Effects of isoleucine, lysine, valine, and a group of non-essential amino acids on mammary amino acid metabolism in lactating dairy cows.

Intracellular amino acids (AA) regulate milk protein synthesis within the mammary glands by modifying mammary plasma flow (MPF) and AA transporter activity. Amino acid transporters catalyze translocation using Na+-gradient, substrate gradient (uniporters), and exchange mechanisms; further, they exhibit specificity for individual AA or groups of AA with similar side-chain properties within each transport system. Non-essential AA are actively transported through Na+-dependent transporters and, thus, are often utilized as intracellular currencies for EAA transport through exchange transporters. Therefore, it was hypothesized that individual EAA supplementation would compete with other EAA for shared transporters, and supplementation with Ala, Gln, and Gly would stimulate EAA transport through exchange transporters. Ten primiparous lactating dairy cows were divided into 2 groups based on milk production and were randomly assigned to treatment sequences within 2 balanced 5 × 5 Latin Squares by group. Period length was 14 d. Treatments were 9-d jugular infusions of 1) saline; 2) 34.5 g Val/d; 3) 32.7 g Ala/d: 40 g Gln/d: 26.7 g Gly/d (AQG); 4) 43 g Lys/d; or 5) 33.5 g Ile/d. All cows were fed a common base diet formulated to contain 15.0% CP. Ile, Lys, or AQG infusions did not affect milk protein or milk production; however, Val infusion decreased both. The effects of Val infusion on milk protein production appeared to be partially driven by decreased DMI. The decline in milk protein percentage indicated that milk lactose production was also affected. Additionally, Val infusion increased MPF efficiency (MPF/Milk; L/L) by approximately 44%. Val infusion tended to decrease or decreased mammary net uptakes of Lys, Leu, Met, and total AA. Ile infusion tended to increase its mammary net uptakes but did not affect any other AA. Lys and AQG infusions did not affect any mammary net uptakes. Val infusion tended to decrease Phe and total NEAA mammary clearance rates. AQG infusion stimulated Tyr clearance rates and tended to decline System N mammary clearance rates. Mammary uptake to milk protein output ratios (U:O) of BCAA did not differ from 1 for Val-infused cows, which indicated that little intramammary catabolism was occurring. Additionally, the average NEAA U:O in response to all treatments except Val was 0.70, but Val-infused cows had NEAA U:O that averaged 0.09 indicating increased synthesis within the glands. The effects of Val on mammary net clearance rates of multiple EAA support the incorporation of AA limitations in ration optimizers to prevent AA imbalances. It is possible that over-supplementation of EAA other than Val may also decrease DMI and mammary activity. Identifying efficiency apexes for each of the EAA will allow more precise diet formulation and supplementation, leading to improved production efficiency.

A blinded study using laser induced endogenous fluorescence spectroscopy to differentiate ex vivo spine tumor, healthy muscle, and healthy bone.

Ten patients undergoing surgical resection for spinal tumors were selected. Samples of tumor, muscle, and bone were resected, de-identified by the treating surgeon, and then scanned with the TumorID technology ex vivo. This study investigates whether TumorID technology is able to differentiate three different human clinical fresh tissue specimens: spine tumor, normal muscle, and normal bone. The TumorID technology utilizes a 405 nm excitation laser to target endogenous fluorophores, thereby allowing for the detection of tissue based on emission spectra. Metabolic profiles of tumor and healthy tissue vary, namely NADH (bound and free emission peak, respectively: 487 nm, 501 nm) and FAD (emission peak: 544) are endogenous fluorophores with distinct concentrations in tumor and healthy tissue. Emission spectra analyzed consisted of 74 scans of spine tumor, 150 scans of healthy normal bone, and 111 scans of healthy normal muscle. An excitation wavelength of 405 nm was used to obtain emission spectra from tissue as previously described. Emission spectra consisted of approximately 1400 wavelength intensity pairs between 450 and 750 nm. Kruskal-Wallis tests were conducted comparing AUC distributions for each treatment group, α = 0.05. Spectral signatures varied amongst the three different tissue types. All pairwise comparisons among tissues for Free NADH were statistically significant (Tumor vs. Muscle: p = 0.0006, Tumor vs. Bone: p < 0.0001, Bone vs. Muscle: p = 0.0357). The overall comparison of tissues for FAD (506.5-581.5 nm) was also statistically significant (p < 0.0001), with two pairwise comparisons being statistically significant (Tumor vs. Muscle: p < 0.0001, Tumor vs. Bone: p = 0.0045, Bone vs. Muscle: p = 0.249). These statistically significant differences were maintained when stratifying tumor into metastatic carcinoma (N = 57) and meningioma (N = 17). TumorID differentiates tumor tissue from normal bone and normal muscle providing further clinical evidence of its efficacy as a tissue identification tool. Future studies should evaluate TumorID's ability to serve as an adjunctive tool for intraoperative assessment of surgical margins and surgical decision-making.

