Discovery of Antimicrobial Phosphonopeptide Natural Products from Bacillus velezensis by Genome Mining.

Phosphonate natural products are renowned for inhibitory activities which underly their development as antibiotics and pesticides. Although most phosphonate natural products have been isolated from Streptomyces, bioinformatic surveys suggest that many other bacterial genera are replete with similar biosynthetic potential. While mining actinobacterial genomes, we encountered a contaminated Mycobacteroides data set which included a biosynthetic gene cluster predicted to produce novel phosphonate compounds. Sequence deconvolution revealed that the contig containing this cluster, as well as many others, belonged to a contaminating Bacillus and is broadly conserved among multiple species, including the epiphyte Bacillus velezensis. Isolation and structure elucidation revealed a new di- and tripeptide composed of l-alanine and a C-terminal l-phosphonoalanine which we name phosphonoalamides E and F. These compounds exhibit broad-spectrum antibacterial activity, including strong inhibition against the agricultural pests responsible for vegetable soft rot (Erwinia rhapontici), onion rot (Pantoea ananatis), and American foulbrood (Paenibacillus larvae). This work expands our knowledge of phosphonate metabolism and underscores the importance of including underexplored microbial taxa in natural product discovery. IMPORTANCE Phosphonate natural products produced by bacteria have been a rich source of clinical antibiotics and commercial pesticides. Here, we describe the discovery of two new phosphonopeptides produced by B. velezensis with antibacterial activity against human and plant pathogens, including those responsible for widespread soft rot in crops and American foulbrood. Our results provide new insight on the natural chemical diversity of phosphonates and suggest that these compounds could be developed as effective antibiotics for use in medicine or agriculture.

Dichotomous effects of cellular expression of STAT3 on tumor growth of HNSCC.

STAT3 signaling has been shown to regulate cellular function and cytokine production in the tumor microenvironment (TME). Within the head and neck squamous cell carcinoma (HNSCC) TME, we previously showed that therapeutic targeting of STAT3 in combination with radiation resulted in improved tumor growth delay. However, given the independent regulatory effects STAT3 has on anti-tumor immunity, we aimed to decipher the effects of individually targeting STAT3 in the cancer cell, regulatory T cells (Tregs), and natural killer (NK) cell compartments in driving tumor growth and resistance to therapy in HNSCCs. We utilized a CRISPR knockout system for genetic deletion of STAT3 within the cancer cell as well as two genetic knockout mouse models, FoxP3-Cre/STAT3 fl and NKp46-Cre/STAT3 fl, for Tregs and NK cell targeting, respectively. Our data revealed differences in development of resistance to treatment with STAT3 CRISPR knockout in the cancer cell, driven by differential recruitment of immune cells. Knockout of STAT3 in Tregs overcomes this resistance and results in Treg reprogramming and recruitment and activation of antigen-presenting cells. In contrast, knockout of STAT3 in the NK cell compartment results in NK cell inactivation and acceleration of tumor growth. These data underscore the complex interplay between the cancer cell and the immune TME and carry significant implications for drug targeting and design of combination approaches in HNSCCs.

Biosynthesis of Argolaphos Illuminates the Unusual Biochemical Origins of Aminomethylphosphonate and Nε-Hydroxyarginine Containing Natural Products.

Phosphonate natural products have a history of successful application in medicine and biotechnology due to their ability to inhibit essential cellular pathways. This has inspired efforts to discover phosphonate natural products by prioritizing microbial strains whose genomes encode uncharacterized biosynthetic gene clusters (BGCs). Thus, success in genome mining is dependent on establishing the fundamental principles underlying the biosynthesis of inhibitory chemical moieties to facilitate accurate prediction of BGCs and the bioactivities of their products. Here, we report the complete biosynthetic pathway for the argolaphos phosphonopeptides. We uncovered the biochemical origins of aminomethylphosphonate (AMPn) and Nε-hydroxyarginine, two noncanonical amino acids integral to the antimicrobial function of argolaphos. Critical to this pathway were dehydrogenase and transaminase enzymes dedicated to the conversion of hydroxymethylphosphonate to AMPn. The interconnected activities of both enzymes provided a solution to overcome unfavorable energetics, empower cofactor regeneration, and mediate intermediate toxicity during these transformations. Sequential ligation of l-arginine and l-valine was afforded by two GCN5-related N-acetyltransferases in a tRNA-dependent manner. AglA was revealed to be an unusual heme-dependent monooxygenase that hydroxylated the Nε position of AMPn-Arg. As the first biochemically characterized member of the YqcI/YcgG protein family, AglA enlightens the potential functions of this elusive group, which remains biochemically distinct from the well-established P450 monooxygenases. The widespread distribution of AMPn and YqcI/YcgG genes among actinobacterial genomes suggests their involvement in diverse metabolic pathways and cellular functions. Our findings illuminate new paradigms in natural product biosynthesis and realize a significant trove of AmPn and Nε-hydroxyarginine natural products that await discovery.

