Characterization of two Vernier-Tuned Distributed Bragg Reflector (VT-DBR) Lasers used in Swept Source Optical Coherence Tomography (SS-OCT)

Insight Photonic Solutions Inc. has continued to develop their patented VT-DBR laser design; these wavelength tunable lasers promise marked image-quality and acquisition time improvements in SS-OCT applications. To be well suited for SS-OCT, tunable lasers must be capable of producing a highly linear wavelength sweep across a tuning range well-matched to the medium being imaged; many different tunable lasers used in SS-OCT are compared to identify the optimal solution. This work electrically and spectrally characterizes two completely new all-semiconductor VT-DBR designs to compare, as well. The Neptune VT-DBR, an O-band laser, operates around the 1310 nm range and is a robust solution for many OCT applications. The VTL-2 is the first 1060 nm VT-DBR laser to be demonstrated. It offers improved penetration through water over earlier designs which operate at longer wavelengths (e.g. - 1550 nm and 1310 nm), making it an optimal solution for the relatively deep imaging requirements of the human eye; the non-invasive nature of OCT makes it the ideal imaging technology for ophthalmology. Each laser has five semiconductor P-N junction segments that collectively enable precise akinetic wavelength-tuning (i.e. - the tuning mechanism has no moving parts). In an SS-OCT system utilizing one of these laser packages, the segments are synchronously driven with high speed current signals that achieve the desired wavelength, power, and sweep pattern of the optical output. To validate the laser’s fast tuning response time necessary for its use in SS-OCT, a circuit model of each tuning section is created; each laser section is modeled as a diode with a significant lead inductance. The dynamic resistance, effective capacitance, and lead inductance of this model are measured as a function of bias current and the response time corresponding to each bias condition is determined. Tuning maps, spectral linewidths, and side-mode suppression ratio (SMSR) measurements important to SS-OCT performance are also collected. Measured response times vary from 700 ps to 2 ns for the Neptune and 1.2 to 2.3 ns for the VTL-2. Linewidth measurements range from 9 MHz to 124 MHz for the Neptune and 300 kHz to 2 MHz for the VTL-2. SMSR measurements greater than 38 dB and 40 dB were observed for the Neptune and VTL-2, respectively. Collectively, these results implicate the VT-DBR lasers as ideal tunable sources for use in SS-OCT applications

Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1

Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr83 and Glu73, respectively. When Glu73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu73 residue

Can Implicit Measures Augment Suicide Detection in Youth? The Feasibility and Acceptability of the Death Implicit Association Test Among Pediatric Medical Impatients

Background: Medically ill youth are at increased suicide risk, necessitating early detection. This study aimed to assess the feasibility of administering the Death Implicit Association Test (Death IAT) to pediatric medical inpatients. Methods: Participants completed measures including the Ask Suicide-Screening Questions (ASQ) and the Death IAT. Results: Over 90% of participants found the Death IAT to be acceptable and more than 75% of participants were comfortable completing the task. There was a small, but statistically significant, improvement from pre-survey to post-survey reports of mood (t(174) = 3.02, p = 0.003, d = 0.15). Participants who endorsed a past suicide attempt on the ASQ had significantly higher “suicide” trial D-scores than those without a past suicide attempt (Wilcoxon W = 1312; p = 0.048; d = 0.61). Conclusions: Implementing an IAT measure among pediatric medical inpatients was feasible and acceptable. In exploratory analyses, “suicide” trial IAT D-scores were associated with past suicide attempts, suggesting future studies should examine whether implicit measures may be useful in hospital settings to augment detection of youth suicide risk

Turnover intention and job tenure of US fundraisers

Fundraisers secure financial resources that organizations need to achieve their missions. Raising money, particularly large gifts, can follow years of relationship building with individual donors. When fundraisers leave these efforts can be set back substantially, making fundraiser turnover particularly worrisome and worthy of exploration. This analysis addressed the issue with US survey data (n = 1663) and examinination of three research questions. What are the job tenure and intent to leave of fundraisers? How is fundraiser job tenure affected by intent to leave? What relationships do job tenure and intent to leave have with fundraisers' individual demographics, position attributes, and organizational characteristics? We found that the study participants had current mean job tenures of 3.6 years (median = 2 years) and mean tenures across their fundraising jobs of 3.9 years (median = 3 years). Twenty percent intended to leave their organization and 7% intended to leave fundraising within the next year. Of the tested variables, salary consistently had the largest effects and was the most significant. Older and more experienced fundraisers had longer tenures. The study provides nuanced information about fundraisers' job-related behaviors, includes careful attention to theory and related research, and presents specific ideas for organizational interventions for increasing fundraiser tenure

