Faculty Bibliography

Background: Cystinuria is a disease of impaired absorption of cystine and dibasic amino acids (DAA) from the intestine and renal tubule leading to formation of cystine kidney stones. However, the metabolic impact of reduced amino acid absorption and excessive loss in the urine is poorly understood. We measured endogenous, gut microbial, and xenobiotic metabolites, providing insight into consequences of the disease and its treatment.
Method(s): Urinary biochemicals were assayed using LC-MS in 293 urine specimens from patients with cystinuria or control urinary phenotypes. Multivariate statistical analyses were conducted to reveal statistically significant biochemical signatures of the disease and products of cysteine-binding thiol drugs (CBTDs). 16s rRNA gene sequencing was performed on fecal samples from 12 wildtype (WT) and 12 cystinuric (Slc3a1 knockout; KO) mice to evaluate their gut microbial composition.
Result(s): Cystinuric urine samples had elevated levels of cysteine-gamma-glutamyl cystine disulfide (glutathione precursor), indole-3-acetic acid (microbial tryptophan metabolism), and novel conjugated forms of putrescine (microbial DAA decomposition). Conversely, taurine (sulfur metabolism), indole-3-acetic acid-glucuronide, and novel urinary metabolite N-methyl pipecolic acid (lysine metabolism) were reduced in cystinuric urine. Where cysteine-bound CBTDs were observed, substantial amounts of "wasted" drug were also detected as CBTD homodimers, non-cysteine disulfides, and mixed drug disulfides. The differentiation of gut microbially-derived metabolites led us to evaluate the gut microbiome diversity and composition in a mouse model of cystinuria revealing clear beta diversity and taxa differentiation between WT and KO mice.
Conclusion(s): Cystinuria is associated with unique urinary metabolic profiles beyond hyperexcretion of cystine and DAA, indicating perturbed metabolic processes and potential gut microbial effects. Study of the gut microbiome of WT and KO mice provides the first evidence for them having distinct taxa, perhaps due to poorly absorbed DAA present in the intestinal lumen. Urinary profiles allow us to characterize the excretion profiles of CBTDs, providing insight which may be helpful to tailor treatment

The human genome encodes thousands of long non-coding RNAs (lncRNAs), the majority of which are poorly conserved and uncharacterized. Here we identify a primate-specific lncRNA (CHROME), elevated in the plasma and atherosclerotic plaques of individuals with coronary artery disease, that regulates cellular and systemic cholesterol homeostasis. LncRNA CHROME expression is influenced by dietary and cellular cholesterol via the sterol-activated liver X receptor transcription factors, which control genes mediating responses to cholesterol overload. Using gain- and loss-of-function approaches, we show that CHROME promotes cholesterol efflux and HDL biogenesis by curbing the actions of a set of functionally related microRNAs that repress genes in those pathways. CHROME knockdown in human hepatocytes and macrophages increases levels of miR-27b, miR-33a, miR-33b and miR-128, thereby reducing expression of their overlapping target gene networks and associated biologic functions. In particular, cells lacking CHROME show reduced expression of ABCA1, which regulates cholesterol efflux and nascent HDL particle formation. Collectively, our findings identify CHROME as a central component of the non-coding RNA circuitry controlling cholesterol homeostasis in humans.

