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SummaryThe maturation of genomic surveillance in the past decade has enabled tracking of the emergence and spread of epidemics at an unprecedented level. During the COVID-19 pandemic, for example, genomic data revealed that local epidemics varied considerably in the frequency of SARS-CoV-2 lineage importation and persistence, likely due to a combination of COVID-19 restrictions and changing connectivity. Here, we show that local COVID-19 epidemics are driven by regional transmission, including across international boundaries, but can become increasingly connected to distant locations following the relaxation of public health interventions. By integrating genomic, mobility, and epidemiological data, we find abundant transmission occurring between both adjacent and distant locations, supported by dynamic mobility patterns. We find that changing connectivity significantly influences local COVID-19 incidence. Our findings demonstrate a complex meaning of ‘local’ when investigating connected epidemics and emphasize the importance of collaborative interventions for pandemic prevention and mitigation.

Ganglioneuromas are rare tumors that occur spontaneously or arise from a poorly differentiated neuroblastic tumor. Although they are typically described in the pediatric population, they can occur in adults. Ganglioneuromas are often discovered incidentally and their typical imaging appearance, although non-specific, is that of a well-defined solid mass. We are presenting a case of a fat-containing adrenal lesion in a 53-year-old male. The extensive lipomatous changes within the lesion led to the presumption that it represented an adrenal myelolipoma. Pathology revealed a ganglioneuroma with extensive lipomatous changes. This is an uncommon presentation of an adrenal ganglioneuroma mimicking an adrenal myelolipoma. The diagnosis of an adrenal ganglioneuroma raises the possibility of syndromic associations for which patients may undergo genetic testing. We provide a review of typical imaging features of an adrenal ganglioneuroma and provide insight into the situations in which a ganglioneuroma can be suggested as a diagnostic consideration.

Customized cationic oil-in-water nanoemulsions (NEs) have been produced to improve the bioavailability of poorly water-soluble drugs, such as triamcinolone acetonide (TA). TA is a synthetic glucocorticoid with anti-inflammatory and antiangiogenic therapeutic properties and it is widely used as an effective treatment in ocular disorders. In this work, TA-NEs were characterized using two different custom-made cationic surfactants, showing a high positive surface charge favouring corneal penetration and a particle size below 300 nm. Both TA-NE formulations demonstrated to be stable at 4 °C during the first months of storage. Furthermore, TA-NEs were able to produce antiangiogenic effects in chicken membranes. The TA-NEs safety profile was evaluated using in vitro and in vivo ocular tolerance tests. Out of the two formulations, the one showing no irritant effects was screened in vivo demonstrating capacity to ameliorate ocular inflammation in New Zealand rabbits significantly, specially to reduce the risk of ocular inflammation processes, with antiangiogenic activity, and can therefore be exploited as a suitable formulation to avoid inflammatory reactions upon ocular surgical procedures, such as cataracts., This work was funded by the Portuguese Science and Technology Foundation (FCT) from the Ministry of Science and Technology (MCTES) for the scholarship SFRH/BD/130555/2017 granted to A. R. Fernandes, and UIDB/04033/2020 (CITAB), co-funded by European Funds (PRODER/COMPETE) and FEDER, under the Partnership Agreement PT2020.

Oxidation of lipids and lipoproteins contributes to inflammation processes that promote the development of eye diseases. This is a consequence of metabolism dysregulation; for instance, that of the dysfunctional peroxisomal lipid metabolism. Dysfunction of lipid peroxidation is a critical factor in oxidative stress that causes ROS-induced cell damage. Targeting the lipid metabolism to treat ocular diseases is an interesting and effective approach that is now being considered. Indeed, among ocular structures, retina is a fundamental tissue that shows high metabolism. Lipids and glucose are fuel substrates for photoreceptor mitochondria; therefore, retina is rich in lipids, especially phospholipids and cholesterol. The imbalance in cholesterol homeostasis and lipid accumulation in the human Bruch's membrane are processes related to ocular diseases, such as AMD. In fact, preclinical tests are being performed in mice models with AMD, making this area a promising field. Nanotechnology, on the other hand, offers the opportunity to develop site-specific drug delivery systems to ocular tissues for the treatment of eye diseases. Specially, biodegradable nanoparticles constitute an interesting approach to treating metabolic eye-related pathologies. Among several drug delivery systems, lipid nanoparticles show attractive properties, e.g., no toxicological risk, easy scale-up and increased bioavailability of the loaded active compounds. This review analyses the mechanisms involved in ocular dyslipidemia, as well as their ocular manifestations. Moreover, active compounds as well as drug delivery systems which aim to target retinal lipid metabolism-related diseases are thoroughly discussed., The research was funded by the Fundação para a Ciência e a Tecnologia (FCT) for the scholarship 2020.04729.BD granted to Raquel da Ana. Eliana B. Souto acknowledges the national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB.