Valine and nonessential amino acids affect bidirectional transport rates of leucine and isoleucine in bovine mammary epithelial cells.

A more complete understanding of the mechanisms controlling AA transport in mammary glands of dairy cattle will help identify solutions to increase nitrogen feeding efficiency on farms. It was hypothesized that Ala, Gln, and Gly (NEAAG), which are actively transported into cells and exchanged for all branched-chain AA (BCAA), may stimulate transport of BCAA, and that Val may antagonize transport of the other BCAA due to transporter competition. Thus, we evaluated the effects of varying concentrations of NEAAG and Val on transport and metabolism of the BCAA Ala, Met, Phe, and Thr by bovine mammary epithelial cells. Primary cultures of bovine mammary epithelial cells were assigned to treatments of low (70% of mean in vivo plasma concentrations of lactating dairy cows) and high (200%) concentrations of Val and NEAAG (LVal and LNEAAG, HVal and HNEAAG, respectively) in a 2 × 2 factorial design. Cells were preloaded with treatment media containing [15N]-labeled AA for 24 h. The [15N]-labeled media were replaced with treatment media containing [13C]-labeled AA. Media and cells were harvested from plates at 0, 0.5, 1, 5, 15, 30, 60, and 240 min after application of the [13C]-labeled AA and assessed for [15N]- and [13C]-AA label concentrations. The data were used to derive transport, transamination, irreversible loss, and protein-synthesis fluxes. All Val fluxes, except synthesis of rapidly exchanging tissue protein, increased with the HVal treatment. Interestingly, the rapidly exchanging tissue protein, transamination, and irreversible-loss rate constants decreased with HVal, indicating that the significant flux increases were primarily driven by mass action with the cells resisting the flux increases by downregulating activity. However, the decreases could also reflect saturation of processes that would drive down the mass-action rate constants. This is supported by decreases in the same rate constants for Ile and Leu with HVal. This could be due to either competition for shared transamination and oxidation reactions or a reduction in enzymatic activity. Also, NEAAG did not affect Val fluxes, but influx and efflux rate constants increased for both Val and Leu with HNEAAG, indicating an activating substrate effect. Overall, AA transport rates generally responded concordantly with extracellular concentrations, indicating the transporters are not substrate-saturated within the in vivo range. However, BCAA transamination and oxidation enzymes may be approaching saturation within in vivo ranges. In addition, System L transport activity appeared to be stimulated by as much as 75% with high intracellular concentrations of Ala, Gln, and Gly. High concentrations of Val antagonized transport activity of Ile and Leu by 68% and 15%, respectively, indicating competitive inhibition, but this was only observable at HNEAAG concentrations. The exchange transporters of System L transport 8 of the essential AA that make up approximately 40% of milk protein, so better understanding this transporter is an important step for increased efficiency.

Sternectomy Reconstruction Using a Customized Three-Dimensional-Printed Polyetheretherketone Implant.

Complex sternal and chest wall reconstruction can be a challenging clinical situation, with the main objectives being restoration of chest wall rigidity, protection of intrathoracic organs, preservation of respiratory function, and reduction of pain and clicking. The treatment of choice is varied, with several different materials available to aid in adequate reconstruction. We present the case of a 60-year-old male with a post-sternectomy defect and debilitating symptoms who underwent reconstruction with a customized, three-dimensional (3D)-printed polyetheretherketone (PEEK) implant and pectoralis muscle flaps. There were no complications in the perioperative period, and the patient reported significant improvement in pain and subjective improvement in chest stability and respiration. The use of PEEK as a reconstructive material for cardiothoracic defects is a viable and safe method that has several important benefits over other utilized materials in the literature. The early success of this case in relieving patient symptoms opens the door for further exploration of PEEK as an alternative for cardiothoracic reconstruction.