In bacteria, K⁺ is used to maintain cell volume and osmotic potential. Homeostasis normally involves a network of constitutively expressed transport systems, but in K⁺ deficient environments, the KdpFABC complex uses ATP to pump K⁺ into the cell. This complex appears to be a hybrid of two types of transporters, with KdpA descending from the superfamily of K⁺ transporters and KdpB belonging to the superfamily of P-type ATPases. Studies of enzymatic activity documented a catalytic cycle with hallmarks of classical P-type ATPases and studies of ion transport indicated that K⁺ import into the cytosol occurred in the second half of this cycle in conjunction with hydrolysis of an aspartyl phosphate intermediate. Atomic structures of the KdpFABC complex from X-ray crystallography and cryo-EM have recently revealed conformations before and after formation of this aspartyl phosphate that appear to contradict the functional studies. Specifically, structural comparisons with the archetypal P-type ATPase, SERCA, suggest that K⁺ transport occurs in the first half of the cycle, accompanying formation of the aspartyl phosphate. Further controversy has arisen regarding the path by which K⁺ crosses the membrane. The X-ray structure supports the conventional view that KdpA provides the conduit, whereas cryo-EM structures suggest that K⁺ moves from KdpA through a long, intramembrane tunnel to reach canonical ion binding sites in KdpB from which they are released to the cytosol. This review discusses evidence supporting these contradictory models and identifies key experiments needed to resolve discrepancies and produce a unified model for this fascinating mechanistic hybrid.
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Background/Purpose : T and B cells come together in ectopic lymphoid aggregates in myositis, suggesting that local T:B cell interactions could play a role in disease. T follicular helper cells are increased in circulation in patients with active myositis. We studied circulating Tfh cells from myositis patients using single-cell RNA-sequencing and examined the proximity of Tfh cells to B cells in patient biopsies. Methods : Tfh cells [CD3 + CXCR5 + PD-1 + CXCR3 neg and CD3 + CXCR5 + PD-1 + CXCR3 pos ] cells were sorted from peripheral blood and subsets were identified by chemokine markers to designate Tfh1 and Tfh2/17 cell subsets. RNA sequencing was performed on individual cells of (3 controls and 3 myositis patients) using Fluidigm C1 HT platform, and data were analyzed using a pseudo-temporal ordering using Monocle. Biopsies were stained using the OPAL standardized sequential immunofluorescence method for PD-1, CXCR5, CD19, and CD4 in human muscle, and machine learning was used to map proximity of B cells to all T-cells compared with Tfh cells. Results : We found various subsets within the Tfh pool, corresponding to Tfh1 and Tfh2/17 cells and some cells that looked to be transitioning between states. Tfh2/17 were enriched in myositis patients vs. controls. The Tfh2/17 cells demonstrated a type I interferon signature, while the Tfh1 cells had a type II interferon and proteasome signature. In tissue, we demonstrate Tfh cells in close proximity to B cells in lymphoid aggregates. Conclusion : Tfh cells are present in myositis biopsies juxtaposed to B cells, suggesting productive T:B interactions in the tissue. Tfh subsets in blood from patients demonstrate distinct pathological signatures when compared to controls

Background/Purpose : Sarilumab blocks interleukin-6 (IL-6) from binding to membrane and soluble IL-6 receptor-alpha. Sarilumab is approved for adults with rheumatoid arthritis (RA) and is being investigated in a Phase 2 trial (NCT02776735) in 2-17-year-old patients (pts) with polyarticular-course juvenile idiopathic arthritis (pcJIA), comprising rheumatoid-factor (RF)-positive and RF-negative polyarticular and extended oligoarticular JIA. This study aimed to evaluate pharmacokinetics (PK), pharmacodynamics (PD), safety, and efficacy of 3 subcutaneous (SC) sarilumab doses in pcJIA. Methods : A 12-week dose-finding study was performed to identify an appropriate sarilumab dose for use in the pcJIA population. Pts were divided by body weight into 2 groups: A (30-60 kg) and B (10-< 30 kg), and received sequential ascending doses of sarilumab, Dose 1 (Group A/B): 2.0/2.5 mg/kg q2w; Dose 2 (Group A/B): 3/4 mg/kg q2w; and Dose 3 (Group A/B): 2.0/2.5 mg/kg qw. pcJIA doses were targeted to achieve similar exposure to adult RA doses (150 mg q2w, 200 mg q2w, and 150 mg qw). Primary outcome was PK; secondary outcomes were safety, PD, and efficacy of sarilumab. Results : 42 pts enrolled (20/22 in Groups A/B); mean age was 13.0/5.2 years. At baseline, mean pcJIA duration, number of active joints, and JADAS27-CRP were 4.6/1.7 years, 17.2/11.0, and 22.2/19.1, in Groups A/B, respectively. As in adult pts, sarilumab exhibited nonlinear PK with target-mediated drug disposition (TMDD). Following repeated SC administrations, exposure increased in a greater than dose-proportional manner and accumulated 1.9-4.5-fold over 12 weeks. Sarilumab exposure was similar in both weight groups for each dose (Figure), and comparable to corresponding adult doses. Treatment-emergent adverse events (AEs) were reported in 36/42 (85.7%) pts (comparable across dose and weight groups); infections (28/42, 66.7%) were the most frequently reported AE. 12 grade 3/4 neutropenias were identified, mostly in Dose 3 (n=6) and in Group B (n=8). None was associated with infection; all resolved in a few days. Overall, 4 pts discontinued due to neutropenia and 1 due to alanine aminotransferase increase. There were no serious AEs, no cases of GI perforation, and no deaths. By Week 12, as observed while on-treatment: all pts attained JIA ACR30; 50%, 62%, and 100% of pts attained JIA ACR70 with Doses 1, 2, and 3, respectively; JADAS27-CRP mean % changes from baseline in Doses 1, 2, and 3 were -74.6%, -73.1%, and -87.9%, respectively. Conclusion : Sarilumab exhibited nonlinear PK with TMDD. Doses tested in pcJIA yielded similar exposure in both weight groups and were comparable to equivalent doses in adults with RA. All dose regimens proved effective for decreasing disease activity. Safety profile was consistent with class effects; higher incidences of neutropenia were observed with Dose 3, and in pts weighing 10-< 30 kg

