Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

The adaptive roles of aneuploidy and polyclonality in Leishmania in response to environmental stress.

Aneuploidy is generally considered harmful, but in some microorganisms, it can act as an adaptive mechanism against environmental stress. Here, we use Leishmania-a protozoan parasite with remarkable genome plasticity-to study the early steps of aneuploidy evolution under high drug pressure (using antimony or miltefosine as stressors). By combining single-cell genomics, lineage tracing with cellular barcodes, and longitudinal genome characterization, we reveal that aneuploidy changes under antimony pressure result from polyclonal selection of pre-existing karyotypes, complemented by further and rapid de novo alterations in chromosome copy number along evolution. In the case of miltefosine, early parasite adaptation is associated with independent point mutations in a miltefosine transporter gene, while aneuploidy changes only emerge later, upon exposure to increased drug levels. Therefore, polyclonality and genome plasticity are hallmarks of parasite adaptation, but the scenario of aneuploidy dynamics depends on the nature and strength of the environmental stress as well as on the existence of other pre-adaptive mechanisms.

Modulation of the Excited States of Ruthenium(II)-perylene Dyad to Access Near-IR Luminescence, Long-Lived Perylene Triplet State and Singlet Oxygen Photosensitization.

Herein, we present a novel ruthenium(II)-perylene dyad (RuPDI-Py) that combines the photophysical properties of pyrrolidine-substituted perylene diimide (PDI-Py) and the ruthenium(II) polypyridine complex [Ru(phen)3]2+. A comprehensive study of excited-state dynamics was carried out using time-resolved and steady-state methods in a dimethyl sulfoxide solution. The RuPDI-Py dyad demonstrated excitation wavelength-dependent photophysical behavior. Upon photoexcitation above 600 nm, the dyad exclusively exhibits the near-infrared (NIR) fluorescence of the 1PDI-Py state at 785 nm (τfl = 1.50 ns). In contrast, upon photoexcitation between 350 and 450 nm, the dyad also exhibits a photoinduced electron transfer from the {[Ru(phen)3]2+} moiety to PDI-Py, generating the charge-separated intermediate state {Ru(III)-(PDI-Py)•-} (4 µs). This state subsequently decays to the long-lived triplet excited state 3PDI-Py (36 µs), which is able to sensitize singlet oxygen (1O2). Overall, tuning 1O2 photoactivation or NIR fluorescence makes RuPDI-Py a promising candidate for using absorbed light energy to perform the desired functions in theranostic applications.

High throughput single-cell genome sequencing gives insights into the generation and evolution of mosaic aneuploidy in Leishmania donovani.

Leishmania, a unicellular eukaryotic parasite, is a unique model for aneuploidy and cellular heterogeneity, along with their potential role in adaptation to environmental stresses. Somy variation within clonal populations was previously explored in a small subset of chromosomes using fluorescence hybridization methods. This phenomenon, termed mosaic aneuploidy (MA), might have important evolutionary and functional implications but remains under-explored due to technological limitations. Here, we applied and validated a high throughput single-cell genome sequencing method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two clonal populations of Leishmania promastigotes representing different stages of MA evolution in vitro. We found that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid founder cell. This possibly involves polyploidization/hybridization at an early stage of population expansion, followed by assorted ploidy reduction. During further stages of expansion, MA increases by moderate and gradual karyotypic alterations, affecting a defined subset of chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies.

High SARS-CoV-2 tropism and activation of immune cells in the testes of non-vaccinated deceased COVID-19 patients.

