A pro-reparative bioelectronic device for controlled delivery of ions and biomolecules.

Wound healing is a complex physiological process that requires precise control and modulation of many parameters. Therapeutic ion and biomolecule delivery has the capability to regulate the wound healing process beneficially. However, achieving controlled delivery through a compact device with the ability to deliver multiple therapeutic species can be a challenge. Bioelectronic devices have emerged as a promising approach for therapeutic delivery. Here, we present a pro-reparative bioelectronic device designed to deliver ions and biomolecules for wound healing applications. The device incorporates ion pumps for the targeted delivery of H+ and zolmitriptan to the wound site. In vivo studies using a mouse model further validated the device's potential for modulating the wound environment via H+ delivery that decreased M1/M2 macrophage ratios. Overall, this bioelectronic ion pump demonstrates potential for accelerating wound healing via targeted and controlled delivery of therapeutic agents to wounds. Continued optimization and development of this device could not only lead to significant advancements in tissue repair and wound healing strategies but also reveal new physiological information about the dynamic wound environment.

Genomic Characterization of Aggressive Breast Cancer in Younger Women.

PURPOSE: Although breast cancer (BC) risk increases with age, BC in younger women is more aggressive with higher mortality compared with older women. We characterize the genomic landscape of BCs in younger women. METHODS: Clinicopathologic, molecular, and genomic differences across age groups (< 40 years, 40-60 years, > 60 years) in female BC patients were investigated in two large cohorts [AACR-GENIE8.1 (n = 11,594) and METABRIC (n = 2509)]. Cox-proportional regression analyzed the prognostic impact of age groups for disease-specific survival (DSS) and recurrence-free survival (RFS) in METABRIC and progression-free survival (PFS) in GENIE cohorts. Chi-squared test was used to assess statistical associations between genomic alterations and age groups. RESULTS: Survival analysis showed that women < 40 years had shorter DSS [hazard ratio (HR): 1.52, p = 0.005], RFS (HR: 1.4, p = 0.006), and PFS (HR: 1.82, p = 0.0003) compared with women 40-60 years, and shorter RFS (HR: 1.5, p = 0.001) and PFS (HR: 2.95, p < 0.0001) compared with women > 60 years. Molecular subtypes in the METABRIC cohort showed women < 40 years were enriched with basal, and HER2+ subtypes, and less enriched with luminal A and B subtype (p < 0.0001). Characterization of genomic alterations in both cohorts demonstrated that BCs in women < 40 years were more enriched with TP53 mutations (FDR < 0.0001), BRCA1 mutations (FDR = 0.01), ERBB2 amplifications (FDR < 0.001), CDK12 amplifications (FDR < 0.001), and PPM1D amplifications (FDR < 0.001). In contrast, BCs in older women (> 60 years) were more enriched with PIK3CA, KMT2C, and CDH1 mutations (FDR < 0.0001). CONCLUSIONS: BCs in young women are associated with shorter survival and more aggressive genomic features, including mutations in TP53 and BRCA1, and amplifications in ERBB2 and CDK12. These findings have the potential to impact clinical trial design and treatment.

ALDH1A3 Coordinates Metabolism With Gene Regulation in Pulmonary Arterial Hypertension.

