A Novel Secretome Biotherapeutic Influences Regeneration in Critical Size Bone Defects.

Severe traumatic injuries often result in critical size bone defects, which are unable to heal without treatment. Autologous grafting is the standard of care but requires additional surgeries for graft procurement. Amnion-derived multipotent progenitor cells release a secretome of biomolecules identified as integral to the process of bone regeneration and angiogenesis. This secretome is currently under development as a biotherapeutic. The efficacy of this secretome biotherapeutic was evaluated in vitro on the proliferation and migration of mesenchymal stem cells and osteoprogenitor cells as well as in vivo using a critical size rat calvarial defect model. The secretome biotherapeutic was loaded onto a collagen scaffold and placed into the defect, which was allowed to heal for 4 and 12 weeks. The secretome biotherapeutic enhanced the proliferation and migration of mesenchymal stem cells and proliferation of osteoprogenitor cells. Further, the secretome biotherapeutic improved new bone volume and connectivity by 12 weeks and significantly improved angiogenesis at 4 weeks and bone density at 4 and 12 weeks with no deleterious effects. The improvement in new bone volume, connectivity, and angiogenesis suggests that the secretome biotherapeutic has beneficial effects for bone healing and a higher dose of the secretome biotherapeutic may further improve regeneration.

Establishment of the Clinician-Scientist Investigator Opportunity Network to Develop Military Medical Research Leaders.

INTRODUCTION: The ability of military clinicians to conduct military medical research is often limited because of competing priorities and a lack of research mentorship. The Clinician-Scientist Investigator Opportunity Network (CSION) was developed with the intent of training clinicians how to engage in requirements-driven research within the DoD. MATERIALS AND METHODS: Three to five academic medical faculties were selected from a pool of applicants each year to participate in a 2-year research fellowship. To be eligible for the CSION program, applicants had to meet the following criteria: (1) Completed residency graduate medical education training, (2) not be currently enrolled as a graduate medical education trainee, and (3) obtained permission from their department leadership to focus 25% of their duty hours on CSION participation to include didactic and research efforts. The remaining 75% of fellows' time was dedicated to clinical duties. Monthly didactics, intensive mentorship, and consistent support were offered to each fellow by the CSION leadership team. Metrics were recorded to include both research and clinical productivity. RESULTS: Between January 2019 and December 2022, 12 CSION fellows graduated from the program (four in the class of 2020, three in 2021, and five in 2022). From 2019 to 2021, the 12 CSION fellows initiated 204 research protocols, generated 489 publications/presentations, and secured 33 research grants. All graduates of the program remain active in clinical research with multiple graduates currently assigned to research positions. CONCLUSIONS: The CSION research education program is a 2-year additional duty research fellowship producing clinician-scientists conducting military-relevant medical research and publications and may be considered a low-cost/highly efficient alternative to achieve the reported benefits of the MD-PhD tract. The expansion of the CSION program may improve the quality of military medical research and health care.

Evaluation of Detection and Efficacy of Decontamination of Respiratory Pathogens Including SARS-CoV-2 on Basic Military Trainee Gas Masks.

INTRODUCTION: Basic military trainee (BMT) gas mask training poses a potential mechanism of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) transmission. After training, gas masks are decontaminated. Insufficient decontamination can lead to viral transmission in the next training class. To our knowledge, the decontamination process has not been validated for efficacy in removing respiratory pathogens such as SARS-CoV-2. MATERIALS AND METHODS: Inactivated strains of SARS-CoV-2, influenza A and B, and Bordetella pertussis were separately inoculated onto gas masks in the emitter area (n = 5). Pathogen detection in swabs collected from gas masks was performed by real-time polymerase chain reaction (RT-PCR) using the BioFire® RP2.1 panel and Biomeme Franklin system. For decontamination efficacy experiments, pathogens were inoculated onto gas masks, and contaminated areas were swabbed before and after decontamination, with detection using both PCR platforms. Lastly, 65 gas masks were swabbed after gas mask training, and again after the trainees and guardians decontaminated the masks, to identify the presence of any respiratory pathogen exhaled onto the gas masks. RESULTS: All four pathogens were detected by both PCR platforms. The BioFire® FilmArray® was more sensitive than the Biomeme platform. Decontamination resulted in undetectable levels of all three viruses. B. pertussis was detected on one mask after decontamination. Experiments with live B. pertussis validated that decontamination eliminated all viable bacteria from gas masks. For BMT sampling, all masks were negative for SARS-CoV-2. One mask tested positive for coronavirus 229E. Once decontaminated, all masks tested negative. CONCLUSIONS: BMT gas masks can be monitored for the presence of respiratory pathogens using RT-PCR. The decontamination process removed all viable respiratory pathogens tested from the gas masks. This study demonstrates that RT-PCR can be used to conduct pathogen surveillance on BMT gas masks after training and that the current decontamination process is effective to eliminate respiratory viruses including SARS-CoV-2.

