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Pseudomonas aeruginosa is an increasingly antibiotic-resistant pathogen that causes severe lung infections, burn wound infections, and diabetic foot infections. P. aeruginosa produces the siderophore pyochelin through the use of a non-ribosomal peptide synthetase (NRPS) biosynthetic pathway. Targeting members of siderophore NRPS proteins is one avenue currently under investigation for the development of new antibiotics against antibiotic-resistant organisms. Here, the crystal structure of the pyochelin adenylation domain PchD is reported. The structure was solved to 2.11 Å when co-crystallized with the adenylation inhibitor 5′-O-(N-salicylsulfamoyl)adenosine (salicyl-AMS) and to 1.69 Å with a modified version of salicyl-AMS designed to target an active site cysteine (4-cyano-salicyl-AMS). In the structures, PchD adopts the adenylation conformation, similar to that reported for AB3403 from Acinetobacter baumannii.Graphical abstract

Most small molecules are unable to rapidly traverse the outer membrane of Gram-negative bacteria and accumulate inside these cells, making the discovery of much-needed drugs against these pathogens challenging. Current understanding of the physicochemical properties that dictate small-molecule accumulation in Gram-negative bacteria is largely based on retrospective analyses of antibacterial agents, which suggest that polarity and molecular weight are key factors. Here we assess the ability of over 180 diverse compounds to accumulate in Escherichia coli . Computational analysis of the results reveals major differences from the retrospective studies, namely that the small molecules that are most likely to accumulate contain an amine, are amphiphilic and rigid, and have low globularity. These guidelines were then applied to convert deoxynybomycin, a natural product that is active only against Gram-positive organisms, into an antibiotic with activity against a diverse panel of multi-drug-resistant Gram-negative pathogens. We anticipate that these findings will aid in the discovery and development of antibiotics against Gram-negative bacteria. The authors use computational modelling and a set of chemically synthesized compounds to define the physicochemical properties required for small-molecule accumulation in Gram-negative bacteria. Rules for small-molecule accumulation in Gram-negative bacteria Most small molecules are unable to cross the outer membrane of Gram-negative bacteria and accumulate inside these cells, which poses a challenge for the discovery of new drugs that target Gram-negative pathogens. By examining a set of chemically diverse small molecules, Paul Hergenrother and colleagues have now defined the physicochemical properties required for small-molecule accumulation in the Gram-negative bacteria Escherichia coli . They find that small molecules containing an amine, and which are amphiphilic, rigid and have low globularity, are most likely to be successful. They then apply these guidelines to convert a compound that targets Gram-positive bacteria only into a broad-spectrum antibiotic that is active against several Gram-negative pathogens.

Most small molecules are unable to rapidly traverse the outer membrane of Gram-negative bacteria and accumulate inside these cells, making the discovery of much-needed drugs for these pathogens very challenging. Current understanding of the physicochemical properties that dictate small-molecule accumulation in Gram-negatives is largely based on retrospective analyses of antibacterials that suggest polarity and molecular weight as key factors. Here we assess the ability of over 180 diverse compounds to accumulate in Escherichia coli. Computational analysis of the results reveals major differences from the retrospective studies, namely that the small molecules that are most likely to accumulate contain an amine, are amphiphilic and rigid, and have low globularity. These guidelines were then applied to convert deoxynybomycin, a natural product that is active only against Gram-positive organisms, into an antibiotic with activity against a diverse panel of multi-drug-resistant Gram-negative pathogens. We anticipate these findings will aid in the discovery and development of antibiotics effective against Gram-negative bacteria.

The US International Trade Commission (ITC) issued an opinion holding that a patent owner failed to satisfy the domestic-industry requirement for two out of three of its asserted patents. The ITC found at least one claim valid and infringed for all three asserted patents, but it also found invalid claims relied on for domestic industry for two of the patents. In reaching its decision, the ITC ignored its past practice of allowing a domestic industry based on an invalid claim. This article, by Justin Hendrix, P. Andrew Riley, and Christine Lehman of Finnegan, Henderson, Farabow, Garrett & Dunner, LLP, discusses the decision and its implications. [PUBLICATION ABSTRACT]