Valinophos Reveals a New Route in Microbial Phosphonate Biosynthesis That Is Broadly Conserved in Nature.

Phosphonate natural products are potent inhibitors of cellular metabolism with an established record of commercialization in medicine and biotechnology. Although genome mining has emerged as an accelerated method for the discovery of new phosphonates, a robust framework of their metabolism is needed to identify the pathways most likely to yield compounds with desired activities. Here we expand our understanding of these natural products by reporting the complete biosynthetic pathway for valinophos, a phosphonopeptide natural product containing the unusual (R)-2,3-dihydroxypropylphosphonate (DHPPA) scaffold. The pathway was defined by several enzymatic transformations and intermediates previously unknown to phosphonate natural products. A dedicated dehydrogenase served as a new phosphoenolpyruvate mutase coupling enzyme. Notably, its reduction of phosphonopyruvate to phosphonolactate defined a new early branchpoint in phosphonate biosynthesis. Functionally interconnected kinase and reductase enzymes catalyzed reactions reminiscent of glycolysis and arginine biosynthesis to produce a transient, but essential, phosphonolactaldehyde intermediate. We demonstrate esterification of l-valine onto DHPPA as a new biochemical activity for ATP-Grasp ligase enzymes. Unexpectedly, a second amino acid ligase then adjoined additional amino acids at the valinyl moiety to produce a suite of DHPPA-dipeptides. The genes for DHPPA biosynthesis were discovered among genomes of bacteria from wide-ranging habitats, suggesting a wealth of unknown compounds that may originate from this core pathway. Our findings establish new biosynthetic principles for natural products and provide definition to unexplored avenues for bioactive phosphonate genome mining.

Exploring feedback-controlled versus open-circuit electrochemical lipolysis in ex vivo and in vivo porcine fat: A feasibility study.

OBJECTIVES: Minimally invasive fat sculpting techniques are becoming more widespread with the development of office-based devices and therapies. Electrochemical lipolysis (ECLL) is a needle-based technology that uses direct current (DC) to electrolyze tissue water creating acid and base in situ. In turn, fat is saponified and adipocyte cell membrane lysis occurs. The electrolysis of water can be accomplished using a simple open-loop circuit (V-ECLL) or by incorporating a feedback control circuit using a potentiostat (P-ECLL). A potentiostat utilizes an operational amplifier with negative feedback to allow users to precisely control voltage at specific electrodes. To date, the variation between the two approaches has not been studied. The aim of this study was to assess current and charge transfer variation and lipolytic effect created by the two approaches in an in vivo porcine model. METHODS: Charge transfer measurements from ex vivo V-ECLL and P-ECLL treated porcine skin and fat were recorded at -1 V P-ECLL, -2 V P-ECLL, -3 V P-ECLL, and -5 V V-ECLL each for 5 min to guide dosimetry parameters for in vivo studies. In follow-up in vivo studies, a sedated female Yorkshire pig was treated with both V-ECLL and P-ECLL across the dorsal surface over a range of dosimetry parameters, including -1.5 V P-ECLL, -2.5 V P-ECLL, -3.5 V P-ECLL, and 5 V V-ECLL each treated for 5 min. Serial biopsies were performed at baseline before treatment, 1, 2, 7, 14, and 28 days after treatment. Tissue was examined using fluorescence microscopy and histology to compare the effects of the two ECLL approaches. RESULTS: Both V-ECLL and P-ECLL treatments induced in-vivo fat necrosis evident by adipocyte membrane lysis, adipocyte denuclearization, and an acute inflammatory response across a 28-day longitudinal study. However, -1.5 V P-ECLL produced a smaller spatial necrotic effect compared to 5 V V-ECLL. In addition, 5 V V-ECLL produced a comparable necrotic effect to that of -2.5 V and -3.5 V P-ECLL. CONCLUSIONS: V-ECLL and P-ECLL at the aforementioned dosimetry parameters both achieved fat necrosis by adipocyte membrane lysis and denuclearization. The -2.5 V and -3.5 V P-ECLL treatments created spatially similar fat necrotic effects when compared to the 5 V V-ECLL treatment. Quantitatively, total charge transfer between dosimetry parameters suggests that -2.5 V P-ECLL and 5 V V-ECLL produce comparable electrochemical reactions. Such findings suggest that a low-voltage closed-loop potentiostat-based system is capable of inducing fat necrosis to a similar extent compared to that of a higher voltage direct current system.