BACKGROUND: Cellular entry of SARS-CoV-2 has been shown to rely on angiotensin-converting enzyme 2 (ACE2) receptors, whose expression in the testis is among the highest in the body. Additionally, the risk of mortality seems higher among male COVID-19 patients, and though much has been published since the first cases of COVID-19, there remain unanswered questions regarding SARS-CoV-2 impact on testes and potential consequences for reproductive health. We investigated testicular alterations in non-vaccinated deceased COVID-19-patients, the precise location of the virus, its replicative activity, and the immune, vascular, and molecular fluctuations involved in the pathogenesis. RESULTS: We found that SARS-CoV-2 testicular tropism is higher than previously thought and that reliable viral detection in the testis requires sensitive nanosensors or RT-qPCR using a specific methodology. Through an in vitro experiment exposing VERO cells to testicular macerates, we observed viral content in all samples, and the subgenomic RNA's presence reinforced the replicative activity of SARS-CoV-2 in testes of the severe COVID-19 patients. The cellular structures and viral particles, observed by transmission electron microscopy, indicated that macrophages and spermatogonial cells are the main SARS-CoV-2 lodging sites, where new virions form inside the endoplasmic reticulum Golgi intermediate complex. Moreover, we showed infiltrative infected monocytes migrating into the testicular parenchyma. SARS-CoV-2 maintains its replicative and infective abilities long after the patient's infection. Further, we demonstrated high levels of angiotensin II and activated immune cells in the testes of deceased patients. The infected testes show thickening of the tunica propria, germ cell apoptosis, Sertoli cell barrier loss, evident hemorrhage, angiogenesis, Leydig cell inhibition, inflammation, and fibrosis. CONCLUSIONS: Our findings indicate that high angiotensin II levels and activation of mast cells and macrophages may be critical for testicular pathogenesis. Importantly, our findings suggest that patients who become critically ill may exhibit severe alterations and harbor the active virus in the testes.

The Culturally Infused Curricular Framework (CICF) for Suicide Prevention Trainings.

Although suicide prevention trainings (SPT) have been a standard approach for suicide prevention for years, researchers have noted a need for more clarity in the definition of core competencies for SPTs, particularly in the areas of diversity and culture. Recent research has identified key theoretically- and empirically-based cultural considerations for suicide prevention, but translation is needed to infuse these standards for culture-related competencies into SPTs. This study performed a systematic literature review with a thematic synthesis analytic approach to establish a set of curricular guidelines for infusion of cultural considerations into SPTs. The study also examined the extent to which existing community trainings already incorporate cultural components. Based on the thematic synthesis of 39 SPT studies from 2010 to 2020 and seminal reviews of the cultural and suicide literature, results identified three overarching categories of cultural curricular competencies (suicide knowledge and awareness, suicide intervention skills, and curriculum delivery) and 14 core cultural curricular subthemes for community trainings (e.g., culturally informed risk factors and warning signs, systemic inequities, etc.). These three categories with 14 core cultural curricular competencies comprise the Culturally Infused Curricular Framework (CICF) for Suicide Prevention Trainings. The majority of trainings (62%) included five or less out of 14 total possible core cultural competencies in their training curricula, pointing to insufficient integration of cultural components in existing community trainings. This study's research-based guideline establishes a culture-inclusive framework to strengthen content and approach of community trainings and suicide prevention across cultural groups.

Pan-Arctic soil moisture control on tundra carbon sequestration and plant productivity.

Long-term atmospheric CO2 concentration records have suggested a reduction in the positive effect of warming on high-latitude carbon uptake since the 1990s. A variety of mechanisms have been proposed to explain the reduced net carbon sink of northern ecosystems with increased air temperature, including water stress on vegetation and increased respiration over recent decades. However, the lack of consistent long-term carbon flux and in situ soil moisture data has severely limited our ability to identify the mechanisms responsible for the recent reduced carbon sink strength. In this study, we used a record of nearly 100 site-years of eddy covariance data from 11 continuous permafrost tundra sites distributed across the circumpolar Arctic to test the temperature (expressed as growing degree days, GDD) responses of gross primary production (GPP), net ecosystem exchange (NEE), and ecosystem respiration (ER) at different periods of the summer (early, peak, and late summer) including dominant tundra vegetation classes (graminoids and mosses, and shrubs). We further tested GPP, NEE, and ER relationships with soil moisture and vapor pressure deficit to identify potential moisture limitations on plant productivity and net carbon exchange. Our results show a decrease in GPP with rising GDD during the peak summer (July) for both vegetation classes, and a significant relationship between the peak summer GPP and soil moisture after statistically controlling for GDD in a partial correlation analysis. These results suggest that tundra ecosystems might not benefit from increased temperature as much as suggested by several terrestrial biosphere models, if decreased soil moisture limits the peak summer plant productivity, reducing the ability of these ecosystems to sequester carbon during the summer.

Active manipulation of radiated fields by a magnetoplasmonic half-wave dipole nanoantenna.