The number of individuals affected by a central nervous system disorder continues to steadily increase. Unfortunately, in many cases the available therapeutic options leaves these diseases undertreated and additional molecular probes are needed to fully understand these conditions. Historically, natural products have served as a rich source of new molecular scaffolds for developing these probes due to their complex structures and unique ability to perturb biological pathways through diverse mechanisms. With this in mind, the kappa opioid receptor (KOR) agonist salvinorin A, the mu opioid receptor (MOR) agonist herkinorin, and the anti-tau diarylheptanoid myricanol were identified as potential probes for investigating substance abuse, pain, and Alzheimer's Disease, respectively. To determine how the unique neoclerodane structure of salvinorin A interacts with the KOR, methods to chemical modify the steric and electronic properties of the furan ring were developed. The resulting structure-activity relationships (SAR) identified three compounds that successfully attenuated drug seeking behavior in an animal model of drug relapse. In a separate study, a simple modification to the A-ring of herkinorin was found to drastically increase the potency and selectivity for the MOR, thus increasing the potential in vivo utility of the probe. Additional SAR studies also resulted in the first sub-nanomolar diterpene MOR agonist. Finally, an enantioselective route to both enantiomers of myricanol was developed to provide the necessary material for more extensive biological investigations into the compound's tau degradation mechanism. The three studies described herein, highlight the importance of using chemical synthesis to modify and build complex natural products scaffolds in order to probe biological systems.

Akuammicine (1), an indole alkaloid isolated from the seeds of Picralima nitida, is a selective agonist of the kappa opioid receptor (κOR). To establish structure-activity relationships (SAR) for this structurally unique κOR ligand, a collection of 26 semisynthetic derivatives of 1 were synthesized. Evaluating these derivatives for their ability to activate the κOR and mu opioid receptor (µOR) revealed key SAR trends and identified derivatives with enhanced κOR potency. Most notably, substitutions to the C10 position of the aryl ring led to a >200-fold improvement in κOR potency and nearly complete selectivity for the κOR over other CNS receptor targets. Activation of the κOR by these analogues also results in the recruitment of β-Arrestin-2, indicating they are balanced agonists and have distinct signaling properties from other recently identified κOR agonists. The discovery of these κOR agonists underscores the potential of using natural products to identify new classes of highly potent and selective ligands and provides new pharmacology tools to probe the κOR.

Growing evidence suggests that inhibition of the α3β4 nicotinic acetylcholine receptor (nAChR) represents a promising therapeutic strategy to treat cocaine use disorder. Recently, aristoquinoline (1), an alkaloid from Aristotelia chilensis, was identified as an α3β4-selective nAChR inhibitor. Here, we prepared 22 derivatives of 1 and evaluated their ability to inhibit the α3β4 nAChR. These studies revealed structure-activity trends and several compounds with increased potency compared to 1 with few off-target liabilities. Additional mechanistic studies indicated that these compounds inhibit the α3β4 nAChR non-competitively, but do not act as channel blockers, suggesting they are negative allosteric modulators. Finally, using a cocaine-primed reinstatement paradigm, we demonstrated that 1 significantly attenuates drug-seeking behavior in an animal model of cocaine relapse. The results from these studies further support a role for the α3β4 nAChR in the addictive properties of cocaine and highlight the possible utility of aristoquinoline derivatives in treating cocaine use disorder.

