Cessation of berry growth coincides with leaf complete stomatal closure at pre-veraison for grapevine (Vitis vinifera) subjected to progressive drought stress.

BACKGROUND AND AIMS: Drought events have devasting impacts on grape berry production. The aim of this study was to investigate berry growth in the context of leaf stomatal closure under progressive drought stress. METHODS: Potted grapevine plants (varieties 'Syrah' and 'Cabernet Sauvignon') were evaluated at pre-verasion (30-45 d after anthesis, DAA) and post-veraison (90-107 DAA). Berry diameter, berry absolute growth rate (AGR), leaf stomatal conductance (Gs) at midday, plant water potential at predawn and midday (ΨPD and ΨMD, respectively), and soil relative water content were measured repeatedly. The ΨPD-threshold of 90 % loss in stomatal conductance (Gs10, i.e. complete stomatal closure) was determined. Data were related to plant dehydration phases I, II and III with corresponding boundaries Θ1 and Θ2, using the water potential curve method. KEY RESULTS: At pre-veraison, berry AGR declined together with leaf Gs in response to soil drying in both varieties. Berry AGR transitioned from positive to negative (shrinkage) values when leaf Gs approached zero. The Gs10-threshold was -0.81 MPa in 'Syrah' and -0.74 MPa in 'Cabernet Sauvignon' and was linked to boundary Θ1. At post-veraison, berry AGR was negligible and negative AGR values were not intensified by increasing drought stress in either variety. CONCLUSION: Leaf complete stomatal closure under progressive drought stress coincides with cessation of berry growth followed by shrinkage at pre-veraison (growth stage 1).

The impact of COVID-19 public health measures on attendance and overdose at supervised injecting facilities in Australia.

OBJECTIVES: The COVID-19 pandemic disrupted service provision of harm reduction and drug treatment services for people who inject drugs in many countries. The two supervised injecting facilities (SIFs) in Sydney and Melbourne were differentially impacted by the pandemic, requiring local procedural changes in each service. We aimed to examine the impact of pandemic responses (including restrictions on movement, known as 'lockdowns') on service use and key parameters such as client reports of drug injected and recorded overdose rates. STUDY DESIGN: Time series analysis of weekly client visits and monthly overdoses occurring at each service. METHODS: Administrative client data from the two SIFs (Sydney data from 1 January 2018 to 30 April 2022; Melbourne data from 1 July 2018 to 30 April 2022) were examined using interrupted time series analyses with lockdown dates in each state entered as interruption terms. We analysed weekly SIF visits overall and by drug type, and monthly rates of opioid overdose at each service. RESULTS: Lockdowns resulted in decreased visits to both services. The number of weekly client visits decreased during the first national lockdown for both the Sydney (trend change = -57.9; 95% CI [-109.4, -6.4]) and Melbourne SIF (near sig trend change = -54.8 [-110.8, 1.05]). Trends in visit numbers increased after lockdowns were lifted in each city; however, visits in Sydney have not returned to the numbers recorded prior to the pandemic. Visits to the Melbourne SIF related to heroin use declined at each lockdown (trend 1 = -42.7 [-81.5, -3.9]; trend 2 = -56.1 [-94.6, -17.7]; trend 3 = -33.8 [-67.4, -0.2]); heroin visits to the Sydney SIF declined during the first lockdown and remained low (trend = -55.6 [-82.8, -28.3]). Methamphetamine visits to the Sydney SIF fluctuated, surpassing heroin visits at several timepoints. Rates of monthly opioid overdoses at both services declined immediately following the start of the first lockdown (Sydney = -16.6 [-26.1, -6.8]; Melbourne = -6.4 [-8.7, -4.1]), with increasing trends recorded at the end of the final lockdown in each jurisdiction (Sydney = 2.8 [0.6, 5.0]; Melbourne = 1.3 [0.72, 3.2]). CONCLUSIONS: Public health restrictions related to the COVID-19 pandemic were associated with reduced client visits to, and overdoses in, Australian SIFs. Variations were noted in the drugs injected, likely reflecting changes in local drug markets. Shifts to other drugs during these periods were evident: methamphetamine in Sydney; co-injection of heroin and diphenhydramine in Melbourne.

