Combination of common mtDNA variants results in mitochondrial dysfunction and a connective tissue dysregulation.

Mitochondrial dysfunction can be associated with a range of clinical manifestations. Here, we report a family with a complex phenotype including combinations of connective tissue, neurological, and metabolic symptoms that were passed on to all surviving children. Analysis of the maternally inherited mtDNA revealed a novel genotype encompassing the haplogroup J - defining mitochondrial DNA (mtDNA) ND5 m.13708G>A (A458T) variant arising on the mtDNA haplogroup H7A background, an extremely rare combination. Analysis of transmitochondrial cybrids with the 13708A-H7 mtDNA revealed a lower mitochondrial respiration, increased reactive oxygen species production (mROS), and dysregulation of connective tissue gene expression. The mitochondrial dysfunction was exacerbated by histamine, explaining why all eight surviving children inherited the dysfunctional histidine decarboxylase allele (W327X) from the father. Thus, certain combinations of common mtDNA variants can cause mitochondrial dysfunction, mitochondrial dysfunction can affect extracellular matrix gene expression, and histamine-activated mROS production can augment the severity of mitochondrial dysfunction. Most important, we have identified a previously unreported genetic cause of mitochondrial disorder arising from the incompatibility of common, nonpathogenic mtDNA variants.

Evaluating the implementation of a community health worker-delivered intervention integrating asthma care in West Philadelphia public schools.

OBJECTIVE: Schools are an important setting because students spend much of their time in school and engage in physical activity during the school day that could exacerbate asthma symptoms. Our objective is to understand the barriers and facilitators to implementing an experimental community health worker-delivered care coordination program for students with asthma within the context of the West Philadelphia Controls Asthma study. METHODS: Surveys (n = 256) and semi-structured interviews (n = 41) were completed with principals, teachers, nurses, and community health workers from 21 public and charter schools in West Philadelphia between January 2019 and September 2021. Survey participants completed the Evidence Based Practice Attitudes Scale, the Implementation Leadership Scale, and Organizational Climate Index. Semi-structured qualitative interview guides were developed, informed by the Consolidated Framework for Implementation Research. RESULTS: Participant responses indicate that they perceived benefits for schools and students related to the community health worker-based care coordination program. Several barriers and facilitators to implementing the program were noted, including challenges associated with incorporating the program into school nurse workflow, environmental triggers in the school environment, and challenges communicating with family members. An important facilitator that was identified was having supportive school administrators and staff who were engaged and saw the benefits of the program. CONCLUSIONS: This work can inform implementation planning for other locales interested in implementing community-based pediatric asthma control programs delivered by community health workers in schools.

Investigation of Receptor Heteromers Using NanoBRET Ligand Binding.

Receptor heteromerization is the formation of a complex involving at least two different receptors with pharmacology that is distinct from that exhibited by its constituent receptor units. Detection of these complexes and monitoring their pharmacology is crucial for understanding how receptors function. The Receptor-Heteromer Investigation Technology (Receptor-HIT) utilizes ligand-dependent modulation of interactions between receptors and specific biomolecules for the detection and profiling of heteromer complexes. Previously, the interacting biomolecules used in Receptor-HIT assays have been intracellular proteins, however in this study we have for the first time used bioluminescence resonance energy transfer (BRET) with fluorescently-labeled ligands to investigate heteromerization of receptors on the cell surface. Using the Receptor-HIT ligand binding assay with NanoBRET, we have successfully investigated heteromers between the angiotensin II type 1 (AT1) receptor and the ß2 adrenergic receptor (AT1-ß2AR heteromer), as well as between the AT1 and angiotensin II type 2 receptor (AT1-AT2 heteromer).

Insights into the Interaction of LVV-Hemorphin-7 with Angiotensin II Type 1 Receptor.

Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-hemorphin-7 (LVV-H7) in human embryonic kidney (HEK293) cells. Here, we examined the molecular binding behavior of LVV-H7 on AT1R and its effect on AngII binding using a nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay in HEK293FT cells, as well as molecular docking and molecular dynamics (MD) studies. Saturation and real-time kinetics supported the positive effect of LVV-H7 on the binding of AngII. While the competitive antagonist olmesartan competed with AngII binding, LVV-H7 slightly, but significantly, decreased AngII's kD by 2.6 fold with no effect on its Bmax. Molecular docking and MD simulations indicated that the binding of LVV-H7 in the intracellular region of AT1R allosterically potentiates AngII binding. LVV-H7 targets residues on intracellular loops 2 and 3 of AT1R, which are known binding sites of allosteric modulators in other GPCRs. Our data demonstrate the allosteric effect of LVV-H7 on AngII binding, which is consistent with the positive modulation of AT1R activity and signaling previously reported. This further supports the pharmacological targeting of AT1R by hemorphins, with implications in vascular and renal physiology.

Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3.

The neuropeptide relaxin-3 and its receptor relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking. A linear relaxin-3 antagonist (R3 B1-22R) based on a modified and truncated relaxin-3 B-chain was recently developed. R3 B1-22R is unstructured in solution; thus, the binding conformation and determinants of receptor binding are unclear. Here, we have designed, chemically synthesized, and pharmacologically characterized more than 60 analogues of R3 B1-22R to develop an extensive understanding of its structure-activity relationships. We show that the key driver for affinity is the nonnative C-terminal Arg23 Additional contributors to binding include amino acid residues that are important also for relaxin-3 binding, including Arg12, Ile15, and Ile19 Intriguingly, amino acid residues that are not exposed in native relaxin-3, including Phe14 and Ala17, also interact with RXFP3. We show that R3 B1-22R has a propensity to form a helical structure, and modifications that support a helical conformation are functionally well-tolerated, whereas helix breakers such as proline residues disrupt binding. These data suggest that the peptide adopts a helical conformation, like relaxin-3, upon binding to RXFP3, but that its smaller size allows it to penetrate deeper into the orthosteric binding site, creating more extensive contacts with the receptor.

Facultative control of matrix production optimizes competitive fitness in Pseudomonas aeruginosa PA14 biofilm models.

As biofilms grow, resident cells inevitably face the challenge of resource limitation. In the opportunistic pathogen Pseudomonas aeruginosa PA14, electron acceptor availability affects matrix production and, as a result, biofilm morphogenesis. The secreted matrix polysaccharide Pel is required for pellicle formation and for colony wrinkling, two activities that promote access to O2. We examined the exploitability and evolvability of Pel production at the air-liquid interface (during pellicle formation) and on solid surfaces (during colony formation). Although Pel contributes to the developmental response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation to the colony edge produced mutants with clear competitive advantages against the wild type in this O2-replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure formation in response to electron acceptor limitation is unique to specific biofilm models and that the facultative control of Pel production is required for PA14 to maintain optimum benefit in different types of communities.

From whence it came: Mitochondrial mRNA leaves, a protein returns.

Small peptides have previously been reported to be encoded in mitochondrial rRNA and translated by cytosolic ribosomes. In this issue of Cell Metabolism, Hu et al. use mass spectrometry to identify a cytosolically translated protein, encoded instead in mitochondrial mRNA, that is surprisingly targeted back into the mitochondrial matrix.

Visualizing subcellular changes in the NAD(H) pool size versus redox state using fluorescence lifetime imaging microscopy of NADH.

NADH autofluorescence imaging is a promising approach for visualizing energy metabolism at the single-cell level. However, it is sensitive to the redox ratio and the total NAD(H) amount, which can change independently from each other, for example with aging. Here, we evaluate the potential of fluorescence lifetime imaging microscopy (FLIM) of NADH to differentiate between these modalities.We perform targeted modifications of the NAD(H) pool size and ratio in cells and mice and assess the impact on NADH FLIM. We show that NADH FLIM is sensitive to NAD(H) pool size, mimicking the effect of redox alterations. However, individual components of the fluorescence lifetime are differently impacted by redox versus pool size changes, allowing us to distinguish both modalities using only FLIM. Our results emphasize NADH FLIM's potential for evaluating cellular metabolism and relative NAD(H) levels with high spatial resolution, providing a crucial tool for our understanding of aging and metabolism.