BACKGROUND: Metabolic alterations provide substrates that influence chromatin structure to regulate gene expression that determines cell function in health and disease. Heightened proliferation of smooth muscle cells (SMC) leading to the formation of a neointima is a feature of pulmonary arterial hypertension (PAH) and systemic vascular disease. Increased glycolysis is linked to the proliferative phenotype of these SMC. METHODS: RNA sequencing was applied to pulmonary arterial SMC (PASMC) from PAH patients with and without a BMPR2 (bone morphogenetic receptor 2) mutation versus control PASMC to uncover genes required for their heightened proliferation and glycolytic metabolism. Assessment of differentially expressed genes established metabolism as a major pathway, and the most highly upregulated metabolic gene in PAH PASMC was aldehyde dehydrogenase family 1 member 3 (ALDH1A3), an enzyme previously linked to glycolysis and proliferation in cancer cells and systemic vascular SMC. We determined if these functions are ALDH1A3-dependent in PAH PASMC, and if ALDH1A3 is required for the development of pulmonary hypertension in a transgenic mouse. Nuclear localization of ALDH1A3 in PAH PASMC led us to determine whether and how this enzyme coordinately regulates gene expression and metabolism in PAH PASMC. RESULTS: ALDH1A3 mRNA and protein were increased in PAH versus control PASMC, and ALDH1A3 was required for their highly proliferative and glycolytic properties. Mice with Aldh1a3 deleted in SMC did not develop hypoxia-induced pulmonary arterial muscularization or pulmonary hypertension. Nuclear ALDH1A3 converted acetaldehyde to acetate to produce acetyl coenzyme A to acetylate H3K27, marking active enhancers. This allowed for chromatin modification at NFYA (nuclear transcription factor Y subunit α) binding sites via the acetyltransferase KAT2B (lysine acetyltransferase 2B) and permitted NFY-mediated transcription of cell cycle and metabolic genes that is required for ALDH1A3-dependent proliferation and glycolysis. Loss of BMPR2 in PAH SMC with or without a mutation upregulated ALDH1A3, and transcription of NFYA and ALDH1A3 in PAH PASMC was ß-catenin dependent. CONCLUSIONS: Our studies have uncovered a metabolic-transcriptional axis explaining how dividing cells use ALDH1A3 to coordinate their energy needs with the epigenetic and transcriptional regulation of genes required for SMC proliferation. They suggest that selectively disrupting the pivotal role of ALDH1A3 in PAH SMC, but not endothelial cells, is an important therapeutic consideration.

Disparities in Primary Breast Cancer Stage at Presentation Among Hispanic Subgroups.

BACKGROUND: Although the United States (US) Hispanic population consists of diverse communities, prior breast cancer studies often analyze this group in aggregate. Our aim was to identify differences in breast cancer stage at presentation in the US population, with a particular focus on Hispanic subgroups. METHODS: Data from the National Cancer Database (NCDB) from 2004 to 2017 were used to select women with primary breast cancer; individuals were disaggregated by racial and ethnic subgroup and Hispanic country of origin. Ordinal logistic regression was used to create adjusted odds ratios (aORs) with 95% confidence intervals (CIs), with higher odds representing presentation at later-stage breast cancer. Subgroup analysis was conducted based on tumor receptor status. RESULTS: Overall, among 2,282,691 women (5.2% Hispanic), Hispanic women were more likely to live in low-income and low-educational attainment neighborhoods, and were also more likely to be uninsured. Hispanic women were also more likely to present at later-stage primary breast cancer when compared with non-Hispanic White women (aOR 1.19, 95% CI 1.18-1.21; p < 0.01). Stage disparities were demonstrated when populations were disaggregated by country of origin, particularly for Mexican women (aOR 1.55, 95% CI 1.51-1.60; p < 0.01). Disparities worsened among both racial and country of origin subgroups in women with triple-negative disease. CONCLUSION: Later breast cancer stage at presentation was observed among Hispanic populations when disaggregated by racial subgroup and country of origin. Socioeconomic disparities, as well as uncaptured disparities in access and/or differential care, may drive these observed differences. Future studies with disaggregated data are needed to characterize outcomes in Hispanic communities and develop targeted interventions.

An Analysis of the Evidence Underpinning the American Academy Orthopaedic Surgery Pediatrics Clinical Practice Guidelines.

