Diserinol Isophthalamide: A Novel Reagent for Complexation with Biomolecular Anions in Electrospray Ionization Mass Spectrometry.

Transferring biomolecules from solution to vacuum facilitates a detailed analysis of molecular structure and dynamics by isolating molecules of interest from a complex environment. However, inherent in the ion desolvation process is the loss of solvent hydrogen bonding partners, which are critical for the stability of a condensed-phase structure. Thus, transfer of ions to vacuum can favor structural rearrangement, especially near solvent-accessible charge sites, which tend to adopt intramolecular hydrogen bonding motifs in the absence of solvent. Complexation of monoalkylammonium moieties (e.g., lysine side chains) with crown ethers such as 18-crown-6 can disfavor structural rearrangement of protonated sites, but no equivalent ligand has been investigated for deprotonated groups. Herein we describe diserinol isophthalamide (DIP), a novel reagent for the gas-phase complexation of anionic moieties within biomolecules. Complexation is observed to the C-terminus or side chains of the small model peptides GD, GE, GG, DF-OMe, VYV, YGGFL, and EYMPME in electrospray ionization mass spectrometry (ESI-MS) studies. In addition, complexation is observed with the phosphate and carboxylate moieities of phosphoserine and phosphotyrosine. DIP performs favorably in comparison to an existing anion recognition reagent, 1,1'-(1,2-phenylene)bis(3-phenylurea), that exhibits moderate carboxylate binding in organic solvent. This improved performance in ESI-MS experiments is attributed to reduced steric constraints to complexation with carboxylate groups of larger molecules. Overall, diserinol isophthalamide is an effective complexation reagent that can be applied in future work to study retention of solution-phase structure, investigate intrinsic molecular properties, and examine solvation effects.

Obesity induces gut microbiota alterations and augments acute graft-versus-host disease after allogeneic stem cell transplantation.

The efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by acute and chronic graft-versus-host disease (GVHD). The impact of obesity on allo-HSCT outcomes is poorly understood. Here, we report that obesity had a negative and selective impact on acute gut GVHD after allo-HSCT in mice with diet-induced obesity (DIO). These animals exhibited increased gut permeability, endotoxin translocation across the gut, and radiation-induced gastrointestinal damage after allo-HSCT. After allo-HSCT, both male and female DIO mouse recipients showed increased proinflammatory cytokine production and expression of the GVHD marker ST2 (IL-33R) and MHC class II molecules; they also exhibited decreased survival associated with acute severe gut GVHD. This rapid-onset, obesity-associated gut GVHD depended on donor CD4+ T cells and occurred even with a minor MHC mismatch between donor and recipient animals. Retrospective analysis of clinical cohorts receiving allo-HSCT transplants from unrelated donors revealed that recipients with a high body mass index (BMI, >30) had reduced survival and higher serum ST2 concentrations compared with nonobese transplant recipients. Assessment of both DIO mice and allo-HSCT recipients with a high BMI revealed reduced gut microbiota diversity and decreased Clostridiaceae abundance. Prophylactic antibiotic treatment protected DIO mouse recipients from endotoxin translocation across the gut and increased inflammatory cytokine production, as well as gut pathology and mortality, but did not protect against later development of chronic skin GVHD. These results suggest that obesity-induced alterations of the gut microbiota may affect GVHD after allo-HSCT in DIO mice, which could be ameliorated by prophylactic antibiotic treatment.

Thymic Epithelial Cell Support of Thymopoiesis Does Not Require Klotho.

Age-related thymic involution is characterized by a decrease in thymic epithelial cell (TEC) number and function parallel to a disruption in their spatial organization, resulting in defective thymocyte development and proliferation as well as peripheral T cell dysfunction. Deficiency of Klotho, an antiaging gene and modifier of fibroblast growth factor signaling, causes premature aging. To investigate the role of Klotho in accelerated age-dependent thymic involution, we conducted a comprehensive analysis of thymopoiesis and peripheral T cell homeostasis using Klotho-deficient (Kl/Kl) mice. At 8 wk of age, Kl/Kl mice displayed a severe reduction in the number of thymocytes (10-100-fold reduction), especially CD4 and CD8 double-positive cells, and a reduction of both cortical and medullary TECs. To address a cell-autonomous role for Klotho in TEC biology, we implanted neonatal thymi from Klotho-deficient and -sufficient mice into athymic hosts. Kl/Kl thymus grafts supported thymopoiesis equivalently to Klotho-sufficient thymus transplants, indicating that Klotho is not intrinsically essential for TEC support of thymopoiesis. Moreover, lethally irradiated hosts given Kl/Kl or wild-type bone marrow had normal thymocyte development and comparably reconstituted T cells, indicating that Klotho is not inherently essential for peripheral T cell reconstitution. Because Kl/Kl mice have higher levels of serum phosphorus, calcium, and vitamin D, we evaluated thymus function in Kl/Kl mice fed with a vitamin D-deprived diet. We observed that a vitamin D-deprived diet abrogated thymic involution and T cell lymphopenia in 8-wk-old Kl/Kl mice. Taken together, our data suggest that Klotho deficiency causes thymic involution via systemic effects that include high active vitamin D levels.

T cell progenitor therapy-facilitated thymopoiesis depends upon thymic input and continued thymic microenvironment interaction.

Infusion of in vitro-derived T cell progenitor (proT) therapy with hematopoietic stem cell transplant aids the recovery of the thymus damaged by total body irradiation. To understand the interaction between proTs and the thymic microenvironment, WT mice were lethally irradiated and given T cell-deficient (Rag1-/-) marrow with WT in vitro-generated proTs, limiting mature T cell development to infused proTs. ProTs within the host thymus led to a significant increase in thymic epithelial cells (TECs) by day 21 after transplant, increasing actively cycling TECs. Upon thymus egress (day 28), proT TEC effects were lost, suggesting that continued signaling from proTs is required to sustain TEC cycling and cellularity. Thymocytes increased significantly by day 21, followed by a significant improvement in mature T cell numbers in the periphery by day 35. This protective surge was temporary, receding by day 60. Double-negative 2 (DN2) proTs selectively increased thymocyte number, while DN3 proTs preferentially increased TECs and T cells in the spleen that persisted at day 60. These findings highlight the importance of the interaction between proTs and TECs in the proliferation and survival of TECs and that the maturation stage of proTs has unique effects on thymopoiesis and peripheral T cell recovery.