Virulence Comparison of a Comprehensive Panel of Xylella fastidiosa Pierce's Disease Isolates from California.

Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, has been found in all major grape-growing regions in California, U.S.A. Large collections of X. fastidiosa isolates are available from these areas, which enable comparative studies of pathogen genetic traits and virulence. Owing to the significant resource requirements for experiments with X. fastidiosa in grapevine, however, most studies use only a single isolate to evaluate disease, and it is not clear how much variability between isolates impacts disease development in experimental or natural settings. In this study, a comprehensive panel of X. fastidiosa isolates from all California grape-growing regions was tested for virulence in susceptible grapevine and in the model host plant, tobacco. Seventy-one isolates were tested, 29 in both grapevine and tobacco. The results of this study highlight the inherent variability of inoculation experiments with X. fastidiosa, including variation in disease severity in plants inoculated with a single isolate, and variability between experimental replicates. There were limited differences in virulence between isolates that were consistent across experimental replicates, or across different host plants. This suggests that choice of isolate within the X. fastidiosa subsp. fastidiosa Pierce's disease group may not make any practical difference when testing in susceptible grape varieties, and that pathogen evolution has not significantly changed virulence of Pierce's disease isolates within California. The location of isolation also did not dictate relative disease severity. This information will inform experimental design for future studies of X. fastidiosa in grapevine and provide important context for genomic research.

High Growing Season Temperatures Limit Winter Recovery of Grapevines from Xylella fastidiosa Infection - Implications for Epidemiology in Hot Climates.

Management of widespread plant pathogens is challenging as climatic differences among crop-growing regions may alter key aspects of pathogen spread and disease severity. Xylella fastidiosa is a xylem-limited bacterial pathogen that is transmitted by xylem sap-feeding insects. Geographic distribution of X. fastidiosa is limited by winter climate, and vines infected with X. fastidiosa can recover from infection when held at cold temperatures. California has a long history of research on Pierce's disease and significant geographic and climatic diversity among grape-growing regions. This background in combination with experimental disease studies under controlled temperature conditions can inform risk assessment for X. fastidiosa spread and epidemic severity across different regions and under changing climate conditions. California's grape-growing regions have considerable differences in summer and winter climate. In northern and coastal regions, summers are mild and winters are cool, conditions which favor winter recovery of infected vines. In contrast, in inland and southern areas, summers are hot and winters mild, reducing likelihood of winter recovery. Here, winter recovery of three table grape cultivars (Flame, Scarlet Royal, and Thompson Seedless) and three wine grape cultivars (Sauvignon Blanc, Cabernet Sauvignon, and Zinfandel) were evaluated under temperature conditions representative of the San Joaquin Valley, an area with hot summers and mild winters that has been severely impacted by Pierce's disease and contains a large portion of California grape production. Mechanically inoculated vines were held in the greenhouse under one of three warming treatments to represent different seasonal inoculation dates prior to being moved into a cold chamber. Winter recovery under all treatments was generally limited but with some cultivar variation. Given hot summer temperatures of many grape-growing regions worldwide, as well as increasing global temperatures overall, winter recovery of grapevines should not be considered a key factor limiting X. fastidiosa spread and epidemic severity in the majority of cases.

Sociodemographic Characteristics of Patients Undergoing Surgery for Metastatic Disease of the Spine.

