On the intersystem crossing rate in a Platinum(II) donor-bridge-acceptor triad.

The rates of ultrafast intersystem crossing in acceptor-bridge-donor molecules centered on Pt(II) acetylides are investigated. Specifically, a Pt(II) trans-acetylide triad NAP--Pt--Ph-CH2-PTZ [1], with acceptor 4-ethynyl-N-octyl-1,8-naphthalimide (NAP) and donor phenothiazine (PTZ), is examined in detail. We have previously shown that optical excitation in [1] leads to a manifold of singlet charge-transfer states, S*, which evolve via a triplet charge-transfer manifold into a triplet state 3NAP centered on the acceptor ligand and partly to a charge-separated state 3CSS (NAP--Pt-PTZ+). A complex cascade of electron transfer processes was observed, but intersystem crossing (ISC) rates were not explicitly resolved due to lack of spin selectivity of most ultrafast spectroscopies. Here we revisit the question of ISC with a combination and complementary analysis of (i) transient absorption, (ii) ultrafast broadband fluorescence upconversion, FLUP, which is only sensitive to emissive states, and (iii) femtosecond stimulated Raman spectroscopy, FSR. Raman resonance conditions allow us to observe S* and 3NAP exclusively by FSR, through vibrations which are pertinent only to these two states. This combination of methods enabled us to extract the intersystem crossing rates that were not previously accessible. Multiple timescales (1.6 ps to ∼20 ps) are associated with the rise of triplet species, which can now be assigned conclusively to multiple ISC pathways from a manifold of hot charge-transfer singlet states. The analysis is consistent with previous transient infrared spectroscopy data. A similar rate of ISC, up to 20 ps, is observed in the trans-acetylide NAP--Pt--Ph [2] which maintains two acetylide groups across the platinum center but lacks a donor unit, whilst removal of one acetylide group in mono-acetylide NAP--Pt-Cl [3] leads to >10-fold deceleration of the intersystem crossing process. Our work provides insight on the intersystem crossing dynamics of the organo-metallic complexes, and identifies a general method based on complementary ultrafast spectroscopies to disentangle complex spin, electronic and vibrational processes following photoexcitation.

Is glutamate associated with fear extinction and cognitive behavior therapy outcome in OCD? A pilot study.

Cognitive behavioral therapy (CBT) including exposure and response prevention is a well-established treatment for obsessive-compulsive disorder (OCD) and is based on the principles of fear extinction. Fear extinction is linked to structural and functional variability in the ventromedial prefrontal cortex (vmPFC) and has been consistently associated with glutamate neurotransmission. The relationship between vmPFC glutamate and fear extinction and its effects on CBT outcome have not yet been explored in adults with OCD. We assessed glutamate levels in the vmPFC using 3T magnetic resonance spectroscopy, and fear extinction (learning and recall) using skin conductance responses during a 2-day experimental paradigm in OCD patients (n = 17) and in healthy controls (HC; n = 13). Obsessive-compulsive patients (n = 12) then received manualized CBT. Glutamate in the vmPFC was negatively associated with fear extinction recall and positively associated with CBT outcome (with higher glutamate levels predicting a better outcome) in OCD patients. Glutamate levels in the vmPFC in OCD patients were not significantly different from those in HC, and were not associated with OCD severity. Our results suggest that glutamate in the vmPFC is associated with fear extinction recall and CBT outcome in adult OCD patients.

PET imaging of dopamine-D2 receptor internalization in schizophrenia.

Recent genetic, molecular and post-mortem studies suggest impaired dopamine (DA)-D2 receptor (D2R) trafficking in patients with schizophrenia (SZ). Imaging and preclinical studies have shown agonist-induced D2R internalization can be imaged with positron emission tomography (PET) using D2R radiotracers combined with psychostimulant challenge. This is feasible if radiotracer binding is measured when postchallenge DA levels have returned to baseline, following the initial competition phase between DA and radiotracer for binding to D2R. Here we used 'late-phase' imaging after challenge to test the hypothesis that impaired D2R internalization in SZ leads to blunted late-phase displacement, or a faster return to baseline, in patients compared with healthy controls (HCs). We imaged 10 patients with SZ and 9 HCs with PET and [11C]raclopride at baseline and two times (3-5 and 6-10 h) following 0.5 mg kg-1 dextroamphetamine. We measured binding potential relative to non-displaceable compartment (BPND) and derived percent reduction from baseline (ΔBPND) for each postamphetamine scan. To test the hypothesis that time course of return of striatal BPND to baseline differed between SZ and HCs, we implemented a linear model with ΔBPND as dependent variable, time after amphetamine as repeated measure and time after amphetamine and diagnostic group as fixed effects. Neither diagnostic group nor interaction of diagnostic group-by-time after amphetamine significantly affected striatal ΔBPND (F=1.38, P=0.26; F=0.51, P=0.61). These results show similar pattern of return of BPND to baseline as a function of time in patients with SZ and HC, suggesting that striatal D2R internalization as measured by our imaging paradigm is normal in patients with SZ.

