Identifying Improvements in Treating Extremity Musculoskeletal Injuries During Prolonged Care.

INTRODUCTION: In prolonged care scenarios, where medical evacuations are significantly delayed, the treatment and transport of casualties with extremity musculoskeletal injuries will drain combat units' human resources. Developing enhanced splinting techniques to restore casualty mobility and function can alleviate this drain. To guide this development, a panel of tactical combat and wilderness medicine experts was assembled to determine which extremity musculoskeletal injuries had the greatest impact on unit capabilities, and the materials available for splinting these injuries. INFORMATION GATHERING: Unstructured consultations with panel members yielded preliminary lists of injuries and materials. These lists were consolidated and redistributed to panel members for final evaluation where they ranked the injuries based on frequency and human resource cost and assessed the accessibility of materials. Responses for the final evaluation were statistically analyzed using Wilcoxon rank-sum tests and Placket Luce models. LESSONS LEARNED: Aggregated responses indicated that panel members thought that knee and ankle ligamentous injuries and radial head fractures were the most frequently occurring injuries, although closed distal femoral fractures, below knee amputations, and open tibia fractures would require the most demand for injury care. Assessing the combined impact of frequency and human resource cost indicated that knee and ankle ligamentous injuries and closed tibia fractures had the greatest impact on unit readiness. Responses also indicated that a variety of materials would be available for applying or improvising splints. CONCLUSION: Although the combined impact of knee and ankle ligamentous injuries were ranked the highest, limitations in relative rankings and the existence of effective low-cost treatments for these injuries suggest that greater gains in unit effectiveness would come from focusing on developing solutions for fractures with higher human resource cost, such as leg and arm fractures. This information can be used to develop enhanced splints that can preserve unit readiness in the field.

Biomolecular condensates as stress sensors and modulators of bacterial signaling.

Microbes exhibit remarkable adaptability to environmental fluctuations. Signaling mechanisms, such as two-component systems and secondary messengers, have long been recognized as critical for sensing and responding to environmental cues. However, recent research has illuminated the potential of a physical adaptation mechanism in signaling-phase separation, which may represent a ubiquitous mechanism for compartmentalizing biochemistry within the cytoplasm in the context of bacteria that frequently lack membrane-bound organelles. This review considers the broader prospect that phase separation may play critical roles as rapid stress sensing and response mechanisms within pathogens. It is well established that weak multivalent interactions between disordered regions, coiled-coils, and other structured domains can form condensates via phase separation and be regulated by specific environmental parameters in some cases. The process of phase separation itself acts as a responsive sensor, influenced by changes in protein concentration, posttranslational modifications, temperature, salts, pH, and oxidative stresses. This environmentally triggered phase separation can, in turn, regulate the functions of recruited biomolecules, providing a rapid response to stressful conditions. As examples, we describe biochemical pathways organized by condensates that are essential for cell physiology and exhibit signaling features. These include proteins that organize and modify the chromosome (Dps, Hu, SSB), regulate the decay, and modification of RNA (RNase E, Hfq, Rho, RNA polymerase), those involved in signal transduction (PopZ, PodJ, and SpmX) and stress response (aggresomes and polyphosphate granules). We also summarize the potential of proteins within pathogens to function as condensates and the potential and challenges in targeting biomolecular condensates for next-generation antimicrobial therapeutics. Together, this review illuminates the emerging significance of biomolecular condensates in microbial signaling, stress responses, and regulation of cell physiology and provides a framework for microbiologists to consider the function of biomolecular condensates in microbial adaptation and response to diverse environmental conditions.

Testing the Reliability of Optical Coherence Tomography to Measure Epidermal Thickness and Distinguish Volar and Nonvolar Skin.

In persons with limb loss, prosthetic devices cause skin breakdown, largely because residual limb skin (nonvolar) is not intended to bear weight such as palmoplantar (volar) skin. Before evaluation of treatment efficacy to improve skin resiliency, efforts are needed to establish normative data and assess outcome metric reliability. The purpose of this study was to use optical coherence tomography to (i) characterize volar and nonvolar skin epidermal thickness and (ii) examine the reliability of optical coherence tomography. Four orientations of optical coherence tomography images were collected on 33 volunteers (6 with limb loss) at 2 time points, and the epidermis was traced to quantify thickness by 3 evaluators. Epidermal thickness was greater (P < .01) for volar skin (palm) (265.1 ± 50.9 µm, n = 33) than for both nonvolar locations: posterior thigh (89.8 ± 18.1 µm, n = 27) or residual limb (93.4 ± 27.4 µm, n = 6). The inter-rater intraclass correlation coefficient was high for volar skin (0.887-0.956) but low for nonvolar skin (thigh: 0.292-0.391, residual limb: 0.211-0.580). Correlation improved when comparing only 2 evaluators who used the same display technique (palm: 0.827-0.940, thigh: 0.633-0.877, residual limb: 0.213-0.952). Despite poor inter-rater agreement for nonvolar skin, perhaps due to challenges in identifying the dermal-epidermal junction, this study helps to support the utility of optical coherence tomography to distinguish volar from nonvolar skin.