We demonstrate a concept for the active manipulation of radiated fields by a magnetoplasmonic half-wave dipole nanoantenna. Our idea comprises a two arms nanoantenna, made of metallic ferromagnetic cobalt-silver alloy (Co6Ag94), inspired by the analogous radio frequency half-wave dipole antenna design. Numerical results, obtained under the magnetization saturation condition, indicate a tilting of the radiated beam depending on the magnitude and sense of the magnetization of the ferromagnetic material. Significantly, we obtained tilting angles as large as ±9.7∘ around the y axis for the magnetization placed along the x or z axes, respectively. Results in this work not only open up a new, to the best of our knowledge, way to dynamically manipulate the beam steering at the chip-scale, but also contribute to unveil novel magneto-optical effects at the nanoscale.

Dawn and dusk peaks of outer segment phagocytosis, and visual cycle function require Rab28.

RAB28 is a farnesylated, ciliary G-protein. Patient variants in RAB28 are causative of autosomal recessive cone-rod dystrophy (CRD), an inherited human blindness. In rodent and zebrafish models, the absence of Rab28 results in diminished dawn, photoreceptor, outer segment phagocytosis (OSP). Here, we demonstrate that Rab28 is also required for dusk peaks of OSP, but not for basal OSP levels. This study further elucidated the molecular mechanisms by which Rab28 controls OSP and inherited blindness. Proteomic profiling identified factors whose expression in the eye or whose expression at dawn and dusk peaks of OSP is dysregulated by loss of Rab28. Notably, transgenic overexpression of Rab28, solely in zebrafish cones, rescues the OSP defect in rab28 KO fish, suggesting rab28 gene replacement in cone photoreceptors is sufficient to regulate Rab28-OSP. Rab28 loss also perturbs function of the visual cycle as retinoid levels of 11-cRAL, 11cRP, and atRP are significantly reduced in larval and adult rab28 KO retinae (p < .05). These data give further understanding on the molecular mechanisms of RAB28-associated CRD, highlighting roles of Rab28 in both peaks of OSP, in vitamin A metabolism and in retinoid recycling.

Connectivity patterns of the core resting-state networks associated with apathy in late-life depression.

BACKGROUND: Apathy is associated with reduced antidepressant response and dementia in late-life depression (LLD). However, the functional cerebral basis of apathy is understudied in LLD. We investigated the functional connectivity of 5 resting-state networks (RSN) hypothesized to underlie apathy in LLD. METHODS: Resting-state functional MRI data were collected from individuals with LLD who did not have dementia as well as healthy older adults between October 2019 and April 2022. Apathy was evaluated using the diagnostic criteria for apathy (DCA), the Apathy Evaluation Scale (AES) and the Apathy Motivation Index (AMI). Subnetworks whose connectivity was significantly associated with each apathy measure were identified via the threshold-free network-based statistics. Regions that were consistently associated with apathy across the measures were reported as robust findings. RESULTS: Our sample included 39 individuals with LLD who did not have dementia and 26 healthy older adults. Compared with healthy controls, individuals with LLD had an altered intra-RSN and inter-RNS connectivity in the default mode, the cingulo-opercular and the frontoparietal networks. All 3 apathy measurements showed associations with modified intra-RSN connectivity in these networks, except for the DCA in the cingulo-opercular network. The AMI scores showed stronger associations with the cingulo-opercular and frontoparietal networks, whereas the AES had stronger associations with the default mode network and the goal-oriented behaviour network. LIMITATIONS: The study was limited by the small number of participants without apathy according to the DCA, which may have reduced the statistical power of between-group comparisons. Additionally, the reliance on specific apathy measures may have influenced the observed overlap in brain regions. CONCLUSION: Our findings indicate that apathy in LLD is consistently associated with changes in both intra-RSN and inter-RSN connectivity of brain regions implicated in goal-oriented behaviours. These results corroborate previous findings of altered functional RSN connectivity in severe LLD.

Rod Photoreceptors Avoid Saturation in Bright Light by the Movement of the G Protein Transducin.