The early-onset torsion dystonia-associated protein, torsinA, is a homeostatic regulator of endoplasmic reticulum stress response.

Early-onset torsion dystonia is the most severe heritable form of dystonia, a human movement disorder that typically starts during a developmental window in early adolescence. Deletion in the DYT1 gene, encoding the torsinA protein, is responsible for this dominantly inherited disorder, which is non-degenerative and exhibits reduced penetrance among carriers. Here, we explore the hypothesis that deficits in torsinA function result in an increased vulnerability to stress associated with protein folding and processing in the endoplasmic reticulum (ER), where torsinA is located. Using an in vivo quantitative readout for the ER stress response, we evaluated the consequences of torsinA mutations in transgenic nematodes expressing variants of human torsinA. This analysis revealed that, normally, torsinA serves a protective function to maintain a homeostatic threshold against ER stress. Furthermore, we show that the buffering capacity of torsinA is greatly diminished by the DYT1-associated deletion or mutations that prevent its translocation to the ER, block ATPase activity, or increase the levels of torsinA in the nuclear envelope versus ER. Combinations of transgenic Caenorhabditis elegans designed to mimic clinically relevant genetic modifiers of disease susceptibility also exhibit a direct functional correlation to changes in the ER stress response. Furthermore, using mouse embryonic fibroblasts (MEFs) from torsinA knockout mice, we demonstrated that loss of endogenous torsinA results in enhanced sensitivity to ER stress. This study extends our understanding of molecular mechanisms underlying dystonia, and establishes a new functional paradigm to evaluate therapeutic strategies to compensate for reduced torsinA activity in the ER as a means to restore homeostatic balance and neuronal function.

Comparative evaluation of mesenchymal stromal cell growth and osteogenic differentiation on a shape memory polymer scaffold.

Trauma-induced, critical-size bone defects pose a clinical challenge to heal. Albeit autografts are the standard-of-care, they are limited by their inability to be shaped to various defect geometries and often incur donor site complications. Herein, the combination of a "self-fitting" shape memory polymer (SMP) scaffold and seeded mesenchymal stromal cells (MSCs) was investigated as an alternative. The porous SMP scaffold, prepared from poly(ε-caprolactone) diacrylate (PCL-DA) and coated with polydopamine, provided conformal shaping and cell adhesion. MSCs from five tissues, amniotic (AMSCs), chorionic tissue (CHSCs), umbilical cord (UCSCs), adipose (ADSCs), and bone marrow (BMSCs) were evaluated for viability, density, and osteogenic differentiation on the SMP scaffold. BMSCs exhibited the fastest increase in cell density by day 3, but after day 10, CHSCs, UCSCs, and ADSCs approached similar cell density. BMSCs also showed the greatest calcification among the cell types, followed closely by ADSCs, CHSCs and AMSCs. Alkaline phosphatase (ALP) activity peaked at day 7 for AMSCs, UCSCs, ADSCs and BMSCs, and at day 14 for CHSCs, which had the greatest overall ALP activity. Of all the cell types, only scaffolds cultured with ADSCs in osteogenic media had increased hardness and local modulus as compared to blank scaffolds after 21 days of cell culture and osteogenic differentiation. Overall, ADSCs performed most favorably on the SMP scaffold. The SMP scaffold was able to support key cellular behaviors of MSCs and could potentially be a viable, regenerative alternative to autograft.

Fresh frozen plasma attenuates lung injury in a novel model of prolonged hypotensive resuscitation.