INTRODUCTION: Some patients, particularly those who are socioeconomically deprived, are diagnosed with primary and/or metastatic cancer only after presenting to the emergency department. Our objective was to determine sociodemographic characteristics of patients undergoing surgery for metastatic spine disease at our institution. METHODS: This retrospective case series included patients 18 years and older who presented to the emergency department with metastatic spine disease requiring surgery. Demographics and survival data were collected. Sociodemographic characteristics were estimated using the Social Deprivation Index (SDI) and Area Deprivation Index (ADI) for the state of California. Univariate log-rank tests and Kaplan-Meier curves were used to assess differences in survival for predictors of interest. RESULTS: Between 2015 and 2021, 64 patients underwent surgery for metastatic disease of the spine. The mean age was 61.0 ± 12.5 years, with 60.9% being male (n = 39). In this cohort, 89.1% of patients were non-Hispanic (n = 57), 71.9% were White (n = 46), and 62.5% were insured by Medicare/Medicaid (n = 40). The mean SDI and ADI were 61.5 ± 28.0 and 7.7 ± 2.2, respectively. 28.1% of patients (n = 18) were diagnosed with primary cancer for the first time while 39.1% of patients (n = 25) were diagnosed with metastatic cancer for the first time. During index hospitalization, 37.5% of patients (n = 24) received palliative care consult. The 3-month, 6-month, and all-time mortality rates were 26.7% (n = 17), 39.5% (n = 23), and 50% (n = 32), respectively, with 10.9% of patients (n = 7) dying during their admission. Payor plan was significant at 3 months ( P = 0.02), and palliative consultation was significant at 3 months ( P = 0.007) and 6 months ( P = 0.03). No notable association was observed with SDI and ADI in quantiles or as continuous variables. DISCUSSION: In this study, 28.1% of patients were diagnosed with cancer for the first time. Three-month and 6-month mortality rates for patients undergoing surgery were 26.7% and 39.5%, respectively. Furthermore, mortality was markedly associated with palliative care consultation and insurance status, but not with SDI and ADI. LEVEL OF EVIDENCE: Retrospective case series, Level III evidence.

A Comparison of Prognostic Models to Facilitate Surgical Decision-Making for Patients With Spinal Metastatic Disease.

STUDY DESIGN: Retrospective cohort. OBJECTIVE: Compare the performance of and provide cutoff values for commonly used prognostic models for spinal metastases, including Revised Tokuhashi, Tomita, Modified Bauer, New England Spinal Metastases Score (NESMS), and Skeletal Oncology Research Group model, at three- and six-month postoperative time points. SUMMARY OF BACKGROUND DATA: Surgery may be recommended for patients with spinal metastases causing fracture, instability, pain, and/or neurological compromise. However, patients with less than three to six months of projected survival are less likely to benefit from surgery. Prognostic models have been developed to help determine prognosis and surgical candidacy. Yet, there is a lack of data directly comparing the performance of these models at clinically relevant time points or providing clinically applicable cutoff values for the models. MATERIALS AND METHODS: Sixty-four patients undergoing surgery from 2015 to 2022 for spinal metastatic disease were identified. Revised Tokuhashi, Tomita, Modified Bauer, NESMS, and Skeletal Oncology Research Group were calculated for each patient. Model calibration and discrimination for predicting survival at three months, six months, and final follow-up were evaluated using the Brier score and Uno's C, respectively. Hazard ratios for survival were calculated for the models. The Contral and O'Quigley method was utilized to identify cutoff values for the models discriminating between survival and nonsurvival at three months, six months, and final follow-up. RESULTS: Each of the models demonstrated similar performance in predicting survival at three months, six months, and final follow-up. Cutoff scores that best differentiated patients likely to survive beyond three months included the Revised Tokuhashi score=10, Tomita score=four, Modified Bauer score=three, and NESMS=one. CONCLUSION: We found comparable efficacy among the models in predicting survival at clinically relevant time points. Cutoff values provided herein may assist surgeons and patients when deciding whether to pursue surgery for spinal metastatic disease. LEVEL OF EVIDENCE: 4.

Is Less Really More? Economic Evaluation of Minimally Invasive Surgery.

