PITPß promotes COPI vesicle fission through lipid transfer and membrane contact formation.

Lipid transfer through membrane contact has been implicated to support vesicular transport, but a mechanistic understanding of this process remains to be achieved. Here, examining Coat Protein I (COPI) transport, we find that phosphatidylcholine (PC) with short acyl chains (sPC), which is needed to support COPI vesicle fission, is delivered through membrane contact from the endoplasmic reticulum (ER) to the Golgi complex at sites of COPI vesicle formation. Phosphatidylinositol transfer protein beta (PITPß) plays a central role in this delivery by not only catalyzing PC transfer, but also forming membrane contact. By combining cell-based studies with reconstitution approaches, we achieve spatial and temporal detail in explaining how sPC delivery occurs. Our findings advance the mechanistic understanding of how membrane contact is needed for vesicular transport in a model pathway and shed new insights into how PITPß acts.

Ether lipids influence cancer cell fate by modulating iron uptake.

Cancer cell fate has been widely ascribed to mutational changes within protein-coding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with a high metastatic ability and cancer stem cell-like traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, leading directly to significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro three-dimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

Phosphoglycerate kinase 1 acts as a cargo adaptor to promote EGFR transport to the lysosome.

The epidermal growth factor receptor (EGFR) plays important roles in multiple cellular events, including growth, differentiation, and motility. A major mechanism of downregulating EGFR function involves its endocytic transport to the lysosome. Sorting of proteins into intracellular pathways involves cargo adaptors recognizing sorting signals on cargo proteins. A dileucine-based sorting signal has been identified previously for the sorting of endosomal EGFR to the lysosome, but a cargo adaptor that recognizes this signal remains unknown. Here, we find that phosphoglycerate kinase 1 (PGK1) is recruited to endosomal membrane upon its phosphorylation, where it binds to the dileucine sorting signal in EGFR to promote the lysosomal transport of this receptor. We also elucidate two mechanisms that act in concert to promote PGK1 recruitment to endosomal membrane, a lipid-based mechanism that involves phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and a protein-based mechanism that involves hepatocyte growth factor receptor substrate (Hrs). These findings reveal an unexpected function for a metabolic enzyme and advance the mechanistic understanding of how EGFR is transported to the lysosome.

The Effect of Preoperative Exposure to Benzodiazepines on Opioid Consumption After One and Two-level Anterior Cervical Discectomy and Fusion.

STUDY DESIGN: Retrospective cohort. OBJECTIVE: Investigate the relationship between preoperative benzodiazepine exposure and postoperative opioid use in patients undergoing primary 1 or 2-level anterior cervical discectomy and fusion (ACDF). BACKGROUND: Little is known about the effect of preoperative benzodiazepine exposure on postoperative opioid use in spine surgery. PATIENTS AND METHODS: Patients undergoing primary 1 or 2-level ACDF at a single institution from February 2020 to November 2021 were identified through electronic medical records. The prescription drug monitoring program was utilized to record the name, dosage, and quantity of preoperative benzodiazepines/opioids filled within 60 days before surgery and postoperative opioids 6 months after surgery. Patients were classified as benzodiazepine naïve or exposed according to preoperative usage, and postoperative opioid dose and duration were compared between groups. Regression analysis was performed for outcomes that demonstrated statistical significance, adjusting for preoperative opioid use, age, sex, and body mass index. RESULTS: Sixty-seven patients comprised the benzodiazepine-exposed group whereas 90 comprised the benzodiazepine-naïve group. There was no significant difference in average daily morphine milligram equivalents between groups (median: 96.0 vs 65.0, P = 0.11). The benzodiazepine-exposed group received postoperative opioids for a longer duration (median: 32.0 d vs 12.0 d, P = 0.004) with more prescriptions (median: 2.0 vs 1.0, P = 0.004) and a greater number of pills (median: 110.0 vs 59.0, P = 0.007). On regression analysis, preoperative benzodiazepine use was not significantly associated with postoperative opioid duration [incidence rate ratio (IRR): 0.93, P = 0.74], number of prescriptions (IRR: 1.21, P = 0.16), or number of pills (IRR: 0.89, P = 0.58). CONCLUSIONS: While preoperative benzodiazepine users undergoing primary 1 or 2-level ACDF received postoperative opioids for a longer duration compared with a benzodiazepine naïve cohort, preoperative benzodiazepine use did not independently contribute to this observation. These findings provide insight into the relationship between preoperative benzodiazepine use and postoperative opioid consumption. LEVEL OF EVIDENCE: Level III.