Effects of dietary starch and ruminally undegraded protein on glucogenic precursors in lactating dairy cows.

Gluconeogenesis is a large contributor to the blood supply of glucose carbons. The impact of varying dietary starch and ruminally degraded protein (RDP) on glucose entry, and the contributions of propionate and lactate to total plasma glucose entry were evaluated. Six cannulated, lactating, Holstein cows were fed one of four treatment diets arranged as a 2 × 2 factorial within a 4 × 4 partially replicated Latin Square design: (1) 8% RDP (LRDP) and 16% starch (LSt), (2) LRDP and 30% starch (HSt), (3) 11% RDP (HRDP) and LSt, or (4) HRDP and HSt. On d 12 of each period, 2-[13C]-sodium propionate (0.15 g/h) was ruminally infused for 4 h; on d 13, 1,2-[13C2]-glucose (0.2 g/h) was infused into the jugular vein for 1 h followed by 1-[13C]-lactate (0.1 g/h) for 1 h. Blood samples were serially collected starting prior to the infusions, and analyzed for plasma glucose, propionate, and lactate isotopic ratios. A one-compartment, glucose carbon model with inputs from lactate, propionate, and other glucogenic precursors (Oth, primarily absorbed glucose plus amino acids) was fitted to the isotope ratio data to derive glucose entry rates and conversion of the precursors to glucose. Milk protein production additively increased when HSt and HRDP were fed (P = 0.05 and P = 0.02, respectively). Plasma glucose and propionate concentrations increased with HSt (P = 0.04 and P = 0.01, respectively) and LRDP (P = 0.02 and P < 0.01, respectively). Total glucose and Oth entry increased (P = 0.03 and P = 0.03, respectively) with HSt, indicating greater glucose absorption from the small intestine or conversion of amino acids to glucose in the liver. However, neither entry rate was affected by RDP. The lack of an RDP effect suggests the increase in microbial outflow in response to RDP did not significantly alter glucose precursor supplies. Entry rates of propionate and lactate carbon to glucose carbon were not affected by treatment suggesting that neither starch nor RDP significantly affected fermentation or lactate production. Derivation of absolute entry rates and contributions to glucose using isotopic tracers is complicated by single carbon removals in the pentose phosphate (PPP), tri-carboxylic acid (TCA), and gluconeogenic pathways, and label randomization with the PPP and TCA pathways. Multiple tracers must be used to avoid assumptions regarding the proportional entries. These results provide insights on glucose supply and contributors, and draw attention to significant label cycling when utilizing isotope techniques.

Geospatial Analysis of Prehospital Triage and Early Potential Preventable Traumatic Deaths.

Severely injured patients often depend on prompt prehospital triage for survival. This study aimed to examine the under-triage of preventable or potentially preventable traumatic deaths. A retrospective review of Harris County, TX, revealed 1848 deaths within 24 hours of injury, with 186 being preventable or potentially preventable (P/PP). The analysis evaluated the geospatial relationship between each death and the receiving hospital. Out of the 186 P/PP deaths, these were more commonly male, minority, and penetrating mechanisms when compared with NP deaths. Of the 186 PP/P, 97 patients were transported to hospital care, 35 (36%) were transported to Level III, IV, or non-designated hospitals. Geospatial analysis revealed an association between the location of initial injury and proximity to receiving Level III, IV, and non-designated centers. Geospatial analysis supports proximity to the nearest hospital as one of the primary reasons for under-triage.

Impact of angiotensin system inhibitors on esophageal cancer survival.