Visualization of ex vivo rabbit olfactory mucosa and foramina with three-dimensional optical coherence tomography.

There is increasing interest in developing a minimally invasive imaging modality to safely evaluate dynamic microscopic changes of the olfactory mucosa and cribriform foramina. Herein, we utilized three-dimensional (3D) optical coherence tomography (OCT) to characterize the ex vivo stratified substructure of olfactory mucosa in rabbits and create 3D reconstructed images of olfactory foramina. Olfactory mucosa and cribriform plates from four New Zealand White rabbits were dissected and imaged using two swept-source OCT systems: (1) 1.3-µm (µm) center wavelength, 100-nm bandwidth, 200-kHz sweep rate, and (2) 1.7-µm center wavelength, 120-nm bandwidth, 90-kHz sweep rate. Volumetric OCT images were compiled to create a 3D reconstruction of the cribriform plate. The ability of OCT to distinguish the olfactory mucosa substructure and foramina was compared to histology. To estimate imaging penetration depth of each system, the first-order exponential decays of depth-resolved intensity were calculated and compared using a paired t-test. Three-dimensional OCT depicted the stratified layered structures within the olfactory mucosa correlating with histology. The epithelium and lamina propria were measured to be 32 µm and 107 µm in 1.3-µm OCT compared to 30 µm and 105 µm in histology. Olfactory foramina were visualized via 3D reconstruction. The 1.7-µm system provided greater depth penetration compared to the 1.3-µm system, allowing for improved foramina visualization. We have shown that OCT can be used to image non-pathologic olfactory mucosa and foramina. Implications for this work include diagnostic and therapeutic potentials for neurorhinological and neurodegenerative diseases.

Promising Implants in Rhinoplasty.

Many dilemmas in rhinoplasty tempt surgeons to use exogenous materials. We have long looked toward implants to decrease operative time, to achieve a more reliable result, or when there is a paucity of autologous material. More than ever, the innovative and highly lucrative field of nasal implantology is developing technologically advanced products. This article looks at some popular nasal implant choices with a look toward what might be on the horizon.

Electrosurgery Turbinate Reduction Revisited: Can Comparable Volumetric Heating be Achieved Without Feedback Control?

BACKGROUND AND OBJECTIVES: Temperature-controlled radiofrequency inferior turbinate ablation (TCRFA) uses a feedback system to control thermal injury and achieve precise volumetric heating to induce specific scar formation. However, it requires costly single-use proprietary consumables. Comparable volumetric tissue heating may be achieved for a fraction of the cost by adjusting the power settings on traditional monopolar electrosurgery devices that use low-cost needle tips. This pre-clinical study aims to determine the optimized power parameters to achieve electrosurgical coagulum volume similar to that of TCRFA. STUDY DESIGN/MATERIALS AND METHODS: An electrosurgery submucosal diathermy (SMD) system (cut mode, 4-32 W, 5-120 seconds) and a temperature-controlled radiofrequency ablation system (standard clinical parameters for treating inferior turbinate hypertrophy) were used to coagulate egg white and chicken breast. Coagulum major and minor axis were measured, and lesion volume was approximated as prolate spheroid. RESULTS: No significant difference in volume was found between the temperature-controlled system and the electrosurgery system at 8 W for 30 seconds, 8 W for 60 seconds, 16 W for 30 seconds, 32 W for 5 seconds, and 32 W for 15 seconds. The time to achieve equivalent lesion size was significantly less in the SMD system when compared to the temperature-controlled system (P < 0.05). CONCLUSION: Electrosurgery handpieces may achieve similar lesion volume effects as the temperature feedback-controlled, single-use handpieces when set to the optimized parameters. SMD handpieces are significantly more cost and time effective than proprietary devices, and they are easily used in the office. SMD devices may be a more affordable alternative to temperature-controlled systems with comparable lesion volume effect and may be valuable for office-based therapy. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Myeloid-Specific Gene Deletion of Protein Phosphatase 2A Magnifies MyD88- and TRIF-Dependent Inflammation following Endotoxin Challenge.