Studying the Role of Lipid Geometry in COPI Vesicle Formation.

The Coat Protein I (COPI) complex forms vesicles from Golgi membrane for retrograde transport among the Golgi stacks, and also from the Golgi to the endoplasmic reticulum (ER). We have been elucidating the mechanistic details of COPI vesicle formation through a reconstitution system that involves the incubation of Golgi membrane with purified components. This approach has enabled us recently to gain new insight into how certain lipids are critical for the fission stage of COPI vesicle formation. Lipid geometry has been proposed to act in the formation of transport carriers by promoting membrane curvature. However, evidence for this role has come from studies using simplified membranes, while confirmation in the more physiologic setting of native membranes has been challenging, as such membranes contain a complex composition of lipids and proteins. We have recently refined the COPI reconstitution system to overcome this experimental obstacle. This has led us to identify an unanticipated type of lipid geometry needed for COPI vesicle fission. This chapter describes the approach that we have developed to enable this discovery. The methodologies include: (i) preparation Golgi membrane from cells that are deficient in a particular lipid enzyme activity and (ii) functional rescue of this deficiency by introducing the product of the lipid enzyme, with experiments being performed at the in vitro level to gain mechanistic clarity and at the in vivo level to confirm physiologic relevance.

Structural elucidation of how ARF small GTPases induce membrane tubulation for vesicle fission.

The ADP-Ribosylation Factor (ARF) small GTPases have been found to act in vesicle fission through a direct ability to tubulate membrane. Here, we have used cryo-electron microscopy (EM) to solve the structure of an ARF6 protein lattice assembled on tubulated membrane to 3.9 Å resolution. ARF6 forms tetramers that polymerize into helical arrays to form this lattice. We identify, and confirm functionally, protein contacts critical for this lattice formation. The solved structure also suggests how the ARF amphipathic helix is positioned in the lattice for membrane insertion, and how a GTPase-activating protein (GAP) docks onto the lattice to catalyze ARF-GTP hydrolysis in completing membrane fission. As ARF1 and ARF6 are structurally conserved, we have also modeled ARF1 onto the ARF6 lattice, which has allowed us to pursue the reconstitution of Coat Protein I (COPI) vesicles to confirm more definitively that the ARF lattice acts in vesicle fission. Our findings are notable for having achieved the first detailed glimpse of how a small GTPase bends membrane and having provided a molecular understanding of how an ARF protein acts in vesicle fission.

Trafficking-defective mutant PROKR2 cycles between endoplasmic reticulum and Golgi to attenuate endoplasmic reticulum stress.

G protein-coupled receptors (GPCRs) play crucial roles in numerous physiological and pathological processes. Mutations in GPCRs that result in loss of function or alterations in signaling can lead to inherited or acquired diseases. Herein, studying prokineticin receptor 2 (PROKR2), we initially identify distinct interactomes for wild-type (WT) versus a mutant (P290S) PROKR2 that causes hypogonadotropic hypogonadism. We then find that both the WT and mutant PROKR2 are targeted for endoplasmic reticulum (ER)-associated degradation, but the mutant is degraded to a greater extent. Further analysis revealed that both forms can also leave the ER to reach the Golgi. However, whereas most of the WT is further transported to the cell surface, most of the mutant is retrieved to the ER. Thus, the post-ER itinerary plays an important role in distinguishing the ultimate fate of the WT versus the mutant. We have further discovered that this post-ER itinerary reduces ER stress induced by the mutant PROKR2. Moreover, we extend the core findings to another model GPCR. Our findings advance the understanding of disease pathogenesis induced by a mutation at a key residue that is conserved across many GPCRs and thus contributes to a fundamental understanding of the diverse mechanisms used by cellular quality control to accommodate misfolded proteins.

GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis.

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.

Connecting COPD GWAS Genes: FAM13A Controls TGFß2 Secretion by Modulating AP-3 Transport.

Chronic obstructive pulmonary disease (COPD) is a common, complex disease and a major cause of morbidity and mortality. Although multiple genetic determinants of COPD have been implicated by genome-wide association studies (GWASs), the pathophysiological significance of these associations remains largely unknown. From a COPD protein-protein interaction network module, we selected a network path between two COPD GWAS genes for validation studies: FAM13A (family with sequence similarity 13 member A)-AP3D1-CTGF- TGFß2. We find that TGFß2, FAM13A, and AP3D1 (but not CTGF) form a cellular protein complex. Functional characterization suggests that this complex mediates the secretion of TGFß2 through an AP-3 (adaptor protein 3)-dependent pathway, with FAM13A acting as a negative regulator by targeting a late stage of this transport that involves the dissociation of coat-cargo interaction. Moreover, we find that TGFß2 is a transmembrane protein that engages the AP-3 complex for delivery to the late endosomal compartments for subsequent secretion through exosomes. These results identify a pathophysiological context that unifies the biological network role of two COPD GWAS proteins and reveal novel mechanisms of cargo transport through an intracellular pathway.

Tetrahydroxanthohumol, a xanthohumol derivative, attenuates high-fat diet-induced hepatic steatosis by antagonizing PPARγ.

We previously reported xanthohumol (XN), and its synthetic derivative tetrahydro-XN (TXN), attenuates high-fat diet (HFD)-induced obesity and metabolic syndrome in C57Bl/6J mice. The objective of the current study was to determine the effect of XN and TXN on lipid accumulation in the liver. Non-supplemented mice were unable to adapt their caloric intake to 60% HFD, resulting in obesity and hepatic steatosis; however, TXN reduced weight gain and decreased hepatic steatosis. Liver transcriptomics indicated that TXN might antagonize lipogenic PPARγ actions in vivo. XN and TXN inhibited rosiglitazone-induced 3T3-L1 cell differentiation concomitant with decreased expression of lipogenesis-related genes. A peroxisome proliferator activated receptor gamma (PPARγ) competitive binding assay showed that XN and TXN bind to PPARγ with an IC50 similar to pioglitazone and 8-10 times stronger than oleate. Molecular docking simulations demonstrated that XN and TXN bind in the PPARγ ligand-binding domain pocket. Our findings are consistent with XN and TXN acting as antagonists of PPARγ.

Lumbar Pedicle Morphology and Vertebral Dimensions in Isthmic and Degenerative Spondylolisthesis-A Comparative Study.