Racial, Gender, and Age Dynamics in Michigan's Urban and Rural Farmers Markets: Reducing Food Insecurity, and the Impacts of a Pandemic.

In recent decades, the number of farmers markets has increased dramatically across the country. Though farmers markets have been described as White spaces, they can play important roles in reducing food insecurity. It is particularly true in Michigan, where farmers markets were crucial collaborators in pioneering programs such as Double-Up Food Bucks that help low-income residents and people of color gain access to fresh, healthy, locally grown food. This article examines the questions: (1) What are the demographic characteristics of farmers market managers, vendors, and customers? (2) How do these influence market activities? (3) To what extent do farmers markets participate in programs to reduce food insecurity? (4) To what extent do farmers markets serve low-income residents and people of color? And (5) How has the Coronavirus Pandemic (COVID-19) affected farmers' markets? This article discusses the findings of a 2020 study that examined the extent to which Michigan's farmers markets served low-income customers and people of color, and participated in food assistance programs. The study examined 79 farmers markets and found that 87.3% of the farmers market managers are White. On average, roughly 79% of the markets' vendors are White, and almost 18% are people of color. Most of the vendors in the markets participate in nutrition assistance programs. Market managers estimate that about 76% of their customers are White, and about 23% are people of color. Farmers markets operated by people of color attract more customers and vendors of color than those administered by White market managers. Almost half of the farmers markets started operations later than usual in 2020 because of the pandemic. More than a third of the markets reported that their funding declined during the pandemic. Moreover, the number of vendors fell at two-thirds of the markets; customers dipped by more than 40%. On the other hand, the number of people requesting food assistance during the pandemic increased in more than half of the markets.

Electromyographic differences of the gluteus maximus, gluteus medius, biceps femoris, and vastus lateralis between the barbell hip thrust and barbell glute bridge.

Hip extensor muscles are critical to sport performance as events requiring sprinting and forceful landings are highly dependent on these muscles. Despite biomechanical differences between the barbell hip thrust (BHT) and the barbell glute bridge (BGB), both are biomechanically efficient ways to load this musculature for training purposes. Research investigating the differences in muscular activity between the BHT and BGB has yet been conducted. The aim of this study was to investigate, through surface electromyography, if one exercise is more optimal than the other in producing greater muscle activation for specific hip extensor muscles. Ten male participants completed a two-part study protocol. Results revealed the BHT elicited significantly greater muscle activity within the vastus lateralis for peak and mean outcomes; however, the BGB elicited significantly greater muscle activity in the upper and lower gluteus maximus for peak and mean outcomes and mean outcome in the gluteus medius. Current findings suggest, the BGB is, at minimum, a superior substitute for the BHT for eliciting a larger magnitude of activity in the gluteus maximus. Future studies between the two exercises are warranted to discern which produces greater hypertrophy and whether adaption of the BHT or BGB transfers more optimally to sport performance.

Root pressure-volume curve traits capture rootstock drought tolerance.

BACKGROUND AND AIMS: Living root tissues significantly constrain plant water uptake under drought, but we lack functional traits to feasibly screen diverse plants for variation in the drought responses of these tissues. Water stress causes roots to lose volume and turgor, which are crucial to root structure, hydraulics and growth. Thus, we hypothesized that root pressure-volume (p-v) curve traits, which quantify the effects of water potential on bulk root turgor and volume, would capture differences in rootstock drought tolerance. METHODS: We used a greenhouse experiment to evaluate relationships between root p-v curve traits and gas exchange, whole-plant hydraulic conductance and biomass under drought for eight grapevine rootstocks that varied widely in drought performance in field trials (101-14, 110R, 420A, 5C, 140-Ru, 1103P, Ramsey and Riparia Gloire), grafted to the same scion variety (Vitis vinifera 'Chardonnay'). KEY RESULTS: The traits varied significantly across rootstocks, and droughted vines significantly reduced root turgor loss point (πtlp), osmotic potential at full hydration (πo) and capacitance (C), indicating that roots became less susceptible to turgor loss and volumetric shrinkage. Rootstocks that retained a greater root volume (i.e. a lower C) also maintained more gas exchange under drought. The rootstocks that previous field trials have classified as drought tolerant exhibited significantly lower πtlp, πo and C values in well-watered conditions, but significantly higher πo and πtlp values under water stress, than the varieties classified as drought sensitive. CONCLUSIONS: These findings suggest that acclimation in root p-v curve traits improves gas exchange in persistently dry conditions, potentially through impacts on root hydraulics or root to shoot chemical signalling. However, retaining turgor and volume in previously unstressed roots, as these roots deplete wet soil to moderately negative water potentials, could be more important to drought performance in the deep, highly heterogenous rooting zones which grapevines develop under field conditions.