The roundworm C. elegans reversibly arrests larval development during starvation [1], but extended early-life starvation reduces reproductive success [2, 3]. Maternal dietary restriction (DR) buffers progeny from starvation as young larvae, preserving reproductive success [4]. However, the developmental basis of reduced fertility following early-life starvation is unknown, and it is unclear how maternal diet modifies developmental physiology in progeny. We show here that extended starvation in first-stage (L1) larvae followed by unrestricted feeding results in a variety of developmental abnormalities in the reproductive system, including proliferative germ-cell tumors and uterine masses that express neuronal and epidermal cell fate markers. We found that maternal DR and reduced maternal insulin/insulin-like growth factor (IGF) signaling (IIS) increase oocyte provisioning of vitellogenin lipoprotein, reducing penetrance of starvation-induced abnormalities in progeny, including tumors. Furthermore, we show that maternal DR and reduced maternal IIS reduce IIS in progeny. daf-16/FoxO and skn-1/Nrf, transcriptional effectors of IIS, are required in progeny for maternal DR and increased vitellogenin provisioning to suppress starvation-induced abnormalities. daf-16/FoxO activity in somatic tissues is sufficient to suppress starvation-induced abnormalities, suggesting cell-nonautonomous regulation of reproductive system development. This work reveals that early-life starvation compromises reproductive development and that vitellogenin-mediated intergenerational insulin/IGF-to-insulin/IGF signaling mediates adaptation to nutrient availability.

Pre-clinical animal studies have shown that triiodothyronine (T3) replacement therapy improves cardiac contractile function after myocardial infarction (MI). We hypothesized that T3 treatment could prevent adverse post-infarction cardiomyocyte remodeling by maintaining transverse-tubule (TT) structures, thus improving calcium dynamics and contractility. METHODS: Myocardial infarction (MI) or sham surgeries were performed on female Sprague-Dawley rats (aged 12 wks), followed by treatment with T3 (5μg/kg/d) or vehicle in drinking water for 16 wks (n = 10-11/group). After in vivo echocardiographic and hemodynamic analyses, left ventricular myocytes were isolated by collagenase digestion and simultaneous calcium and contractile transients in single cardiomyocytes were recorded using IonOptix imaging. Live cardiomyocytes were stained with AlexaFluor-488 conjugated wheat germ agglutinin (WGA-488) or di-8-ANEPPS, and multiple z-stack images per cell were captured by confocal microscopy for analysis of TT organization. RTqPCR and immunoblot approaches determined expression of TT proteins. RESULTS: Echocardiography and in vivo hemodynamic measurements showed significant improvements in systolic and diastolic function in T3- vs vehicle-treated MI rats. Isolated cardiomyocyte analysis showed significant dysfunction in measurements of myocyte relengthening in MI hearts, and improvements with T3 treatment: max relengthening velocity (Vmax, um/s), 2.984 ± 1.410 vs 1.593 ± 0.325, p < 0.05 and time to Vmax (sec), 0.233 ± 0.037 vs 0.314 ± 0.019, p < 0.001; MI + T3 vs MI + Veh, respectively. Time to peak contraction was shortened by T3 treatment (0.161 ± 0.021 vs 0.197 ± 0.011 s., p < 0.01; MI + T3 vs MI + Veh, respectively). Analysis of TT periodicity of WGA- or ANEPPS-stained cardiomyocytes indicated significant TT disorganization in MI myocytes and improvement with T3 treatment (transverse-oriented tubules (TE%): 9.07 ± 0.39 sham, 6.94 ± 0.67 MI + Veh and 8.99 ± 0.38 MI + T3; sham vs MI + Veh, p < 0.001; MI + Veh vs MI + T3, p < 0.01). Quantitative RT-PCR showed that reduced expression of BIN1 (Bridging integrator-1), Jph2 (junctophilin-2), RyR2 (ryanodine receptor) and Ca_v1.2 (L-type calcium channel) in the failing myocardium were increased by T3 and immunoblot analysis further supporting a potential T3 effect on the TT-associated proteins, BIN1 and Jph2. In conclusion, low dose T3 treatment initiated immediately after myocardial infarction attenuated adverse TT remodeling, improved calcium dynamics and contractility, thus supporting the potential therapeutic utility of T3 treatment in heart failure.