bispecific antibodies (bsAbs) were generated that consist of anti-Her2 IgG or Fab site-specifically conjugated to anti-CD3 Fab using the genetically encoded noncanonical amino acid. These bsAbs varied in valency or in the presence or absence of an Fc domain. Different valencies did not significantly affect antitumor efficacy, whereas the presence of an Fc domain enhanced cytotoxic activity, but triggered antigen-independent T-cell activation. We show that the bsAbs can efficiently redirect T cells to kill all Her2 expressing cancer cells, including Her2 1+ cancers, both in vitro and in rodent xenograft models. This work increases our understanding of the structural features that affect bsAb activity, and underscores the potential of bsAbs as a promising therapeutic option for breast cancer patients with low or heterogeneous Her2 expression.

A genetically encoded alkyne directs palladium-mediated protein labeling on live mammalian cell surface.

The merging of site-specific incorporation of small bioorthogonal functional groups into proteins via amber codon suppression with bioorthogonal chemistry has created exciting opportunities to extend the power of organic reactions to living systems. Here we show that a new alkyne amino acid can be site-selectively incorporated into mammalian proteins via a known orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and directs an unprecedented, palladium-mediated cross-coupling reaction-driven protein labeling on live mammalian cell surface. A comparison study with the alkyne-encoded proteins in vitro indicated that this terminal alkyne is better suited for the palladium-mediated cross-coupling reaction than the copper-catalyzed click chemistry.

A CXCR4-targeted site-specific antibody-drug conjugate.

A chemically defined anti-CXCR4-auristatin antibody-drug conjugate (ADC) was synthesized that selectively eliminates tumor cells overexpressing the CXCR4 receptor. The unnatural amino acid p-acetylphenylalanine (pAcF) was site-specifically incorporated into an anti-CXCR4 immunoglobulin G (IgG) and conjugated to an auristatin through a stable, non-cleavable oxime linkage to afford a chemically homogeneous ADC. The full-length anti-CXCR4 ADC was selectively cytotoxic to CXCR4(+) cancer cells in vitro (half maximal effective concentration (EC50 )≈80-100 pM). Moreover, the anti-CXCR4 ADC eliminated pulmonary lesions from human osteosarcoma cells in a lung-seeding tumor model in mice. No significant overt toxicity was observed but there was a modest decrease in the bone-marrow-derived CXCR4(+) cell population. Because CXCR4 is highly expressed in a majority of metastatic cancers, a CXCR4-auristatin ADC may be useful for the treatment of a variety of metastatic malignancies.

Storable N-phenylcarbamate palladacycles for rapid functionalization of an alkyne-encoded protein.

Here we report the synthesis of storable N-phenylcarbamate palladacycles that showed robust reactivity in the cross-coupling reaction with an alkyne-encoded protein with a second-order rate constant approaching 19 770 ± 930 M(-1) s(-1).

Fast and sequence-specific palladium-mediated cross-coupling reaction identified from phage display.

Fast and specific bioorthogonal reactions are highly desirable because they provide efficient tracking of biomolecules that are present in low abundance and/or involved in fast dynamic process in living systems. Toward this end, classic strategy involves the optimization of substrate structures and reaction conditions in test tubes, testing their compatibility with biological systems, devising synthetic biology schemes to introduce the modified substrates into living cells or organisms, and finally validating the superior kinetics for enhanced capacity in tracking biomolecules in vivo--a lengthy process often mired by unexpected results. Here, we report a streamlined approach in which the "microenvironment" of a bioorthogonal chemical reporter is exploited directly in biological systems via phage-assisted interrogation of reactivity (PAIR) to optimize not only reaction kinetics but also specificity. Using the PAIR strategy, we identified a short alkyne-containing peptide sequence showing fast kinetics (k2=13,000±2000 M(-1) s(-1)) in a palladium-mediated cross-coupling reaction. Site-directed mutagenesis studies suggested that the residues surrounding the alkyne moiety facilitate the assembly of a key palladium-alkyne intermediate along the reaction pathway. When this peptide sequence was inserted into the extracellular domain of epidermal growth factor receptor (EGFR), this reactive sequence directed the specific labeling of EGFR in live mammalian cells.

Storable palladacycles for selective functionalization of alkyne-containing proteins.

We report the facile preparation of palladacycles as storable arylpalladium(II) reagents from acetanilides via cyclopalladation. The palladacycles exhibit good stability in PBS buffer and are capable of functionalizing a metabolically encoded HPG-containing protein, thus providing a new type of biocompatible organometallic reagent for selectively functionalizing the alkyne-encoded proteins.

Upper lethal temperatures of Northern Bobwhite embryos and the thermal properties of their eggs.

