Expanding landscape of coronary microvascular disease in co-morbid conditions: Metabolic disease and beyond.

Coronary microvascular disease (CMD) and impaired coronary blood flow control are defects that occur early in the pathogenesis of heart failure in cardiometabolic conditions, prior to the onset of atherosclerosis. In fact, recent studies have shown that CMD is an independent predictor of cardiac morbidity and mortality in patients with obesity and metabolic disease. CMD is comprised of functional, structural, and mechanical impairments that synergize and ultimately reduce coronary blood flow in metabolic disease and in other co-morbid conditions, including transplant, autoimmune disorders, chemotherapy-induced cardiotoxicity, and remote injury-induced CMD. This review summarizes the contemporary state-of-the-field related to CMD in metabolic and these other co-morbid conditions based on mechanistic data derived mostly from preclinical small- and large-animal models in light of available clinical evidence and given the limitations of studying these mechanisms in humans. In addition, we also discuss gaps in current understanding, emerging areas of interest, and opportunities for future investigations in this field.

A water structure indicator suitable for generic contexts: Two-liquid behavior at hydration and nanoconfinement conditions and a molecular approach to hydrophobicity and wetting.

In a recent work, we have briefly introduced a new structural index for water that, unlike previous indicators, was devised specifically for generic contexts beyond bulk conditions, making it suitable for hydration and nanoconfinement settings. In this work, we shall study this metric in detail, demonstrating its ability to reveal the existence of a fine-tuned interplay between the local structure and energetics in liquid water. This molecular principle enables the establishment of an extended hydrogen bond network, while simultaneously allowing for the existence of network defects by compensating for uncoordinated sites. By studying different water models and different temperatures encompassing both the normal liquid and the supercooled regime, this molecular mechanism will be shown to underlie the two-state behavior of bulk water. In addition, by studying functionalized self-assembled monolayers and diverse graphene-like surfaces, we shall show that this principle is also operative at hydration and nanoconfinement conditions, thus generalizing the validity of the two-liquid scenario of water to these contexts. This approach will allow us to define conditions for wettability, providing an accurate measure of hydrophobicity and a reliable predictor of filling and drying transitions. Hence, it might open the possibility of elucidating the active role of water in the broad fields of biophysics and materials science. As a preliminary step, we shall study the hydration structure and hydrophilicity of graphene-like systems (parallel graphene sheets and carbon nanotubes) as a function of the confinement dimensionality.

Optimizing Antimony Speciation Analysis via Frontal Chromatography-ICP-MS to Explore the Release of PET Additives.

Antimony (Sb) contamination poses significant environmental and health concerns due to its toxic nature and widespread presence, largely from anthropogenic activities. This study addresses the urgent need for an accurate speciation analysis of Sb, particularly in water sources, emphasizing its migration from polyethylene terephthalate (PET) plastic materials. Current methodologies primarily focus on total Sb content, leaving a critical knowledge gap for its speciation. Here, we present a novel analytical approach utilizing frontal chromatography coupled with inductively coupled plasma mass spectrometry (FC-ICP-MS) for the rapid speciation analysis of Sb(III) and Sb(V) in water. Systematic optimization of the FC-ICP-MS method was achieved through multivariate data analysis, resulting in a remarkably short analysis time of 150 s with a limit of detection below 1 ng kg-1. The optimized method was then applied to characterize PET leaching, revealing a marked effect of the plastic aging and manufacturing process not only on the total amount of Sb released but also on the nature of leached Sb species. This evidence demonstrates the effectiveness of the FC-ICP-MS approach in addressing such an environmental concern, benchmarking a new standard for Sb speciation analysis in consideration of its simplicity, cost effectiveness, greenness, and broad applicability in environmental and health monitoring.

A Comprehensive Review of the Current State of Robot-assisted Laparoscopic Salvage Prostatectomy.