Protein phosphatase 2A (PP2A) is a member of the intracellular serine/threonine phosphatases. Innate immune cell activation triggered by pathogen-associated molecular patterns is mediated by various protein kinases, and PP2A plays a counter-regulatory role by deactivating these kinases. In this study, we generated a conditional knockout of the α isoform of the catalytic subunit of PP2A (PP2ACα). After crossing with myeloid-specific cre-expressing mice, effective gene knockout was achieved in various myeloid cells. The myeloid-specific knockout mice (lyM-PP2Afl/fl) showed higher mortality in response to endotoxin challenge and bacterial infection. Upon LPS challenge, serum levels of TNF-α, KC, IL-6, and IL-10 were significantly increased in lyM-PP2Afl/fl mice, and increased phosphorylation was observed in MAPK pathways (p38, ERK, JNK) and the NF-κB pathway (IKKα/ß, NF-κB p65) in bone marrow-derived macrophages (BMDMs) from knockout mice. Heightened NF-κB activation was not associated with degradation of IκBα; instead, enhanced phosphorylation of the NF-κB p65 subunit and p38 phosphorylation-mediated TNF-α mRNA stabilization appear to contribute to the increased TNF-α expression. In addition, increased IL-10 expression appears to be due to PP2ACα-knockout-induced IKKα/ß hyperactivation. Microarray experiments indicated that the Toll/IL-1R domain-containing adaptor inducing IFN-ß/ TNFR-associated factor 3 pathway was highly upregulated in LPS-treated PP2ACα-knockout BMDMs, and knockout BMDMs had elevated IFN-α/ß production compared with control BMDMs. Serum IFN-ß levels from PP2ACα-knockout mice treated with LPS were also greater than those in controls. Thus, we demonstrate that PP2A plays an important role in regulating inflammation and survival in the setting of septic insult by targeting MyD88- and Toll/IL-1R domain-containing adaptor inducing IFN-ß-dependent pathways.

Computational analysis of six optical coherence tomography systems for vocal fold imaging: A comparison study.

OBJECTIVES: There have been many advancements in laryngeal imaging using optical coherence tomography (OCT), with varying system design and probes for use in research, office, and operating room settings. We evaluated the performance of six distinct OCT systems in imaging porcine vocal folds (cords) using computational image processing and segmentation. METHODS: Porcine vocal folds were scanned using six OCT systems. Imaging system and probe performance were quantitatively assessed for signal penetration, layer differentiation, and epithelium (EP) measurement. Fitted exponential decay curves with corresponding α constant and intensity thresholding segmentation were utilized to quantify the aforementioned parameters. RESULTS: The smallest average α constant and deepest signal penetration was of the SS-OCT 1700 nm 90 kHz microscope system (α = -1.74), followed by the SS-OCT 1310 nm 200 kHz VCSEL microscope system (α = -1.99), and SS-OCT 1310 nm 50 kHz rigid forward viewing endoscope system (α = -2.23). The EP was not readily visualized for three out of six systems, but was detected using automated segmentation. Average EP thickness (mean ± SD) was calculated as 55.79 ± 31.86 µm which agrees favorably with previous literature. CONCLUSION: Comparisons of OCT systems are challenging, as they encompass different probe design, optical path, and lasers, depending on application. Practical evaluation of different systems using computer based quantitative image processing and segmentation revealed basic, constructive information, such as EP measurements. To further validate the comparisons of system performance with clinical usability, in vivo human laryngeal imaging will be conducted. Further development of automated image processing and segmentation can be useful in rapid analysis of information. Lasers Surg. Med. 51:412-422, 2019. © 2019 Wiley Periodicals, Inc.