Rod photoreceptors can be saturated by exposure to bright background light, so that no flash superimposed on the background can elicit a detectable response. This phenomenon, called increment saturation, was first demonstrated psychophysically by Aguilar and Stiles and has since been shown in many studies to occur in single rods. Recent experiments indicate, however, that rods may be able to avoid saturation under some conditions of illumination. We now show in ex vivo electroretinogram and single-cell recordings that in continuous and prolonged exposure even to very bright light, the rods of mice from both sexes recover as much as 15% of their dark current and that responses can persist for hours. In parallel to recovery of outer segment current is an ∼10-fold increase in the sensitivity of rod photoresponses. This recovery is decreased in transgenic mice with reduced light-dependent translocation of the G protein transducin. The reduction in outer-segment transducin together with a novel mechanism of visual-pigment regeneration within the rod itself enable rods to remain responsive over the whole of the physiological range of vision. In this way, rods are able to avoid an extended period of transduction channel closure, which is known to cause photoreceptor degeneration.SIGNIFICANCE STATEMENT Rods are initially saturated in bright light so that no flash superimposed on the background can elicit a detectable response. Frederiksen and colleagues show in whole retina and single-cell recordings that, if the background light is prolonged, rods slowly recover and can continue to produce significant responses over the entire physiological range of vision. Response recovery occurs by translocation of the G protein transducin from the rod outer to the inner segment, together with a novel mechanism of visual-pigment regeneration within the rod itself. Avoidance of saturation in bright light may be one of the principal mechanisms the retina uses to keep rod outer-segment channels from ever closing for too long a time, which is known to produce photoreceptor degeneration.

Effects of functional electro-stimulation combined with blood flow restriction in affected muscles by spinal cord injury.

Muscle atrophy is a great consequence of spinal cord injuries (SCI) due to immobility. SCI's detrimental effects on large muscle groups may lead to secondary effects such as glucose intolerance, increased risk of metabolic syndrome, and diabetes. Exercising with blood flow restriction (BFR) has been proposed as an effective method to induce hypertrophy using low training loads, with little or no muscle damage. This study investigated acute and chronic effects of low-intensity functional electrical stimulation (FES) combined with BFR on muscles affected by spinal cord injury. The acute effects of one bout of FES with (FES + BFR group) and without BFR (FES group) on muscle thickness (MT) and edema formation were compared. The chronic effects on MT and edema following 8 weeks of twice weekly training with and without BFR were also compared. The FES + BFR group showed MT and edema increases compared to the FES only group (p< 0.05). The FES + BFR showed a chronic MT increase after 4 weeks of training (p <0.05), with no further MT increases from the 4th to the 8th week (p>0.05). Following 3 weeks of detraining, MT decreased to baseline. No MT changes were observed in the FES (p>0.05). The FES + BF stimuli induced MT increases on the paralyzed skeletal muscles of SCI. The acute effects suggest that FES causes a greater metabolite accumulation and edema when combined with BFR. The early increases in MT can be attributed to edema, whereas after the 4th week, it is likely to be related to muscle hypertrophy. Register Clinical Trial Number on ReBeC: RBR-386rm8.

Assessing Remotely Piloted Aerial Systems in the characterization of rocky shores for oil spills environmental sensitivity mapping, northern São Paulo littoral, Brazil.

The Environmental Sensitivity Index (ESI) for oil spills was developed to assist coordinators to evaluate oil spill impact along shorelines and also to coordinate the allocation of resources during and after the incident, aiming to reduce environmental damage and consequences. Recently, Remotely Piloted Aerial Systems (RPAS) are being used in a wide range of areas, since they complement traditional remote sensing data (e.g., satellite images) and offer a rapidly, precise, detail and high-resolution images that fit well for environmental studies. Herein, the use of high-resolution RPAS images for ESI analysis of rocky shores in the Brazilian territory was performed. Using RPAS images, with their higher-resolution compared with ArcGIS Pro Basemap satellite images, increased the detailed of ESI analysis for oil spills, increasing the number of regions in the rocky shore that are more sensitive to oil spills. The RPAS images were able to decrease the number of areas that were less sensitive to oil spills, and increase areas that are more sensitive to oil spills. This increase is important, since they were not detected in the ESI analysis using conventional ArcGIS Pro Basemap satellite images. The RPAS images permit to delineate precisely rocky shores, improving ESI interpretation in rocky shores.

Mentored student research: A case study evaluation of benefits and best practices.