Spinal matrix metalloproteinase-9 contributes to physical dependence on morphine in mice.

Preventing and reversing opioid dependence continues to be a clinical challenge and underlying mechanisms of opioid actions remain elusive. We report that matrix metalloproteinase-9 (MMP-9) in the spinal cord contributes to development of physical dependence on morphine. Chronic morphine exposure and naloxone-precipitated withdrawal increase activity of spinal MMP-9. Spinal inhibition or targeted mutation of MMP-9 suppresses behavioral signs of morphine withdrawal and the associated neurochemical alterations. The increased MMP-9 activity is mainly distributed in the superficial dorsal horn and colocalized primarily with neurons and small numbers of astrocytes and microglia. Morphine exposure and withdrawal increase phosphorylation of NR1 and NR2B receptors, ERK1/2, calmodulin-dependent kinase II, and cAMP response element binding proteins; and such phosphorylation is suppressed by either spinal inhibition or targeted mutation of MMP-9. Further, spinal administration of exogenous MMP-9 induces morphine withdrawal-like behavioral signs and mechanical allodynia, activates NR1 and NR2 receptors, and downregulates integrin-beta1, while a function-neutralizing antibody against integrin-beta1 suppresses MMP-9-induced phosphorylation of NR1 and NR2B. Morphine withdrawal-induced MMP-9 activity is also reduced by an nNOS inhibitor. Thus, we hypothesize that spinal MMP-9 may contribute to the development of morphine dependence primarily through neuronal activation and interaction with NR1 and NR2B receptors via integrin-beta1 and NO pathways. The other gelatinase, MMP-2, is not involved in morphine dependence. Inhibiting spinal MMP-9 or MMP-2 reduces chronic and/or acute morphine tolerance. This study suggests a novel therapeutic approach for preventing, minimizing, or reversing opioid dependence and tolerance.

Characteristics of Patients Who Attended Behavioral Health Services After Primary Care Referral With Referral Management Support.

OBJECTIVE: This study examined whether documented disparities in access to behavioral health specialty care persisted in a novel integrated primary care model situated in a large health system when triage and referral management supports were provided by a centralized resource center for patients with behavioral health needs. METHODS: Patients triaged and referred to specialty behavioral health care who did or did not attend a specialty care visit (N=1,450) were compared in terms of various demographic and clinical characteristics by using binary logistic regression. RESULTS: Among patients with attendance data, financially unstable individuals were more likely than financially stable counterparts to miss their first appointment with a specialty behavioral health provider after referral from primary care. Previously documented attendance disparities based on race, ethnicity, and illness severity were not observed. CONCLUSIONS: These findings can inform targeted strategies to increase attendance among patients with financial insecurity and reduce disparities in outpatient behavioral health services.

Cell-Matrix Interactions Regulate Functional Extracellular Vesicle Secretion from Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) are cell-secreted particles with broad potential to treat tissue injuries by delivering cargo to program target cells. However, improving the yield of functional EVs on a per cell basis remains challenging due to an incomplete understanding of how microenvironmental cues regulate EV secretion at the nanoscale. We show that mesenchymal stromal cells (MSCs) seeded on engineered hydrogels that mimic the elasticity of soft tissues with a lower integrin ligand density secrete ∼10-fold more EVs per cell than MSCs seeded on a rigid plastic substrate, without compromising their therapeutic activity or cargo to resolve acute lung injury in mice. Mechanistically, intracellular CD63+ multivesicular bodies (MVBs) transport faster within MSCs on softer hydrogels, leading to an increased frequency of MVB fusion with the plasma membrane to secrete more EVs. Actin-related protein 2/3 complex but not myosin-II limits MVB transport and EV secretion from MSCs on hydrogels. The results provide a rational basis for biomaterial design to improve EV secretion while maintaining their functionality.

A chameleonic macrocyclic peptide with drug delivery applications.