Voiding Trial in Office after Unsuccessful Voiding Trial in Postoperative Unit: How Many More Days Is Enough?

STUDY OBJECTIVE: To determine the incidence of a successful in-office voiding trial after minimally invasive hysterectomy. DESIGN: A retrospective cohort study. SETTING: A tertiary care academic center. PATIENTS: All patients undergoing minimally invasive hysterectomies (vaginal, laparoscopic, or robotic) from January 2013 to July 2018 who have an unsuccessful voiding trial in the postoperative unit. INTERVENTIONS: A voiding trial. MEASUREMENTS AND MAIN RESULTS: Of 558 outpatient hysterectomies (with same-day discharge) performed in the time period of interest, 174 patients were discharged home with a Foley catheter (31%). Of those patients, 37% presented for a repeat in-office voiding trial at less than 3 postoperative days, 31% presented at 3 postoperative days, and 31.6% presented at more than 3 postoperative days. Eighty-six percent of patients presenting for their first in-office voiding trial were successful at voiding. There were no differences noted in age, gravity, parity, the use of hormone replacement therapy, menopausal status, smoking, hypertension, or diabetes in patients who passed their first in-office voiding trial compared with those who did not. Vaginal hysterectomy was the most common route of hysterectomy and was performed in 57% of cases followed by robotic hysterectomy in 32%. There were no differences noted in the indication or route of hysterectomy between patients who pass or fail their first in-office voiding trial. At the time of hysterectomy, 47% of patients had concomitant female pelvic medicine and reconstructive surgery procedures performed (midurethral sling, periurethral injections, or colporrhaphy). The incidence of urinary tract infections in this patient cohort was 12%, but the incidence was significantly higher in patients who failed compared with those who passed their first in-office voiding trial (37.3% vs 7.3%, p <.001). After adjusting for age, hysterectomy route, and concomitant female pelvic medicine and reconstructive surgery procedures performed, the number of postoperative days at the time of the first in-office voiding trial does not predict success. CONCLUSION: The timing of the repeat in-office voiding trial in posthysterectomy patients who fail their initial voiding trial in the postanesthesia care unit was not related to success. The incidence of urinary tract infections is higher in patients who fail their repeat voiding trial because recatheterization and a longer duration of catherization are necessary.

Photobiomodulation promotes adenoviral gene transduction in auditory cells.

Gene therapy is the delivery of a therapeutic gene into target cells to treat disorders by replacing disease-causing mutated genes with healthy ones. Gene therapy of the inner ear has been recently described, with applications for sensorineural hearing loss. However, gene delivery to the location of the inner ear, and thus efficacy of therapy, is challenging. Photobiomodulation (PBM) with a low-level laser has been suggested to have a therapeutic effect and has the potential to augment gene therapy. To investigate whether PBM improves the rate of adenovirus (Ad)-mediated viral delivery, we compared low-level laser therapy (LLLT) and non-LLLT HEI-OC1 cells treated with an Ad viral vector carrying green fluorescent protein (GFP). Cultured HEI-OC1 cells were divided into six groups: no treatment control, LLLT only, 1 µL Ad-GFP, 3 µL Ad-GFP, 1 µL Ad-GFP + LLLT, and 3 µL Ad-GFP + LLLT (LLLT: 808 nm at 15 mW for 15 min). Cells were irradiated twice: at 2 h and again at 24 h. A nonparametric Mann-Whitney U test was used to statistically analyze differences between the control and treatment groups. The viral inoculations used in this study did not change the amount of viable HEI-OC1 cells (N = 4-8). The 1 µL Ad-GFP + LLLT and 3 µL Ad-GFP + LLLT groups showed an increased density of GFP-positive cells compared to 1 µL and 3 µL Ad-GFP cells (N = 5-8, 1 µL: p = 0.0159; 3 µL: p = 0.0168,). The quantitative analysis of the epifluorescence of the 1 µL Ad-GFP + LLLT, and 3 µL Ad-GFP + LLLT groups revealed increased GFP expression/cell compared to 1 µL and 3 µL Ad-GFP cells (N = 6-15, 1 µL: p = 0.0082; 3 µL: p = 0.0012). The RT-qPCR results were consistent (N = 4-5, p = 0.0159). These findings suggest that PBM may enhance the gene delivery of Ad-mediated viral transduction, and the combination of the two may be a promising tool for gene therapy for sensorineural hearing loss.