Structural and Functional Analysis of Phytotoxin Toxoflavin-Degrading Enzyme

Pathogenic bacteria synthesize and secrete toxic low molecular weight compounds as virulence factors. These microbial toxins play essential roles in the pathogenicity of bacteria in various hosts, and are emerging as targets for antivirulence strategies. Toxoflavin, a phytotoxin produced by Burkholderia glumae BGR1, has been known to be the key factor in rice grain rot and wilt in many field crops. Recently, toxoflavin-degrading enzyme (TxDE) was identified from Paenibacillus polymyxa JH2, thereby providing a possible antivirulence strategy for toxoflavin-mediated plant diseases. Here, we report the crystal structure of TxDE in the substrate-free form and in complex with toxoflavin, along with the results of a functional analysis. The overall structure of TxDE is similar to those of the vicinal oxygen chelate superfamily of metalloenzymes, despite the lack of apparent sequence identity. The active site is located at the end of the hydrophobic channel, 9 Å in length, and contains a Mn(II) ion interacting with one histidine residue, two glutamate residues, and three water molecules in an octahedral coordination. In the complex, toxoflavin binds in the hydrophobic active site, specifically the Mn(II)-coordination shell by replacing a ligating water molecule. A functional analysis indicated that TxDE catalyzes the degradation of toxoflavin in a manner dependent on oxygen, Mn(II), and the reducing agent dithiothreitol. These results provide the structural features of TxDE and the early events in catalysis

Persistence survey of Toxic Shock Syndrome toxin-1 producing Staphylococcus aureus and serum antibodies to this superantigen in five groups of menstruating women

Background: Menstrual Toxic Shock Syndrome (mTSS) is thought to be associated with the vaginal colonization with specific strains of Staphylococcus aureus TSST-1 in women who lack sufficient antibody titers to this toxin. There are no published studies that examine the seroconversion in women with various colonization patterns of this organism. Thus, the aim of this study was to evaluate the persistence of Staphylococcus aureus colonization at three body sites (vagina, nares, and anus) and serum antibody to toxic shock syndrome toxin-producing Staphylococcus aureus among a small group of healthy, menstruating women evaluated previously in a larger study. Methods: One year after the completion of that study, 311 subjects were recalled into 5 groups. Four samples were obtained from each participant at several visits over an additional 6-11 month period: 1) an anterior nares swab; 2) an anal swab; 3) a vagina swab; and 4) a blood sample. Gram stain, a catalase test, and a rapid S. aureus-specific latex agglutination test were performed to phenotypically identify S. aureus from sample swabs. A competitive ELISA was used to quantify TSST-1 production. Human TSST-1 IgG antibodies were determined from the blood samples using a sandwich ELISA method. Results: We found only 41% of toxigenic S. aureus and 35.5% of non-toxigenic nasal carriage could be classified as persistent. None of the toxigenic S. aureus vaginal or anal carriage could be classified as persistent. Despite the low persistence of S. aureus colonization, subjects colonized with a toxigenic strain were found to display distributions of antibody titers skewed toward higher titers than other subjects. Seven percent (5/75) of subjects became seropositive during recall, but none experienced toxic shock syndrome-like symptoms. Conclusions: Nasal carriage of S. aureus appears to be persistent and the best predicator of subsequent colonization, whereas vaginal and anal carriage appear to be more transient. From these findings, it appears that antibody titers in women found to be colonized with toxigenic S. aureus remained skewed toward higher titers whether or not the colonies were found to be persistent or transient in nature. This suggests that colonization at some point in time is sufficient to elevate antibody titer levels and those levels appear to be persistent. Results also indicate that women can become seropositive without experiencing signs or symptoms of toxic shock syndrome

Structural Insights into Viral Determinants of Nematode Mediated Grapevine fanleaf virus Transmission

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector

NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D Domain Swapping

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges