Environmental DNA (eDNA) removal rates in streams differ by particle size under varying substrate and light conditions.

The use of environmental DNA (eDNA) as a sampling tool offers insights into the detection of invasive and/or rare aquatic species and enables biodiversity assessment without traditional sampling approaches, which are often labor-intensive. However, our understanding of the environmental factors that impact eDNA removal (i.e., how rapidly eDNA is removed from the water column by the combination of decay and physical removal) in flowing waters is limited. This limitation constrains predictions about the location and density of target organisms after positive detection. To address this question, we spiked Common Carp (Cyprinus carpio) eDNA into recirculating mesocosms (n = 24) under varying light (shaded versus open) and benthic substrate conditions (no substrate, bare substrate, and biofilm-colonized substrate). We then collected water samples from each mesocosm at four time points (40 min, 6 h, 18 h, and 48 h), and sequentially filtered the samples through 10, 1.0, and 0.2 µm filters to quantify removal rates for different eDNA particle sizes under varying light and substrate conditions. Combining all size classes, total eDNA removal rates were higher for mesocosms with biofilm-colonized substrate compared to those with no substrate or bare (i.e., no biofilm) substrate, which is consistent with previous findings linking biofilm colonization with increased eDNA removal and degradation. Additionally, when biofilm was present, light availability increased eDNA removal; eDNA levels fell below detection after 6-18 h for open mesocosms versus 18-48 h for shaded mesocosms. Among size classes, larger particles (>10 µm) were removed faster than small particles (1.0-0.2 µm). These results suggest that changes in the distribution of eDNA size classes over time (e.g., with downstream transport) and with differing environmental conditions could be used to predict the location of target organisms in flowing waters, which will advance the use of eDNA as a tool for species monitoring and management.

Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture.

Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells. Cell culture was established in stirred-tank microcarrier bioreactors, and an efficient affinity chromatography method was developed for the purification of harvested CVA21 through binding of the viral capsids to an immobilized glutathione (GSH) ligand. Bioreactor temperature during infection was investigated to maximize titer, and a decrease in temperature from 37°C to 34°C yielded a two-three-fold increase in infectivity. After purification of the 34°C harvests, the GSH affinity chromatography elution not only maintained a >two-fold increase in infectivity and viral genomes but also increased the proportion of empty capsids compared to 37°C harvests. Using material generated from both infection temperature setpoints, chromatographic parameters and mobile phase compositions were studied at the laboratory scale to maximize infectious particle yields and cell culture impurity clearance. Empty capsids that co-eluted with full capsids from 34°C infection temperature harvests were poorly resolved across the conditions tested, but subsequent polishing anion exchange and cation exchange chromatography steps were developed to clear residual empty capsids and other impurities. Oncolytic CVA21 production was scaled-up 75-fold from the laboratory scale and demonstrated across seven batches in 250 L single-use microcarrier bioreactors and purified with customized, prepacked, single-use 1.5 L GSH affinity chromatography columns. The large-scale bioreactors controlled at 34°C during infection maintained a three-fold increase in productivity in the GSH elution, and excellent clearance of host cell and media impurities was observed across all batches. This study presents a robust method for the manufacture of an oncolytic virus immunotherapy application that may be implemented for the scalable production of other viruses and viral vectors which interact with glutathione.

Elective laminectomy and excision of the thoracic spine neoplasm: an evaluation of early outcomes.

BACKGROUND: Laminectomy with excision of spinal neoplasms is commonly performed. The current study examines risk profiles associated with elective laminectomies of benign, malignant primary, and secondary/metastatic neoplasms of the thoracic spine. METHODS: Adult patients undergoing elective thoracic laminectomy and excision of spinal neoplasm were abstracted from ACS-NSQIP years 2011-2014. Patients were classified into three cohorts: benign primary, malignant primary, secondary/metastatic. Univariate and multivariable analyses compared operation time, early complications, hospital length of stay (HLOS), and discharge destination across cohorts. RESULTS: One-hundred sixty patients were included, aged 58.0±14.8-years. Mean operation time was 209.23±101.52 minutes and cohorts did not differ significantly on multivariable analysis. Mean HLOS was 6.10±7.14 days and did not differ by cohort. Early complications were observed in 15.6% of patients; secondary/metastatic patients associated with increased odds of >1 unit of blood transfusion, but not overall complications. Failure to be discharged home occurred in 31.8% of patients (benign primary: 30.0%, malignant primary: 66.6%, secondary/metastatic: 10.5%; P<0.001). Malignant primary tumors associated with increased multivariable odds of failure to be discharged home (OR 3.63, 95% CI [1.09, 12.10], P=0.036). Tumor location (extradural, intradural/extramedullary, intramedullary) was not a significant predictor of outcomes. A higher number of concurrent fusions were observed for secondary/metastatic laminectomies (benign primary: 7.3%; malignant primary: 5.5%; secondary/metastatic: 36.8%; P<0.001). CONCLUSIONS: Comparable performance on operation time and HLOS between neoplasm cohorts suggests broad indication of resectional laminectomies for elective treatment. Primary malignant thoracic spine tumors may require higher levels of early postdischarge care. Future studies are needed to confirm these findings.

Anterior Versus Transforaminal Lumbar Interbody Fusion: Perioperative Risk Factors and 30-Day Outcomes.

BACKGROUND: Operative management of lower back pain often necessitates anterior lumbar interbody fusion (ALIF) or transforaminal lumbar interbody fusion (TLIF). Specific pathoanatomic advantages and indications exist for both approaches, and few studies to date have characterized comparative early outcomes. METHODS: Adult patients undergoing elective ALIF or TLIF operations were abstracted from the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) years 2011-2014. Univariate analyses were performed by surgery cohort for each outcome and adjusted for demographic/clinical variables (age ≥ 65, sex, race, body mass index, American Society of Anesthesiologists physical classification score, functional status, inpatient/outpatient status, smoking, hypertension, Charlson Comorbidity Index) using multivariable regression. Means, standard errors, mean differences (B), odds ratios (ORs), and associated 95% confidence intervals (CIs) are reported. Significance was assessed at P < .05. RESULTS: Of 8263 subjects (ALIF: 4325, TLIF: 3938), ALIF subjects were younger, less obese, less physically impaired, and had significantly lower rates of hypertension, diabetes, coagulopathy, and previous cardiac surgery. On multivariable analysis, ALIF associated with shorter operative time (B = -11.80 minutes, 95% CI [-16.48, -7.12]; P < .001). Transforaminal lumbar interbody fusion was associated with increased incidence of urinary tract infections (UTIs; OR = 1.57, 95% CI [1.10, 2.26]; P = .013) and of blood transfusions (OR = 1.19, 95% CI [1.04, 1.37]; P = .012). Multivariate analysis also demonstrated TLIF associated with shorter hospital length of stay (B = -0.27 days, 95% CI [-0.54, -0.01]; P = .041), and fewer cases of pneumonia (OR = 0.55, 95% CI [0.32, 0.94]; P = .029) and prolonged ventilator dependency (OR = 0.33, 95% CI [0.12, 0.84]; P = .021). CONCLUSIONS: Comparatively, ALIF patients experienced decreased operative time and decreased incidence of postoperative UTIs and blood transfusions. Anterior lumbar interbody fusion patients were more likely to suffer postoperative pulmonary complications and longer hospital stays. Our data support the notion that both anterior and transforaminal surgical approaches perform comparably in context of 30-day perioperative outcomes.

A First-in-Human, Phase I Study of Neural Stem Cell Transplantation for Chronic Spinal Cord Injury.

We tested the feasibility and safety of human-spinal-cord-derived neural stem cell (NSI-566) transplantation for the treatment of chronic spinal cord injury (SCI). In this clinical trial, four subjects with T2-T12 SCI received treatment consisting of removal of spinal instrumentation, laminectomy, and durotomy, followed by six midline bilateral stereotactic injections of NSI-566 cells. All subjects tolerated the procedure well and there have been no serious adverse events to date (18-27 months post-grafting). In two subjects, one to two levels of neurological improvement were detected using ISNCSCI motor and sensory scores. Our results support the safety of NSI-566 transplantation into the SCI site and early signs of potential efficacy in three of the subjects warrant further exploration of NSI-566 cells in dose escalation studies. Despite these encouraging secondary data, we emphasize that this safety trial lacks statistical power or a control group needed to evaluate functional changes resulting from cell grafting.