INTRODUCTION: Angiotensin system inhibitors are associated with improved prognosis in patients with gastrointestinal and hepatobiliary cancers. Data suggest that renin-angiotensin system signaling stimulates the tumor's immune microenvironment to impact overall survival. The goal of this study is to investigate the role of angiotensin system inhibitor use on the overall survival and disease-free survival of esophageal cancer patients. METHODS: Retrospective review of esophagectomy patients with esophageal adenocarcinoma and squamous cell cancer at a single institution tertiary care center from 2007 to 2018 was performed. Outcomes include overall survival and disease-free survival. Patient characteristics were compared with t test and χ2 test. Survival was analyzed with Kaplan-Meier and Cox proportional-hazards regression. RESULTS: One hundred seventy-one patients were identified and 123 underwent esophagectomy for cancer. No significant differences in patient demographics were found between angiotensin system inhibitor users and non-angiotensin system inhibitor users except for the rates of hypertension (40% vs 94%, P < .01) and diabetes (16% vs 47%, P < .01). Distributions of tumor neoadjuvant therapy, adjuvant therapy, pathology, staging, margins, and surgical approach were similar. Postoperatively, there was no difference in major adverse cardiovascular events or infection rates. This study did not find any differences in overall survival and disease-free survival between angiotensin system inhibitor users and non-angiotensin system inhibitor users. CONCLUSION: Angiotensin system inhibitors have been shown to improve survival and decrease relative risk for several types of cancers; however, our data do not support the same effect on esophageal cancer patients undergoing curative intent surgery. Further research is needed to investigate potential nuances in angiotensin system inhibitor dose, chronicity of use, esophageal pathology, and applicability to nonsurgical candidates.

Overcoming the out-of-plane bending issue in an aromatic hydrocarbon: the anharmonic vibrational frequencies of c-(CH)C3H2.

The challenges associated with the out-of-plane bending problem in multiply-bonded hydrocarbon molecules can be mitigated in quartic force field analyses by varying the step size in the out-of-plane coordinates. Carbon is a highly prevalent element in astronomical and terrestrial environments, but this major piece of its spectra has eluded theoretical examinations for decades. Earlier explanations for this problem focused on method and basis set issues, while this work seeks to corroborate the recent diagnosis as a numerical instability problem related to the generation of the potential energy surface. Explicit anharmonic frequencies for c-(CH)C3H2+ are computed using a quartic force field and the CCSD(T)-F12b method with cc-pVDZ-F12, cc-pVTZ-F12, and aug-cc-pVTZ basis sets. The first of these is shown to offer accuracy comparable to that of the latter two with a substantial reduction in computational time. Additionally, c-(CH)C3H2+ is shown to have two fundamental frequencies at the onset of the interstellar unidentified infrared bands, at 5.134 and 6.088 µm or 1947.9 and 1642.6 cm-1, respectively. This suggests that the results in the present study should assist in the attribution of parts of these aromatic bands, as well as provide data in support of the laboratory or astronomical detection of c-(CH)C3H2+.

Deep long-period earthquakes generated by second boiling beneath Mauna Kea volcano.

Deep long-period earthquakes (DLPs) are an enigmatic type of volcanic seismicity that sometimes precedes eruptions but mostly occurs at quiescent volcanoes. These earthquakes are depleted in high-frequency content and typically occur near the base of the crust. We observed a near-periodic, long-lived sequence of more than one million DLPs in the past 19 years beneath the dormant postshield Mauna Kea volcano in Hawai'i. We argue that this DLP sequence was caused by repeated pressurization of volatiles exsolved through crystallization of cooling magma stalled beneath the crust. This "second boiling" of magma is a well-known process but has not previously been linked to DLP activity. Our observations suggest that, rather than portending eruptions, global DLP activity may more commonly be indicative of stagnant, cooling magma.

Molecular oxygen generation from the reaction of water cations with oxygen atoms.

The oxywater cation (H2OO+), previously shown to form barrierlessly in the gas phase from water cations and atomic oxygen, is proposed here potentially to possess a 2A″ ←4A″ excitation leading to the H2⋯O2 + complex. This complex could then easily decompose into molecular hydrogen and the molecular oxygen cation. The present quantum chemical study shows that the necessary electronic transition takes place in the range of 1.92 eV (645 nm), in the orange-red range of the visible and solar spectrum, and dissociation of the complex only requires 5.8 kcal/mol (0.25 eV). Such a process for the abiotic, gas phase formation of O2 would only need to be photocatalyzed by visible wavelength photons. Hence, such a process could produce O2 at the mesosphere/stratosphere boundary as climate change is driving more water into the upper atmosphere, in the comet 67P/Churyumov-Gerasimenko where surprisingly high levels of O2 have been observed, or at gas-surface (ice) interfaces.