Rationale: Early, accurate diagnosis of interstitial lung disease (ILD) informs prognosis and therapy, especially in idiopathic pulmonary fibrosis (IPF). Current diagnostic methods are imperfect. High-resolution computed tomography has limited resolution, and surgical lung biopsy (SLB) carries risks of morbidity and mortality. Endobronchial optical coherence tomography (EB-OCT) is a low-risk, bronchoscope-compatible modality that images large lung volumes in vivo with microscopic resolution, including subpleural lung, and has the potential to improve the diagnostic accuracy of bronchoscopy for ILD diagnosis. Objectives: We performed a prospective diagnostic accuracy study of EB-OCT in patients with ILD with a low-confidence diagnosis undergoing SLB. The primary endpoints were EB-OCT sensitivity/specificity for diagnosis of the histopathologic pattern of usual interstitial pneumonia (UIP) and clinical IPF. The secondary endpoint was agreement between EB-OCT and SLB for diagnosis of the ILD fibrosis pattern. Methods: EB-OCT was performed immediately before SLB. The resulting EB-OCT images and histopathology were interpreted by blinded, independent pathologists. Clinical diagnosis was obtained from the treating pulmonologists after SLB, blinded to EB-OCT. Measurements and Main Results: We enrolled 31 patients, and 4 were excluded because of inconclusive histopathology or lack of EB-OCT data. Twenty-seven patients were included in the analysis (16 men, average age: 65.0 yr): 12 were diagnosed with UIP and 15 with non-UIP ILD. Average FVC and Dl(CO) were 75.3% (SD, 18.5) and 53.5% (SD, 16.4), respectively. Sensitivity and specificity of EB-OCT was 100% (95% confidence interval, 75.8–100.0%) and 100% (79.6–100%), respectively, for both histopathologic UIP and clinical diagnosis of IPF. There was high agreement between EB-OCT and histopathology for diagnosis of ILD fibrosis pattern (weighted κ: 0.87 [0.72–1.0]). Conclusions: EB-OCT is a safe, accurate method for microscopic ILD diagnosis, as a complement to high-resolution computed tomography and an alternative to SLB.

The eye is a very complex organ comprising several physiological and physical barriers that compromise drug absorption into deeper layers. Nanoemulsions are promising delivery systems to be used in ocular drug delivery due to their innumerous advantages, such as high retention time onto the site of application and the modified release profile of loaded drugs, thereby contributing to increasing the bioavailability of drugs for the treatment of eye diseases, in particular those affecting the posterior segment. In this review, we address the main factors that govern the development of a suitable nanoemulsion formulation for eye administration to increase the patient’s compliance to the treatment. Appropriate lipid composition and type of surfactants (with a special emphasis on cationic compounds) are discussed, together with manufacturing techniques and characterization methods that are instrumental for the development of appropriate ophthalmic nanoemulsions., This work was funded by the Portuguese Science and Technology Foundation (FCT) from the Ministry of Science and Technology (MCTES), European Social Fund (FSE) of the EU, for the scholarship SFRH/BD/130555/2017 granted to A. R. Fernandes, and for the projects UIDB/04469/2020 (CEB strategic fund) and UIDB/04033/2020 (CITAB), co-funded by European Funds (PRODER/COMPETE) and FEDER, under the Partnership Agreement PT2020., info:eu-repo/semantics/publishedVersion

We report on the stellar occultation by (523764) 2014 WC510 observed on 2018 December 1 UT. This occultation campaign was part of the Research and Education Collaborative Occultation Network (RECON), a network of small telescopes spread over 2000 km in western USA and Canada. Light curves from six stations revealed three groups of two or more consecutive flux drops correlated in time between adjacent stations. A Bayesian model comparison reveals that a model with a double object occulting a double star is favored over alternative models considered. For the statistically favored model, we determined that the primary component of the object has a diameter d p = 181 ± 16 km and the secondary d s = 138 ± 32 km, assuming identical geometric albedo between the two components. The two components have a projected separation of 349 ± 26 km. Adopting an absolute magnitude for the system of H V = 7.2 from the Minor Planet Center, we derive a geometric albedo of p V = 5.1% ± 1.7%. This is the smallest resonant object with an occultation size measurement and with a detected secondary from a ground-based stellar occultation, filling a region of the size versus separation parameter space of binary objects that is largely unexplored. The results show the capabilities of the unique design of the RECON experiment sensitive to small objects and close binaries. 2014 WC510 is presently at a low galactic latitude where the high surface density of stars will provide good occultation opportunities in the upcoming years.