BACKGROUND: The pedicle screw is the most common device used to achieve fixation in fusion of spondylolistheses. Safe and accurate placement with this technique relies on a thorough understanding of the bony anatomy. There is a paucity of literature comparing the surgically relevant osseous anatomy in patients with a degenerative spondylolisthesis (DS) and an isthmic spondylolisthesis (IS). The goal of this study was to determine the differences in the osseous anatomy in patients with a DS and those with an IS. METHODS: A retrospective comparative cohort study was conducted on patients with a single-level, symptomatic L4-L5 DS or a single-level, symptomatic L5-S1 IS. Magnetic resonance imaging for these patients was reviewed. Morphometries of the pedicle and vertebral body were analyzed by 2 independent observers for the levels from L3 to S1, and radiographic parameters were compared between groups. RESULTS: A total of 572 levels in 143 patients were studied, including 103 patients with a DS and 40 with an IS. After accounting for confounders, IS and DS had an independent effect on transverse vertebral body width, pedicle height and width, and sagittal pedicle angle. Patients with an IS had a smaller pedicle height (P < .001) and pedicle width (P = .001) than patients with DS. In addition, the angulation of the pedicles varied on the basis of the diagnosis. CONCLUSIONS: The osseous anatomy is significantly different in patients with a DS than with an IS. Patients with an IS have smaller pedicles in the lumbar spine. Also, the L4 and L5 pedicles are more caudally angulated and the S1 pedicle is less medialized. LEVEL OF EVIDENCE: 3. CLINICAL RELEVANCE: Understanding the differences in pedicle anatomy is important for the safe placement of pedicle screws.

Complications and Revision Rates in Minimally Invasive Robotic-Guided Versus Fluoroscopic-Guided Spinal Fusions: The MIS ReFRESH Prospective Comparative Study.

STUDY DESIGN: Prospective, multicenter, partially randomized. OBJECTIVE: Assess rates of complications, revision surgery, and radiation between Mazor robotic-guidance (RG) and fluoro-guidance (FG). SUMMARY OF BACKGROUND DATA: Minimally invasive surgery MIS ReFRESH is the first study designed to compare RG and FG techniques in adult minimally invasive surgery (MIS) lumbar fusions. METHODS: Primary endpoints were analyzed at 1 year follow-up. Analysis of variables through Cox logistic regression and a Kaplan-Meier Survival Curve of surgical complications. RESULTS: Nine sites enrolled 485 patients: 374 (RG arm) and 111 (FG arm). 93.2% of patients had more than 1 year f/u. There were no differences for sex, Charlson Comorbidity Index, diabetes, or tumor. Mean age of RG patients was 59.0 versus 62.5 for FG (P = 0.009) and body mass index (BMI) was 31.2 versus 28.1 (P< 0.001). Percentage of smokers was almost double in the RG (15.2% vs. 7.2%, P = 0.029). Surgical time was similar (skin-to-skin time/no. of screws) at 24.9 minutes RG and 22.9 FG (P = 0.550). Fluoroscopy during surgery/no. of screws was 15.5 seconds RG versus 35.4 seconds FG, (15 seconds average reduction). Fluoroscopy time during instrumentation/no. of screws was 3.6 seconds RG versus 17.8 seconds FG showing an 80% average reduction of fluoro time/screw in RG (P < 0.001). Within 1 year follow-up, there were 39 (10.4%) surgical complications RG versus 39 (35.1%) FG, and 8 (2.1%) revisions RG versus 7 (6.3%) FG. Cox regression analysis including age, sex, BMI, CCI, and no. of screws, demonstrated that the hazard ratio (HR) for complication was 5.8 times higher FG versus RG (95% CI: 3.5-9.6, P < 0.001). HR for revision surgery was 11.0 times higher FG versus RG cases (95% CI 2.9-41.2, P < 0.001). CONCLUSION: Mazor robotic-guidance was found to have a 5.8 times lower risk of a surgical complication and 11.0 times lower risk for revision surgery. Surgical time was similar between groups and robotic-guidance reduced fluoro time per screw by 80% (approximately 1 min/case).Level of Evidence: 2.

Structural insights into membrane remodeling by SNX1.

The sorting nexin (SNX) family of proteins deform the membrane to generate transport carriers in endosomal pathways. Here, we elucidate how a prototypic member, SNX1, acts in this process. Performing cryoelectron microscopy, we find that SNX1 assembles into a protein lattice that consists of helical rows of SNX1 dimers wrapped around tubular membranes in a crosslinked fashion. We also visualize the details of this structure, which provides a molecular understanding of how various parts of SNX1 contribute to its ability to deform the membrane. Moreover, we have compared the SNX1 structure with a previously elucidated structure of an endosomal coat complex formed by retromer coupled to a SNX, which reveals how the molecular organization of the SNX in this coat complex is affected by retromer. The comparison also suggests insight into intermediary stages of assembly that results in the formation of the retromer-SNX coat complex on the membrane.