Uncovering supramolecular chirality codes for the design of tunable biomaterials.

In neurodegenerative diseases, polymorphism and supramolecular assembly of ß-sheet amyloids are implicated in many different etiologies and may adopt either a left- or right-handed supramolecular chirality. Yet, the underlying principles of how sequence regulates supramolecular chirality remains unknown. Here, we characterize the sequence specificity of the central core of amyloid-ß 42 and design derivatives which enable chirality inversion at biologically relevant temperatures. We further find that C-terminal modifications can tune the energy barrier of a left-to-right chiral inversion. Leveraging this design principle, we demonstrate how temperature-triggered chiral inversion of peptides hosting therapeutic payloads modulates the dosed release of an anticancer drug. These results suggest a generalizable approach for fine-tuning supramolecular chirality that can be applied in developing treatments to regulate amyloid morphology in neurodegeneration as well as in other disease states.

The development of rating scales to evaluate experiential prosthetic foot preference for people with lower limb amputation.

BACKGROUND: Selection of a foot is an important aspect of prosthetic prescription and vital to maximizing mobility and functional goals after lower limb amputation. Development of a standardized approach to soliciting user experiential preferences is needed to improve evaluation and comparison of prosthetic feet. OBJECTIVE: To develop rating scales to assess prosthetic foot preference and to evaluate use of these scales in people with transtibial amputation after trialing different prosthetic feet. DESIGN: Participant-blinded, repeated measures crossover trial. SETTING: Veterans Affairs and Department of Defense Medical Centers, laboratory setting. PARTICIPANTS: Seventy-two male prosthesis users with unilateral transtibial amputation started, and 68 participants completed this study. INTERVENTIONS: Participants trialed three mobility-level appropriate commercial prosthetic feet briefly in the laboratory. MAIN OUTCOME MEASURES: "Activity-specific" rating scales were developed to assess participants' ability with a given prosthetic foot to perform typical mobility activities (eg, walking at different speeds, on inclines, and stairs) and "global" scales to rate overall perceived energy required to walk, satisfaction, and willingness to regularly use the prosthetic foot. Foot preference was determined by comparing the rating scale scores, after laboratory testing. RESULTS: The greatest within-participant differences in scores among feet were observed in the "incline" activity, where 57% ± 6% of participants reported 2+ point differences. There was a significant association (p < .05) between all "activity-specific" rating scores (except standing) and each "global" rating score. CONCLUSIONS: The standardized rating scales developed in this study could be used to assess prosthetic foot preference in both the research and clinical settings to guide prosthetic foot prescription for people with lower limb amputation capable of a range of mobility levels.

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions.

Bacterial ribonucleoprotein bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here, we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We find 111 BR-body-enriched proteins showing that BR-bodies are more complex than previously assumed. We identify five BR-body-enriched proteins that undergo RNA-dependent phase separation in vitro with a complex network of condensate mixing. We observe that some RNP condensates co-assemble with preferred directionality, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body-associated proteins have the capacity to dissolve the condensate. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing.

RNase E biomolecular condensates stimulate PNPase activity.

Bacterial Ribonucleoprotein bodies (BR-bodies) play an essential role in organizing RNA degradation via phase separation in the cytoplasm of bacteria. BR-bodies mediate multi-step mRNA decay through the concerted activity of the endoribonuclease RNase E coupled with the 3'-5' exoribonuclease Polynucleotide Phosphorylase (PNPase). In vivo, studies indicated that the loss of PNPase recruitment into BR-bodies led to a significant build-up of RNA decay intermediates in Caulobacter crescentus. However, it remained unclear whether this is due to a lack of colocalized PNPase and RNase E within BR-bodies or whether PNPase's activity is stimulated within the BR-body. We reconstituted RNase E's C-terminal domain with PNPase towards a minimal BR-body in vitro to distinguish these possibilities. We found that PNPase's catalytic activity is accelerated when colocalized within the RNase E biomolecular condensates, partly due to scaffolding and mass action effects. In contrast, disruption of the RNase E-PNPase protein-protein interaction led to a loss of PNPase recruitment into the RNase E condensates and a loss of ribonuclease rate enhancement. We also found that RNase E's unique biomolecular condensate environment tuned PNPase's substrate specificity for poly(A) over poly(U). Intriguingly, a critical PNPase reactant, phosphate, reduces RNase E phase separation both in vitro and in vivo. This regulatory feedback ensures that under limited phosphate resources, PNPase activity is enhanced by recruitment into RNase E's biomolecular condensates.

Scaffold-Scaffold Interaction Facilitates Cell Polarity Development in Caulobacter crescentus.