Age-related macular degeneration (AMD) is defined as a degenerative, progressive and multifactorial disorder that affects the macula with a complex etiology. The retinal pigment epithelium is a monolayer of cells that has the function to separate the surface of the choroid from the neural retina that is involved in the signal transduction leading to vision. The blood-aqueous barrier and the blood retinal barrier limit the permeation of drugs into the retina and thereby reducing their efficacy. Triamcinolone acetonide (TA) is widely used as anti-inflammatory and immunomodulatory drug that promotes the inhibition of the inflammatory processes. The factors that stimulate or inhibit angiogenesis in AMD create a local balance that is responsible for the growth of sub-retinal neovascularization. In AMD, the main angiogenic stimulus is the vascular endothelial growth factor (VEGF). In this work, nanoemulsions with cationic surfactants (mono- and dicationic DABCO and quinuclidine) were produced to deliver TA, and were found to reduce the production of tumor necrosis factor alpha (TNF-), which stimulates the choroidal neovascularization development by upregulating the VEGF production, and consequently decreased the VEGF levels. Our results support the potential use of mono- and dicationic DABCO and quinuclidine-based cationic nanoemulsions for the delivery of TA in the treatment of AMD., This work was funded by the Portuguese Science and Technology Foundation (FCT) from the Ministry of Science and Technology (MCTES) for the scholarship SFRH/BD/130555/2017 granted to A. R. Fernandes, and for the projects UIDB/04469/2020 (CEB strategic fund) and UIDB/04033/2020 (CITAB), co-funded by European Funds (PRODER/COMPETE) and FEDER, under the Partnership Agreement PT2020., info:eu-repo/semantics/publishedVersion

Background The aim of this study was to determine the relative frequency of primary cutaneous lymphoma (PCL) in Argentina according to the new World Health Organization (WHO)-European Organization for the Research and Treatment of Cancer (EORTC) classification system. Methods A total of 416 patients from 21 dermatology services were included during a 5-year period (2010-2015); these patients were classified using WHO-EORTC criteria. Results There were 231 (55.2%) males and 185 (44.8%) females; the male-to-female ratio was 1.35. The median age of the patients was 57 years (range, 0-90 years). Most patients were Caucasian (79%), and only 16% of patients were registered as Amerindian. Most patients (387/416, 93%) had cutaneous T-cell lymphoma (CTCL); 28 patients (6.7%) were diagnosed with cutaneous B-cell lymphoma (CBCL). The most frequent CTCL subtypes, in decreasing order of prevalence, were mycosis fungoides (MF), including its variants (75.7%); CD30+ primary cutaneous lymphoproliferative disorders (7.2%); and Sezary syndrome (SS) (3.1%). Cutaneous follicle center lymphoma was the most common CBCL subtype (2.9%). In the subset of patients ≤20 years of age, the most common condition was MF (57%), followed by extranodal NK-T nasal-type lymphoma (14%). Conclusions This study revealed relatively higher rates of MF and lower rates of CBCL in Argentinean patients that have been reported in American and European countries.

We present observations of five stellar occultations for (11351) Leucus and reports from two efforts on (21900) Orus. Both objects are prime mission candidate targets for the Lucy Discovery mission. Combined results for Leucus indicate a very dark surface with p V = 0.037 ± 0.001, which is derived from the average of the multichord occultations. Our estimate of the triaxial ellipsoidal shape is for axial diameters of 63.8 × 36.6 × 29.6 km assuming that the spin pole is normal to the line of sight. The actual shape of the object is only roughly elliptical in profile at each epoch. Significant topography is seen with horizontal scales up to 30 km and vertical scales up to 5 km. The most significant feature is a large depression on the southern end of the object as seen from a terrestrial viewpoint. For this work we developed a method to correct for differential refraction, accounting for the difference in color between the target object and the reference stars for astrometry derived from ground-based images.