STUDY DESIGN: Review. OBJECTIVE: A comparative overview of cost-effectiveness between minimally invasive versus and equivalent open spinal surgeries. METHODS: A literature search using PubMed was performed to identify articles of interest. To maximize the capture of studies in our initial search, we combined variants of the terms "cost," "minimally invasive," "spine," "spinal fusion," "decompression" as either keywords or MeSH terms. PearlDiver database was queried for open and minimally invasive surgery (MIS; endoscopic or percutaneous) reimbursements between Q3 2015 and Q2 2018. RESULTS: In general, MIS techniques appeared to decrease blood loss, shorten hospital lengths of stay, mitigate complications, decrease perioperative pain, and enable quicker return to daily activities when compared to equivalent open surgical techniques. With regard to cost, primarily as a result of these latter benefits, MIS was associated with lower costs of care when compared to equivalent open techniques. However, cost reporting was sparse, and relevant methodology was inconsistent throughout the spine literature. Within the PearlDiver data sets, MIS approaches had lower reimbursements than open approaches for both lumbar posterior fusion and discectomy. CONCLUSIONS: Current data suggests that overall cost-savings may be incurred with use of MIS techniques. However, data reporting on costs lacks in uniformity, making it difficult to formulate any firm conclusions regarding any incremental improvements in cost-effectiveness that may be incurred when utilizing MIS techniques when compared to equivalent open techniques.

Ceramic Biologics for Bony Fusion-a Journey from First to Third Generations.

PURPOSE OF REVIEW: To provide information on characteristics and use of various ceramics in spine fusion and future directions. RECENT FINDINGS: In most recent years, focus has been shifted to the use of ceramics in minimally invasive surgeries or implementation of nanostructured surface modification features to promote osteoinductive properties. In addition, effort has been placed on the development of bioactive synthetics. Core characteristic of bioactive synthetics is that they undergo change to simulate a beneficial response within the bone. This change is based on chemical reaction and various chemical elements present in the bioactive ceramics. Recently, a synthetic 15-amino acid polypeptide bound to an anorganic bone material which mimics the cell-binding domain of type-I collagen opened a possibility for osteogenic and osteoinductive roles of this hybrid graft material. Ceramics have been present in the spine fusion arena for several decades; however, their use has been limited. The major obstacle in published literature is small sample size resulting in low evidence and a potential for bias. In addition, different physical and chemical properties of various ceramics further contribute to the limited evidence. Although ceramics have several disadvantages, they still hold a great promise as a value-based graft material with being easily available, relatively inexpensive, and non-immunogenic.

Syndesmotic Screw Removal in a Clinic Setting Is Safe and Cost-effective.

Background. There is no consensus in the literature regarding the necessity of syndesmotic screw removal, but the majority of surgeons prefer screw removal in the operating room. Purpose. The aim of this study is to analyze the safety and cost-effectiveness of syndesmotic screw removal in the clinic. Methods. A retrospective chart review was performed on all acute, traumatic ankle fractures that required syndesmotic stabilization over 5 years at a level 1 trauma center. Radiographs were evaluated for maintenance of syndesmotic reduction. Orthopaedic clinic visits and operating room costs were calculated. Results. Of 269 patients, syndesmotic screws were successfully removed in the clinic in 170 patients and retained in 99 patients. Two superficial infections (1.2%) developed following screw removal. The superficial infection rate was 3.3% (2 of 60) in patients who did not receive antibiotics compared with 0% (0 of 110) in patients who received antibiotics (P = .12). No patient lost syndesmotic reduction after screw removal. Cost savings of $13 829 per patient were achieved by syndesmotic screw removal in the clinic. Conclusion. Our study demonstrates that syndesmotic screw removal in the clinic is safe, does not result in tibiofibular diastasis, is cost-effective, and results in substantial financial savings. Level of Evidence: Level IV.

Regulated ex vivo regional gene therapy for bone repair using an inducible caspase-9 suicide gene system.

In order to adapt ex vivo regional gene therapy for clinical applications in orthopaedic surgery, safety issues must be considered. In this study we developed a suicide approach using a dual gene expression two step transcriptional amplification lentiviral vector (LV-TSTA) encoding BMP-2 and an inducible caspase 9 (iC9) system that selectively induces apoptosis upon activation with a chemical inducer of dimerization (CID). Transduction of rat bone marrow stromal cells (RBMSCs) with LV-TSTA-iC9/BMP-2 led to abundant BMP-2 production (90.3 ± 7.9 ng/24 h/106 cells) in vitro and stimulated bone formation in a mouse muscle pouch in the absence of CID. Moreover it was shown that CID could be used to selectively induce apoptosis in iC9-transduced cells both in vitro and in vivo. Double exposure to serial dilutions of CID decreased in vitro production of BMP-2 by 85-87% and Luc activity by 97-99% in iC9/BMP-2 or iC9/Luc-transduced cells respectively. Early administration of CID (Days 0-1 post-op) in mice implanted with iC9/BMP-2-transduced RBMSCs was effective in blocking bone formation, indicating that CID was toxic to the transduced cells. In iC9/Luc-implanted mice, late administration of two doses of CID (Days 27-28 post-op) significantly reduced the luciferase signal. The current study provides proof of concept for the potential clinical application of regulated gene therapy to promote bone repair.