High Prevalence of Spinal Cord Cavernous Malformations in the Familial Cerebral Cavernous Malformations Type 1 Cohort.

BACKGROUND AND PURPOSE: Cavernous malformations occur most often in the brain but can occur in the spinal cord. Small studies of patients with familial cerebral cavernous malformations suggested a prevalence of spinal cord cavernous malformations of 20%-42%. We aimed to review our familial cohort and prospectively estimate the prevalence of spinal cord cavernous malformations. MATERIALS AND METHODS: We initially reviewed our familial cerebral cavernous malformations cohort for spinal cord cavernous malformations and reviewed clinical spine MR imaging examinations for sequence sensitivity. We then prospectively performed research MR imaging of the spinal cord in 29 patients from the familial cohort to estimate the prevalence. RESULTS: Gradient-based sequences identified the most spinal cord cavernous malformations on clinical MR images, forming the basis for developing our screening MR imaging. Screening spinal cord MR imaging demonstrated a prevalence of 72.4%, and a positive correlation with patient age and number of cerebral cavernous malformations. CONCLUSIONS: Spinal cord cavernous malformations occur commonly in the familial cerebral cavernous malformation population. Gradient-based sequences are the most sensitive and should be used when spinal cord cavernous malformations are suspected. This study establishes the prevalence in the familial population at around 70% and supports the idea that this condition is a progressive systemic disease that affects the entire central nervous system.

Tree carbon allocation explains forest drought-kill and recovery patterns.

The mechanisms governing tree drought mortality and recovery remain a subject of inquiry and active debate given their role in the terrestrial carbon cycle and their concomitant impact on climate change. Counter-intuitively, many trees do not die during the drought itself. Indeed, observations globally have documented that trees often grow for several years after drought before mortality. A combination of meta-analysis and tree physiological models demonstrate that optimal carbon allocation after drought explains observed patterns of delayed tree mortality and provides a predictive recovery framework. Specifically, post-drought, trees attempt to repair water transport tissue and achieve positive carbon balance through regrowing drought-damaged xylem. Furthermore, the number of years of xylem regrowth required to recover function increases with tree size, explaining why drought mortality increases with size. These results indicate that tree resilience to drought-kill may increase in the future, provided that CO2 fertilisation facilitates more rapid xylem regrowth.

An innovative long final year assistantship in general practice: description and evaluation.

We describe and evaluate an innovative immersive 15 week final year assistantship in general practice. Evaluation data was taken from five years of routinely collected School data and available national comparative data. The assistantship aims to enable students to consolidate knowledge and hone their skills through central participation in the care of large numbers of patients with acute and long term conditions. We estimate that most students consulted with over 450 patients during the assistantship. Students report that they became useful to their practice teams, had multiple episodes of feedback on their performance which they found useful and, in the school exit survey, reported that they were highly prepared for practice. 9.4 per cent of students reported that the assistantship was 'too long' and, especially those who completed the assistantship in the second semester, they were out of hospital for too long before F1. Some described a learning 'plateau' after the 10th week which was addressed by modifications to the assistantship. Nevertheless, in national surveys, our graduates' self-reported preparedness for practice is high, a perception shared by their F1 supervisors. General practice can make a valuable contribution to the education of senior medical students and contribute to their preparedness for practice.

Comparative genomics and transcriptomics of Pichia pastoris.