BACKGROUND AND OBJECTIVE: Salvage robot assisted radical prostatectomy (sRARP) is performed for patients with biochemical or biopsy proven, localized prostate cancer recurrences after radiation or ablative therapies. Traditionally, sRARP has been avoided by lower volume surgeons due to technical demand and high complication rates. Post-radiation sRARP outcomes studies exist but remain few in number. With increasing use of whole gland and focal ablative therapies, updates on sRARP in this setting are needed. The aim of this narrative review is to provide an overview of recently reviewed studies on the oncologic outcomes, functional outcomes, and complications after post-radiation and post-ablative sRARP. Tips and tricks are provided to guide surgeons who may perform sRARP. MATERIALS AND METHODS: We performed a non-systematic literature search of PubMed and MEDLINE for the most relevant articles pertaining to the outlined topics from 2010-2022 without limitation on study design. Only case reports, editorial comments, letters, and manuscripts in non-English languages were excluded. Key Content and Findings: Salvage robotic radical prostatectomy is performed in cases of biochemical recurrence after radiation or ablative therapies. Oncologic outcomes after sRARP are worse compared to primary surgery (pRARP) though improvements have been made with the robotic approach when compared to open salvage prostatectomy. Higher pre-sRARP PSA levels and more advanced pathologic stage portend worse oncologic outcomes. Patients meeting low-risk, EAU-biochemical recurrence criteria have improved oncologic outcomes compared to those with high-risk BCR. While complication rates in sRARP are higher compared to pRARP, Retzius sparing approaches may reduce complication rates, particularly rectal injuries. In comparison to the traditional open approach, sRARP is associated with a lower rate of bladder neck contracture. In terms of functional outcomes, potency rates after sRARP are poor and continence rates are low, though Retzius sparing approaches demonstrate acceptable recovery of urinary continence by 1 year, post-operatively. CONCLUSIONS: Advances in the robotic platform and improvement in robotic experience have resulted in acceptable complication rates after sRARP. However, oncologic and functional outcomes after sRARP in both the post-radiation and post-ablation settings are worse compared to pRARP. Thus, when engaging in shared decision making with patients regarding the initial management of localized prostate cancer, patients should be educated regarding oncologic and functional outcomes and complications in the case of biochemically recurrent prostate cancer that may require sRARP.

Glucose Phosphotransferase System Modulates Pyruvate Metabolism, Bacterial Fitness, and Microbial Ecology in Oral Streptococci.

Spontaneous mutants with defects in the primary glucose phosphotransferase permease (manLMNO) of Streptococcus sanguinis SK36 showed enhanced fitness at low pH. Transcriptomics and metabolomics with a manL deletion mutant (SK36/manL) revealed redirection of pyruvate to production of acetate and formate, rather than lactate. These observations were consistent with measurements of decreased lactic acid accumulation and increased excretion of acetate, formate, pyruvate, and H2O2. Genes showing increased expression in SK36/manL included those encoding carbohydrate transporters, extracellular glycosidases, intracellular polysaccharide metabolism, and arginine deiminase and pathways for metabolism of acetoin, ethanolamine, ascorbate, and formate, along with genes required for membrane biosynthesis and adhesion. Streptococcus mutans UA159 persisted much better in biofilm cocultures with SK36/manL than with SK36, an effect that was further enhanced by culturing the biofilms anaerobically but dampened by adding arginine to the medium. We posited that the enhanced persistence of S. mutans with SK36/manL was in part due to excess excretion of pyruvate by the latter, as addition of pyruvate to S. mutans-S. sanguinis cocultures increased the proportions of UA159 in the biofilms. Reducing the buffer capacity or increasing the concentration of glucose benefited UA159 when cocultured with SK36, but not with SK36/manL, likely due to the altered metabolism and enhanced acid tolerance of the mutant. When manL was deleted in S. mutans or Streptococcus gordonii, the mutants presented altered fitness characteristics. Our study demonstrated that phosphotransferase system (PTS)-dependent modulation of central metabolism can profoundly affect streptococcal fitness and metabolic interactions, revealing another dimension in commensal-pathogen relationships influencing dental caries development. IMPORTANCE Dental caries is underpinned by a dysbiotic microbiome and increased acid production. As beneficial bacteria that can antagonize oral pathobionts, oral streptococci such as S. sanguinis and S. gordonii can ferment many carbohydrates, despite their relative sensitivity to low pH. We characterized the molecular basis for why mutants of glucose transporter ManLMNO of S. sanguinis showed enhanced production of hydrogen peroxide and ammonia and improved persistence under acidic conditions. A metabolic shift involving more than 300 genes required for carbohydrate transport, energy production, and envelope biogenesis was observed. Significantly, manL mutants engineered in three different oral streptococci displayed altered capacities for acid production and interspecies antagonism, highlighting the potential for targeting the glucose-PTS to modulate the pathogenicity of oral biofilms.