Head-to-tail cyclized peptides are intriguing natural products with unusual properties. The PawS-Derived Peptides (PDPs) are ribosomally synthesized as part of precursors for seed storage albumins in species of the daisy family, and are post-translationally excised and cyclized during proteolytic processing. Here we report a PDP twice the typical size and with two disulfide bonds, identified from seeds of Zinnia elegans. In water, synthetic PDP-23 forms a unique dimeric structure in which two monomers containing two ß-hairpins cross-clasp and enclose a hydrophobic core, creating a square prism. This dimer can be split by addition of micelles or organic solvent and in monomeric form PDP-23 adopts open or closed V-shapes, exposing different levels of hydrophobicity dependent on conditions. This chameleonic character is unusual for disulfide-rich peptides and engenders PDP-23 with potential for cell delivery and accessing novel targets. We demonstrate this by conjugating a rhodamine dye to PDP-23, creating a stable, cell-penetrating inhibitor of the P-glycoprotein drug efflux pump.

Development of Relaxin-3 Agonists and Antagonists Based on Grafted Disulfide-Stabilized Scaffolds.

Relaxin-3 is a neuropeptide with important roles in metabolism, arousal, learning and memory. Its cognate receptor is the relaxin family peptide-3 (RXFP3) receptor. Relaxin-3 agonist and antagonist analogs have been shown to be able to modulate food intake in rodent models. The relaxin-3 B-chain is sufficient for receptor interactions, however, in the absence of a structural support, linear relaxin-3 B-chain analogs are rapidly degraded and thus unsuitable as drug leads. In this study, two different disulfide-stabilized scaffolds were used for grafting of important relaxin-3 B-chain residues to improve structure and stability. The use of both Veronica hederifolia Trypsin inhibitor (VhTI) and apamin grafting resulted in agonist and antagonist analogs with improved helicity. VhTI grafted peptides showed poor binding and low potency at RXFP3, on the other hand, apamin variants retained significant activity. These variants also showed improved half-life in serum from ~5 min to >6 h, and thus are promising RXFP3 specific pharmacological tools and drug leads for neuropharmacological diseases.

Nutritional and psychological considerations after bariatric surgery.

The anatomic and physiological changes resulting from bariatric surgery can affect both nutrition and psychological attitudes. Modifications of the gastrointestinal tract lead to a diminished ability to absorb nutrients, electrolytes, and bile salts, as well as deficiencies in iron, calcium, and other vitamins and minerals. Dehydration, lactose intolerance, and protein calorie malnutrition are other common sequelae. Alterations in bone metabolism increase long-term risk for osteopenia and osteoporosis. Noncompliance with postsurgical nutritional regimens has been estimated to occur in from one third to almost two thirds of cases and can exacerbate these complications. Psychological issues are often present in patients with morbid obesity and can affect surgical outcomes. These issues include mood and personality disorders, destructive eating behaviors, and poor body image. Nearly one third of patients undergoing bariatric surgery also have a history of substance abuse disorder. The literature suggests that although the mental health of patients improves as a result of bariatric surgery, the benefits may be transient, and problems such as negative personality profiles, detrimental eating patterns, and negative body image persist to some extent. Identification of presurgical psychiatric problems can help identify those patients more likely to achieve lasting weight loss when surgery is combined with long-term follow-up to minimize medical and psychological complications.

Reorienting Orientation: Introducing the Social Determinants of Health to First-Year Medical Students.