Ent-homocyclopiamine B, a Prenylated Indole Alkaloid of Biogenetic Interest from the Endophytic Fungus Penicillium concentricum.

Ent-homocyclopiamine B (1), a new prenylated indole alkaloid bearing an alicyclic nitro group along with 2-methylbutane-1,2,4-triol (2) were isolated from an endophytic fungus Penicillium concentricum of the liverwort Trichocolea tomentella (Trichocoleaceae). The structure of 1 was elucidated through extensive spectroscopic analyses and comparison with data reported for a structurally related nitro-bearing Penicillium metabolite, clopiamine C (3), which contain an indolizidine ring instead of the quinolizine ring in 1. The new compound, ent-homocyclopiamine B, exhibited slight growth inhibition against Gram-positive bacteria. Based on the reported biosynthesis of related compounds and the isolation of the mevalonic acid derived compound 2-methyl-1,2,4-butanetriol (2), we proposed that ent-homocylopiamine B (1) was biosynthesized from lysine and prenyl group-producing mevalonic pathway.

The human kindness curriculum: An innovative preclinical initiative to highlight kindness and empathy in medicine.

Background: Prior studies have shown a marked drop in empathy among students during their third (clinical) year of medical school. Curricula developed to address this problem have varied greatly in content and have not always been subjected to validated measures of impact. Methods: In 2015, we initiated a Human Kindness (HK) curriculum for the initial 2 years of medical school. This mandatory 12-h curriculum (6 h/year) included an innovative series of lectures and patient interactions with regard to compassion and empathy in the clinical setting. Both quantitative (Jefferson Scale of Empathy [JSE]) and qualitative data were collected prospectively to evaluate the impact of the HK curriculum. Results: In the initial Pilot Year, neither 1st (Group 1) nor 2nd (Group 2) year medical students showed pre-post changes in JSE scores. Substantial changes were made to the curriculum based on faculty and student evaluations. In the following Implementation Year, both the new 1st (Group 3) and the now 2nd year (Group 4) students, who previously experienced the Pilot Year, showed significant improvements in post-course JSE scores; this improvement remained valid across subanalyses of gender, age, and student career focus (e.g., internal medicine, surgery, etc.). Despite the disappointingly flat initial Pilot Year JSE scores, the 3rd year students (Group 2) who experienced only the Pilot Year of the curriculum (i.e., 2nd year students at the time of the Pilot Year) had subsequent JSE scores that did not show the typical decline associated with the clinical years. Students generally evaluated the HK curriculum positively and rated it as being important to their medical education and development as a physician. Discussion: A required preclinical curriculum focused on HK resulted in significant improvements in medical student empathy; this improvement was maintained during the 1st clinical year of training.

mTOR and ROS regulation by anethole on adipogenic differentiation in human mesenchymal stem cells.

BACKGROUND: Adipocyte differentiation of human mesenchymal stem cells (hMSCs) is dependent on mitochondrial metabolism and reactive oxygen species (ROS) to initiate adipocyte differentiation. Although anethole has been known as an anti-oxidant and lipid peroxidation inhibitor, there is little investigated about its role in adipogenic differentiation. METHODS: The effects on cytotoxicity and proliferation of anethole in hMSCs were measured by the MTT assay. The anti-adipogenic effect of anethole on hMSCs was analyzed by Oil Red O staining and western blot analysis. The anti-oxidant activity of anethole on hMSC was assessed by flowcytometry and fluorescence staining using 2',7' -dichlorofluorescin diacetate (DCFDA). The western blotting was used to detect of phospho-Akt, phospho-mTOR, phospho-p70S6K, PPARγ, and phsopho-AMP-activated kinase (AMPK). RESULTS: Anethole suppressed the adipogenic differentiation of hMSCs through down-regulation of Akt-mTOR-p70S6K-PPARγ and up-regulation of AMPK. Anethole affected oxidative conditions through ROS generation. Anethole also rescued AMPK activity and reduced activation of mTOR-p70S6K-PPARγ under oxidative conditions in presence of exogenous hydrogen peroxide. CONCLUSION: ROS and mTOR regulation is a crucial factor in adipogenic differentiation, anethole has an important role in regulating activities of mTOR/PPARγ and ROS control in adipogenic differentiation of hMSCs.