Full list of authors: Souami, D.; Braga-Ribas, F.; Sicardy, B.; Morgado, B.; Ortiz, J. L.; Desmars, J.; Camargo, J. I. B.; Vachier, F.; Berthier, J.; Carry, B.; Anderson, C. J.; Showers, R.; Thomason, K.; Maley, P. D.; Thomas, W.; Buie, M. W.; Leiva, R.; Keller, J. M.; Vieira-Martins, R.; Assafin, M.; Santos-Sanz, P.; Morales, N.; Duffard, R.; Benedetti-Rossi, G.; Gomes-Júnior, A. R.; Boufleur, R.; Pereira, C. L.; Margoti, G.; Pavlov, H.; George, T.; Oesper, D.; Bardecker, J.; Dunford, R.; Kehrli, M.; Spencer, C.; Cota, J. M.; Garcia, M.; Lara, C.; McCandless, K. A.; Self, E.; Lecacheux, J.; Frappa, E.; Dunham, D.; Emilio, M. .--Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., Context. We present results from the first recorded stellar occultation by the large trans-Neptunian object (174567) Varda that was observed on September 10, 2018. Varda belongs to the high-inclination dynamically excited population, and has a satellite, Ilmarë, which is half the size of Varda. Aims. We determine the size and albedo of Varda and constrain its 3D shape and density. Methods. Thirteen different sites in the USA monitored the event, five of which detected an occultation by the main body. A best-fitting ellipse to the occultation chords provides the instantaneous limb of the body, from which the geometric albedo is computed. The size and shape of Varda are evaluated, and its bulk density is constrained using Varda's mass as is known from previous works. Results. The best-fitting elliptical limb has semi-major (equatorial) axis of (383 ± 3) km and an apparent oblateness of 0.066 ± 0.047, corresponding to an apparent area-equivalent radius R′equiv = (370±7) km and geometric albedo pv = 0.099 ± 0.002 assuming a visual absolute magnitude HV = 3.81 ± 0.01. Using three possible rotational periods for the body (4.76, 5.91, and 7.87 h), we derive corresponding MacLaurin solutions. Furthermore, given the low-amplitude (0.06 ± 0.01) mag of the single-peaked rotational light-curve for the aforementioned periods, we consider the double periods. For the 5.91 h period (the most probable) and its double (11.82 h), we find bulk densities and true oblateness of ρ = (1.78 ± 0.06) g cm-3, ϵ = 0.235 ± 0.050, and ρ = (1.23 ± 0.04) g cm-3, ϵ = 0.080 ± 0.049. However, it must be noted that the other solutions cannot be excluded just yet. © D. Souami et al. 2020., This campaign was carried out within the "Lucky Star" umbrella that agglomerates the efforts of the Paris, Granada and Rio teams. It is funded by the European Research Council under the European Community's H2020 (2014-2020/ERC Grant Agreement No. 669416). The following authors acknowledge the respective CNPq grants: F.B.-R. 309578/2017-5; R.V.M. 304544/2017-5, 401903/2016-8; J.I.B.C. 308150/2016-3 and 305917/2019-6; M.A. 427700/2018-3, 310683/2017-3, 473002/2013-2. This study was financed in part by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior -Brasil (CAPES) -Finance Code 001 and the National Institute of Science and Technology of the e-Universe project (INCT do e-Universo, CNPq grant 465376/2014-2). G.B.R. acknowledges CAPES-FAPERJ/PAPDRJ grant E26/203.173/2016, MA FAPERJ grant E-26/111.488/2013 and ARGJr FAPESP grant 2018/11239-8. J.L.O., P.S.-S., N.M., and R.D. acknowledge financial support from the State Agency for Research of the Spanish MCIU through the "Center of Excellence Severo Ochoa" award for the Instituto de Astrofisica de Andalucia (SEV-2017-0709). P.S.-S. acknowledges financial support by the Spanish grant AYA-RTI2018-098657-J-I00 "LEO-SBNAF" (MCIU/AEI/FEDER, UE). Observations from the RECON network were provided by students, teachers, and community members, including Xavier Banaga, Jesus Bustos, Amanda Carrillo, Dorey W. Conway, Kenneth Conway, Danielle D. Laguna, Andrew E. McCandless, Kaitlin McArdle, and Jared T. White, Jr. The observers listed in this paper are but a small fraction of the total RECON network and their dedication to this project is deeply appreciated. Funding for RECON was provided by grants from NSF AST-1413287, AST-1413072, AST-1848621, and AST-1212159. This work has made use of data from the European Space Agency (ESA) mission Gaia (https://www.cosmos.esa.int/gaia), processed by the Gaia Data Processing and Analysis Consortium (DPAC, https://www.cosmos.esa.int/web/gaia/dpac/consortium).Funding for the DPAC has been provided by national institutions, in particular the institutions participating in the Gaia Multilateral Agreement. This research has made use of the VizieR catalogue access tool, CDS, Strasbourg, France (DOI: 10.26093/cds/vizier). The original description of the VizieR service was published in Ochsenbein et al. (2000).