PET imaging of dopamine-D2 receptor internalization in schizophrenia.

This corrects the article DOI: 10.1038/mp.2017.107.

Deficits in striatal dopamine release in cannabis dependence.

Most drugs of abuse lead to a general blunting of dopamine release in the chronic phase of dependence, which contributes to poor outcome. To test whether cannabis dependence is associated with a similar dopaminergic deficit, we examined striatal and extrastriatal dopamine release in severely cannabis-dependent participants (CD), free of any comorbid conditions, including nicotine use. Eleven CD and 12 healthy controls (HC) completed two positron emission tomography scans with [11C]-(+)-PHNO, before and after oral administration of d-amphetamine. CD stayed inpatient for 5-7 days prior to the scans to standardize abstinence. Magnetic resonance spectroscopy (MRS) measures of glutamate in the striatum and hippocampus were obtained in the same subjects. Percent change in [11C]-(+)-PHNO-binding potential (ΔBPND) was compared between groups and correlations with MRS glutamate, subclinical psychopathological and neurocognitive parameters were examined. CD had significantly lower ΔBPND in the striatum (P=0.002, effect size (ES)=1.48), including the associative striatum (P=0.003, ES=1.39), sensorimotor striatum (P=0.003, ES=1.41) and the pallidus (P=0.012, ES=1.16). Lower dopamine release in the associative striatum correlated with inattention and negative symptoms in CD, and with poorer working memory and probabilistic category learning performance in both CD and HC. No relationships to MRS glutamate and amphetamine-induced subclinical positive symptoms were detected. In conclusion, this study provides evidence that severe cannabis dependence-without the confounds of any comorbidity-is associated with a deficit in striatal dopamine release. This deficit extends to other extrastriatal areas and predicts subclinical psychopathology.

Whey protein gel composites in the diet of goats increased the omega-3 and omega-6 content of milk fat.

Previously, feeding whey protein gels containing polyunsaturated fatty acids (PUFA) reduced their rumen biohydrogenation and increased their concentration in milk fat of Holstein cows. Our objective was to test the efficacy of whey protein isolate (WPI) gels produced in a steam tunnel as a method to alter the fatty acid (FA) composition of the milk lipids. Four primiparous Lamancha goats in midlactation were fed three diets in a 3 × 4 Latin square design. The WPI gels were added to a basal concentrate mix that contained one of three lipid sources: (i) 100% soya bean oil (S) to create (WPI/S), (ii) a 1:1 (wt/wt) mixture of S and linseed (L) oil to create (WPI/SL), or (iii) 100% L to create (WPI/L). Periods were 22 days with the first 10 days used as an adjustment phase followed by a 12-day experimental phase. During the adjustment phase, all goats received a rumen available source of lipid, yellow grease, to provide a baseline for milk FA composition. During the experimental phase, each goat received its assigned WPI. Milk FA concentration of C18:2 n-6 and C18:3 n-3 reached 9.3 and 1.64 g/100 g FA, respectively, when goats were fed WPI/S. Feeding WPI/SL increased the C18:2 n-6 and C18:3 n-3 concentration to 6.22 and 4.36 g/100 g FA, and WPI/L increased C18:2 n-6 and C18:3 n-3 to 3.96 and 6.13 g/100 g FA respectively. The adjusted transfer efficiency (%) of C18:3 n-3 to milk FA decreased significantly as dietary C18:3 n-3 intake increased. Adjusted transfer efficiency for C18:2 n-6 did not change with increasing intake of C18:2 n-6. The WPI gels were effective at reducing rumen biohydrogenation of PUFA; however, we observed a change in the proportion increase of C18:3 n-3 in milk FA suggesting possible regulation of n-3 FA to the lactating caprine mammary gland.