Northern Bobwhite eggs in the southern United States are often exposed to ambient temperatures in excess of their normal incubation temperature when unattended during their typical extended preincubation period. In drought years, typified by high ambient temperatures, Bobwhite eggs are often exposed to temperatures > 45°C, well-above the upper lethal temperature of most other birds. Because the upper lethal temperature of Bobwhite embryos is not currently known, simulated clutches of eggs were exposed to preincubation temperatures ranging from 39 to 52°C for exposure times of 1, 3, or 6 h. The upper lethal temperatures and the temperatures resulting in ≥ 50% death of Northern Bobwhite embryos were recorded in addition to the time to thermal equilibrium of Bobwhite eggs. The upper lethal temperature for 1, 3, and 6 h of preincubation exposure was 51, 49, and 46°C, respectively. The temperatures resulting in ≥ 50% death were 46, 44, and 40°C for eggs exposed to elevated temperatures for 1, 3, and 6 h, respectively. The mean time for the inner-egg temperature to reach the ambient temperature was 38 ± 1 min (± SE). The thermal tolerances of Northern Bobwhite embryos were much higher than expected, and among the highest reported for birds, indicating an adaptation to the naturally occurring temperature extremes that often occur in the Bobwhite's semi-arid southern range. However, as the temperature increased above the incubation temperature, hatching success declined, showing that increased thermal tolerance has a cost. Although Bobwhite producers, managers, and researchers will find this information useful, it seems most interesting that high temperatures could plausibly have contributed to the population decline observed in the Bobwhite's semi-arid range.

Copper-free Sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells.

Bioorthogonal reactions suitable for functionalization of genetically or metabolically encoded alkynes, for example, copper-catalyzed azide-alkyne cycloaddition reaction ("click chemistry"), have provided chemical tools to study biomolecular dynamics and function in living systems. Despite its prominence in organic synthesis, copper-free Sonogashira cross-coupling reaction suitable for biological applications has not been reported. In this work, we report the discovery of a robust aminopyrimidine-palladium(II) complex for copper-free Sonogashira cross-coupling that enables selective functionalization of a homopropargylglycine (HPG)-encoded ubiquitin protein in aqueous medium. A wide range of aromatic groups including fluorophores and fluorinated aromatic compounds can be readily introduced into the HPG-containing ubiquitin under mild conditions with good to excellent yields. The suitability of this reaction for functionalization of HPG-encoded ubiquitin in Escherichia coli was also demonstrated. The high efficiency of this new catalytic system should greatly enhance the utility of Sonogashira cross-coupling in bioorthogonal chemistry.

Photoinducible bioorthogonal chemistry: a spatiotemporally controllable tool to visualize and perturb proteins in live cells.

Visualization in biology has been greatly facilitated by the use of fluorescent proteins as in-cell probes. The genes coding for these wavelength-tunable proteins can be readily fused with the DNA coding for a protein of interest, which enables direct monitoring of natural proteins in real time inside living cells. Despite their success, however, fluorescent proteins have limitations that have only begun to be addressed in the past decade through the development of bioorthogonal chemistry. In this approach, a very small bioorthogonal tag is embedded within the basic building blocks of the cell, and then a variety of external molecules can be selectively conjugated to these pretagged biomolecules. The result is a veritable palette of biophysical probes for the researcher to choose from. In this Account, we review our progress in developing a photoinducible, bioorthogonal tetrazole-alkene cycloaddition reaction ("photoclick chemistry") and applying it to probe protein dynamics and function in live cells. The work described here summarizes the synthesis, structure, and reactivity studies of tetrazoles, including their optimization for applications in biology. Building on key insights from earlier reports, our initial studies of the reaction have revealed full water compatibility, high photoactivation quantum yield, tunable photoactivation wavelength, and broad substrate scope; an added benefit is the formation of fluorescent cycloadducts. Subsequent studies have shown fast reaction kinetics (up to 11.0 M(-1) s(-1)), with the rate depending on the HOMO energy of the nitrile imine dipole as well as the LUMO energy of the alkene dipolarophile. Moreover, through the use of photocrystallography, we have observed that the photogenerated nitrile imine adopts a bent geometry in the solid state. This observation has led to the synthesis of reactive, macrocyclic tetrazoles that contain a short "bridge" between two flanking phenyl rings. This photoclick chemistry has been used to label proteins rapidly (within ∼1 min) both in vitro and in E. coli . To create an effective interface with biology, we have identified both a metabolically incorporable alkene amino acid, homoallylglycine, and a genetically encodable tetrazole amino acid, p-(2-tetrazole)phenylalanine. We demonstrate the utility of these two moieties, respectively, in spatiotemporally controlled imaging of newly synthesized proteins and in site-specific labeling of proteins. Additionally, we demonstrate the use of the photoclick chemistry to perturb the localization of a fluorescent protein in mammalian cells.

Azirine ligation: fast and selective protein conjugation via photoinduced azirine-alkene cycloaddition.

We report a new bioorthogonal ligation reaction between p-nitrodiphenylazirine and dimethyl fumarate. This photoinduced azirine-alkene cycloaddition provides a rapid (~2 min) and highly selective route to protein conjugation at neutral pH and room temperature in biological medium.

Bioorthogonal chemistry: recent progress and future directions.