Automating neurosurgical tumor resection surgery: Volumetric laser ablation of cadaveric porcine brain with integrated surface mapping.

OBJECTIVE: Current surgical instruments for soft tissue resection including neurosurgical procedures rely on the accuracy and precision of the human operator and are fundamentally constrained by the human hand. Automated surgical action with the integration of intraoperative data sources can enable highly accurate and fast tissue manipulation using laser ablation. This study presents the first experiments with a prototype designed for automated tumor resection via laser ablation. We demonstrate targeted soft tissue resection in porcine brain with an integrated device that combines 3D scanning capabilities with a steerable surgical laser and discuss implications for future automated robotic neurosurgical procedures. STUDY DESIGN AND METHODS: A device consisting of a two-axis galvanometer for steering a cutting laser and a 3D surface profiler is used to perform volumetric removal of tissue of ex vivo porcine brain. Three-dimensional surface profiles are gathered between cuts and used to estimate ablation rate. RESULTS: Volumetric ablation of porcine brain tissue is performed and subsequently surface profiled. The average ablation rates across the area cutting areas were 2.6 mm3 /s and 3.7 mm3 /s for the initial and subsequent cuts, respectively. A Kruskal-Wallis and post-hoc Tukey test show statistical significance between the initial and subsequent cuts. Accuracy between cuts when benchmarked against a human surgeon varied from 47 to 88%. CONCLUSION: A feed-forward volumetric resection is demonstrated with sensing and cutting housed within a single device, thereby opening the potential for automated soft tissue resection as necessary during the surgical removal of pathologic tissues. High variance around target cut depths motivates future work in developing a closed-loop ablation tool as well as characterization of laser-tissue interactions for predictive modelling. Objective Lasers Surg. 50:1017-1024, 2018. © 2018 Wiley Periodicals, Inc.

Relative seismic velocity variations correlate with deformation at Kilauea volcano.

Seismic noise interferometry allows the continuous and real-time measurement of relative seismic velocity through a volcanic edifice. Because seismic velocity is sensitive to the pressurization state of the system, this method is an exciting new monitoring tool at active volcanoes. Despite the potential of this tool, no studies have yet comprehensively compared velocity to other geophysical observables on a short-term time scale at a volcano over a significant length of time. We use volcanic tremor (~0.3 to 1.0 Hz) at Kilauea as a passive source for interferometry to measure relative velocity changes with time. By cross-correlating the vertical component of day-long seismic records between ~230 station pairs, we extract coherent and temporally consistent coda wave signals with time lags of up to 120 s. Our resulting time series of relative velocity shows a remarkable correlation between relative velocity and the radial tilt record measured at Kilauea summit, consistently correlating on a time scale of days to weeks for almost the entire study period (June 2011 to November 2015). As the summit continually deforms in deflation-inflation events, the velocity decreases and increases, respectively. Modeling of strain at Kilauea suggests that, during inflation of the shallow magma reservoir (1 to 2 km below the surface), most of the edifice is dominated by compression-hence closing cracks and producing faster velocities-and vice versa. The excellent correlation between relative velocity and deformation in this study provides an opportunity to understand better the mechanisms causing seismic velocity changes at volcanoes, and therefore realize the potential of passive interferometry as a monitoring tool.

In vivo endoscopic Doppler optical coherence tomography imaging of the colon.