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice.

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

A matched cohort comparison of cervical disc arthroplasty versus anterior cervical discectomy and fusion: Evaluating perioperative outcomes.

OBJECTIVE: Cervical disc arthroplasty (CDA) is a recent alternative to anterior cervical discectomy and fusion (ACDF) in patients suffering cervical disc herniation and degeneration. To date, a systematic analysis of their comparative advantages and risks following elective surgery remains elusive. METHODS: Adult patients undergoing elective CDA or ACDF were extracted from the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database years 2011-2014. A total of 19,369 patients were matched 1:1 by age, sex, functional status, charlson comorbidity index, ASA classification, BMI classification, and number of vertebral levels operated on during surgery. This matching process led to a final sample of 588 subjects (294 CDA, 294 ACDF). Multivariable regression was performed for five outcome measures: operation time, early complications, reoperation rates, hospital length of stay (HLOS), and discharge destination. Mean differences (B), odds ratios (OR) and associated 95% confidence intervals (CI) are reported. RESULTS: Compared to ACDF, CDA was associated with decreased mean operation time (B=-18.78-min, 95% CI [-29.13, -8.42]; p<0.001), decreased HLOS (B=-0.44-days [-0.77, -0.11]; p=0.009), and increased likelihood of discharge to home (OR=5.39 [1.14-25.43]; p=0.033). No differences in reoperation rates and complications were found. CONCLUSION: In a matched cohort analysis, CDA performs comparably to ACDF and is associated with decreased operation time and HLOS, and increased likelihood of discharge to home, without differences in 30-day complications or reoperation rates. Future prospective studies are warranted.

A Review of Stem Cell Therapy for Spinal Cord Injury: Large Animal Models and the Frontier in Humans.

OBJECTIVE: To review the literature of spinal cord injury and stem cell therapy for large animal models and incorporate those results into an understanding of stem cell therapy for human cord injury. METHODS: Review of the literature. RESULTS: Eleven canine studies were identified and 3 sub-human primate studies were identified showing variable results. CONCLUSIONS: Stem cell therapy is a promising therapeutic option for patients with spinal cord injury; however, the technology has many un-answered questions and further research is needed.

Lateral mass screw stimulation thresholds in posterior cervical instrumentation surgery: a predictor of medial deviation.

OBJECTIVE Normative data exists for stimulus-evoked pedicle screw electromyography (EMG) current thresholds in the lumbar spine, and is routinely referenced during spine surgeries to detect a screw breach, prevent injury of neural elements, and ensure the most biomechanically sound instrumentation construct. To date, similar normative data for cervical lateral mass screws is limited, thus the utility of lateral mass screw testing remains unclear. To address this disparity, in this study the authors describe cumulative lateral mass screw stimulation threshold data in patients undergoing posterior cervical instrumentation with lateral mass screws. These data are correlated with screw placement on postoperative imaging, and a novel correlation is discovered with direct clinical implications. METHODS Using a ball-tip probe, 154 lateral mass screws in 21 patients were electrically tested intraoperatively. In each case, for each screw, the lowest (or threshold) current at which the first polyphasic stimulus-evoked EMG response was reproducibly observed by a neurophysiologist was recorded. All patients underwent postoperative CT. Screw position within the lateral mass was first measured in the axial and sagittal planes for each lateral mass screw using the CT images. Screw placement was also evaluated by 2 independent physicians, blinded to current threshold data, on a binary scale of acceptability. The predictive capacity of screw EMG threshold data was evaluated via multivariable regression analyses and receiver operating characteristic (ROC) analyses. Predictive capacity was examined with respect to screw position within the lateral mass, as well as screw acceptability. RESULTS Lateral mass screw EMG thresholds did not appear to differ significantly for screws considered "acceptable" versus "unacceptable" according to the radiographic criteria. Accordingly, ROC analysis confirmed that EMG current threshold data were of minimal utility in predicting screw radiographic acceptability. However, EMG threshold was significantly predictive of screw medial distance from the spinal canal. A screw stimulating below 7.5 mA correctly identified a screw as being within 2 mm of the spinal canal with 75% sensitivity and 92% specificity (positive predictive value 20%, negative predictive value 99.3%), independent of its distance relative to other lateral mass landmarks. EMG current threshold was not significantly predictive of screw deviation in the superior or inferior directions, and was inversely predictive of screw deviations in the lateral direction. CONCLUSIONS In the context of uncertainty regarding the utility of cervical lateral mass EMG current threshold data, this study found that EMG current thresholds correspond significantly, and exclusively, with screw distance from the spinal canal. This association appears independent of other criteria for screw misplacement. As such, the authors recommend that EMG current thresholds be referenced in the case of a suspected medial breach as an effective means to rule out screw placement too medial to the spinal canal.

Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.

Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients.

Traumatic L5 Posterolateral Spondyloptosis: A Case Report and Review of the Literature.

Traumatic retrolisthesis of the lumbar spine is a rare clinical entity. Only a few case reports have shown retrolisthesis of the fractured fragment over the inferior vertebral body. Fracture dislocations of the spine are unstable injuries that require operative fixation to restore alignment and prevent progressive deformity. We present the case of a traumatic L5-S1 fracture dislocation with retrolisthesis of the L5 vertebral body over the superior aspect of S1 managed with anterior, middle, and posterior column reconstruction. The patient presented with paraplegia and bowel and bladder incontinence. Retrolisthesis fracture dislocations injuries are rare, and as such, there are no guidelines regarding their management. In our case, we performed an L5 vertebrectomy with anterior, middle, and posterior column reconstruction via a posterior approach using a lumbosacral-pelvic construct. The patient did not regain function in his distal lower extremities postoperatively.

Nonextraction treatment of a Class II malocclusion and impacted maxillary central incisor.

The multidisciplinary treatment of a patient with a Class II malocclusion and an impacted maxillary central incisor is presented. Nonextraction correction of the distoclusion was achieved through mandibular growth and protraction of the mandibular first molars. Closed-eruption surgical exposure of the incisor, combined with soft-tissue revision posteruption, resulted in significantly improved facial esthetics.

Tensile bond strength of acrylic resin denture teeth to a microwave- or heat-processed denture base.

STATEMENT OF PROBLEM: Fracture of acrylic resin prosthetic teeth from acrylic resin denture bases can be a problem for some patients. The optimal combination of acrylic resin denture tooth, denture base material, and processing method is not known. Purpose. The objective of this study was to compare the tensile bond strengths of heat- and microwave-polymerized acrylic resins among 4 types of acrylic resin denture teeth. MATERIAL AND METHODS: Heat-polymerized (Lucitone 199) and microwave-polymerized (Acron MC) acrylic resins were used. Four types of acrylic resin denture teeth (IPN, SLM, Vitapan, and SR-Orthotyp-PE) were milled to a fixed diameter according to ADA specification no. 15. Ten specimens of each tooth type were processed to each of the denture base materials according to the manufacturers' instructions. Ten additional resin control specimens without teeth also were fabricated. Specimens were thermocycled and tested for strength until fracture with a custom alignment device. Data were analyzed with analysis of variance and Duncan's multiple range test. A scanning electron microscope was used to identify adhesive and cohesive failures within debonded specimens. RESULTS: The mean force required to fracture the specimens ranged from 5.3 +/- 3.01 to 21.6 +/- 5.2 MPa for the microwave-polymerized base and 11.2 +/- 3.0 to 39.1 +/- 5.1 MPa for the heat-polymerized base. The most common failure was cohesive within the denture tooth. With each base material, Orthotyp and IPN teeth exhibited the highest bond strengths; SLM and Orthotyp bond strengths were similar. In general, heat-polymerized groups failed cohesively within the denture base resin or the tooth, and microwave-polymerized groups failed adhesively at either the ridge lap or occlusal surface of the denture tooth. CONCLUSION: Within the limitations of this study, the results suggest that the type of denture base material and denture tooth selected for use may influence the tensile bond strength of the tooth to the base. Selection of more compatible combinations of base and resin teeth may reduce the number of prosthesis fractures and resultant repairs.