Faculty Bibliography

Advances in -omics analyses have tremendously enhanced our understanding of the role of the microbiome in human health and disease. Most research is focused on the bacteriome, but scientists have now realized the significance of the virome and microbial dysbiosis as well, particularly in noninfectious diseases such as cancer. In this review, we summarize the role of mycobiome in tumorigenesis, with a dismal prognosis, and attention to pancreatic ductal adenocarcinoma (PDAC). We also discuss bacterial and mycobial interactions to the host's immune response that is prevalently responsible for resistance to cancer therapy, including immunotherapy. We reported that the Malassezia species associated with scalp and skin infections, colonize in human PDAC tumors and accelerate tumorigenesis via activating the C3 complement-mannose-binding lectin (MBL) pathway. PDAC tumors thrive in an immunosuppressive microenvironment with desmoplastic stroma and a dysbiotic microbiome. Host-microbiome interactions in the tumor milieu pose a significant threat in driving the indolent immune behavior of the tumor. Microbial intervention in multimodal cancer therapy is a promising novel approach to modify an immunotolerant ("cold") tumor microenvironment to an immunocompetent ("hot") milieu that is effective in eliminating tumorigenesis.

OBJECTIVES/OBJECTIVE:To evaluate the outcomes of patients who underwent soft tissue flap coverage during treatment of a tibia fracture nonunion. DESIGN/METHODS:Retrospective analysis on prospectively collected data. SETTING/METHODS:Academic medical center. PATIENTS/PARTICIPANTS/METHODS:157 patients were treated for a fracture nonunion following a tibia fracture over a 15-year period. Sixty-six had sustained an open tibial fracture initially and 25 of these patients underwent soft tissue flaps for their open tibia fracture nonunion. INTERVENTION/METHODS:Manipulation of soft tissue flaps, either placement or elevation for graft placement in ununited previously open tibial fractures. MAIN OUTCOME MEASUREMENTS/METHODS:Bony healing, time to union, ultimate soft tissue status, postoperative complications, and functional outcome scores using the Short Musculoskeletal Functional Assessment (SMFA). This group was compared to a group of open tibial fracture nonunions that did not undergo soft tissue transfer. RESULTS:Bony healing was achieved in 24/25 patients (96.0%) who received flaps at a mean time to union of 8.7 ± 3.3 months compared to 39/41 patients (95.1%) at a mean 7.5 ± 3.2 months (p > 0.05) in the non-coverage group. Healing rate and time to union did not differ between groups. At latest follow-up, the flap coverage group reported a mean SMFA index of 17.1 compared to an SMFA index of 27.7 for the non-coverage group (p = 0.037). CONCLUSIONS:Utilization of soft tissue flaps in the setting of open tibia shaft nonunion repair surgery are associated with a high union rate (>90%). Coverage with or manipulation of soft tissue flaps did not result in improved bony healing rate or time to union compared to those who did not require flaps. However, soft tissue flap coverage was associated with higher functional scores at long-term follow-up. LEVEL OF EVIDENCE/METHODS:Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

The adaptable transcriptional response to changes in food availability not only ensures animal survival but also lets embryonic development progress. Interestingly, the CNS is preferentially protected from periods of malnutrition, a phenomenon known as "brain sparing." However, the mechanisms that mediate this response remain poorly understood. To get a better understanding of this, we used Drosophila melanogaster as a model, analyzing the transcriptional response of neural stem cells (neuroblasts) and glia of the blood-brain barrier (BBB) from larvae of both sexes during nutrient restriction using targeted DamID. We found differentially expressed genes in both neuroblasts and glia of the BBB, although the effect of nutrient deficiency was primarily observed in the BBB. We characterized the function of a nutritional sensitive gene expressed in the BBB, the serine protease homolog, scarface (scaf). Scaf is expressed in subperineurial glia in the BBB in response to nutrition. Tissue-specific knockdown of scaf increases subperineurial glia endoreplication and proliferation of perineurial glia in the blood-brain barrier. Furthermore, neuroblast proliferation is diminished on scaf knockdown in subperineurial glia. Interestingly, reexpression of Scaf in subperineurial glia is able to enhance neuroblast proliferation and brain growth of animals in starvation. Finally, we show that loss of scaf in the blood-brain barrier increases sensitivity to drugs in adulthood, suggesting a physiological impairment. We propose that Scaf integrates the nutrient status to modulate the balance between neurogenesis and growth of the BBB, preserving the proper equilibrium between the size of the barrier and the brain.SIGNIFICANCE STATEMENT The Drosophila BBB separates the CNS from the open circulatory system. The BBB glia are not only acting as a physical segregation of tissues but participate in the regulation of the metabolism and neurogenesis during development. Here we analyze the transcriptional response of the BBB glia to nutrient deprivation during larval development, a condition in which protective mechanisms are switched on in the brain. Our findings show that the gene scarface reduces growth in the BBB while promoting the proliferation of neural stem, assuring the balanced growth of the larval brain. Thus, Scarface would link animal nutrition with brain development, coordinating neurogenesis with the growth of the BBB.