BACKGROUND AND OBJECTIVE: Colorectal cancer (CRC) remains the second deadliest cancer in the United States. Several screening methods exist; however, detection of small polyps remains a challenge. Optical coherence tomography (OCT) has been demonstrated to be capable of detecting lesions as small as 1 mm in the mouse colon, but detection is based on measuring a doubling of the mucosa thickness. The colon microvasculature may be an attractive biomarker of early tumor development because tumor vessels are characterized by irregular structure and dysfunction. Our goal was to develop an endoscopic method of detecting and segmenting colon vessels using Doppler OCT to enable future studies for improving early detection and development of novel chemopreventive agents. METHOD: We conducted in vivo colon imaging in an azoxymethane (AOM)-treated mouse model of colorectal cancer using a miniature endoscope and a swept-source OCT system at 1,040 nm with a 16 kHz sweep rate. We applied the Kasai autocorrelation algorithm to laterally oversampled OCT B-scans to resolve vascular flow in the mucosa and submucosa. Vessels were segmented by applying a series of image processing steps: (i) intensity thresholding; (ii) two-dimensional matched filtering; and (iii) histogram segmentation. RESULTS: We observed differences in the vessels sizes and spatial distribution in a mature adenoma compared to surrounding undiseased tissue and compared the results with histology. We also imaged flow in four young mice (two AOM-treated and two control) showing no significant differences, which is expected so early after carcinogen exposure. We also present flow images of adenoma in a living mouse and a euthanized mouse to demonstrate that no flow is detected after euthanasia. CONCLUSION: We present, to the best of our knowledge, the first Doppler OCT images of in vivo mouse colon collected with a fiber-based endoscope. We also describe a fast and robust image processing method for segmenting vessels in the colon. These results suggest that Doppler OCT is a promising imaging modality for vascular imaging in the colon that requires no exogenous contrast agents. Lasers Surg. Med. 49:249-257, 2017. © 2016 Wiley Periodicals, Inc.

Reversible lysine-specific demethylase 1 antagonist HCI-2509 inhibits growth and decreases c-MYC in castration- and docetaxel-resistant prostate cancer cells.

BACKGROUND: Lysine-specific demethylase 1 (LSD1 or KDM1A) overexpression correlates with poor survival and castration resistance in prostate cancer. LSD1 is a coregulator of ligand-independent androgen receptor signaling promoting c-MYC expression. We examined the antitumor efficacy of LSD1 inhibition with HCI-2509 in advanced stages of prostate cancer. METHODS: Cell survival, colony formation, histone methylation, c-MYC level, c-MYC expression, cell cycle changes and in vivo efficacy were studied in castration-resistant prostate cancer cells upon treatment with HCI-2509. In vitro combination studies, using HCI-2509 and docetaxel, were performed to assess the synergy. Cell survival, colony formation, histone methylation and c-myc levels were studied in docetaxel-resistant prostate cancer cells treated with HCI-2509. RESULTS: HCI-2509 is cytotoxic and inhibits colony formation in castration-resistant prostate cancer cells. HCI-2509 treatment causes a dose-dependent increase in H3K9me2 (histone H3lysine 9) levels, a decrease in c-MYC protein, inhibition of c-MYC expression and accumulation in the G0/G1 phase of the cell cycle in these cells. PC3 xenografts in mice have a significant reduction in tumor burden upon treatment with HCI-2509 with no associated myelotoxicity or weight loss. More synergy is noted at sub-IC50 (half-maximal inhibitory concentration) doses of docetaxel and HCI-2509 in PC3 cells than in DU145 cells. HCI-2509 has growth-inhibitory efficacy and decreases the c-myc level in docetaxel-resistant prostate cancer cells. CONCLUSIONS: LSD1 inhibition with HCI-2509 decreases the c-MYC level in poorly differentiated prostate cancer cell lines and has a therapeutic potential in castration- and docetaxel-resistant prostate cancer.

Mutation Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Reduces Low-Sensitivity α4ß2, and Increases α5α4ß2, Nicotinic Receptor Surface Expression.