Introduction: Medical students rarely learn about the intersection of socioeconomic and environmental effects on access to health care and maintenance of health. Case-based discussion can cohesively highlight the social determinants of health to complement preclinical education. Our modules can foster future interest in working with vulnerable populations, help students recognize barriers to care, and identify strategies to help these patients. Methods: The Social Determinants of Health Orientation Program (SDHOP) introduced students to the nonbiomedical factors that contribute to patients' health. Key topics were presented in small discussion groups led by faculty facilitators. The subjects addressed included access to care; immigration/language barriers; lesbian, gay, bisexual, and transgender health; human trafficking; race/ethnicity; and women's health. Results: The SDHOP initiative was integrated into the formal curriculum and successfully implemented in its first year at our institution. Pre- and postsurveys were administered to assess student satisfaction with the course, as well as changes in knowledge and attitude regarding the topics covered. Of the 186 SDHOP participants, 111 medical students responded to both surveys and reported improvements in both knowledge of and comfort level with these topics and specific related terms. Ninety-one percent rated the overall quality of SDHOP and its individual modules as good or excellent. Discussion: SDHOP contributes to medical education by providing an all-inclusive model for teaching students about the social determinants of health. Our results suggest that presenting these topics in a small-group discussion model improves medical student cultural competency and comfort level with patients of diverse backgrounds.

A LASSO Method to Identify Protein Signature Predicting Post-transplant Renal Graft Survival.

Identifying novel biomarkers to predict renal graft survival is important in post-transplant clinical practice. Serum creatinine, currently the most popular surrogate biomarker, offers limited information of the underlying allograft profiles. It is known to perform unsatisfactorily to predict renal function. In this paper, we apply a LASSO machine-learning algorithm in the Cox proportional hazards model to identify promising proteins that are associated with the hazard of allograft loss after renal transplantation, motivated by a clinical pilot study that collected 47 patients receiving renal transplants at the University of Michigan Hospital. We assess the association of 17 proteins previously identified by Cibrik et al. [5] with allograft rejection in our regularized Cox regression analysis, where the LASSO variable selection method is applied to select important proteins that predict the hazard of allograft loss. We also develop a post-selection inference to further investigate the statistical significance of the proteins on the hazard of allograft loss, and conclude that two proteins KIM-1 and VEGF-R2 are important protein markers for risk prediction.

Biomechanical properties of the superficial fascial system.

BACKGROUND: Surgical repair of the superficial fascial system (SFS) has been claimed to both increase wound strength and enhance surgical outcome through anchoring of deeper tissues. OBJECTIVE: The authors assessed the biomechanical properties of the SFS to determine whether repair of the SFS layer improved early and long-term postoperative wound strength. METHODS: Four complementary studies were conducted to study the dermis and SFS junctional architecture and connective tissue content: gross dissection using a dehydrating agent (Pen-Fix; Richard-Allan Scientific, Kalamazoo, MI), a histologic study with hemotoxylin and eosin staining, soft tissue radiography, and immunofluorescence staining. Freshly excised human abdominal and lower back/buttock tissues underwent a midline incision, followed by repair using dermal sutures only (DRM), dermal sutures plus SFS sutures (DRM/SFS) or repair of the SFS only (SFS). Fresh swine abdominal tissues were similarly excised and repaired. Biomechanical tests were undertaken to compare the ex vivo human and swine tissues. Three types of closure-dermal sutures only (DRM), dermal sutures plus permanent 0-braided nylon suture in the SFS (DRM/SFS/N), and dermal sutures plus absorbable 0-vicryl suture in the SFS (DRM/SFS/V) were also tested in an in vivo swine model. RESULTS: Immunofluorescence studies showed collagen and elastin content and ratios to be comparable in the dermis and SFS. In ex vivo studies of human abdominal and back tissues, cyclic creep did not vary significantly among the different types of repair. DRM/SFS repair had a significantly higher failure load than dermal repair alone in both human abdominal and back tissues. In the in vivo swine study, normal tissue had a significantly higher failure load than all repair groups. The wounds where SFS had been repaired in addition to dermis exhibited an increased tensile strength and, among these, the wounds closed with SFS repair with a nonabsorbable suture exhibited greater tensile strength compared to absorbable suture repair. However, no statistically significant difference was noted, due to the small sample size. CONCLUSIONS: We have determined, using an ex vivo model, that repair of the SFS layer in addition to dermis repair significantly increases the initial biomechanical strength of wound repair. This has the potential to decrease early wound dehiscence. In our in vivo model, the use of a nonabsorbable suture to approximate the SFS demonstrated a trend toward increased long-term wound strength. We believe our studies provide scientific data documenting that SFS is a key contributory strength layer in the early postoperative period, and is likely to be a strength layer even in the later stages of wound healing.