BACKGROUND: Randomized control trials (RCTs) serve as evidentiary support for recommendations underpinning clinical practice guidelines (CPGs) with the goal of optimizing patient care. A knowledge gap exists within scientific literature when evaluating the quality of RCTs used as evidence in the American Academy of Orthopaedic Surgery (AAOS) pediatric CPGs. We aim to evaluate the reporting quality and risk of bias in RCTs underlying AAOS Pediatric CPG recommendations. METHODS: We located all AAOS Pediatric CPGs. We then extracted all RCTs from the CPG reference sections. All included RCTs were evaluated using the Consolidated Standards of Reporting Trials (CONSORT) checklist and Cochrane Collaboration risk of bias assessment tool (RoB 2.0). Descriptive statistics were recorded, and bivariate analysis was used to account for variance in CONSORT scores. A Mann-Whitney U test was completed to compare CONSORT studies published before and after 2010. RESULTS: Three CPGs and 23 RCTs met inclusion criteria. Mean CONSORT adherence was 69.8% (21.6/31). The lowest adhered to CONSORT items were 10, 23, and 24, while items 2a, 13a, and 18 displayed the highest adherence. Ten RCTs (43.5%, 10/23) had "low" risk of bias, 5 RCTs (21.7%, 5/23) were of "some concerns," and 8 RCTs (34.8%, 8/23) received a "high" designation for risk of bias. There were no statistically significant associations in the bivariate regression analysis or Mann-Whitney U test. CONCLUSIONS: Our results suggest that CONSORT adherence within RCTs used as evidence in AAOS Pediatric CPGs is substandard-relying on evidence that, in some cases, is >20 years old. Many of the RCTs cited as supporting evidence have a "high" risk of bias. Altogether, these CPGs may need to be updated or expanded to include more recent evidence relevant to pediatric orthopaedic surgery.

Natural biopolymers as proton conductors in bioelectronics.

Bioelectronic devices sense or deliver information at the interface between living systems and electronics by converting biological signals into electronic signals and vice-versa. Biological signals are typically carried by ions and small molecules. As such, ion conducting materials are ideal candidates in bioelectronics for an optimal interface. Among these materials, ion conducting polymers that are able to uptake water are particularly interesting because, in addition to ionic conductivity, their mechanical properties can closely match the ones of living tissue. In this review, we focus on a specific subset of ion-conducting polymers: proton (H+ ) conductors that are naturally derived. We first provide a brief introduction of the proton conduction mechanism, and then outline the chemical structure and properties of representative proton-conducting natural biopolymers: polysaccharides (chitosan and glycosaminoglycans), peptides and proteins, and melanin. We then highlight examples of using these biopolymers in bioelectronic devices. We conclude with current challenges and future prospects for broader use of natural biopolymers as proton conductors in bioelectronics and potential translational applications.

Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform.

Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism-evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.

WhiB6 regulation of ESX-1 gene expression is controlled by a negative feedback loop in Mycobacterium marinum.

ESX (ESAT-6 system) export systems play diverse roles across mycobacterial species. Interestingly, genetic disruption of ESX systems in different species does not result in an accumulation of protein substrates in the mycobacterial cell. However, the mechanisms underlying this observation are elusive. We hypothesized that the levels of ESX substrates were regulated by a feedback-control mechanism, linking the levels of substrates to the secretory status of ESX systems. To test this hypothesis, we used a combination of genetic, transcriptomic, and proteomic approaches to define export-dependent mechanisms regulating the levels of ESX-1 substrates in Mycobacterium marinum WhiB6 is a transcription factor that regulates expression of genes encoding ESX-1 substrates. We found that, in the absence of the genes encoding conserved membrane components of the ESX-1 system, the expression of the whiB6 gene and genes encoding ESX-1 substrates were reduced. Accordingly, the levels of ESX-1 substrates were decreased, and WhiB6 was not detected in M. marinum strains lacking genes encoding ESX-1 components. We demonstrated that, in the absence of EccCb1, a conserved ESX-1 component, substrate gene expression was restored by constitutive, but not native, expression of the whiB6 gene. Finally, we found that the loss of WhiB6 resulted in a virulent M. marinum strain with reduced ESX-1 secretion. Together, our findings demonstrate that the levels of ESX-1 substrates in M. marinum are fine-tuned by negative feedback control, linking the expression of the whiB6 gene to the presence, not the functionality, of the ESX-1 membrane complex.

A whole genome approach for discovering the genetic basis of blood group antigens: independent confirmation for P1 and Xga.

BACKGROUND: Although P1 and Xga are known to be associated with the A4GALT and XG genes, respectively, the genetic basis of antigen expression has been elusive. Recent reports link both P1 and Xga expression with nucleotide changes in the promotor regions and with antigen-negative phenotypes due to disruption of transcription factor binding. STUDY DESIGN AND METHODS: Whole genome sequencing was performed on 113 individuals as part of the MedSeq Project with serologic RBC antigen typing for P1 (n = 77) and Xga (n = 15). Genomic data were analyzed by two approaches, nucleotide frequency correlation and serologic correlation, to find A4GALT and XG changes associated with P1 and Xga expression. RESULTS: For P1, the frequency approach identified 29 possible associated nucleotide changes, and the serologic approach revealed four among them correlating with the P1+/P1- phenotype: chr22:43,115,523_43,115,520AAAG/delAAAG (rs66781836); chr 22:43,114,551C/T (rs8138197); chr22:43,114,020 T/G (rs2143918); and chr22:43,113,793G/T (rs5751348). For Xga , the frequency approach identified 82 possible associated nucleotide changes, and among these the serologic approach revealed one correlating with the Xg(a+)/Xg(a-) phenotype: chrX:2,666,384G/C (rs311103). CONCLUSION: A bioinformatics analysis pipeline was created to identify genetic changes responsible for RBC antigen expression. This study, in progress before the recently published reports, independently confirms the basis for P1 and Xga . Although this enabled molecular typing of these antigens, the Y chromosome PAR1 region interfered with Xga typing in males. This approach could be used to identify and confirm the genetic basis of antigens, potentially replacing the historical approach using family pedigrees as genomic sequencing becomes commonplace.

Development and validation of a video-based cold snare polypectomy assessment tool (with videos).

BACKGROUND AND AIMS: Polyps <1 cm are the most commonly found polyps during colonoscopy. Cold snare removal is preferred given the significantly higher rate of incomplete resection associated with piecemeal biopsy forceps resection. There are currently no standardized tools to assess competence in cold snare polypectomy. This study aimed to develop and validate a cold snare polypectomy assessment tool (CSPAT). METHODS: Experts in cold snare polypectomy used a Delphi method to develop the CSPAT. Metrics with a greater than 85% agreement as being "important" or "very important" were included in the CSPAT. The tool included evaluation of polyp inspection, positioning, appropriate ensnarement of tissue to ensure a rim of normal tissue, tissue retrieval, and postpolypectomy site inspection. Experts in cold snare polypectomy used the CSPAT to evaluate preselected videos that were previously evaluated using the Direct Observation of Polypectomy Skills (DOPyS) tool. Interobserver agreement was evaluated. CSPAT scores were compared with DOPyS scores to assess content validity. RESULTS: Sixteen experts developed the 12-item CSPAT, and 13 experts reviewed 55 videos. There was a moderate degree of agreement in 10 metrics (κ = .52-.59) and a substantial degree of agreement (κ = .61-.63) in the other 2. There was a strong correlation between the mean of individual metrics 1 to 12 compared with the global competence assessment (ρ = .88, P < .001). There was a moderate correlation between the average overall DOPyS score and the overall CSPAT competence score (ρ = .56, P < .001). CONCLUSIONS: We have developed and validated a customized CSPAT that can be easily applied to video-based assessments to assess competence in training and among practicing endoscopists.

Distinct binding of BRCA2 BRC repeats to RAD51 generates differential DNA damage sensitivity.

BRCA2 is a multi-faceted protein critical for the proper regulation of homology-directed repair of DNA double-strand breaks. Elucidating the mechanistic features of BRCA2 is crucial for understanding homologous recombination and how patient-derived mutations impact future cancer risk. Eight centrally located BRC repeats in BRCA2 mediate binding and regulation of RAD51 on resected DNA substrates. Herein, we dissect the biochemical and cellular features of the BRC repeats tethered to the DNA binding domain of BRCA2. To understand how the BRC repeats and isolated domains of BRCA2 contribute to RAD51 binding, we analyzed both the biochemical and cellular properties of these proteins. In contrast to the individual BRC repeat units, we find that the BRC5-8 region potentiates RAD51-mediated DNA strand pairing and provides complementation functions exceeding those of BRC repeats 1-4. Furthermore, BRC5-8 can efficiently repair nuclease-induced DNA double-strand breaks and accelerate the assembly of RAD51 repair complexes upon DNA damage. These findings highlight the importance of the BRC5-8 domain in stabilizing the RAD51 filament and promoting homology-directed repair under conditions of cellular DNA damage.