The application process is relatively simple. Not every party who has litigated against the United States is entitled to an award of attorney fees and expenses. The first qualification is success on the merits. EAJA authorizes a \"prevailing party\" to recover attorney fees from the government, unless the court finds the government's position \"was substantially justified.\" 28 U.S.C. § 2412(d)(1)(A). An applicant is a prevailing party for EAJA purposes if that party established \"entitlement to some relief on the merits of his claims, either in the trial court or on appeal.\" Hanrahan v. Hampton, 446 U.S. 754, 757 (1980). An appellant attains prevailing party status when the appeal achieves \"some form of 'judicially sanctioned change in the legal relationship of the parties.'\" Cycholl v. Principi, 15 Vet. App. 355, 357 (2001) (quoting Sumner v. Principi, 15 Vet. App. 256, 260-61 (2001)). An appellant is a prevailing party in a case \"where the plaintiff secures a remand requiring further agency proceedings because of alleged error by the agency . .. without regard to the outcome of the agency proceedings where there has been no retention of jurisdiction by the court.\" Haipern v. Principi, 384 F.3d 1297, 1306 (Fed. Cir. 2004) (quoting Former Emps. of Motorola Ceramic Prods, v. United States, 336 F.3d 1360, 1366 (Fed. Cir. 2003)). To apply for EAJA fees, you must file an application and a supporting affidavit by lead counsel within 30 days after judgment in the action becomes final. 28 U.S.C. § 2412(d)(1)(B). When those 30 days begin to run depends on several factors. 38 U.S.C. § 7291. In the case of a JMR, the CAVC judgment is final on the date the court grants the joint motion. Bowers v. Brown, 8 Vet. App. 25, 27 (1995) (holding that in the case of a JMR, no separate judgment will be entered, and the 30-day period for filing an EAJA application \"commences to run the day following the entry of the order which 'constitutes the mandate' under Rule 41(b)\"). The timing of the EAJA application is significantly different when the CAVC decides the appeal in either a memorandum, i.e., a one-judge, or a panel decision. In either circumstance, the judgment will issue 21 days after the date of the decision, if no motion for reconsideration, rehearing, panel decision, or en banc review is filed. See U.S. Vet. App. R. 35. Entry of judgment begins the 60-day period during which either party may appeal to the Federal Circuit. U.S. Vet. App. R. 36. When the 60-day appeal period expires and neither party has appealed, only then does the 30-day period for filing a fee application begin to run. If there is an appeal to the Federal Circuit or a petition for certiorari to the Supreme Court, the application must await conclusion of those proceedings. 38 U.S.C. § 7292. No extensions of time are available for submitting an EAJA application. If fees are claimed for the work of more than one person, all time must be integrated chronologically. U.S. Vet. App. R. 39(f). There is no requirement that a lawyer have entered an appearance in the appeal in order to recover fees for time expended by that lawyer. If you chose to deduct or reduce time expended on the appeal, you should list that time in a column adjacent to the related time entry. Time expended but shown to have been deducted or reduced helps establish that you exercised \"billing judgment\" in claiming fees from the government and that the amount claimed is \"reasonable.\" See 28 U.S.C. § 2412(b) (providing that \"a court may award reasonable fees\"); Baldridge v. Nicholson, 19 Vet. App. 227, 234 (2005) (citing Hensley v. Eckerhart, 461 U.S. 424, 434 (1983)) (\"[A]n applicant for EAJA fees must treat the government as though it were a private client by exercising the same kind of billing judgment that an attorney would use in preparing a billing statement for a private client.\"). Time spent prepare ing and seeking an extension of time due to your needs is not reimbursable under the EAJA. See Hensley v. Principi, 16 Vet. App. 491, 499 (2002) (reducing attorneytime hours where, \"had the appellant's counsel more efficiently managed his workload,\" extensions of time might not have been necessary). Hensley v. Principi suggests that extensions shown to have resulted from the appellee's conduct may be reimbursable. See id. Likewise, secretarial time does not qualify for reimbursement. The CPI that should be used for an EAJA fee application can be found on the website for the Bureau of Labor Statistics (BLS) at www.bls.gov. On the BLS website, select \"Subjects,\" then \"Consumer Price Index,\" and finally \"CPI Databases.\" Then select \"Top Picks\" in the row labeled \"All Urban Consumers.\" On the next page, select your area and press \"Retrieve Data.\" To find the appropriate CPI for your calculation, select the rate listed under the month and year you prepared the opening brief or JMR. This will be considered the midpoint for the fee calculation. If no CPI is listed for that month, use the CPI from the closest prior month. For example, if you are located in the San Francisco area and you wrote the opening brief in March 2013, your CPI is 242.677 from the CPI listed under February 2013. Next determine the CPI for March 1996 by going to \"Change Output Options\" near the top of the page, selecting 1996 in the \"From\" dropdown box, and pressing the blue \"GO\" button. Find the CPI for March 1996 from the page then displayed (152.9 for the San Francisco area). With this information, you can calculate the cost-of-living adjusted hourly rate for the fee application using the following formula:

Magnetic resonance imaging (MRI) of the knee is the preferred method for diagnosing knee injuries. However, interpretation of knee MRI is time-intensive and subject to diagnostic error and variability. An automated system for interpreting knee MRI could prioritize high-risk patients and assist clinicians in making diagnoses. Deep learning methods, in being able to automatically learn layers of features, are well suited for modeling the complex relationships between medical images and their interpretations. In this study we developed a deep learning model for detecting general abnormalities and specific diagnoses (anterior cruciate ligament [ACL] tears and meniscal tears) on knee MRI exams. We then measured the effect of providing the model's predictions to clinical experts during interpretation. Our dataset consisted of 1,370 knee MRI exams performed at Stanford University Medical Center between January 1, 2001, and December 31, 2012 (mean age 38.0 years; 569 [41.5%] female patients). The majority vote of 3 musculoskeletal radiologists established reference standard labels on an internal validation set of 120 exams. We developed MRNet, a convolutional neural network for classifying MRI series and combined predictions from 3 series per exam using logistic regression. In detecting abnormalities, ACL tears, and meniscal tears, this model achieved area under the receiver operating characteristic curve (AUC) values of 0.937 (95% CI 0.895, 0.980), 0.965 (95% CI 0.938, 0.993), and 0.847 (95% CI 0.780, 0.914), respectively, on the internal validation set. We also obtained a public dataset of 917 exams with sagittal T1-weighted series and labels for ACL injury from Clinical Hospital Centre Rijeka, Croatia. On the external validation set of 183 exams, the MRNet trained on Stanford sagittal T2-weighted series achieved an AUC of 0.824 (95% CI 0.757, 0.892) in the detection of ACL injuries with no additional training, while an MRNet trained on the rest of the external data achieved an AUC of 0.911 (95% CI 0.864, 0.958). We additionally measured the specificity, sensitivity, and accuracy of 9 clinical experts (7 board-certified general radiologists and 2 orthopedic surgeons) on the internal validation set both with and without model assistance. Using a 2-sided Pearson's chi-squared test with adjustment for multiple comparisons, we found no significant differences between the performance of the model and that of unassisted general radiologists in detecting abnormalities. General radiologists achieved significantly higher sensitivity in detecting ACL tears (p-value = 0.002; q-value = 0.019) and significantly higher specificity in detecting meniscal tears (p-value = 0.003; q-value = 0.019). Using a 1-tailed t test on the change in performance metrics, we found that providing model predictions significantly increased clinical experts' specificity in identifying ACL tears (p-value < 0.001; q-value = 0.006). The primary limitations of our study include lack of surgical ground truth and the small size of the panel of clinical experts. Our deep learning model can rapidly generate accurate clinical pathology classifications of knee MRI exams from both internal and external datasets. Moreover, our results support the assertion that deep learning models can improve the performance of clinical experts during medical imaging interpretation. Further research is needed to validate the model prospectively and to determine its utility in the clinical setting.

Dehydrins, plant proteins that are upregulated during dehydration stress conditions, have modular sequences that can contain three conserved motifs (the Y-, S-, and K-segments). The presence and order of these motifs are used to classify dehydrins into one of five architectures: Kn, SKn, KnS, YnKn, and YnSKn, where the subscript n describes the number of copies of that motif. In this study, an architectural and phylogenetic analysis was performed on 426 dehydrin sequences that were identified in 53 angiosperm and 3 gymnosperm genomes. It was found that angiosperms contained all five architectures, while gymnosperms only contained Kn and SKn dehydrins. This suggests that the ancestral dehydrin in spermatophytes was either Kn or SKn, and the Y-segment containing dehydrins first arose in angiosperms. A high-level split between the YnSKn dehydrins from either the Kn or SKn dehydrins could not be confidently identified, however, two lower level architectural divisions appear to have occurred after different duplication events. The first likely occurred after a whole genome duplication, resulting in the duplication of a Y3SK2 dehydrin; the duplicate subsequently lost an S- and K- segment to become a Y3K1 dehydrin. The second split occurred after a tandem duplication of a Y1SK2 dehydrin, where the duplicate lost both the Y- and S- segment and gained four K-segments, resulting in a K6 dehydrin. We suggest that the newly arisen Y3K1 dehydrin is possibly on its way to pseudogenization, while the newly arisen K6 dehydrin developed a novel function in cold protection.