Cardiac epigenetic changes in VEGF signaling genes associate with myocardial microvascular rarefaction in experimental chronic kidney disease.

Chronic kidney disease (CKD) is common in patients with heart failure and often results in left ventricular diastolic dysfunction (LVDD). However, the mechanisms responsible for cardiac damage in CKD-LVDD remain to be elucidated. Epigenetic alterations may impose long-lasting effects on cellular transcription and function, but their exact role in CKD-LVDD is unknown. We investigate whether changes in cardiac site-specific DNA methylation profiles might be implicated in cardiac abnormalities in CKD-LVDD. CKD-LVDD and normal control pigs (n = 6 each) were studied for 14 wk. Renal and cardiac hemodynamics were quantified by multidetector CT and echocardiography. In randomly selected pigs (n = 3/group), cardiac site-specific 5-methylcytosine (5mC) immunoprecipitation (MeDIP)- and mRNA-sequencing (seq) were performed, followed by integrated (MeDiP-seq/mRNA-seq analysis), and confirmatory ex vivo studies. MeDIP-seq analysis revealed 261 genes with higher (fold change > 1.4; P < 0.05) and 162 genes with lower (fold change < 0.7; P < 0.05) 5mC levels in CKD-LVDD versus normal pigs, which were primarily implicated in vascular endothelial growth factor (VEGF)-related signaling and angiogenesis. Integrated MeDiP-seq/mRNA-seq analysis identified a select group of VEGF-related genes in which 5mC levels were higher, but mRNA expression was lower in CKD-LVDD versus normal pigs. Cardiac VEGF signaling gene and VEGF protein expression were blunted in CKD-LVDD compared with controls and were associated with decreased subendocardial microvascular density. Cardiac epigenetic changes in VEGF-related genes are associated with impaired angiogenesis and cardiac microvascular rarefaction in swine CKD-LVDD. These observations may assist in developing novel therapies to ameliorate cardiac damage in CKD-LVDD.NEW & NOTEWORTHY Chronic kidney disease (CKD) often leads to left ventricular diastolic dysfunction (LVDD) and heart failure. Using a novel translational swine model of CKD-LVDD, we characterize the cardiac epigenetic landscape, identifying site-specific 5-methylcytosine changes in vascular endothelial growth factor (VEGF)-related genes associated with impaired angiogenesis and cardiac microvascular rarefaction. These observations shed light on the mechanisms of cardiac microvascular damage in CKD-LVDD and may assist in developing novel therapies for these patients.

Renal ischemia alters the transcriptomic and epigenetic profile of inflammatory genes in swine scattered tubular-like cells.