BACKGROUND: Hemorrhagic shock remains a leading cause of early death among severely injured in both civilian and military settings. As future military operations will require strategies allowing prolonged field care of the injured, we sought to develop an in vivo model of prolonged hypotensive resuscitation (PHR) and to evaluate the role of plasma-based resuscitation in this model. We hypothesized that resuscitation with fresh frozen plasma (FFP) would mitigate lung injury when compared with Hextend in a rodent model of PHR. METHODS: Mice underwent laparotomy and hemorrhagic shock (mean arterial blood pressure, 35 ± 5 mm Hg × 90 minutes) followed by PHR with either FFP or Hextend to maintain a mean arterial blood pressure of 55 mm Hg to 60 mm Hg for 6 hours. Sham animals underwent cannulation only. At the end of 6 hours, animals were euthanized, and lung tissue harvested for measurement of histopathologic injury, inflammation and permeability using hematoxylin and eosin staining, myeloperoxidase immunofluorescence staining and Evans Blue dye. Pulmonary syndecan-1 immunostaining was assessed as an indicator of endothelial cell integrity. RESULTS: All animals in the FFP, Hextend, and sham groups survived to the end of resuscitation. Resuscitation with FFP mitigated lung histopathologic injury compared with Hextend (histologic injury score of 4.38 ± 2.07 vs. 7.5 ± 0.93, scale of 0-9, p = 0.002) and was comparable to shams (histologic injury score of 4.0 ± 1.93, scale of 0-9, p = 0.99). Fresh frozen plasma also reduced lung inflammation (0.116 ± 0.044 vs. 0.308 ± 0.054 relative fluorescence of myeloperoxidase, p = 0.002) and restored pulmonary syndecan-1 (0.514 ± 0.061 vs. 0.059 ± 0.021, relative syndecan-1 fluorescence, p < 0.001) when compared with Hextend. Consistently, FFP mitigated lung hyperpermeability compared with Hextend (7.30 ± 1.34 µg vs. 14.91 ± 5.55 µg Evans blue/100 mg lung tissue, p = 0.005). CONCLUSION: We have presented a novel model of PHR of military relevance to the prolonged field care environment. In this model, FFP maintains its pulmonary protective effects using a PHR strategy compared with Hextend, which supports the need for further development and implementation of plasma-based resuscitation in the forward environment. LEVEL OF EVIDENCE: Basic science.

Localized low-dose rhBMP-2 is effective at promoting bone regeneration in mandibular segmental defects.

At least 26% of recent battlefield injuries are to the craniomaxillofacial (CMF) region. Recombinant human bone morphogenetic protein 2 (rhBMP-2) is used to treat CMF open fractures, but several complications have been associated with its use. This study tested the efficacy and safety of a lower (30% recommended) dose of rhBMP-2 to treat mandibular fractures. rhBMP-2 delivered via a polyurethane (PUR) and hydroxyapatite/ß-tricalcium phosphate (Mastergraft®) scaffold was evaluated in a 2 cm segmental mandibular defect in minipigs. Bone regeneration was analyzed at 4, 8, and 12 weeks postsurgery using clinical computed tomography (CT) and rhBMP-2, and inflammatory marker concentrations were analyzed in serum and surgery-site drain effluent. CT scans revealed that pigs treated with PUR-Mastergraft® + rhBMP-2 had complete bone bridging, while the negative control group showed incomplete bone-bridging (n = 6). Volumetric analysis of regenerated bone showed that the PUR-Mastergraft® + rhBMP-2 treatment generated significantly more bone than control by 4 weeks, a trend that continued through 12 weeks. Variations in inflammatory analytes were detected in drain effluent samples and saliva but not in serum, suggesting a localized healing response. Importantly, the rhBMP-2 group did not exhibit an excessive increase in inflammatory analytes compared to control. Treatment with low-dose rhBMP-2 increases bone regeneration capacity in pigs with mandibular continuity defects and restores bone quality. Negative complications from rhBMP-2, such as excessive inflammatory analyte levels, were not observed. Together, these results suggest that treatment with low-dose rhBMP-2 is efficacious and may improve safety when treating CMF open fractures. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1491-1503, 2019.

Nonhuman Primate (Rhesus Macaque) Models of Severe Pressure-Targeted Hemorrhagic and Polytraumatic Hemorrhagic Shock.