e21174 Background: Mutations (mt) in the KRAS gene are common oncogenic drivers in advanced NSCLC. The KRAS mt subtypes and certain co-mutations (co-mt) have prognostic and predictive implications. New drugs promise to uniquely target KRAS G12C, raising interest in studying the unique clinical and molecular characteristics of this subtype. Methods: We retrospectively identified 56 KRAS mt advanced NSCLC patients (pts) that underwent Next-Generation Sequencing (NGS) from 06/2015 to 12/2020. We used a commercial NGS panel that tested for > 300 genomic alterations, including substitutions, insertions, deletions and copy number alterations. For statistical analysis, we divided the patients into G12C and non-G12C groups. Results: In our cohort of 56 KRAS mt pts, median age was 67 (range: 58-74) with a predominance of females (63%) and heavy smokers (89%). KRAS G12C was the most common subtype 38%; G12V 14%, G12D 11%, G12S 11%, G13D 7% and others < 5% each. G12C, G12D and G13D groups had a higher proportion of PD-L1+ tumors (84%, 100%, and 100% respectively, p = 0.02) compared to other KRAS subtypes. Pts in G12C group were on average older (median age 71 vs 61, p = 0.02) than non-G12C. Most frequent co-mt in G12C were TP53 (33%), STK11 (29%), TET2 (19%), RB1 (14%), CDKN2A/B (14%) , MCL1 (14%) and ASXL1 (14%) ; for non-G12C, they were TP53 (54%), CDKN2A/B (37%), STK11 (34%) and RBM10 (17%). CDKN2A/B co-mt (37% vs 14%, p = 0.08) was significantly more frequent in non-G12C group and TET2 in G12C (19% vs 0%, p = 0.016). Non-G12C group more frequently had high TMB (17% vs 0%, p = 0.07) compared to G12C. No difference in survival was seen between G12C and non-G12C groups. We observed no difference in PFS (p = 0.31) or OS (p = 0.64) between smokers and no/light-smokers with KRAS mt. Co-mt with KEAP1 and SMARCA4 were significantly associated with survival in KRAS mt. Compared to KRAS+/KEAP1wt, KRAS +/ KEAP1+ pts had poor PFS (median 1.1 vs 7.5 m, p < 0.0001) and OS (1.1 vs. 27.8 m, p < 0.0001) measured from start of initial therapy. KRAS+/SMARCA4+ had worse PFS (1.0 vs 6.9 m, p < 0.0001) and OS (1.4 vs. 27.8, p = 0.0001) compared to KRAS+/SMARCA4wt. KRAS mt pts with STK11/KEAP1 that were treated with immunotherapy-based regimens had shorter PFS (1.1 vs 7.6 m, p = 0.001) and OS (1.4 vs 90.9 m, p = 0.0007) compared to those treated with chemotherapy alone. Conclusions: Our data shows that pts with KRAS co-mt with STK11/KEAP1 had worse PFS and OS with the addition of immunotherapy compared to chemotherapy alone, highlighting the potential implications of these co-mt patterns on treatment outcomes. The types of co-mts are similar between KRAS G12C and non-G12C, with the exception of CDKN2A/B (less likely) and TET2 (more likely). Larger data sets are warranted to confirm our observations and determine if these co-mts may create a predictive model for individualized therapy for KRAS mt pts, potentially independent of current predictive markers.

Following the discovery of small molecule acyl piperazine ROMK inhibitors, the acyl octahydropyrazino[2,1-c][1,4]oxazine series was identified. This series displays improved ROMK/hERG selectivity, and as a consequence, the resulting ROMK inhibitors do not evoke QTc prolongation in an in vivo cardiovascular dog model. Further efforts in this series led to the discovery of analogs with improved pharmacokinetic profiles. This new series also retained comparable ROMK potency compared to earlier leads.

ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.