The ability to use covalent chemistry to label biomolecules selectively in their native habitats has greatly enhanced our understanding of biomolecular dynamics and function beyond what is possible with genetic tools alone. To attain the exquisite selectivity that is essential in this covalent approach a "bottom-up" two-step strategy has achieved many successes recently. In this approach, a bioorthogonal chemical functionality is built into life's basic building blocks-amino acids, nucleosides, lipids, and sugars-as well as coenzymes; after the incorporation, an array of biophysical probes are selectively appended to the tagged biomolecules via a suitable bioorthogonal reaction. While much has been accomplished in the expansion of non-natural building blocks carrying unique chemical moieties, the dearth of robust bioorthogonal reactions has limited both the scope and utility of this promising approach. Here, we summarize the recent progress in the development of bioorthogonal reactions and their applications in various biological systems. A major emphasis has been placed on the mechanistic and kinetic studies of these reactions with the hope that continuous improvements can be made with each reaction in the future. In view of the gap between the capabilities of the current repertoire of bioorthogonal reactions and the unmet needs of outstanding biological problems, we also strive to project the future directions of this rapidly developing field.

Discovery of long-wavelength photoactivatable diaryltetrazoles for bioorthogonal 1,3-dipolar cycloaddition reactions.

Several long-wavelength (365 nm) photoactivatable diaryltetrazoles were discovered by screening a series of substituted diaryltetrazoles and subsequently showed excellent reactivity in the photoactivated 1,3-dipolar cycloaddition reactions toward electron-deficient and conjugated alkenes in organic solvents as well as an alkene-containing protein in the aqueous buffer.

Cost and utilization impact of a clinical pathway for patients undergoing pancreaticoduodenectomy.

BACKGROUND: When implemented in several common surgical procedures, clinical pathways have been reported to reduce costs and resource utilization, while maintaining or improving patient care. However, there is little data to support their use in more complex surgery. The objective of this study was to determine the effects of clinical pathway implementation in patients undergoing elective pancreaticoduodenectomy (PD) on cost and resource utilization. METHODS: Outcome data from before and after the development of a clinical pathway were analyzed. The clinical pathway standardized the preoperative outpatient care, critical care, and postoperative floor care of patients who underwent PD. An independent department determined total costs for each patient, which included all hospital and physician costs, in a blinded review. Outcomes that were examined included perioperative mortality, postoperative morbidity, length of stay, readmissions, and postoperative clinic visits. RESULTS: From January, 1996 to December, 1998, 148 consecutive patients underwent PD or total pancreatectomy; 68 before pathway development (PrePath) and 80 after pathway implementation (PostPath). There were no significant differences in patient demographics, comorbid conditions, underlying diagnosis, or use of neoadjuvant therapy between the two groups. Mean total costs were significantly reduced in PostPath patients compared with PrePath patients ($36,627 vs. $47,515; P = .003). Similarly, mean length of hospital stay was also significantly reduced in PostPath patients (13.5 vs. 16.4 days; P = .001). The total cost differences could not be attributed solely to differences in room and board costs. Cost and length-of-stay differences remained when outliers were excluded from the analysis. Despite these findings, there were no significant differences between PrePath and PostPath patients in terms of perioperative mortality (3% vs. 1%), readmissions within 1 month of discharge (15% vs. 11%), or mean number of clinic visits within 90 days of discharge (3.3 vs. 3.4 visits). CONCLUSIONS: The establishment of a clinical pathway for PD patients dramatically reduced costs and resource utilization without any apparent detrimental effect on quality of patient care. These findings support the implementation of clinical pathways for PD patients, as well as investigation into pathway care for other complex surgical procedures.

The impact of clinical pathways on the practice of head and neck oncologic surgery: the University of Texas M. D. Anderson Cancer Center Experience.

OBJECTIVE: To assess the impact of clinical pathways on the practice of head and neck oncologic surgery in an academic center. DESIGN: Cross-sectional study. SETTING: Cancer treatment center. PATIENTS: The study population consisted of 3 groups of patients who underwent unilateral neck dissection and were treated in the Department of Head and Neck Surgery of the University of Texas M. D. Anderson Cancer Center, Houston. Additional procedures which may have been performed were direct laryngoscopy, rigid esophagoscopy, and/or dental extractions. Ninety-six patients treated during 1993-1994 prior to the implementation of the clinical pathway (historical control group) were compared with 94 patients treated during 1996-1998, 64 who were not (contemporaneous nonpathway group) and 30 who were managed on the clinical pathway (pathway group). Patients from 1995 were excluded since the pathway was in the planning stages then. MAIN OUTCOME MEASURES: Median length of stay; median total costs of care. RESULTS: The median length of hospital stay of the historical control, contemporaneous nonpathway, and pathway groups decreased from 4.0 to 2.0 days (P<.001). The total median costs of care were less in the pathway group as compared with the historical control group ($6,227 and $8,459, respectively, P<.001) and also less in the contemporaneous nonpathway group compared with the historical control group (S6885 and $8,459, respectively, P<.001). Mean and median length of hospital stay and costs were lower in the pathway group as compared with the nonpathway group but not significantly (P = .11 and P = .07, respectively) The contemporaneous nonpathway and pathway groups did not differ in complications or readmissions. CONCLUSIONS: Development and implementation of this clinical pathway played a statistically significant role in decreasing length of hospital stay and total costs of care associated with neck dissection between nonpathway and pathway patients. Thus, a more cost-effective practice environment has resulted for all of our patients.