Combined immunodeficiency due to a mutation in the γ1 subunit of the coat protein I complex.

The coat protein I (COPI) complex mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER). Five siblings with persistent bacterial and viral infections and defective humoral and cellular immunity had a homozygous p.K652E mutation in the γ1 subunit of COPI (γ1-COP). The mutation disrupts COPI binding to the KDEL receptor and impairs the retrieval of KDEL-bearing chaperones from the Golgi to the ER. Homozygous Copg1K652E mice had increased ER stress in activated T and B cells, poor antibody responses, and normal numbers of T cells that proliferated normally, but underwent increased apoptosis upon activation. Exposure of the mutants to pet store mice caused weight loss, lymphopenia, and defective T cell proliferation that recapitulated the findings in the patients. The ER stress-relieving agent tauroursodeoxycholic acid corrected the immune defects of the mutants and reversed the phenotype they acquired following exposure to pet store mice. This study establishes the role of γ1-COP in the ER retrieval of KDEL-bearing chaperones and thereby the importance of ER homeostasis in adaptive immunity.

Reduction in complication and revision rates for robotic-guided short-segment lumbar fusion surgery: results of a prospective, multi-center study.

Studies evaluating robotic guidance in lumbar fusion are limited primarily to evaluation of screw accuracy and perioperative complications. This is the first study to evaluate granular differences in short and long-term complication and revision rate profiles between robotic (RG) fluoroscopic (FG) guidance for minimally invasive short-segment lumbar fusions. A retrospective analysis of a prospective, multi-center database was performed. Complications were subdivided into surgical (further subcategorized into adjacent segment disease, new-onset back pain, radiculopathy, motor-deficit, hardware failure, pseudoarthrosis), wound, and medical complications. Complication and revision rates were compared between RG and FG groups cumulatively at 30, 90 days, and 1 year. 374 RG and 111 FG procedures were performed. RG was associated with an 86.25, 83.20, and 69.42% cumulative reduction in complication rate at 30, 90 days, and 1 year, respectively, compared to FG (p < 0.001). At all follow-up points, new-onset radiculopathy and medical complications were most prevalent in both groups. The greatest reductions in complication rates were seen for new-onset back pain (88.13%; p = 0.001) and wound complications (95.05%; p < 0.001) at 30 days, new-onset motor deficits (90.11%; p = 0.004) and wound complications (85.16%; p < 0.001) at 90 days, and new-onset motor deficits (85.16%; p = 0.002), wound (85.16%; p < 0.001), and medical complications (75.72%; p < 0.001) at 1 year. RG was associated with a 92.58% (p = 0.002) reduction in revision rate at 90 days and a 66.08% (p = 0.026) reduction at 1 year. RG was associated with significant reductions in postoperative complication rates at all follow-up time points and significant reductions in revision rates at 90 days and 1 year.

Fluoroscopy time analysis of a prospective, multi-centre study comparing robotic- and fluoroscopic-guided placement of percutaneous pedicle screw instrumentation for short segment minimally invasive lumbar fusion surgery.