AAV ancestral reconstruction library enables selection of broadly infectious viral variants.

Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties and to gain insights into AAV's evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes and, in general, ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.

Systematic variation in gene expression patterns in human cancer cell lines.

We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.

A gene expression database for the molecular pharmacology of cancer.

We used cDNA microarrays to assess gene expression profiles in 60 human cancer cell lines used in a drug discovery screen by the National Cancer Institute. Using these data, we linked bioinformatics and chemoinformatics by correlating gene expression and drug activity patterns in the NCI60 lines. Clustering the cell lines on the basis of gene expression yielded relationships very different from those obtained by clustering the cell lines on the basis of their response to drugs. Gene-drug relationships for the clinical agents 5-fluorouracil and L-asparaginase exemplify how variations in the transcript levels of particular genes relate to mechanisms of drug sensitivity and resistance. This is the first study to integrate large databases on gene expression and molecular pharmacology.

Improving blood product management in placenta accreta patients with severe bleeding: institutional experience.

BACKGROUND: Placenta accrete spectrum (PAS) is a significant risk factor for postpartum hemorrhage and effective blood product management is critical in ensuring patient safety. In PAS patients undergoing cesarean section (CS) blood transfusion management guided by the combined clinical experience of the anesthesiologist and surgeon with point-of-care coagulation testing appears safe and effective. We describe and evaluate our experience and identify potential areas for improvement with blood product management in this patient population. METHODS: A retrospective chart review of peri-operative demographic, anesthetic, and obstetric data was conducted for all patients with PAS undergoing CS between 2012 and 2018 at our center. To facilitate a practical evaluation of blood product management, we divided patients into two groups based on the severity of bleeding. RESULTS: A total of 221 parturients with PAS underwent CS, with 133 in group 1 requiring excessive amounts of transfusion and 88 in group 2 requiring management similar to other uncomplicated CS cases. There were no deaths or instances of disseminated intravascular coagulation, and intensive care unit admission occurred in five cases (2.2%). Patients in group 1 had higher mean nadir values of intra-operative hemoglobin and platelet count. We observed a high rate of missing data for peri-operative measurement of lactate and fibrinogen, PAS grade documentation, and temperature monitoring. CONCLUSION: Given no significant morbidity or mortality, clinical judgment in experienced centers appears safe for the management of PAS patients undergoing CS. The adoption of an institutional protocol and point-of-care coagulation testing could decrease over-transfusion and associated complications.

Directed evolution of novel adeno-associated viruses for therapeutic gene delivery.

Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical trials for numerous disease targets, such as muscular dystrophy, hemophilia, Parkinson's disease, Leber's congenital amaurosis and macular degeneration. Despite its considerable promise and emerging clinical success, several challenges impede the broader implementation of AAV gene therapy, including the prevalence of neutralizing antibodies in the human population, low transduction of a number of therapeutically relevant cell and tissue types, an inability to overcome physical and cellular barriers in vivo and a relatively limited carrying capacity. These challenges arise as the demands we place on AAV vectors are often different from or even at odds with the properties nature bestowed on their parent viruses. Viral-directed evolution-the iterative generation of large, diverse libraries of viral mutants and selection for variants with specific properties of interest-offers an approach to address these problems. Here we outline progress in creating novel classes of AAV variant libraries and highlight the successful isolation of variants with novel and advantageous in vitro and in vivo gene delivery properties.

Experience from the selection and nutritional preparation for Expedition ICE MAIDEN: the first successful all-female unassisted Antarctic traverse.