BACKGROUND: Scattered tubular-like cells (STCs) are differentiated renal tubular cells that during recovery from ischemic injury dedifferentiate to repair other injured renal cells. Renal artery stenosis (RAS), often associated with chronic inflammatory injury, compromises the integrity and function of STCs, but the underlying mechanisms remain unknown. We hypothesized that RAS alters the transcriptomic and epigenetic profile of inflammatory genes in swine STCs. METHODS: STCs were harvested from pig kidneys after 10 weeks of RAS or sham (n=6 each). STC mRNA profiles of inflammatory genes were analyzed using high-throughput mRNA-sequencing (seq) and their DNA methylation (5mC) and hydroxymethylation (5hmC) profiles by DNA immunoprecipitation and next-generation sequencing (MeDIP-seq) (n=3 each), followed by an integrated (mRNA-seq/MeDIP-seq) analysis. STC protein expression of candidate differentially expressed (DE) genes and common proinflammatory proteins were subsequently assessed in vitro before and after epigenetic (Bobcat339) modulation. RESULTS: mRNA-seq identified 57 inflammatory genes up-regulated in RAS-STCs versus Normal-STCs (>1.4 or <0.7-fold, P<0.05), of which 14% exhibited lower 5mC and 5% higher 5hmC levels in RAS-STCs versus Normal-STCs, respectively. Inflammatory gene and protein expression was higher in RAS-STCs compared with Normal-STCs but normalized after epigenetic modulation. CONCLUSIONS: These observations highlight a novel modulatory mechanism of this renal endogenous repair system and support development of epigenetic or anti-inflammatory therapies to preserve the reparative capacity of STCs in individuals with RAS.

Correlations between defect propensity and dynamical heterogeneities in supercooled water.

A salient feature of supercooled liquids consists in the dramatic dynamical slowdown they undergo as temperature decreases while no significant structural change is evident. These systems also present dynamical heterogeneities (DH): certain molecules, spatially arranged in clusters, relax various orders of magnitude faster than the others. However, again, no static quantity (such as structural or energetic measures) shows strong direct correlations with such fast-moving molecules. In turn, the dynamic propensity approach, an indirect measure that quantifies the tendency of the molecules to move in a given structural configuration, has revealed that dynamical constraints, indeed, originate from the initial structure. Nevertheless, this approach is not able to elicit which structural quantity is, in fact, responsible for such a behavior. In an effort to remove dynamics from its definition in favor of a static quantity, an energy-based propensity has also been developed for supercooled water, but it could only find positive correlations between the lowest-energy and the least-mobile molecules, while no correlations could be found for those more relevant mobile molecules involved in the DH clusters responsible for the system's structural relaxation. Thus, in this work, we shall define a defect propensity measure based on a recently introduced structural index that accurately characterizes water structural defects. We shall show that this defect propensity measure provides positive correlations with dynamic propensity, being also able to account for the fast-moving molecules responsible for the structural relaxation. Moreover, time dependent correlations will show that defect propensity represents an appropriate early-time predictor of the long-time dynamical heterogeneity.

Turning an energy-based defect detector into a multi-molecule structural indicator for water.

Recent studies have provided conclusive evidence for the existence of a liquid-liquid critical point in numerical models of water. Such a scenario implies the competition between two local molecular arrangements of different densities: a high-density liquid (HDL) and a low-density liquid (LDL). Within this context, the development of accurate structural indicators to properly characterize the two interconverting local structures is demanded. In a previous study, we introduced a reliable energy-based structural descriptor that properly discriminates water molecules into tetrahedrally arranged molecules (T molecules) and distorted molecules (D molecules). The latter constitute defects in terms of hydrogen bond (HB) coordination and have been shown to represent a minority component, even at high temperatures above the melting point. In addition, the D molecules tend to form high-quality HBs with three T molecules and to be surrounded by T and D molecules at further distances. Thus, it became evident that, while the LDL state might consist of a virtually pure T state, the HDL state would comprise mixed molecular arrangements including the D molecules. Such a need to abandon the single-molecule description requires the investigation of the degree of structural information to be incorporated in order to build an appropriate multi-molecule indicator. Hence, in this work, we shall study the effect of the local structural constraints on the water molecules in order to discriminate the different molecular arrangements into two disjoint classes. This will enable us to build a multi-molecule structural indicator for water whose performance will then be investigated within the water's supercooled regime.

Evidence for aggregation-independent, PrPC-mediated Aß cellular internalization.