Human Asymptomatic Epitopes Identified from the Herpes Simplex Virus Tegument Protein VP13/14 (UL47) Preferentially Recall Polyfunctional Effector Memory CD44high CD62Llow CD8+ TEM Cells and Protect Humanized HLA-A*02:01 Transgenic Mice against Ocular Herpesvirus Infection.

Herpes simplex virus 1 (HSV-1) infection is widespread among humans. The HSV-1 virion protein 13/14 (VP13/14), also known as UL47, is a tegument antigen targeted by CD8+ T cells from HSV-seropositive individuals. However, whether VP13/14-specific CD8+ T cells play a role in the natural protection seen in asymptomatic (ASYMP) individuals (individuals who have never had a clinical herpetic disease) has not been elucidated. Using predictive computer-assisted algorithms, we identified 10 potential HLA-A*02:01-restricted CD8+ T-cell epitopes from the 693-amino-acid sequence of the VP13/14 protein. Three out of 10 epitopes exhibited a high to moderate affinity of binding to soluble HLA-A*02:01 molecules. The phenotype and function of CD8+ T cells specific for each epitope were compared in HLA-A*02:01-positive ASYMP individuals and symptomatic (SYMP) individuals (individuals who have frequent clinical herpetic diseases) using determination of a combination of tetramer frequency and the levels of granzyme B, granzyme K, perforin, gamma interferon, tumor necrosis factor alpha, and interleukin-2 production and CD107a/b cytotoxic degranulation. High frequencies of multifunctional CD8+ T cells directed against three epitopes, VP13/14 from amino acids 286 to 294 (VP13/14286-294), VP13/14 from amino acids 504 to 512 (VP13/14504-512), and VP13/14 from amino acids 544 to 552 (VP13/14544-552), were detected in ASYMP individuals, while only low frequencies were detected in SYMP individuals. The three epitopes also predominantly recalled more CD45RAlow CD44high CCR7low CD62Llow CD8+ effector memory T cells (TEM cells) in ASYMP individuals than SYMP individuals. Moreover, immunization of HLA-A*02:01 transgenic mice with the three CD8+ TEM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8+ TEM cells associated with strong protective immunity against ocular herpesvirus infection and disease. Our findings outline the phenotypic and functional features of protective HSV-specific CD8+ T cells that should guide the development of a safe and effective T-cell-based herpes simplex vaccine. IMPORTANCE: Although most herpes simplex virus 1 (HSV-1)-infected individuals shed the virus in their body fluids following reactivation from latently infected sensory ganglia, the majority never develop a recurrent herpetic disease and remain asymptomatic (ASYMP). In contrast, small proportions of individuals are symptomatic (SYMP) and develop frequent bouts of recurrent disease. The present study demonstrates that naturally protected ASYMP individuals have a higher frequency of effector memory CD8+ T cells (CD8+ TEM cells) specific to three epitopes derived from the HSV-1 tegument protein VP13/14 (VP13/14286-294,VP13/14504-512, and VP13/14544-552) than SYMP patients. Moreover, immunization of humanized HLA-A*02:01 transgenic mice with the three CD8+ TEM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8+ T cells associated with strong protective immunity against ocular herpesvirus infection and disease. The findings support the emerging concept of the development of a safe and effective asymptomatic herpes simplex vaccine that is selectively based on CD8+ T-cell epitopes from ASYMP individuals.

A Macrocyclic Chelator That Selectively Binds Ln4+ over Ln3+ by a Factor of 1029.