Wnt/Ryk signaling contributes to neuropathic pain by regulating sensory neuron excitability and spinal synaptic plasticity in rats.

Treating neuropathic pain continues to be a major clinical challenge and underlying mechanisms of neuropathic pain remain elusive. We have recently demonstrated that Wnt signaling, which is important in developmental processes of the nervous systems, plays critical roles in the development of neuropathic pain through the ß-catenin-dependent pathway in the spinal cord and the ß-catenin-independent pathway in primary sensory neurons after nerve injury. Here, we report that Wnt signaling may contribute to neuropathic pain through the atypical Wnt/Ryk signaling pathway in rats. Sciatic nerve injury causes a rapid-onset and long-lasting expression of Wnt3a, Wnt5b, and Ryk receptors in primary sensory neurons, and dorsal horn neurons and astrocytes. Spinal blocking of the Wnt/Ryk receptor signaling inhibits the induction and persistence of neuropathic pain without affecting normal pain sensitivity and locomotor activity. Blocking activation of the Ryk receptor with anti-Ryk antibody, in vivo or in vitro, greatly suppresses nerve injury-induced increased intracellular Ca and hyperexcitability of the sensory neurons, and also the enhanced plasticity of synapses between afferent C-fibers and the dorsal horn neurons, and activation of the NR2B receptor and the subsequent Ca-dependent signals CaMKII, Src, ERK, PKCγ, and CREB in sensory neurons and the spinal cord. These findings indicate a critical mechanism underlying the pathogenesis of neuropathic pain and suggest that targeting the Wnt/Ryk signaling may be an effective approach for treating neuropathic pain.

WNT signaling underlies the pathogenesis of neuropathic pain in rodents.

Treating neuropathic pain is a major clinical challenge, and the underlying mechanisms of neuropathic pain remain elusive. We hypothesized that neuropathic pain-inducing nerve injury may elicit neuronal alterations that recapitulate events that occur during development. Here, we report that WNT signaling, which is important in developmental processes of the nervous system, plays a critical role in neuropathic pain after sciatic nerve injury and bone cancer in rodents. Nerve injury and bone cancer caused a rapid-onset and long-lasting expression of WNTs, as well as activation of WNT/frizzled/ß-catenin signaling in the primary sensory neurons, the spinal dorsal horn neurons, and astrocytes. Spinal blockade of WNT signaling pathways inhibited the production and persistence of neuropathic pain and the accompanying neurochemical alterations without affecting normal pain sensitivity and locomotor activity. WNT signaling activation stimulated production of the proinflammatory cytokines IL-18 and TNF-α and regulated the NR2B glutamate receptor and Ca2+-dependent signals through the ß-catenin pathway in the spinal cord. These findings indicate a critical mechanism underlying the pathogenesis of neuropathic pain and suggest that targeting the WNT signaling pathway may be an effective approach for treating neuropathic pain, including bone cancer pain.

Completing the metamorphosis: building a center of excellence in postbariatric plastic surgery.

Postbariatric body contouring is an expanding and rapidly evolving specialty within plastic surgery. To best address the many clinical issues related to the safe and effective care of the postbariatric patient, the authors have established a dedicated multidisciplinary treatment center. Team building efforts have brought together attending plastic surgeons who specialize in body contouring, nurses focused on body contouring after weight loss, and an administrative staff well trained in the issues related to managing these specific types of cases. Through core partnerships, nutritionists, lifestyle counselors, and psychologic professionals are present in the clinic to evaluate patients alongside the plastic surgeon. A dedicated physician assistant and body-contouring fellow work in both the clinic and the operating room (OR). This team approach has allowed for the establishment of defined screening procedures and patient care algorithms. Outreach and patient education initiatives have allowed the center to forge strong alliances with the regional bariatric surgeons and make patients aware of the role of plastic surgery as they start the process of weight loss. A strong research mission is demonstrated by an IRB-approved clinical registry and database that facilitate outcomes studies.