Evidence linking amyloid beta (Aß) cellular uptake and toxicity has burgeoned, and mechanisms underlying this association are subjects of active research. Two major, interconnected questions are whether Aß uptake is aggregation-dependent and whether it is sequence-specific. We recently reported that the neuronal uptake of Aß depends significantly on peptide chirality, suggesting that the process is predominantly receptor-mediated. Over the past decade, the cellular prion protein (PrPC) has emerged as an important mediator of Aß-induced toxicity and of neuronal Aß internalization. Here, we report that the soluble, nonfibrillizing Aß (1-30) peptide recapitulates full-length Aß stereoselective cellular uptake, allowing us to decouple aggregation from cellular, receptor-mediated internalization. Moreover, we found that Aß (1-30) uptake is also dependent on PrPC expression. NMR-based molecular-level characterization identified the docking site on PrPC that underlies the stereoselective binding of Aß (1-30). Our findings therefore identify a specific sequence within Aß that is responsible for the recognition of the peptide by PrPC, as well as PrPC-dependent cellular uptake. Further uptake stereodifferentiation in PrPC-free cells points toward additional receptor-mediated interactions as likely contributors for Aß cellular internalization. Taken together, our results highlight the potential of targeting cellular surface receptors to inhibit Aß cellular uptake as an alternative route for future therapeutic development for Alzheimer's disease.

Influence of brushing motions on the shaping of oval canals by rotary and reciprocating instruments.

OBJECTIVE: The effects of brushing on shaping with three different instruments were assessed in oval canals. DESIGN: Mandibular incisors were assigned to 6 groups (n = 12/group) according to the system, each one with or without brushing: Reciproc Blue, VDW.Rotate, and Race EVO. Micro-computed tomography was performed before and after preparation. RESULTS: Brushing strokes caused no increase in canal volume, surface area, and structure model index independently of the system (p > 0.05), except for RaCe EVO in the full canal surface area (p < 0.05). Brushing did not increase the prepared areas (p > 0.05), except for Reciproc in the apical canal (p < 0.05). Reciproc with no brushing exhibited less pericervical dentin than with brushing (p < 0.05), while RaCe EVO with brushing resulted in less remaining dentin (p < 0.05). CONCLUSIONS: The brushing motion had no effects on the overall shaping performance of the 3 instruments tested. An exception was the increase in prepared surface area in the apical canal segment when the Reciproc instrument was used with brushing strokes.

Multifunctional Switch Based on Spin-Labeled Gold Nanoparticles.

The fabrication of multifunctional switches is a fundamental step in the development of nanometer-scale molecular spintronic devices. The anchoring of active organic radicals on gold nanoparticles (AuNPs) surface is little studied and the realization of AuNPs-based switches remains extremely challenging. We report the first demonstration of a surface molecular switch based on AuNPs decorated with persistent perchlorotriphenylmethyl (PTM) radicals. The redox properties of PTM are exploited to fabricate electrochemical switches with optical and magnetic responses, showing high stability and reversibility. Electronic interaction between the radicals and the gold surface is investigated by UV-vis, showing a very broad absorption band in the near-infrared (NIR) region, which becomes more intense when PTMs are reduced to anionic phase. By using multiple experimental techniques, we demonstrate that this interaction is likely favored by the preferentially flat orientation of PTM ligands on the metallic NP surface, as confirmed by first-principles simulations.

A single system detects and protects the beneficial oral bacterium Streptococcus sp. A12 from a spectrum of antimicrobial peptides.