BACKGROUND: We endeavored to develop clinically translatable nonhuman primate (NHP) models of severe polytraumatic hemorrhagic shock. METHODS: NHPs were randomized into five severe pressure-targeted hemorrhagic shock (PTHS) ±â€Šadditional injuries scenarios: 30-min PTHS (PTHS-30), 60-min PTHS (PTHS-60), PTHS-60 + soft tissue injury (PTHS-60+ST), PTHS-60+ST + femur fracture (PTHS-60+ST+FF), and decompensated PTHS+ST+FF (PTHS-D). Physiologic parameters were recorded and blood samples collected at five time points with animal observation through T = 24 h. Results presented as mean ±â€ŠSEM; statistics: log transformation followed by two-way ANOVA with Bonferroni multiple comparisons, Wilcoxon nonparametric test for comparisons, and the Friedmans' one-way ANOVA; significance: P < 0.05. RESULTS: Percent blood loss was 40% ±â€Š2, 59% ±â€Š3, 52% ±â€Š3, 49% ±â€Š2, and 54% ±â€Š2 for PTHS-30, PTHS-60, PTHS-60+ST, PTHS-60+ST+FF, and PTHS-D, respectively. All animals survived to T = 24 h except one in each of the PTHS-60 and PTHS-60+ST+FF groups and seven in the PTHS-D group. Physiologic, coagulation, and inflammatory parameters demonstrated increasing derangements with increasing model severity. CONCLUSION: NHPs exhibit a high degree of resilience to hemorrhagic shock and polytrauma as evidenced by moderate perturbations in metabolic, coagulation, and immunologic outcomes with up to 60 min of profound hypotension regardless of injury pattern. Extending the duration of PTHS to the point of decompensation in combination with polytraumatic injury, evoked derangements consistent with those observed in severely injured trauma patients which would require ICU care. Thus, we have successfully established a clinically translatable NHP trauma model for use in testing therapeutic interventions to trauma.

Inflammatory Profile in Response to Uncontrolled Hemorrhage in a Non-Human Primate (Rhesus Macaque) Model.

BACKGROUND: Uncontrolled hemorrhage (UH), the leading cause of potentially survivable combat-related death, elicits a deleterious inflammatory response. Our group previously reported an increased secretion of pro-inflammatory cytokines in a novel non-human primate model of UH; however, to better understand the molecular profile of the inflammatory response to UH, we performed a comprehensive evaluation of inflammation at the proteomic and transcriptomic level. METHODS: Anesthetized rhesus macaques (n = 8) underwent UH by 60% left lobe hepatectomy T = 0 min. At T = 5 min, animals received 11 mL of 5% albumin followed by normal saline infusion to a total resuscitation volume of 20 mL/kg by T = 120 min. Blood (T = 0, 5, 20, 120, 480 min) was collected for qPCR and multiplex cytokine quantification. Results from each non-human primate (NHP) per time-point are shown. Statistical analysis by one-way ANOVA with repeated measures, P <0.05 was considered significant. RESULTS: Luminex analysis in serum revealed significant up-regulation compared with baseline of 8 cytokines/chemokines starting T = 120 min postinjury and significant down-regulation of 4 cytokines/chemokines as early as T = 20 min postinjury. Gene expression analysis in white blood cells uncovered 10 genes that were up-regulated greater than 3-fold compared with baseline and 29 genes that were down-regulated greater than 3-fold. CONCLUSION: The present study confirms the presence of systemic inflammation after UH at the proteomic and transcriptomic level providing insight into the inflammatory mediators that are involved as well as their kinetics following UH. The data demonstrates that NHP hemorrhage models may be suitable for evaluating therapeutics to control inflammation following hemorrhage.

Structural features and chaperone activity of the NudC protein family.

The NudC family consists of four conserved proteins with representatives in all eukaryotes. The archetypal nudC gene from Aspergillus nidulans is a member of the nud gene family that is involved in the maintenance of nuclear migration. This family also includes nudF, whose human orthologue, Lis1, codes for a protein essential for brain cortex development. Three paralogues of NudC are known in vertebrates: NudC, NudC-like (NudCL), and NudC-like 2 (NudCL2). The fourth distantly related member of the family, CML66, contains a NudC-like domain. The three principal NudC proteins have no catalytic activity but appear to play as yet poorly defined roles in proliferating and dividing cells. We present crystallographic and NMR studies of the human NudC protein and discuss the results in the context of structures recently deposited by structural genomics centers (i.e., NudCL and mouse NudCL2). All proteins share the same core CS domain characteristic of proteins acting either as cochaperones of Hsp90 or as independent small heat shock proteins. However, while NudC and NudCL dimerize via an N-terminally located coiled coil, the smaller NudCL2 lacks this motif and instead dimerizes as a result of unique domain swapping. We show that NudC and NudCL, but not NudCL2, inhibit the aggregation of several target proteins, consistent with an Hsp90-independent heat shock protein function. Importantly, and in contrast to several previous reports, none of the three proteins is able to form binary complexes with Lis1. The availability of structural information will be of help in further studies on the cellular functions of the NudC family.