BACKGROUND: Pichia pastoris has emerged as an important alternative host for producing recombinant biopharmaceuticals, owing to its high cultivation density, low host cell protein burden, and the development of strains with humanized glycosylation. Despite its demonstrated utility, relatively little strain engineering has been performed to improve Pichia, due in part to the limited number and inconsistent frameworks of reported genomes and transcriptomes. Furthermore, the co-mingling of genomic, transcriptomic and fermentation data collected about Komagataella pastoris and Komagataella phaffii, the two strains co-branded as Pichia, has generated confusion about host performance for these genetically distinct species. Generation of comparative high-quality genomes and transcriptomes will enable meaningful comparisons between the organisms, and potentially inform distinct biotechnological utilies for each species. RESULTS: Here, we present a comprehensive and standardized comparative analysis of the genomic features of the three most commonly used strains comprising the tradename Pichia: K. pastoris wild-type, K. phaffii wild-type, and K. phaffii GS115. We used a combination of long-read (PacBio) and short-read (Illumina) sequencing technologies to achieve over 1000X coverage of each genome. Construction of individual genomes was then performed using as few as seven individual contigs to create gap-free assemblies. We found substantial syntenic rearrangements between the species and characterized a linear plasmid present in K. phaffii. Comparative analyses between K. phaffii genomes enabled the characterization of the mutational landscape of the GS115 strain. We identified and examined 35 non-synonomous coding mutations present in GS115, many of which are likely to impact strain performance. Additionally, we investigated transcriptomic profiles of gene expression for both species during cultivation on various carbon sources. We observed that the most highly transcribed genes in both organisms were consistently highly expressed in all three carbon sources examined. We also observed selective expression of certain genes in each carbon source, including many sequences not previously reported as promoters for expression of heterologous proteins in yeasts. CONCLUSIONS: Our studies establish a foundation for understanding critical relationships between genome structure, cultivation conditions and gene expression. The resources we report here will inform and facilitate rational, organism-wide strain engineering for improved utility as a host for protein production.

Long-range magnetic interactions and proximity effects in an amorphous exchange-spring magnet.

Low-dimensional magnetic heterostructures are a key element of spintronics, where magnetic interactions between different materials often define the functionality of devices. Although some interlayer exchange coupling mechanisms are by now well established, the possibility of direct exchange coupling via proximity-induced magnetization through non-magnetic layers is typically ignored due to the presumed short range of such proximity effects. Here we show that magnetic order can be induced throughout a 40-nm-thick amorphous paramagnetic layer through proximity to ferromagnets, mediating both exchange-spring magnet behaviour and exchange bias. Furthermore, Monte Carlo simulations show that nearest-neighbour magnetic interactions fall short in describing the observed effects and long-range magnetic interactions are needed to capture the extent of the induced magnetization. The results highlight the importance of considering the range of interactions in low-dimensional heterostructures and how magnetic proximity effects can be used to obtain new functionality.

Drought tolerance as a driver of tropical forest assembly: resolving spatial signatures for multiple processes.

Spatial patterns in trait variation reflect underlying community assembly processes, allowing us to test hypotheses about their trait and environmental drivers by identifying the strongest correlates of characteristic spatial patterns. For 43 evergreen tree species (> 1 cm dbh) in a 20-ha seasonal tropical rainforest plot in Xishuangbanna, China, we compared the ability of drought-tolerance traits, other physiological traits, and commonly measured functional traits to predict the spatial patterns expected from the assembly processes of habitat associations, niche-overlap-based competition, and hierarchical competition. We distinguished the neighborhood-scale (0-20 m) patterns expected from competition from larger-scale habitat associations with a wavelet method. Species' drought tolerance and habitat variables related to soil water supply were strong drivers of habitat associations, and drought tolerance showed a significant spatial signal for influencing competition. Overall, the traits most strongly associated with habitat, as quantified using multivariate models, were leaf density, leaf turgor loss point (π(tlp); also known as the leaf wilting point), and stem hydraulic conductivity (r2 range for the best fit models = 0.27-0.36). At neighborhood scales, species spatial associations were positively correlated with similarity in π(tlp), consistent with predictions for hierarchical competition. Although the correlation between π(tlp) and interspecific spatial associations was weak (r2 < 0.01), this showed a persistent influence of drought tolerance on neighborhood interactions and community assembly. Quantifying the full impact of traits on competitive interactions in forests may require incorporating plasticity among individuals within species, especially among specific life stages, and moving beyond individual traits to integrate the impact of multiple traits on whole-plant performance and resource demand.