A tetravalent cerium macrocyclic complex (CeLK4) was prepared with an octadentate terephthalamide ligand comprised of hard catecholate donors and characterized in the solution state by spectrophotometric titrations and electrochemistry and in the crystal by X-ray diffraction. The solution-state studies showed that L exhibits a remarkably high affinity toward Ce4+, with log ß110 = 61(2) and ΔG = -348 kJ/mol, compared with log ß110 = 32.02(2) for the analogous Pr3+ complex. In addition, L exhibits an unusual preference for forming CeL4- relative to formation of the analogous actinide complex, ThL4-, which has ß110 = 53.7(5). The extreme stabilization of tetravalent cerium relative to its trivalent state is also evidenced by the shift of 1.91 V in the redox potential of the Ce3+/Ce4+ couple of the complex (measured at -0.454 V vs SHE). The unprecedented behavior prompted an electronic structure analysis using L3- and M5,4-edge X-ray absorption near-edge structure (XANES) spectroscopies and configuration interaction calculations, which showed that 4f-orbital bonding in CeLK4 has partial covalent character due to ligand-to-metal charge transfer (LMCT) in the ground state. The experimental results are presented in the context of earlier measurements on tetravalent cerium compounds, indicating that the amount of LMCT for CeLK4 is similar to that observed for [Et4N]2[CeCl6] and CeO2 and significantly less than that for the organometallic sandwich compound cerocene, (C8H8)2Ce. A simple model to rationalize changes in 4f orbital bonding for tri- and tetravalent lanthanide and actinide compounds is also provided.

Distinct pathways of humoral and cellular immunity induced with the mucosal administration of a nanoemulsion adjuvant.

Nasal administration of an oil-in-water nanoemulsion (NE) adjuvant W805EC produces potent systemic and mucosal, Th-1- and Th-17-balanced cellular responses. However, its molecular mechanism of action has not been fully characterized and is of particular interest because NE does not contain specific ligands for innate immune receptors. In these studies, we demonstrate that W805EC NE adjuvant activates innate immunity, induces specific gene transcription, and modulates NF-κB activity via TLR2 and TLR4 by a mechanism that appears to be distinct from typical TLR agonists. Nasal immunization with NE-based vaccine showed that the TLR2, TLR4, and MyD88 pathways and IL-12 and IL-12Rß1 expression are not required for an Ab response, but they are essential for the induction of balanced Th-1 polarization and Th-17 cellular immunity. NE adjuvant induces MHC class II, CD80, and CD86 costimulatory molecule expression and dendritic cell maturation. Further, upon immunization with NE, adjuvant mice deficient in the CD86 receptor had normal Ab responses but significantly reduced Th-1 cellular responses, whereas animals deficient in both CD80 and CD86 or lacking CD40 failed to produce either humoral or cellular immunity. Overall, our data show that intranasal administration of Ag with NE induces TLR2 and TLR4 activation along with a MyD88-independent Ab response and a MyD88-dependent Th-1 and Th-17 cell-mediated immune response. These findings suggest that the unique properties of NE adjuvant may offer novel opportunities for understanding previously unrecognized mechanisms of immune activation important for generating effective mucosal and systemic immune responses.

Variability of Uncrossmatched Blood Use by Helicopter EMS Programs in the United States.

STUDY OBJECTIVE: Some helicopter emergency medical services (HEMS) maintain an independent supply of blood for use during transport, although practice is variable and not well described. We aimed to characterize the blood-carrying practices by HEMS programs across the United States. METHODS: Online surveys were sent to the leadership of the 261 HEMS programs nationwide listed in the 2011 Atlas and Database of Air Medical Services (ADAMS) database. We examined blood-carrying practices in aggregate, including typical transport time, proportion of scene transports, and local population density. A GIS (Geographic Information System) and multivariable logistic regression models were used to estimate the impact of characteristics of local practice on each program's decision to carry blood. RESULTS: A total of 235 (91%) programs responded to the survey, representing 857 of the 929 (92.2%) HEMS rotor wing aircraft nationwide. Fifty-nine (25.3%) programs independently carried blood. A higher proportion of interfacility transports (OR 1.023; 95% CI 1.010-1.036) and decreased local population density (OR 1.006; 95% CI 1.001-1.011) were associated with increased odds of carrying blood. Transport time (OR 1.006; 95% CI 0.991-1.020) and number of transports (OR 1.000; CI 1.000-1.000) were not associated with a program's blood carrying practices. There was no effect of local practices on a program's decision to carry blood (OR 1.002; 95% CI 0.980-1.026). CONCLUSION: There is great variability in the utilization of blood by HEMS programs in the United States. Programs that serve more rural areas and programs with a larger percentage of interfacility transports are more likely to independently carry blood.