BACKGROUND: As minimally invasive spine surgery becomes more widespread, concerns regarding radiation exposure to surgeons and patients alike have become a growing concern. Robotic guidance has been developed as a way to increase the accuracy of instrumentation while decreasing radiation burden. METHODS: A retrospective analysis of a large, multi-centre, prospective study comparing robotic-guided (RG) to fluoroscopic-guided (FG) (Multi-centre, Partially Randomized, Controlled Trial of MIS Robotic vs. Freehand in Short Adult Degenerative Spinal Fusion Surgeries) was performed to evaluate for differences in radiation exposure between study groups. RESULTS: RG was associated with 78.3% (p < 0.001) and 79.8% (p < 0.001) reduction in total and per screw fluoroscopy times, respectively, as compared to FG. RG was also associated with a 50.8% (p < 0.001) reduction in total operative fluoroscopy time. CONCLUSIONS: RG was associated with significantly lower fluoroscopy times compared to FG. This suggests that utilization of robotic navigation systems may result in decreased operative radiation exposure, which is a growing concern for surgeons performing minimally invasive spine surgery.

ß-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.

ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

Coordination of Grp1 recruitment mechanisms by its phosphorylation.

The action of guanine nucleotide exchange factors (GEFs) on the ADP-ribosylation factor (ARF) family of small GTPases initiates intracellular transport pathways. This role requires ARF GEFs to be recruited from the cytosol to intracellular membrane compartments. An ARF GEF known as General receptor for 3-phosphoinositides 1 (Grp1) is recruited to the plasma membrane through its pleckstrin homology (PH) domain that recognizes phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here, we find that the phosphorylation of Grp1 induces its PH domain to recognize instead phosphatidylinositol 4-phosphate (PI4P). This phosphorylation also releases an autoinhibitory mechanism that results in the coil-coil (CC) domain of Grp1 engaging two peripheral membrane proteins of the recycling endosome. Because the combination of these actions results in Grp1 being recruited preferentially to the recycling endosome rather than to the plasma membrane, our findings reveal the complexity of recruitment mechanisms that need to be coordinated in localizing an ARF GEF to an intracellular compartment to initiate a transport pathway. Our elucidation is also remarkable for having revealed that phosphoinositide recognition by a PH domain can be switched through its phosphorylation.

The protein kinase Akt acts as a coat adaptor in endocytic recycling.

Coat proteins have a central role in vesicular transport by binding to cargoes for their sorting into intracellular pathways. Cargo recognition is mediated by components of the coat complex known as adaptor proteins1-3. We previously showed that Arf-GAP with coil-coil, ANK repeat and PH domain-containing protein 1 (ACAP1) functions as an adaptor for a clathrin coat complex that has a function in endocytic recycling4-6. Here, we show that the protein kinase Akt acts as a co-adaptor in this complex, and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling. In addition to advancing the understanding of endocytic recycling, we uncover a fundamentally different function in which a kinase acts, as Akt in this case is an effector rather than a regulator in a cellular event.

Effects of Operating Room Size on Surgical Site Infection Following Lumbar Fusion Surgery.

BACKGROUND: Surgical site infections (SSIs) represent a devastating complication after spine surgery. Many factors have been identified, but the influence of operating room (OR) size on infection rate has not been assessed. METHODS: Two thousand five hundred and twenty-three patients who underwent open lumbar spine fusion at a single institution between 2010 and 2016 were included. Patients were dichotomized into large versus small groups based on OR volume. Bivariate logistic regression and a final multivariate model following a multicollinearity check were used to calculate odds of infection for all variables. RESULTS: A total of 63 patients (2.5%) developed SSIs with 46 (73%) in the larger OR group and 17 (27%) in the smaller OR group. The rate of SSIs in larger ORs was 3.02% compared with 1.81% in smaller ORs. Significant parameters impacting SSI in bivariate analysis included an earlier year of surgery, BMI > 30, more comorbidities, more levels decompressed and fused, smoking, and larger OR volumes. Multivariate analysis identified BMI > 30, Elixhauser scores, smoking, and increasing levels decompressed as significant predictors. Topical vancomycin was found to significantly decrease rate of infection in both analyses. CONCLUSIONS: OR size (large versus small) was ultimately not a significant predictor of infection related to rates of SSIs, although it did show a clinical trend toward significance, suggesting association. Future prospective analysis is warranted. LEVEL OF EVIDENCE: 3.