Comparative study of the neurotrophic effects elicited by VEGF-B and GDNF in preclinical in vivo models of Parkinson's disease.

Vascular endothelial growth factor B (VEGF-B) has recently been shown to be a promising novel neuroprotective agent for several neurodegenerative conditions. In the current study we extended previous work on neuroprotective potential for Parkinson's disease (PD) by testing an expanded dose range of VEGF-B (1 and 10 µg) and directly comparing both neuroprotective and neurorestorative effects of VEGF-B in progressive unilateral 6-hydroxydopamine (6-OHDA) PD models to a single dose of glial cell line-derived neurotrophic factor (GDNF, 10 µg), that has been established by several groups as a standard in both preclinical PD models. In the amphetamine-induced rotational tests the treatment with 1 and 10 µg VEGF-B resulted in significantly improved motor function of 6-OHDA-lesioned rats compared to vehicle-treated 6-OHDA-lesioned rats in the neuroprotection paradigm. Both doses of VEGF-B caused an increase in tyrosine hydroxylase (TH)-positive cell and fiber count in the substantia nigra (SN) and striatum in the neuroprotective experiment. The effect size was comparable to the effects seen with GDNF. In the neurorestoration paradigm, VEGF-B injection had no significant effect in either the behavioral or the immunohistochemical analyses, whereas GDNF injection significantly improved the amphetamine-induced rotational behavior and reduced TH-positive neuronal cell loss in the SN. We also present a strong positive correlation (p=1.9e-50) of the expression of VEGF-B with nuclear-encoded mitochondrial genes involved in fatty acid metabolism in rat midbrain, pointing to the mitochondria as a site of action of VEGF-B. GDNF showed a positive correlation with nuclear-encoded mitochondrial genes that was not nearly as strong (p=0.018). VEGF-B counteracted rotenone-induced reduction of (a) fatty acid transport protein 1 and 4 levels and (b) both Akt protein and phosphorylation levels in SH-SY5Y cells. We further verified VEGF-B expression in the human SN pars compacta of healthy controls and PD patients, in neuronal cells that show co-expression with neuromelanin. These results have demonstrated that VEGF-B has potential as a neuroprotective agent for PD therapy and should be further investigated.

Corrector VX-809 stabilizes the first transmembrane domain of CFTR.

Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis (CF). A potential CF therapy would be to repair CFTR processing mutants. It has been demonstrated that processing mutants of P-glycoprotein (P-gp), CFTR's sister protein, can be efficiently repaired by a drug-rescue mechanism. Many arginine suppressors that mimic drug-rescue have been identified in the P-gp transmembrane (TM) domains (TMDs) that rescue by forming hydrogen bonds with residues in adjacent helices to promote packing of the TM segments. To test if CFTR mutants could be repaired by a drug-rescue mechanism, we used truncation mutants to test if corrector VX-809 interacted with the TMDs. VX-809 was selected for study because it is specific for CFTR, it is the most effective corrector identified to date, but it has limited clinical benefit. Identification of the VX-809 target domain will help to develop correctors with improved clinical benefits. It was found that VX-809 rescued truncation mutants lacking the NBD2 and R domains. When the remaining domains (TMD1, NBD1, TMD2) were expressed as separate polypeptides, VX-809 only increased the stability of TMD1. We then performed arginine mutagenesis on TM6 in TMD1. Although the results showed that TM6 had distinct lipid and aqueous faces, CFTR was different from P-gp as no arginine promoted maturation of CFTR processing mutants. The results suggest that TMD1 contains a VX-809 binding site, but its mechanism differed from P-gp drug-rescue. We also report that V510D acts as a universal suppressor to rescue CFTR processing mutants.