The commensal bacterium Streptococcus sp. A12 has multiple properties that may promote the stability of health-associated oral biofilms, including overt antagonism of the dental caries pathogen Streptococcus mutans. A LanFEG-type ABC transporter, PcfFEG, confers tolerance to the lantibiotic nisin and enhances the ability of A12 to compete against S. mutans. Here, we investigated the regulation of pcfFEG and adjacent genes for a two-component system, pcfRK, to better understand antimicrobial peptide resistance by A12. Induction of pcfFEG-pcfRK was the primary mechanism to respond rapidly to nisin. In addition to nisin, PcfFEG conferred tolerance by A12 to a spectrum of lantibiotic and non-lantibiotic antimicrobial peptides produced by a diverse collection of S. mutans isolates. Loss of PcfFEG resulted in the altered spatio-temporal arrangement of A12 and S. mutans in a dual-species biofilm model. Deletion of PcfFEG or PcfK resulted in constitutive activation of pcfFEG and expression of pcfFEG was inhibited by small peptides in the pcfK mutant. Transcriptional profiling of pcfR or pcfK mutants combined with functional genomics revealed peculiarities in PcfK function and a novel panel of genes responsive to nisin. Collectively, the results provide fundamental insights that strengthen the foundation for the design of microbial-based therapeutics to control oral infectious diseases.

Cardiac micro-RNA and transcriptomic profile of a novel swine model of chronic kidney disease and left ventricular diastolic dysfunction.

Chronic kidney disease (CKD) is an independent risk factor for the development of heart failure, but the underlying mechanisms remain unknown. Using a novel translational swine model of CKD and cardiac dysfunction, we hypothesize that CKD alters the cardiac miRNA and transcriptomic profile that associate with cardiac remodeling and metabolic processes implicated in the development of left ventricular diastolic dysfunction (CKD-LVDD). CKD-LVDD and normal control pigs (n = 6 each) were studied for 14 wk. Renal and cardiac hemodynamics were quantified by multidetector CT and echocardiography. In randomly selected pigs (n = 3/group), cardiac miRNA- and mRNA-sequencing (seq) was performed, validated (qPCR), and followed by confirmatory ex vivo studies. Differential expression analysis identified nine miRNAs and 125 mRNAs upregulated and 17 miRNAs and 172 mRNAs downregulated [fold-change ≥ 2, and false discovery rate (FDR) ≤ 0.05] in CKD-LVDD versus normal controls. Integrated miRNA-/mRNA-seq analysis identified 71 overlappings downregulated mRNA targets of miRNAs upregulated, and 39 overlappings upregulated mRNA targets of miRNAs downregulated in CKD-LVDD versus controls. Functional analysis showed that these genes were primarily implicated in processes associated with cardiac remodeling, including ubiquitination, ATP and fatty acid synthesis, and extracellular matrix remodeling. In agreement, hearts of CKD-LVDD pigs exhibited abnormal diastolic relaxation, mitochondrial injury, moderate LV fibrosis, and myocardial lipid accumulation. Our work comprehensively characterizes the cardiac micro-RNA and transcriptomic profile of a translational model of CKD-LVDD. Our data may set the foundation for new targeted studies to further elucidate LVDD pathophysiology and assist to develop therapeutic interventions.NEW & NOTEWORTHY Chronic kidney disease (CKD) is a progressive disorder in which more than 50% of deaths are attributed to cardiovascular disease. Using a swine model of CKD that develops left ventricular dysfunction (CKD-LVDD), we characterize the cardiac micro-RNA and transcriptomic profile, identifying dysregulated genes associated with cardiac remodeling and fatty acid metabolism that might be post-transcriptionally regulated early in the disease. These findings pinpointed pathological pathways that may open new avenues toward therapeutic research to reduce cardiovascular morbidity in CKD.

Activation of TnSmu1, an integrative and conjugative element, by an ImmR-like transcriptional regulator in Streptococcus mutans.

Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. Recently, as part of efforts to determine hypothetical gene functions, we have discovered an important regulatory module encoded on an ICE known as TnSmu1 on the Streptococcus mutans chromosome. The regulatory module consists of a cI-like repressor with a helix-turn-helix DNA binding domain immR Smu (immunity repressor) and a metalloprotease immA Smu (anti-repressor). It is not possible to create an in-frame deletion mutant of immR Smu and repression of immR Smu with CRISPRi (CRISPR interference) causes substantial cell defects. We used a bypass of essentiality (BoE) screen to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can restore the viability of an immR Smu mutant. Deletion of immR Smu also leads to production of a circular intermediate form of TnSmu1, which is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ImmRSmu and broader effects on S. mutans UA159 physiology, we used CRISPRi and RNA-seq. Strongly induced genes included all the TnSmu1 mobile element, genes involved in amino acid metabolism, transport systems and a type I-C CRISPR-Cas system. Lastly, bioinformatic analysis shows that the TnSmu1 mobile element and its associated genes are well distributed across S. mutans isolates. Taken together, our results show that activation of TnSmu1 is controlled by the immRA Smu module, and that activation is deleterious to S. mutans, highlighting the complex interplay between mobile elements and their host.