Novel amplification targets for rapid detection and differentiation of Xylella fastidiosa subspecies fastidiosa and multiplex in plant and insect tissues.

Xylella fastidiosa is an insect-transmitted bacterial plant pathogen which causes a variety of economically important diseases worldwide. Molecular identification of X. fastidiosa is used for quarantine screening, surveillance, and research applications; many of which require subspecies level differentiation of pathogen isolates. This study describes quantitative PCR (qPCR) and isothermal amplification assays which can rapidly identify X. fastidiosa isolates belonging to the fastidiosa and multiplex subspecies. The TaqMan qPCR primers described here are used to differentiate X. fastidiosa strains by subspecies in plant and insect tissue in a single reaction, with the inclusion of a general amplification control probe to identify potential false negative samples. This TaqMan qPCR protocol can identify between 103 and 104 cfu/ml concentrations of X. fastidiosa at the subspecies level in a variety of sample types. Additionally, loop-mediated isothermal amplification (LAMP) targets were designed for faster detection of X. fastidiosa subspecies fastidiosa and multiplex, applicable to a field setting. These assays are effective for strain differentiation in artificially and naturally inoculated plant material, and in field collected insect vectors.

Spine Degenerative Conditions and Their Treatments: National Trends in the United States of America.

STUDY DESIGN: Retrospective database study. OBJECTIVE: Low back and neck pain are among the top leading causes of disability worldwide. The aim of our study was to report the current trends on spine degenerative disorders and their treatments. METHODS: Patients diagnosed with lumbar or cervical spine conditions within the orthopedic subset of Medicare and Humana databases (PearlDiver). From the initial cohorts we identified subgroups based on the treatment: fusion or nonoperative within 1 year from diagnosis. Poisson regression was used to determine demographic differences in diagnosis and treatment approaches. RESULTS: Within the Medicare database there were 6 206 578 patients diagnosed with lumbar and 3 156 215 patients diagnosed with cervical degenerative conditions between 2006 and 2012, representing a 16.5% (lumbar) decrease and 11% (cervical) increase in the number of diagnosed patients. There was an increase of 18.5% in the incidence of fusion among lumbar patients. For the Humana data sets there were 1 160 495 patients diagnosed with lumbar and 660 721 patients diagnosed with cervical degenerative disorders from 2008 to 2014. There was a 33% (lumbar) and 42% (cervical) increases in the number of diagnosed patients. However, in both lumbar and cervical groups there was a decrease in the number of surgical and nonoperative treatments. CONCLUSIONS: There was an overall increase in both lumbar and cervical conditions, followed by an increase in lumbar fusion procedures within the Medicare database. There is still a burning need to optimize the spine care for the elderly and people in their prime work age to lessen the current national economic burden.

Gene Therapy for Bone Repair Using Human Cells: Superior Osteogenic Potential of Bone Morphogenetic Protein 2-Transduced Mesenchymal Stem Cells Derived from Adipose Tissue Compared to Bone Marrow.