The early-onset torsion dystonia-associated protein, torsinA, displays molecular chaperone activity in vitro.

TorsinA is a member of the AAA+ ATPase family of proteins and, notably, is the only known ATPase localized to the ER lumen. It has been suggested to act as a molecular chaperone, while a mutant form associated with early-onset torsion dystonia, a dominantly inherited movement disorder, appears to result in a net loss of function in vivo. Thus far, no studies have examined the chaperone activity of torsinA in vitro. Here we expressed and purified both wild-type (WT) and mutant torsinA fusion proteins in bacteria and examined their ability to function as molecular chaperones by monitoring suppression of luciferase and citrate synthase (CS) aggregation. We also assessed their ability to hold proteins in an intermediate state for refolding. As measured by light scattering and SDS-PAGE, both WT and mutant torsinA effectively, and similarly, suppressed protein aggregation compared to controls. This function was not further enhanced by the presence of ATP. Further, we found that while neither form of torsinA could protect CS from heat-induced inactivation, they were both able to reactivate luciferase when ATP and rabbit reticulocyte lysate were added. This suggests that torsinA holds luciferase in an intermediate state, which can then be refolded in the presence of other chaperones. These data provide conclusive evidence that torsinA acts as a molecular chaperone in vitro and suggests that early-onset torsion dystonia is likely not a consequence of a loss in torsinA chaperone activity but might be an outcome of insufficient torsinA localization at the ER to manage protein folding or trafficking.

Chemical enhancement of torsinA function in cell and animal models of torsion dystonia.

Movement disorders represent a significant societal burden for which therapeutic options are limited and focused on treating disease symptomality. Early-onset torsion dystonia (EOTD) is one such disorder characterized by sustained and involuntary muscle contractions that frequently cause repetitive movements or abnormal postures. Transmitted in an autosomal dominant manner with reduced penetrance, EOTD is caused in most cases by the deletion of a glutamic acid (DeltaE) in the DYT1 (also known as TOR1A) gene product, torsinA. Although some patients respond well to anticholingerics, therapy is primarily limited to either neurosurgery or chemodenervation. As mutant torsinA (DeltaE) expression results in decreased torsinA function, therapeutic strategies directed toward enhancement of wild-type (WT) torsinA activity in patients who are heterozygous for mutant DYT1 may restore normal cellular functionality. Here, we report results from the first-ever screen for candidate small molecule therapeutics for EOTD, using multiple activity-based readouts for torsinA function in Caenorhabditis elegans, subsequent validation in human DYT1 patient fibroblasts, and behavioral rescue in a mouse model of DYT1 dystonia. We exploited the nematode to rapidly discern chemical effectors of torsinA and identified two classes of antibiotics, quinolones and aminopenicillins, which enhance WT torsinA activity in two separate in vivo assays. Representative molecules were assayed in EOTD patient fibroblasts for improvements in torsinA-dependent secretory function, which was improved significantly by ampicillin. Furthermore, a behavioral defect associated with an EOTD mouse knock-in model was also rescued following administration of ampicillin. These combined data indicate that specific small molecules that enhance torsinA activity represent a promising new approach toward therapeutic development for EOTD, and potentially for other diseases involving the processing of mutant proteins.

Phospholipids of the differentiating species of Tetrahymena.

The whole-cell phospholipid composition of the six known polymorphic species of Tetrahymena has been examined by [(3)H]acetate and [(3)H]myristic acid radiolabeling, and by gas-liquid chromatography of total phospholipid-bound fatty acids. Five of the polymorphic species contained similar phospholipid profiles following radiolabeling in that phosphatidylethanolamine (PE) was the predominant phospholipid; however, in cells of Tetrahymena patula LFF, aminoethylphosphonolipid was present in amounts nearly equal to PE. Tetrahymena patula LFF contained an unusually large percentage of sphingolipid (16.2% by [(3)H]acetate radiolabeling). Substantial differences were found in the fatty acid profiles of the polymorphic species, which included the degree of fatty acid unsaturation and relative weight percentages of odd-chain fatty acids. Tetrahymena vorax contained a low ratio of unsaturated C(18) fatty acids to saturated C(18) fatty acids as compared with all other species examined. The differentiating species generally contained a lesser percentage of monoenoic fatty acids and a lower ratio of unsaturated C(16) fatty acids to saturated C(16) fatty acids as compared with the two monomorphic species examined.