A number of mutations in α4ß2-containing (α4ß2*) nicotinic acetylcholine (ACh) receptors (nAChRs) are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), including one in the ß2 subunit called ß2V287L. Two α4ß2* subtypes with different subunit stoichiometries and ACh sensitivities co-exist in the brain, a high-sensitivity subtype with (α4)2(ß2)3 subunit stoichiometry and a low-sensitivity subtype with (α4)3(ß2)2 stoichiometry. The α5 nicotinic subunit also co-assembles with α4ß2 to form a high-sensitivity α5α4ß2 nAChR. Previous studies suggest that the ß2V287L mutation suppresses low-sensitivity α4ß2* nAChR expression in a knock-in mouse model and also that α5 co-expression improves the surface expression of ADNFLE mutant nAChRs in a cell line. To test these hypotheses further, we expressed mutant and wild-type (WT) nAChRs in oocytes and mammalian cell lines, and measured the effects of the ß2V287L mutation on surface receptor expression and the ACh response using electrophysiology, a voltage-sensitive fluorescent dye, and superecliptic pHluorin (SEP). The ß2V287L mutation reduced the EC50 values of high- and low-sensitivity α4ß2 nAChRs expressed in Xenopus oocytes for ACh by a similar factor and suppressed low-sensitivity α4ß2 expression. In contrast, it did not affect the EC50 of α5α4ß2 nAChRs for ACh. Measurements of the ACh responses of WT and mutant nAChRs expressed in mammalian cell lines using a voltage-sensitive fluorescent dye and whole-cell patch-clamping confirm the oocyte data. They also show that, despite reducing the maximum response, ß2V287L increased the α4ß2 response to a sub-saturating ACh concentration (1 µM). Finally, imaging SEP-tagged α5, α4, ß2, and ß2V287L subunits showed that ß2V287L reduced total α4ß2 nAChR surface expression, increased the number of ß2 subunits per α4ß2 receptor, and increased surface α5α4ß2 nAChR expression. Thus, the ß2V287L mutation alters the subunit composition and sensitivity of α4ß2 nAChRs, and increases α5α4ß2 surface expression.

Identification of synovial fluid microRNA signature in knee osteoarthritis: differentiating early- and late-stage knee osteoarthritis.

OBJECTIVES: This study aimed to identify circulating microRNA (miRNA) signatures in knee synovial fluid (SF) from early-stage and late-stage knee osteoarthritis (OA) patients. METHODS: miRNAs were screened by miRNA-PCR-arrays and validated by Real-time quantitative polymerase chain reaction (RT-qPCR) in SF from early-stage (Kellgren-Lawrence (KL): Grade: I/II) and late-stage OA patients (Grade: III/IV). OA cartilage or synovial explants were cultured to study the source/release of identified miRNAs. Computational-approach was utilized to predict gene/pathway targets. RESULTS: Our screening/validation analysis identified a panel of seven (out of 752) circulating miRNAs (23a-3p, 24-3p, 27a-3p, 27b-3p, 29c-3p, 34a-5p and 186-5p) that were significantly differentially expressed in late-stage vs early-stage OA-SF, irrespective of age, gender and Body Mass Index (BMI). miR-378a-5p was mostly detectable in majority of late-stage OA-SF. Cartilage explants stimulated with IL-1ß showed a significant reduction in miR-23a-3p, 27a-3p and 27b-3p expression with no significant changes in other validated miRNAs. However, IL-1ß-stimulated OA synovial explants exhibited significantly increased expression of miR-23a-3p, 24-3p, 27a-3p, 27b-3p, 29c-3p, 186-5p and 378a-5p, and release of only 23a-3p and 27b-3p in supernatants, suggesting that IL-1ß contributes to the release of 23a-3p and 27b-3p into the SF from synovium. Computational-analysis identified 2 genes (ROQUIN-1 [RC3H1] and quaking-gene [QKI]) that are targeted by six out of eight miRNAs; miR-27b-3p exhibited greatest association with RC3H1 and QKI genes. Indeed, synovial explants treated with miR-27b-3p-mimic show significant suppression of both RC3H1 and QKI genes. CONCLUSIONS: We provide first evidence of the differential expression of circulating miRNAs in early-stage vs late-stage knee OA-SF. Further, we provide source, release and genes/pathways regulated by identified miRNAs.

Multispectral fluorescence imaging of human ovarian and fallopian tube tissue for early-stage cancer detection.