Efforts to mentor undergraduates in research require time, energy, and resources but can yield significant benefits. A review of education research on undergraduate research is presented followed by a case study of how technical and educational principles from the literature have been implemented and practiced in a new underwater acoustics lab. This case study involves the students who have joined this lab over its first three years. The results highlight the importance of several key factors that mentors can implement to improve their students' research experience. In order to meet the steep learning curve that new students face, faculty mentors should establish a culture of research (i.e., well established norms, expectations, and practices associated with being a part of a research group) through clear expectations, open communication, and student-led peer mentorship supported by carefully selected or designed resources (i.e., scaffolding). This paper seeks to share these education research resources with the acoustics community while providing a case study for review as a specific application of these key principles into an acoustics laboratory setting.

Design of Plasmonic Yagi-Uda Nanoantennas for Chip-Scale Optical Wireless Communications.

Optical wireless transmission has recently become a major cutting-edge alternative for on-chip/inter-chip communications with higher transmission speeds and improved power efficiency. Plasmonic nanoantennas, the building blocks of this new nanoscale communication paradigm, require precise design to have directional radiation and improved communication ranges. Particular interest has been paid to plasmonic Yagi-Uda, i.e., the optical analog of the conventional Radio Frequency (RF) Yagi-Uda design, which may allow directional radiation of plasmonic fields. However, in contrast to the RF model, an overall design strategy for the directional and optimized front-to-back ratio of the radiated far-field patterns is lacking. In this work, a guide for the optimized design of Yagi-Uda plasmonic nanoantennas is shown. In particular, five different design conditions are used to study the effects of sizes and spacing between the constituent parts (made of Au). Importantly, it is numerically demonstrated (using the scattered fields) that closely spaced nanoantenna elements are not appropriated for directional light-to-plasmon conversion/radiation. In contrast, if the elements of the nanoantenna are widely spaced, the structure behaves like a one-dimensional array of nanodipoles, producing a funnel-like radiation pattern (not suitable for on-chip wireless optical transmission). Therefore, based on the results here, it can be concluded that the constituent metallic rib lengths must be optimized to exhibit the resonance at the working wavelength, whilst their separations should follow the relation λeff/π, where λeff indicates the effective wavelength scaling for plasmonic nanostructures.

Non-photopic and photopic visual cycles differentially regulate immediate, early, and late phases of cone photoreceptor-mediated vision.

Cone photoreceptors in the retina enable vision over a wide range of light intensities. However, the processes enabling cone vision in bright light (i.e. photopic vision) are not adequately understood. Chromophore regeneration of cone photopigments may require the retinal pigment epithelium (RPE) and/or retinal Müller glia. In the RPE, isomerization of all-trans-retinyl esters to 11-cis-retinol is mediated by the retinoid isomerohydrolase Rpe65. A putative alternative retinoid isomerase, dihydroceramide desaturase-1 (DES1), is expressed in RPE and Müller cells. The retinol-isomerase activities of Rpe65 and Des1 are inhibited by emixustat and fenretinide, respectively. Here, we tested the effects of these visual cycle inhibitors on immediate, early, and late phases of cone photopic vision. In zebrafish larvae raised under cyclic light conditions, fenretinide impaired late cone photopic vision, while the emixustat-treated zebrafish unexpectedly had normal vision. In contrast, emixustat-treated larvae raised under extensive dark-adaptation displayed significantly attenuated immediate photopic vision concomitant with significantly reduced 11-cis-retinaldehyde (11cRAL). Following 30 min of light, early photopic vision was recovered, despite 11cRAL levels remaining significantly reduced. Defects in immediate cone photopic vision were rescued in emixustat- or fenretinide-treated larvae following exogenous 9-cis-retinaldehyde supplementation. Genetic knockout of Des1 (degs1) or retinaldehyde-binding protein 1b (rlbp1b) did not eliminate photopic vision in zebrafish. Our findings define molecular and temporal requirements of the nonphotopic or photopic visual cycles for mediating vision in bright light.