Bithiazole correctors rescue CFTR mutants by two different mechanisms.

Better correctors are needed to repair cystic fibrosis transmembrane conductance regulator (CFTR) processing mutants that cause cystic fibrosis. Determining where the correctors bind to CFTR would aid in the development of new correctors. A recent study reported that the second nucleotide-binding domain (NBD2) was involved in binding of bithiazole correctors. Here, we show that bithiazole correctors could also rescue CFTR mutants that lacked NBD2. These results suggest that bithiazoles rescue CFTR mutants by two different mechanisms.

Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface.

The P-glycoprotein drug pump protects us from toxins. Drug-binding sites in the transmembrane (TM) domains (TMDs) are connected to the nucleotide-binding domains (NBDs) by intracellular helices (IHs). TMD-NBD cross-talk is a key step in the transport mechanism because drug binding stimulates ATP hydrolysis followed by drug efflux. Here, we tested whether the IHs are critical for maturation and TMD-NBD coupling by characterizing the effects of mutations to the IH1 and IH2 interfaces. Although IH1 mutations had little effect, most mutations at the IH2-NBD2 interface inhibited maturation or activity. For example, the F1086A mutation at the IH2-NBD2 interface abolished drug-stimulated ATPase activity. The mutant F1086A, however, retained the ability to bind ATP and drug substrates. The mutant was defective in mediating ATP-dependent conformational changes in the TMDs because binding of ATP no longer promoted cross-linking between cysteines located at the extracellular ends of TM segments 6 and 12. Replacement of Phe-1086 (in NBD2) with hydrophobic but not charged residues yielded active mutants. The activity of the F1086A mutant could be restored when the nearby residue Ala-266 (in IH2) was replaced with aromatic residues. These results suggest that Ala-266/Phe-1086 lies in a hydrophobic IH2-NBD2 "ball-and-socket" joint.

Potential interactions between diadromous fishes of U.K. conservation importance and the electromagnetic fields and subsea noise from marine renewable energy developments.

The considerable extent of construction and operation of marine renewable energy developments (MRED) within U.K. and adjacent waters will lead, among other things, to the emission of electromagnetic fields (EMF) and subsea sounds into the marine environment. Migratory fishes that respond to natural environmental cues, such as the Earth's geomagnetic field or underwater sounds, move through the same waters that the MRED occupy, thereby raising the question of whether there are any effects of MRED on migratory fishes. Diadromous species, such as the Salmonidae and Anguillidae, which undertake large-scale migrations through coastal and offshore waters, are already significantly affected by other human activities leading to national and international conservation efforts to manage any existing threats and to minimize future concerns, including the potential effect of MRED. Here, the current state of knowledge with regard to the potential for diadromous fishes of U.K. conservation importance to be affected by MRED is reviewed. The information on which to base the review was found to be limited with respect to all aspects of these fishes' migratory behaviour and activity, especially with regards to MRED deployment, making it difficult to establish cause and effect relationships. The main findings, however, were that diadromous species can use the Earth's magnetic field for orientation and direction finding during migrations. Juveniles of anadromous brown trout (sea trout) Salmo trutta and close relatives of S. trutta respond to both the Earth's magnetic field and artificial magnetic fields. Current knowledge suggests that EMFs from subsea cables may interact with migrating Anguilla sp. (and possibly other diadromous fishes) if their movement routes take them over the cables, particularly in shallow water (<20 m). The only known effect is a temporary change in swimming direction. Whether this will represent a biologically significant effect, for example delayed migration, cannot yet be determined. Diadromous fishes are likely to encounter EMFs from subsea cables either during the adult movement phases of life or their early life stages during migration within shallow, coastal waters adjacent to natal rivers. The underwater sound from MRED devices has not been fully characterized to determine its acoustic properties and propagation through the coastal waters. MRED that require pile driving during construction appear to be the most relevant to consider. In the absence of a clear understanding of their response to underwater sound, the specific effects on migratory species of conservation concern remain very difficult to determine in relation to MRED. Based on the studies reviewed, it is suggested that fishes that receive high intensity sound in close proximity to construction may be physiologically affected to some degree, whereas those at farther distances, potentially up to several km, may exhibit behaviour responses; the effect of which is unknown and will be dependent on the properties of the received sound and receptor characteristics and condition. Whether there are behavioural effects on the fishes during operation is unknown but any change to the environment and subsequent response by the fishes would need to be considered over the lifetime of the MRED. It is not yet possible to determine if effects relating to sound exposure are biologically significant. The current assumptions of limited effects are built on an incomplete understanding of how the species move around their environment and interact with natural and anthropogenic EMFs and subsea sound. A number of important knowledge gaps exist, principally whether migratory fish species on the whole respond to the EMF and the sound associated with MRED. Future research should address the principal gaps before assuming that any effect on diadromous species results in a biological effect.