Intracerebral Delivery of Brain-Derived Neurotrophic Factor Using HyStem®-C Hydrogel Implants Improves Functional Recovery and Reduces Neuroinflammation in a Rat Model of Ischemic Stroke.

Ischemic stroke is a leading cause of death and disability worldwide. Potential therapeutics aimed at neural repair and functional recovery are limited in their blood-brain barrier permeability and may exert systemic or off-target effects. We examined the effects of brain-derived neurotrophic factor (BDNF), delivered via an extended release HyStem®-C hydrogel implant or vehicle, on sensorimotor function, infarct volume, and neuroinflammation, following permanent distal middle cerebral artery occlusion (dMCAo) in rats. Eight days following dMCAo or sham surgery, treatments were implanted directly into the infarction site. Rats received either vehicle, BDNF-only (0.167 µg/µL), hydrogel-only, hydrogel impregnated with 0.057 µg/µL of BDNF (hydrogel + BDNFLOW), or hydrogel impregnated with 0.167 µg/µL of BDNF (hydrogel + BDNFHIGH). The adhesive removal test (ART) and 28-point Neuroscore (28-PN) were used to evaluate sensorimotor function up to two months post-ischemia. The hydrogel + BDNFHIGH group showed significant improvements on the ART six to eight weeks following treatment and their behavioral performance was consistently greater on the 28-PN. Infarct volume was reduced in rats treated with hydrogel + BDNFHIGH as were levels of microglial, phagocyte, and astrocyte marker immunoexpression in the corpus striatum. These data suggest that targeted intracerebral delivery of BDNF using hydrogels may mitigate ischemic brain injury and restore functional deficits by reducing neuroinflammation.

Noncanoncial signal recognition particle RNAs in a major eukaryotic phylum revealed by purification of SRP from the human pathogen Cryptococcus neoformans.

Despite conservation of the signal recognition particle (SRP) from bacteria to man, computational approaches have failed to identify SRP components from genomes of many lower eukaryotes, raising the possibility that they have been lost or altered in those lineages. We report purification and analysis of SRP in the human pathogen Cryptococcus neoformans, providing the first description of SRP in basidiomycetous yeast. The C. neoformans SRP RNA displays a predicted structure in which the universally conserved helix 8 contains an unprecedented stem-loop insertion. Guided by this sequence, we computationally identified 152 SRP RNAs throughout the phylum Basidiomycota. This analysis revealed additional helix 8 alterations including single and double stem-loop insertions as well as loop diminutions affecting RNA structural elements that are otherwise conserved from bacteria to man. Strikingly, these SRP RNA features in Basidiomycota are accompanied by phylum-specific alterations in the RNA-binding domain of Srp54, the SRP protein subunit that directly interacts with helix 8. Our findings reveal unexpected fungal SRP diversity and suggest coevolution of the two most conserved SRP features-SRP RNA helix 8 and Srp54-in basidiomycetes. Because members of this phylum include important human and plant pathogens, these noncanonical features provide new targets for antifungal compound development.

δPKC interaction with the d subunit of F1Fo ATP synthase impairs energetics and exacerbates ischemia/reperfusion injury in isolated rat hearts.