Evolutionary analysis of SARS-CoV-2 spike protein for its different clades.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become the main target for antiviral and vaccine development. Despite its relevance, e information is scarse about its evolutionary traces. The aim of this study was to investigate the diversification patterns of the spike for each clade of SARS-CoV-2 through different approaches. Two thousand and one hundred sequences representing the seven clades of the SARS-CoV-2 were included. Patterns of genetic diversifications and nucleotide evolutionary rate were estimated for the spike genomic region. The haplotype networks showed a star shape, where multiple haplotypes with few nucleotide differences diverge from a common ancestor. Four hundred seventy-nine different haplotypes were defined in the seven analyzed clades. The main haplotype, named Hap-1, was the most frequent for clades G (54%), GH (54%), and GR (56%) and a different haplotype (named Hap-252) was the most important for clades L (63.3%), O (39.7%), S (51.7%), and V (70%). The evolutionary rate for the spike protein was estimated as 1.08 × 10-3 nucleotide substitutions/site/year. Moreover, the nucleotide evolutionary rate after nine months of the pandemic was similar for each clade. In conclusion, the present evolutionary analysis is relevant as the spike protein of SARS-CoV-2 is the target for most therapeutic candidates; besides, changes in this protein could have consequences on viral transmission, response to antivirals and efficacy of vaccines. Moreover, the evolutionary characterization of clades improves knowledge of SARS-CoV-2 and deserves to be assessed in more detail as re-infection by different phylogenetic clades has been reported.

Phylogenetic analysis of SARS-CoV-2 in the first few months since its emergence.

During the first few months of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution in a new host, contrasting hypotheses have been proposed about the way the virus has evolved and diversified worldwide. The aim of this study was to perform a comprehensive evolutionary analysis to describe the human outbreak and the evolutionary rate of different genomic regions of SARS-CoV-2. The molecular evolution in nine genomic regions of SARS-CoV-2 was analyzed using three different approaches: phylogenetic signal assessment, emergence of amino acid substitutions, and Bayesian evolutionary rate estimation in eight successive fortnights since the virus emergence. All observed phylogenetic signals were very low and tree topologies were in agreement with those signals. However, after 4 months of evolution, it was possible to identify regions revealing an incipient viral lineage formation, despite the low phylogenetic signal since fortnight 3. Finally, the SARS-CoV-2 evolutionary rate for regions nsp3 and S, the ones presenting greater variability, was estimated as 1.37 × 10-3 and 2.19 × 10-3 substitution/site/year, respectively. In conclusion, results from this study about the variable diversity of crucial viral regions and determination of the evolutionary rate are consequently decisive to understand essential features of viral emergence. In turn, findings may allow the first-time characterization of the evolutionary rate of S protein, crucial for vaccine development.

Ruthenium(II) Diphosphine Complexes with Mercapto Ligands That Inhibit Topoisomerase IB and Suppress Tumor Growth In Vivo.

Ruthenium(II) complexes (Ru1-Ru5), with the general formula [Ru(N-S)(dppe)2]PF6, bearing two 1,2-bis(diphenylphosphino)ethane (dppe) ligands and a series of mercapto ligands (N-S), have been developed. The combination of these ligands in the complexes endowed hydrophobic species with high cytotoxic activity against five cancer cell lines. For the A549 (lung) and MDA-MB-231 (breast) cancer cell lines, the IC50 values of the complexes were 288- to 14-fold lower when compared to cisplatin. Furthermore, the complexes were selective for the A549 and MDA-MB-231 cancer cell lines compared to the MRC-5 nontumor cell line. The multitarget character of the complexes was investigated by using calf thymus DNA (CT DNA), human serum albumin, and human topoisomerase IB (hTopIB). The complexes potently inhibited hTopIB. In particular, complex [Ru(dmp)(dppe)2]PF6 (Ru3), bearing the 4,6-diamino-2-mercaptopyrimidine (dmp) ligand, effectively inhibited hTopIB by acting on both the cleavage and religation steps of the catalytic cycle of this enzyme. Molecular docking showed that the Ru1-Ru5 complexes have binding affinity by active sites on the hTopI and hTopI-DNA, mainly via π-alkyl and alkyl hydrophobic interactions, as well as through hydrogen bonds. Complex Ru3 displayed significant antitumor activity against murine melanoma in mouse xenograph models, but this complex did not damage DNA, as revealed by Ames and micronucleus tests.

Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration.

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4-/- mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk-/- but not Abca4-/- mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4-/- background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4-/- mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.