Ex vivo regional gene therapy strategies using animal mesenchymal stem cells genetically modified to overexpress osteoinductive growth factors have been successfully used in a variety of animal models to induce both heterotopic and orthotopic bone formation. However, in order to adapt regional gene therapy for clinical applications, it is essential to assess the osteogenic capacity of transduced human cells and choose the cell type that demonstrates the best clinical potential. Bone-marrow stem cells (BMSC) and adipose-derived stem cells (ASC) were selected in this study for in vitro evaluation, before and after transduction with a lentiviral two-step transcriptional amplification system (TSTA) overexpressing bone morphogenetic protein 2 (BMP-2; LV-TSTA-BMP-2) or green fluorescent protein (GFP; LV-TSTA-GFP). Cell growth, transduction efficiency, BMP-2 production, and osteogenic capacity were assessed. The study demonstrated that BMSC were characterized by a slower cell growth compared to ASC. Fluorescence-activated cell sorting analysis of GFP-transduced cells confirmed successful transduction with the vector and revealed an overall higher but not statistically significant transduction efficiency in ASC versus BMSC (90.2 ± 4.06% vs. 80.4 ± 8.51%, respectively; p = 0.146). Enzyme-linked immunosorbent assay confirmed abundant BMP-2 production by both cell types transduced with LV-TSTA-BMP-2, with BMP-2 production being significantly higher in ASC versus BMSC (239.5 ± 116.55 ng vs. 70.86 ± 24.7 ng; p = 0.001). Quantitative analysis of extracellular deposition of calcium (Alizarin red) and alkaline phosphatase activity showed that BMP-2-transduced cells had a higher osteogenic differentiation capacity compared to non-transduced cells. When comparing the two cell types, ASC/LV-TSTA-BMP-2 demonstrated a significantly higher mineralization potential compared to BMSC/LV-TSTA-BMP-2 7 days post transduction (p = 0.014). In conclusion, this study demonstrates that transduction with LV-TSTA-BMP-2 can significantly enhance the osteogenic potential of both human BMSC and ASC. BMP-2-treated ASC exhibited higher BMP-2 production and greater osteogenic differentiation capacity compared to BMP-2-treated BMSC. These results, along with the fact that liposuction is an easy procedure with lower donor-site morbidity compared to BM aspiration, indicate that adipose tissue might be a preferable source of MSCs to develop a regional gene therapy approach to treat difficult bone-repair scenarios.

Sox9 positive periosteal cells in fracture repair of the adult mammalian long bone.

INTRODUCTION: The phases of fracture healing have been well characterized. However, the exact source and genetic profile of the skeletal progenitors that participate in bone repair is somewhat unclear. Sox9 expression in skeletal elements precedes bone and cartilage formation and a Sox9+ cell type is retained in the adult periosteum. We hypothesized that Sox9+ periosteal cells are multipotent skeletal progenitors normally participating in fracture repair. METHODS: To test this hypothesis we used tamoxifen (TM)-mediated lineage tracing of Sox9+ cells in Sox9CreErt2:Td-Tomato mice. Intact femora were analyzed with immunostaining and RNA sequencing to evaluate the skeletal distribution and gene expression profile of Td-Tomato positive, Sox9-descendent cells in the adult femur. To assess the role of Td-tomato+cells in the fracture healing process, mice underwent a closed mid-diaphyseal femoral fracture. Fractured hind limbs were analyzed by X-ray, histology and immuno-staining at 3, 9 or 56days post-fracture. RESULTS: In the intact adult mouse femur, Td-Tomato-labeled cells were observed in the primary spongiosa, periosteum and endosteum. RNA sequencing showed that Td-Tomato positive periosteal cells were co-enriched for Sox9 transcripts, and mRNAs for osteoblast and chondrocyte specific genes. In a femoral fracture model, we showed that pre-labeled Td-Tomato positive descendent cells were mobilized during the early stages of bone repair (day 3 post-op) contributing to the fracture repair process by differentiating into chondrocytes, osteoblasts and osteocytes. CONCLUSION: A Sox9+ skeletal progenitor population resides in the adult periosteum. Fate tracing studies show that descendants of the Sox9+ periosteal progenitors give rise to chondrocytes, osteoblasts and mature cortical osteocytes in repair of the fractured femur. To our knowledge this is the first report of a reparative Sox9+ progenitor population in the periosteum of the adult long bone. Taken together with developmental studies, our data suggest a broad role for Sox9+ osteochondroprogenitors in development and repair of the mammalian skeleton.