Previously, we demonstrated protection against hypoxic injury in neonatal cardiac myocytes and reduced release of cardiac troponin I from perfused rat hearts by a novel peptide inhibitor [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK-] of the delta protein kinase C (δPKC) interaction with the "d" subunit of mitochondrial F1Fo ATP synthase (dF1Fo). This peptide was developed in our laboratory and contains: an HIV-Tat protein transduction domain; a mitochondrial targeting motif; the δPKC-dF1Fo inhibitor sequence; and a FLAG epitope. In the present study the δPKC-dF1Fo inhibitor attenuated co-immunoprecipitation of δPKC with dF1Fo, improved recovery of contractility, diminished levels of tissue t-carbonyls and 4-hydroxy-2-nonenal (HNE), and reduced 2,3,5-triphenyltetrazolium chloride-monitored infarct size following simulated global ischemia/reperfusion (IR) exposures. Perfusion of hearts with this peptide prior to IR enhanced ATP levels 2.1-fold, improved ADP (state 3)- and FCCP (maximal)-stimulated respiration in mitochondrial oxygen consumption assays, and attenuated Ca(++)-induced mitochondrial swelling following ischemic injury. Mitochondrial membrane potential (assessed by JC-1) was also improved 1.6-fold by the inhibitor in hearts subsequently exposed to IR injury. Brief IR exposures did not cause mitochondrial loss of cytochrome c in the presence or absence of the inhibitor. Additionally, the inhibitor did not modify accumulation of the autophagy marker LC3II after brief IR injury. Our results support the potential for this first-in-class peptide as a translational agent for combating cardiac IR injury.

Assessment of cardiac function in mice lacking the mitochondrial calcium uniporter.

Mitochondrial calcium is thought to play an important role in the regulation of cardiac bioenergetics and function. The entry of calcium into the mitochondrial matrix requires that the divalent cation pass through the inner mitochondrial membrane via a specialized pore known as the mitochondrial calcium uniporter (MCU). Here, we use mice deficient of MCU expression to rigorously assess the role of mitochondrial calcium in cardiac function. Mitochondria isolated from MCU(-/-) mice have reduced matrix calcium levels, impaired calcium uptake and a defect in calcium-stimulated respiration. Nonetheless, we find that the absence of MCU expression does not affect basal cardiac function at either 12 or 20months of age. Moreover, the physiological response of MCU(-/-) mice to isoproterenol challenge or transverse aortic constriction appears similar to control mice. Thus, while mitochondria derived from MCU(-/-) mice have markedly impaired mitochondrial calcium handling, the hearts of these animals surprisingly appear to function relatively normally under basal conditions and during stress.

Cyclophilin D modulates mitochondrial acetylome.

RATIONALE: Mice lacking cyclophilin D (CypD(-/-)), a mitochondrial chaperone protein, have altered cardiac metabolism. As acetylation has been shown to regulate metabolism, we tested whether changes in protein acetylation might play a role in these metabolic changes in CypD(-/-) hearts. OBJECTIVE: Our aim was to test the hypothesis that loss of CypD alters the cardiac mitochondrial acetylome. METHODS AND RESULTS: To identify changes in lysine-acetylated proteins and to map acetylation sites after ablation of CypD, we subjected tryptic digests of isolated cardiac mitochondria from wild-type and CypD(-/-) mice to immunoprecipitation using agarose beads coupled to antiacetyl lysine antibodies followed by mass spectrometry. We used label-free analysis for the relative quantification of the 875 common peptides that were acetylated in wild-type and CypD(-/-) samples and found 11 peptides (10 proteins) decreased and 96 peptides (48 proteins) increased in CypD(-/-) samples. We found increased acetylation of proteins in fatty acid oxidation and branched-chain amino acid metabolism. To evaluate whether this increase in acetylation might play a role in the inhibition of fatty acid oxidation that was previously reported in CypD(-/-) hearts, we measured the activity of l-3-hydroxyacyl-CoA dehydrogenase, which was acetylated in the CypD(-/-) hearts. Consistent with the hypothesis, l-3-hydroxyacyl-CoA dehydrogenase activity was inhibited by ≈50% compared with the wild-type mitochondria. CONCLUSIONS: These results implicate a role for CypD in modulating protein acetylation. Taken together, these results suggest that ablation of CypD leads to changes in the mitochondrial acetylome, which may contribute to altered mitochondrial metabolism in CypD(-/-) mice.