Faculty Bibliography

Background - Mutations in the gene encoding the sodium channel Na_v1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Na_v1.5 expression between cardiomyocyte domains. At the lateral membrane and T-tubules, Na_v1.5 localization and function remain insufficiently characterized. Methods - We used novel single-molecule localization microscopy (SMLM) and computational modeling to define nanoscale features of Na_v1.5 localization and distribution at the lateral membrane (LM), the LM groove, and T-tubules (TT) in cardiomyocytes from wild-type (N = 3), dystrophin-deficient (mdx; N = 3) mice, and mice expressing C-terminally truncated Na_v1.5 (ΔSIV; N = 3). We moreover assessed TT sodium current by recording whole-cell sodium currents in control (N = 5) and detubulated (N = 5) wild-type cardiomyocytes. Results - We show that Na_v1.5 organizes as distinct clusters in the groove and T-tubules which density, distribution, and organization partially depend on SIV and dystrophin. We found that overall reduction in Na_v1.5 expression in mdx and ΔSIV cells results in a non-uniform re-distribution with Na_v1.5 being specifically reduced at the groove of ΔSIV and increased in T-tubules of mdx cardiomyocytes. A TT sodium current could however not be demonstrated. Conclusions - Na_v1.5 mutations may site-specifically affect Na_v1.5 localization and distribution at the lateral membrane and T-tubules, depending on site-specific interacting proteins. Future research efforts should elucidate the functional consequences of this redistribution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PURPOSE/OBJECTIVE:The purpose of this study is to determine if the radiographic humerus union measurement (RHUM) is predictive of union in humeral shaft fractures treated nonoperatively. METHODS:All patients with long bone fracture nonunion presenting to a single surgeon were enrolled in a prospective registry. This registry was queried to identify patients with humeral shaft fractures treated nonoperatively and developed nonunion. The nonunion cohort was matched to a three to one gender- and age-matched control group that were treated nonoperatively for a humeral shaft fracture and achieved union. Two fellowship-trained orthopedic traumatologists blinded to eventual union scored radiographs obtained 12 weeks after injury using the RHUM. A binomial logistic regression determined the effect of the RHUM on the likelihood of developing union. RESULTS:Nine patients with humeral shaft fractures treated nonoperatively with radiographs 12 weeks after injury that developed nonunion were identified. These patients were matched to 27 controls. Logistic regression demonstrated the RHUM was a significant predictor of healing 12 weeks after humeral shaft fracture treated nonoperatively (p = 0.014, odds ratio 9.434, 95% CI for OR 1.586-56.098). All patients with RHUM below 7 went on to nonunion. All patients with RHUM above 8 healed. Three of seven patients (43%) with RHUM of 7 or 8 healed. CONCLUSION/CONCLUSIONS:The RHUM demonstrated an increased likelihood of achieving union 12 weeks after injury. Orthopedic surgeons can counsel patients that fractures with RHUM scores of 6 or below are in danger of developing nonunion and can target interventions appropriately.

Trichorhinophalangeal syndrome type 1 (TRPS1; Online Mendelian Inheritance in Man #190350) is an autosomal dominant disorder caused by mutations in TRPS1. We report a Thai male with TRPS1 who carried a c.1842C>T (p.Arg615Ter) mutation. He had 15 supernumerary teeth, double mental foramina, hypoplastic mandibular condyles with slender condylar necks and unique ultrastructural hair findings. Body hair was absent. The hair in the area of a congenital melanocytic nevus had a greater number of hair cuticles than normal. Occipital hair had abnormal hair follicles and cuticles. The scale edges of the hair cuticles were detached and rolled up. Hypoplastic mandibular condyles with slender condylar necks, double mental foramina and the rolled up edges of hair cuticles have not been reported in patients with TRPS1.

Electronic health record (EHR) technologies have improved the ease of access to structured clinical data. The standard means by which data are collected continues to be manual chart review. The authors compared the accuracy of manual chart review against modern electronic data warehouse queries. A manual chart review of the EHR was performed with medical record numbers and surgical admission dates for the 100 most recent inpatient venous thromboembolic events after total joint arthroplasty. A separate data query was performed with the authors' electronic data warehouse. Data sets were then algorithmically compared to check for matches. Discrepancies between data sets were evaluated to categorize errors as random vs systematic. From 100 unique patient encounters, 27 variables were retrieved. The average transcription error rate was 9.19% (SD, ±5.74%) per patient encounter and 11.04% (SD, ±21.40%) per data variable. The systematic error rate was 7.41% (2 of 27). When systematic errors were excluded, the random error rate was 5.79% (SD, ±7.04%) per patient encounter and 5.44% (SD, ±5.63%) per data variable. Total time and average time for manual data collection per patient were 915 minutes and 10.3±3.89 minutes, respectively. Data collection time for the entire electronic query was 58 seconds. With an error rate of 10%, manual chart review studies may be more prone to type I and II errors. Computer-based data queries can improve the speed, reliability, reproducibility, and scalability of data retrieval and allow hospitals to make more data-driven decisions. [Orthopedics. 2020;43(x):xx-xx.].