INTRODUCTION: Expedition ICE MAIDEN (Ex IM) was the first all-female unsupported crossing of Antarctica. We describe the prerequisite selection and training, comparing those who formed the final team with other participants, and discuss how the expedition diet was established. METHODS: All women serving in the British Army were invited to participate. Following initial assessments, successful women completed three training/selection ski expeditions. Between expeditions 1 and 2, participants completed 6 months rigorous UK-based training. Weight was measured before and after the 6 months UK-based training, expeditions 2 and 3, and body composition by skinfold before and after expedition 2. Participant feedback, body composition and weight changes were applied to modify the expedition diet and provide weight gain targets prior to Ex IM. RESULTS: Following 250 applications, 50 women were assessed and 22, 12 and seven women attended training expeditions 1, 2 and 3, respectively. The final team of six women lost more weight than other participants during UK-based training (mean (SD) change -1.3 (1.5) kg vs -0.5 (1.6) kg, respectively, p=0.046) and during training expedition 2 (-2.8 (0.8) kg vs -1.7 (0.4) kg, respectively, p=0.048), when they also gained more lean mass (+2.1 (0.8) kg vs +0.4 (0.7) kg, respectively, p=0.004). The Ex IM diet provided 5000 kCal/day, comprising approximately 45% carbohydrate, 45% fat and 10% protein. Median (range) weight change between expedition 3 and Ex IM was +8.7 (-1.9 to +14.3) kg. CONCLUSIONS: The selected Ex IM team demonstrated favourable training-associated body composition changes. Training-associated weight loss informed the expeditionary diet design.

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein G mRNA into membrane-bound polyribosome complexes.

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.

Mammalian p55CDC mediates association of the spindle checkpoint protein Mad2 with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events.

We have investigated the function of p55CDC, a mammalian protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila. Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase. Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm. At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase. In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2. p55CDC is required for binding Mad2 with the Cdc27 and Cdc16. Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex. Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events. These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex.

Electric power from differences in salinity: the dialytic battery.

An array of alternating anion and cation exchange membranes can be used to generate electric power from the free energy of mixing of river and sea waters. A simple mathematical model, which predicts experimental results well, is useful in exploring conditions for optimization of the process. Major, but not impossible, improvements in technology would be required to bring the cost of power from the dialytic battery into line with foreseeable energy prices.

Charge-mosaic membranes: dialytic separation of electrolytes from nonelectrolytes and amino acids.

Charge-mosaic membranes were used for dialytic separations of potassium chloride from low-molecular-weight nonelectrolytes and neutral amino acids. The permeability ratio (potassium chloride to uncharged species) ranged from about 6 in the case of methanol to about 86 in that of mannitol. A theoretical model predicts that optimum rates of dialysis should be achieved by dialyzing against salt concentrations other than zero; this prediction was confirmed by experiment. These observations suggest potential applications of mosaics in laboratory separations, industrial processing, and hemodialysis.

Charge-mosaic membranes: enhanced permeability and negative osmosis with a symmetrical salt.

Charge-mosaic membranes are prepared by embedding a single layer of alternating cation and anion exchange beads in silicone resin. Membranes made in an identical manner but containing only one type of exchanger serve as controls. The mosaic membranes are 50 to 100 times more permeable to potassium chloride than the controls; and furthermore they give rise to net volume flow from concentrated to dilute solutions of potassium chloride in the absence of a pressure gradient ("negative osmosis"), whereas the controls exhibit normal osmotic behavior. The negative reflection coefficients of the mosaics suggest potential applications in desalination.

Receptors for histamine can be detected on the surface of selected leukocytes.

Histamine insolubilized by chemical linkage via a protein or polypeptide carrier to Sepharose beads cannot penetrate cells. Even so, the resultant histamine-coated Sepharose binds leukocytes selectively. Despite the low molecular weight of histaminie, the binding is via preformed cell membrane receptors and can be specifically and competitively blocked or reversed by antihistamines.

Liposome-cell interaction: transfer and intracellular release of a trapped fluorescent marker.

When small, unilamellar lipid vesicles containing a high concentration of the fluorescent dye 6-carboxyfluorescein are incubated with either frog retinas or human lymphocytes, fluroescence distributes widely throughout each cell. Since "self-quenching" largely prevents the dye from fluorescing as long as it remains sequestered in vesicles, it is clear that a considerable amount of dye is released from the vesicles and diluted into the much larger volume of the cell.

Liposomes and local hyperthermia: selective delivery of methotrexate to heated tumors.

Liposomes with phase transitions a few degrees above physiological temperature delivered more than four times as much methotrexate to murine tumors heated to 42 degrees C as to unheated control tumors. Most of the accumulated drug appeared to be intracellular and bound to dihydrofolate reductase, the enzyme blocked by methotrexate in its role as an antineoplastic agent.