An Enantiomeric Fragment Pair (EFP) Approach for the Study of Cellular Uptake of Intrinsically Disordered Proteins.

The study of intrinsically disordered and amyloidogenic proteins poses a major challenge to researchers due to the propensity of the system to aggregate and to form amyloid fibrils and deposits. This intrinsic nature limits the way amyloids can be studied and increases the level of complexity of the techniques needed to study the system of interest. Recent reports suggest that cellular recognition and internalization of pre-fibrillary species of amyloidogenic peptides and proteins may initiate some of its toxic actions. Therefore, developing novels tools to facilitate the understanding and determination of the interactions between intrinsically disordered proteins and the cellular membrane is becoming increasingly valuable. Here, we present and propose an approach for the study of the interactions of intrinsically disordered proteins with the cellular surface based on the use of enantiomeric fragment pairs (EFPs). By following a stepwise methodology in which the amyloidogenic peptide or protein is fragmented into specific segments, we show how this approach can be exploited to differentiate between different types of cellular uptake, to determine the degree of receptor-mediated cellular internalization of intrinsically disordered peptides and proteins, and to pinpoint the specific regions within the amino acid sequence responsible for the cellular recognition. Adopting this approach overcomes aggregation-related challenges and offers a particularly well-suited platform for the elucidation of receptor-intermediated recognition, uptake, and toxicity.

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

Renal mitochondrial injury in the pathogenesis of CKD: mtDNA and mitomiRs.

Chronic kidney disease (CKD) is a public health concern that affects over 200 million people worldwide and is associated with a tremendous economic burden. Therefore, deciphering the mechanisms underpinning CKD is crucial to decelerate its progression towards end-stage renal disease (ESRD). Renal tubular cells are populated with a high number of mitochondria, which produce cellular energy and modulate several important cellular processes, including generation of reactive oxygen species (ROS), calcium homeostasis, proliferation, and apoptosis. Over the past few years, increasing evidence has implicated renal mitochondrial damage in the pathogenesis of common etiologies of CKD, such as diabetes, hypertension, metabolic syndrome (MetS), chronic renal ischemia, and polycystic kidney disease (PKD). However, most compelling evidence is based on preclinical studies because renal biopsies are not routinely performed in many patients with CKD. Previous studies have shown that urinary mitochondrial DNA (mtDNA) copy numbers may serve as non-invasive biomarkers of renal mitochondrial dysfunction. Emerging data also suggest that CKD is associated with altered expression of mitochondria-related microRNAs (mitomiRs), which localize in mitochondria and regulate the expression of mtDNA and nucleus-encoded mitochondrial genes. This review summarizes relevant evidence regarding the involvement of renal mitochondrial injury and dysfunction in frequent forms of CKD. We further provide an overview of non-invasive biomarkers and potential mechanisms of renal mitochondrial damage, especially focusing on mtDNA and mitomiRs.

Unitary rotation of pixellated polychromatic images.

Unitary rotations of polychromatic images on finite two-dimensional pixellated screens provide invertibility, group composition, and thus conservation of information. Rotations have been applied on monochromatic image data sets, where we now examine closer the Gibbs-like oscillations that appear due to discrete "discontinuities" of the input images under unitary transformations. Extended to three-color images, we examine here the display of color at the pixels where, due to oscillations, some pixel color values may fall outside their required common numerical range [0,1], between absence and saturation of the red, green, and blue formant colors we choose to represent the images.