The ATPase activity of the P-glycoprotein drug pump is highly activated when the N-terminal and central regions of the nucleotide-binding domains are linked closely together.

The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the homologous halves of P-gp is composed of a transmembrane domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The predicted drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures and EM projection images suggest that the two halves of P-gp are separated by a central cavity that closes upon binding of nucleotide. Binding of drug substrates may induce further structural rearrangements because they stimulate ATPase activity. Here, we used disulfide cross-linking with short (8 Å) or long (22 Å) cross-linkers to identify domain-domain interactions that activate ATPase activity. It was found that cross-linking of cysteines that lie close to the LSGGQ (P517C) and Walker A (I1050C) sites of NBD1 and NBD2, respectively, as well as the cytoplasmic extensions of TM segments 3 (D177C or L175C) and 9 (N820C) with a short cross-linker activated ATPase activity over 10-fold. A pyrylium compound that inhibits ATPase activity blocked cross-linking at these sites. Cross-linking between the NBDs was not inhibited by tariquidar, a drug transport inhibitor that stimulates P-gp ATPase activity but is not transported. Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. The results suggest that trapping P-gp in a conformation in which the NBDs are closely associated likely mimics the structural rearrangements caused by binding of drug substrates that stimulate ATPase activity.

Thiorhodamines containing amide and thioamide functionality as inhibitors of the ATP-binding cassette drug transporter P-glycoprotein (ABCB1).

Twelve thiorhodamine derivatives have been examined for their ability to stimulate the ATPase activity of purified human P-glycoprotein (P-gp)-His(10), to promote uptake of calcein AM and vinblastine into multidrug-resistant, P-gp-overexpressing MDCKII-MDR1 cells, and for their rates of transport in monolayers of multidrug-resistant, P-gp-overexpressing MDCKII-MDR1 cells. The thiorhodamine derivatives have structural diversity from amide and thioamide functionality (N,N-diethyl and N-piperidyl) at the 5-position of a 2-thienyl substituent on the thiorhodamine core and from diversity at the 3-amino substituent with N,N-dimethylamino, fused azadecalin (julolidyl), and fused N-methylcyclohexylamine (half-julolidyl) substituents. The julolidyl and half-julolidyl derivatives were more effective inhibitors of P-gp than the dimethylamino analogues. Amide-containing derivatives were transported much more rapidly than thioamide-containing derivatives.

Multilayer Architectures for Facial Action Unit Recognition.

In expression recognition and many other computer vision applications, the recognition performance is greatly improved by adding a layer of nonlinear texture filters between the raw input pixels and the classifier. The function of this layer is typically known as feature extraction. Popular filter types for this layer are Gabor energy filters (GEFs) and local binary patterns (LBPs). Recent work [1] suggests that adding a second layer of nonlinear filters on top of the first layer may be beneficial. However, it is unclear what is the best architecture of layers and selection of filters. In this paper, we present a thorough empirical analysis of the performance of single-layer and dual-layer texture-based approaches for action unit recognition. For the single hidden layer case, GEFs perform consistently better than LBPs, which may be due to their robustness to jitter and illumination noise as well as to their ability to encode texture at multiple resolutions. For dual-layer case, we confirm that, while small, the benefit of adding this second layer is reliable and consistent across data sets. Interestingly for this second layer, LBPs appear to perform better than GEFs.