With early detection, 5-year survival rates for ovarian cancer exceed 90%, yet no effective early screening method exists. Emerging consensus suggests over 50% of the most lethal form of the disease originates in the fallopian tube. Twenty-eight women undergoing oophorectomy or debulking surgery provided informed consent for the use of surgical discard tissue samples for multispectral fluorescence imaging. Using multiple ultraviolet and visible excitation wavelengths and emissions bands, 12 fluorescence and 6 reflectance images of 47 ovarian and 31 fallopian tube tissue samples were recorded. After imaging, each sample was fixed, sectioned, and stained for pathological evaluation. Univariate logistic regression showed cancerous tissue samples had significantly lower intensity than noncancerous tissue for 17 image types. The predictive power of multiple image types was evaluated using multivariate logistic regression (MLR) and quadratic discriminant analysis (QDA). Two MLR models each using two image types had receiver operating characteristic curves with area under the curve exceeding 0.9. QDA determined 56 image type combinations with perfect resubstituting using as few as five image types. Adaption of the system for future in vivo fallopian tube and ovary endoscopic imaging is possible, which may enable sensitive detection of ovarian cancer with no exogenous contrast agents.

Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward.

Upregulation of ß2 subunit-containing (ß2*) nicotinic acetylcholine receptors (nAChRs) is implicated in several aspects of nicotine addiction, and menthol cigarette smokers tend to upregulate ß2* nAChRs more than nonmenthol cigarette smokers. We investigated the effect of long-term menthol alone on midbrain neurons containing nAChRs. In midbrain dopaminergic (DA) neurons from mice containing fluorescent nAChR subunits, menthol alone increased the number of α4 and α6 nAChR subunits, but this upregulation did not occur in midbrain GABAergic neurons. Thus, chronic menthol produces a cell-type-selective upregulation of α4* nAChRs, complementing that of chronic nicotine alone, which upregulates α4 subunit-containing (α4*) nAChRs in GABAergic but not DA neurons. In mouse brain slices and cultured midbrain neurons, menthol reduced DA neuron firing frequency and altered DA neuron excitability following nAChR activation. Furthermore, menthol exposure before nicotine abolished nicotine reward-related behavior in mice. In neuroblastoma cells transfected with fluorescent nAChR subunits, exposure to 500 nm menthol alone also increased nAChR number and favored the formation of (α4)3(ß2)2 nAChRs; this contrasts with the action of nicotine itself, which favors (α4)2(ß2)3 nAChRs. Menthol alone also increases the number of α6ß2 receptors that exclude the ß3 subunit. Thus, menthol stabilizes lower-sensitivity α4* and α6 subunit-containing nAChRs, possibly by acting as a chemical chaperone. The abolition of nicotine reward-related behavior may be mediated through menthol's ability to stabilize lower-sensitivity nAChRs and alter DA neuron excitability. We conclude that menthol is more than a tobacco flavorant: administered alone chronically, it alters midbrain DA neurons of the nicotine reward-related pathway.

In vivo molecular imaging of colorectal cancer using quantum dots targeted to vascular endothelial growth factor receptor 2 and optical coherence tomography/laser-induced fluorescence dual-modality imaging.

Optical coherence tomography/laser induced fluorescence (OCT/LIF) dual-modality imaging allows for minimally invasive, nondestructive endoscopic visualization of colorectal cancer in mice. This technology enables simultaneous longitudinal tracking of morphological (OCT) and biochemical (fluorescence) changes as colorectal cancer develops, compared to current methods of colorectal cancer screening in humans that rely on morphological changes alone. We have shown that QDot655 targeted to vascular endothelial growth factor receptor 2 (QD655-VEGFR2) can be applied to the colon of carcinogen-treated mice and provides significantly increased contrast between the diseased and undiseased tissue with high sensitivity and specificity ex vivo. QD655-VEGFR2 was used in a longitudinal in vivo study to investigate the ability to correlate fluorescence signal to tumor development. QD655-VEGFR2 was applied to the colon of azoxymethane (AOM-) or saline-treated control mice in vivo via lavage. OCT/LIF images of the distal colon were taken at five consecutive time points every three weeks after the final AOM injection. Difficulties in fully flushing unbound contrast agent from the colon led to variable background signal; however, a spatial correlation was found between tumors identified in OCT images, and high fluorescence intensity of the QD655 signal, demonstrating the ability to detect VEGFR2 expressing tumors in vivo.