Peroxynitrite, a transient reactive oxygen species (ROS), is believed to play a deleterious role in physiological processes. Herein, we report a two-photon ratiometric fluorescent probe that selectively reacts with peroxynitrite yielding a >200-fold change upon reaction. The probe effectively visualized fluctuations in peroxynitrite generation by arginase 1 in vivo and in vitro. This provides evidence that arginase 1 is a critical regulator of peroxynitrite.

Conserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4-7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending 'tug-of-war' that controls a temporally shifting window of accessibility for the transcription initiation machinery.

Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.

Background Proteinuria and glomerular segmental fibrosis are inevitable complications of diabetic nephropathy though their mechanisms are poorly understood. Understanding the clinical characteristics and pathogenesis of proteinuria and glomerular segmental fibrosis in diabetic nephropathy is, therefore, urgently needed for patient management of this severe disease. Methods and Results Diabetes mellitus was induced in podocyte-specific glucocorticoid receptor knockout (GR^PKO) mice and control littermates by administration of streptozotocin. Primary podocytes were isolated and subjected to analysis of Wnt signaling and fatty acid metabolism. Conditioned media from primary podocytes was transferred to glomerular endothelial cells. Histologic analysis of kidneys from diabetic GR^PKO mice showed worsened fibrosis, increased collagen deposition, and glomerulomegaly indicating severe glomerular fibrosis. Higher expression of transforming growth factor-βR1 and β-catenin and suppressed expression of carnitine palmitoyltransferase 1A in nephrin-positive cells were found in the kidneys of diabetic GR^PKO mice. Podocytes isolated from diabetic GR^PKO mice demonstrated significantly higher profibrotic gene expression and suppressed fatty acid oxidation compared with controls. Administration of a Wnt inhibitor significantly improved the fibrotic features in GR^PKO mice. The glomerular endothelium of diabetic GR^PKO mice demonstrated the features of endothelial-to-mesenchymal transition. Moreover, endothelial cells treated with conditioned media from podocytes lacking GR showed increased expression of α-smooth muscle actin, transforming growth factor-βR1 and β-catenin levels. Conclusions These data demonstrate that loss of podocyte GR leads to upregulation of Wnt signaling and disruption in fatty acid metabolism. Podocyte-endothelial cell crosstalk, mediated through GR, is important for glomerular homeostasis, and its disruption likely contributes to diabetic nephropathy.

Rationale: Loss-of-function of the cardiac sodium channel Na_V1.5 causes conduction slowing and arrhythmias. Na_V1.5 is differentially distributed within subcellular domains of cardiomyocytes, with sodium current (I_Na) being enriched at the intercalated discs (ID). Various pathophysiological conditions associated with lethal arrhythmias display ID-specific I_Na reduction, but the mechanisms underlying microdomain-specific targeting of Na_V1.5 remain largely unknown. Objective: To investigate the role of the microtubule (MT) plus-end tracking proteins end binding protein 1 (EB1) and CLIP-associated protein 2 (CLASP2) in mediating Na_V1.5 trafficking and subcellular distribution in cardiomyocytes.Methods and Results: EB1 overexpression in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) resulted in enhanced whole-cell I_Na, increased action potential (AP) upstroke velocity (V_max), and enhanced Na_V1.5 localization at the plasma membrane as detected by multi-color stochastic optical reconstruction microscopy (STORM). Fluorescence recovery after photobleaching (FRAP) experiments in HEK293A cells demonstrated that EB1 overexpression promoted Na_V1.5 forward trafficking. Knockout of MAPRE1 in hiPSC-CMs led to reduced whole-cell I_Na, decreased V_max and AP duration (APD) prolongation. Similarly, acute knockout of the MAPRE1 homolog in zebrafish (mapre1b) resulted in decreased ventricular conduction velocity and V_max as well as increased APD. STORM imaging and macropatch I_Na measurements showed that subacute treatment (2-3 hours) with SB216763 (SB2), a GSK3β inhibitor known to modulate CLASP2-EB1 interaction, reduced GSK3β localization and increased Na_V1.5 and I_Na preferentially at the ID region of wild type murine ventricular cardiomyocytes. By contrast, SB2 did not affect whole cell I_Na or Na_V1.5 localization in cardiomyocytes from Clasp2-deficient mice, uncovering the crucial role of CLASP2 in SB2-mediated modulation of NaV1.5 at the ID. Conclusions: Our findings demonstrate the modulatory effect of the MT plus-end tracking protein EB1 on Na_V1.5 trafficking and function, and identify the EB1-CLASP2 complex as a target for preferential modulation of I_Na within the ID region of cardiomyocytes.

KdpFABC is an oligomeric K⁺ transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K⁺ transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryogenic electron microscopy. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC, and KdpF remain static. We observe a series of spherical densities that we assign as K⁺ or water and which define a pathway for K⁺ transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K⁺ transfer between alternative sites in KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K⁺ to the cytoplasm.