Pathologic diagnosis of cervical neoplasia incorporates the practice of cytopathology, molecular pathology, and surgical pathology. Cervical pathology includes preinvasive squamous lesions (squamous intraepithelial lesions/SIL), invasive squamous cell carcinoma, adenocarcinoma in situ, invasive adenocarcinoma, as well as other less common primary epithelial and mesenchymal tumors. The most common cervical lesions are human papillomavirus (HPV)-associated squamous lesions. A two-tiered diagnostic approach classifying preinvasive lesions into low-grade and high-grade squamous intraepithelial lesions is used in cytopathology and surgical pathology. Low-grade squamous intraepithelial lesions (LSIL), which include condylomata, are the result of productive HPV infection that may be transient and regress. High-grade squamous intraepithelial lesions (HSIL) have a greater risk of progression to invasive carcinoma and require further treatment. Adenocarcinoma in situ, a precursor to invasive adenocarcinoma, is also HPV-associated and may coexist with squamous lesions. HPV testing/genotyping has been incorporated into the Papanicolaou cytology screening and helps stratify patients into those needing further evaluation. The incidence of invasive carcinoma has decreased with the widespread use of cytology testing. A subset of tumors not associated with HPV infection, while rare, may present diagnostic challenges, especially in cytology and small biopsies.

This paper describes the pathway towards the realisation of a 5G Facility that will allow the validation of the major 5G Key Performance Indicators (KPIs). It reflects the approach that the 5GENESIS consortium will adopt in this direction. More precisely, it describes the key design principles of such Facility as well as the targeted use cases for the KPIs validation. The adopted approach for the Facility realisation includes the design of a common implementation blueprint that will be instantiated in five Platforms distributed across Europe. To maximise the diversity and the efficiency of the Facility, complementary performance objectives have been selected for the Platforms, while specific characteristics from different vertical industries have been allocated to each of them.

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.

Mitochondrial organization is often altered to accommodate cellular bioenergetic and biosynthetic demands. Changes in metabolism are a hallmark of a number of diseases, including cancer; however, the interdependence between mitochondrial metabolic function and organization is not well understood. Here, we present a noninvasive, automated and quantitative method to assess mitochondrial organization in three-dimensional (3D) tissues using exclusively endogenous two-photon excited fluorescence (TPEF) and show that mitochondrial organization reflects alterations in metabolic activities. Specifically, we examine the organization of mitochondria within live, engineered epithelial tissue equivalents that mimic normal and precancerous human squamous epithelial tissues. We identify unique patterns of mitochondrial organization in the different tissue models we examine, and we attribute these to differences in the metabolic profiles of these tissues. We find that mitochondria are clustered in tissues with high levels of glycolysis and are more highly networked in tissues where oxidative phosphorylation is more dominant. The most highly networked organization is observed within cells with high levels of glutamine consumption. Furthermore, we demonstrate that mitochondrial organization provides complementary information to traditional morphological hallmarks of cancer development, including variations in nuclear size. Finally, we present evidence that this automated quantitative analysis of endogenous TPEF images can identify differences in the mitochondrial organization of freshly excised normal and pre-cancerous human cervical tissue specimens. Thus, this method could be a promising new modality to assess the role of mitochondrial organization in the metabolic activity of 3D tissues and could be further developed to serve as an early cancer clinical diagnostic biomarker.

The kidney plays a critical role in blood pressure homeostasis as a result of the integrated activity of different mechanisms that ensure proper salt and water reabsorption. Diuretics, developed more than four decades ago, are used to treat hypertension and/or congestive heart failure, although there are therapeutic issues that limit their use. Human and rodent genetic studies provide a large body of evidence which suggests that inhibitors of the kidney potassium channel, ROMK, will represent novel diuretics for the treatment of hypertension. The search for potent and selective ROMK inhibitors has recently yielded compounds that display efficacy in animal models, providing the first pharmacological validation of ROMK as a novel diuretic target.

A sub-class of distinct small molecule ROMK inhibitors were developed from the original lead 1. Medicinal chemistry endeavors led to novel ROMK inhibitors with good ROMK functional potency and improved hERG selectivity. Two of the described ROMK inhibitors were characterized for the first in vivo proof-of-concept biology studies, and results from an acute rat diuresis model confirmed the hypothesis that ROMK inhibitors represent new mechanism diuretic and natriuretic agents.

A series of benzazepinones were synthesized and evaluated for block of Nav1.7 sodium channels. Compound 30 from this series displayed potent channel block, good selectivity versus other targets, and dose-dependent oral efficacy in a rat model of neuropathic pain.