Stresses associated with disease may pathologically remodel the proteome by both increasing interaction strength and altering interaction partners, resulting in proteome-wide connectivity dysfunctions. Chaperones play an important role in these alterations, but how these changes are executed remains largely unknown. Our study unveils a specific N-glycosylation pattern used by a chaperone, Glucose-regulated protein 94 (GRP94), to alter its conformational fitness and stabilize a state most permissive for stable interactions with proteins at the plasma membrane. This "protein assembly mutation' remodels protein networks and properties of the cell. We show in cells, human specimens, and mouse xenografts that proteome connectivity is restorable by inhibition of the N-glycosylated GRP94 variant. In summary, we provide biochemical evidence for stressor-induced chaperone-mediated protein mis-assemblies and demonstrate how these alterations are actionable in disease.

ABC transporters facilitate the movement of diverse molecules across cellular membranes, but how their activity is regulated post-translationally is not well understood. Here we report the crystal structure of MlaFB from E. coli, the cytoplasmic portion of the larger MlaFEDB ABC transporter complex, which drives phospholipid trafficking across the bacterial envelope to maintain outer membrane integrity. MlaB, a STAS domain protein, binds the ABC nucleotide binding domain, MlaF, and is required for its stability. Our structure also implicates a unique C-terminal tail of MlaF in self-dimerization. Both the C-terminal tail of MlaF and the interaction with MlaB are required for the proper assembly of the MlaFEDB complex and its function in cells. This work leads to a new model for how an important bacterial lipid transporter may be regulated by small proteins, and raises the possibility that similar regulatory mechanisms may exist more broadly across the ABC transporter family.

Despite remarkable responses to cancer immunotherapy in a subset of patients, many patients remain resistant to therapies. It is now clear that elevated levels of tumor-infiltrating T cells as well as a systemic anti-tumor immune response are requirements for successful immunotherapies. However, the tumor microenvironment imposes an additional resistance mechanism to immunotherapy. We have developed a practical and improved strategy for cancer immunotherapy using an oncolytic virus and anti-OX40. This strategy takes advantage of a preexisting T cell immune repertoire in vivo, removing the need to know about present tumor antigens. We have shown in this study that the replication-deficient oncolytic Sindbis virus vector expressing interleukin-12 (IL-12) (SV.IL12) activates immune-mediated tumor killing by inducing OX40 expression on CD4 T cells, allowing the full potential of the agonistic anti-OX40 antibody. The combination of SV.IL12 with anti-OX40 markedly changes the transcriptome signature and metabolic program of T cells, driving the development of highly activated terminally differentiated effector T cells. These metabolically reprogrammed T cells demonstrate enhanced tumor infiltration capacity as well as anti-tumor activity capable of overcoming the repressive tumor microenvironment. Our findings identify SV.IL12 in combination with anti-OX40 to be a novel and potent therapeutic strategy that can cure multiple types of low-immunogenic solid tumors.

Homeostasis of neural firing properties is important in stabilizing neuronal circuitry, but how such plasticity might depend on alternative splicing is not known. Here we report that chronic inactivity homeostatically increases action potential duration by changing alternative splicing of BK channels; this requires nuclear export of the splicing factor Nova-2. Inactivity and Nova-2 relocation were connected by a novel synapto-nuclear signaling pathway that surprisingly invoked mechanisms akin to Hebbian plasticity: Ca²⁺-permeable AMPA receptor upregulation, L-type Ca²⁺ channel activation, enhanced spine Ca²⁺ transients, nuclear translocation of a CaM shuttle, and nuclear CaMKIV activation. These findings not only uncover commonalities between homeostatic and Hebbian plasticity but also connect homeostatic regulation of synaptic transmission and neuronal excitability. The signaling cascade provides a full-loop mechanism for a classic autoregulatory feedback loop proposed ∼25 years ago. Each element of the loop has been implicated previously in neuropsychiatric disease.

To date, microglia subsets in the healthy CNS have not been identified. Utilizing autofluorescence (AF) as a discriminating parameter, we identified two novel microglia subsets in both mice and non-human primates, termed autofluorescence-positive (AF⁺) and negative (AF^-). While their proportion remained constant throughout most adult life, the AF signal linearly and specifically increased in AF⁺ microglia with age and correlated with a commensurate increase in size and complexity of lysosomal storage bodies, as detected by transmission electron microscopy and LAMP1 levels. Post-depletion repopulation kinetics revealed AF^- cells as likely precursors of AF⁺ microglia. At the molecular level, the proteome of AF⁺ microglia showed overrepresentation of endolysosomal, autophagic, catabolic, and mTOR-related proteins. Mimicking the effect of advanced aging, genetic disruption of lysosomal function accelerated the accumulation of storage bodies in AF⁺ cells and led to impaired microglia physiology and cell death, suggestive of a mechanistic convergence between aging and lysosomal storage disorders.