Cell polarity development is the prerequisite for cell differentiation and generating biodiversity. In the model bacterium Caulobacter crescentus, the polarization of the scaffold protein PopZ during the predivisional cell stage plays a central role in asymmetric cell division. However, our understanding of the spatiotemporal regulation of PopZ localization remains incomplete. In the current study, a direct interaction between PopZ and the new pole scaffold PodJ is revealed, which plays a primary role in triggering the new pole accumulation of PopZ. The coiled-coil 4-6 domain in PodJ is responsible for interacting with PopZ in vitro and promoting PopZ transition from monopolar to bipolar in vivo. Elimination of the PodJ-PopZ interaction impairs the PopZ-mediated chromosome segregation by affecting both the positioning and partitioning of the ParB-parS centromere. Further analyses of PodJ and PopZ from other bacterial species indicate this scaffold-scaffold interaction may represent a widespread strategy for spatiotemporal regulation of cell polarity in bacteria. IMPORTANCE Caulobacter crescentus is a well-established bacterial model to study asymmetric cell division for decades. During cell development, the polarization of scaffold protein PopZ from monopolar to bipolar plays a central role in C. crescentus asymmetric cell division. Nevertheless, the spatiotemporal regulation of PopZ has remained unclear. Here, we demonstrate that the new pole scaffold PodJ functions as a regulator in triggering PopZ bipolarization. The primary regulatory role of PodJ was demonstrated in parallel by comparing it with other known PopZ regulators, such as ZitP and TipN. Physical interaction between PopZ and PodJ ensures the timely accumulation of PopZ at the new cell pole and the inheritance of the polarity axis. Disruption of the PodJ-PopZ interaction impaired PopZ-mediated chromosome segregation and may lead to a decoupling of DNA replication from cell division during the cell cycle. Together, the scaffold-scaffold interaction may provide an underlying infrastructure for cell polarity development and asymmetric cell division.

Relationship between phantom limb pain, function, and psychosocial health in individuals with lower-limb loss.

INTRODUCTION: The adverse influence of chronic pain on function and psychological health in the general population is well understood. However, the relationship between phantom limb pain (PLP) after limb loss with function and psychological health is less clear. The study purpose was to assess the influences of PLP presence and intensity on function and psychosocial health in individuals with lower-limb loss (LLL). METHODS: One hundred two individuals with major LLL completed a study-specific questionnaire on the presence and intensity of their PLP. The Patient-Reported Outcomes Measurement Information System -29 questionnaire was also administered. RESULTS: Of 102 participants, 64% reported PLP, with a mean intensity of 4.8 ± 2.3 out of 10. Individuals with vs. without PLP demonstrated significantly greater sleep disturbances ( p = 0.03), whereas the differences in function, fatigue, pain interference, depressive symptoms, anxiety, or ability to participate in social roles and activities were not statistically different between groups ( p > 0.05). Of note, mean scores for many of the Patient-Reported Outcomes Measurement Information System-29 short forms among the current sample were similar to the mean of the general population, minimizing the potential clinical impact of PLP on these domains. CONCLUSIONS: Our findings indicate a lack of meaningful associations between PLP presence or intensity with function, and psychosocial health among individuals with LLL. These findings conflict with previous research suggesting an adverse relationship between PLP, function, and psychosocial health after limb loss.

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions.

Bacterial RNP bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We found 111 BR-body enriched proteins, including several RNA binding proteins, many of which are also recruited directly to in vitro reconstituted RNase E droplets, showing BR-bodies are more complex than previously assumed. While most BR-body enriched proteins that were tested cannot phase separate, we identified five that undergo RNA-dependent phase separation in vitro, showing other RNP condensates interface with BR-bodies. RNA degradosome protein clients are recruited more strongly to RNase E droplets than droplets of other RNP condensates, implying that client specificity is largely achieved through direct protein-protein interactions. We observe that some RNP condensates assemble with preferred directionally, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body associated proteins have the capacity to dissolve the condensate. Finally, we find that RNA dramatically stimulates the rate of RNase E phase separation in vitro, explaining the dissolution of BR-bodies after cellular mRNA depletion observed previously. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing.

The relationship between residual limb health, motion within the socket, and prosthetic suspension.

Residual limb health is critical for continued prosthesis use; however, many prosthesis users experience skin-related breakdown. The interface between the residual limb and the prosthetic socket sets the local mechanical environment and plays a role in skin stresses. Motion of the residual limb in the socket adds additional mechanical strain on the limb. This article explores the relationship between motion of the limb in the socket and residual limb health. We evaluated current methods for assessing residual limb health and motion of the residual limb in the socket and compared these evaluations across different prosthetic suspension systems. While few direct studies comparing residual limb health and motion exist, it has been shown that elevated vacuum suspension systems result in both improved residual limb health compared to passive suction and pin-lock systems and decreased motion compared to passive suction, pin-lock, knee sleeve, and anatomical suspension systems. While motion and health have not been directly linked, elevated vacuum suspension may demonstrate a relationship that reduced motion of the residual limb in the socket improves residual limb health. Further evaluation in this area is necessary to more completely and directly understand the relationship between residual limb motion and residual limb health.

Exoskeletal solutions to enable mobility with a lower leg fracture in austere environments.

The treatment and evacuation of people with lower limb fractures in austere environments presents unique challenges that assistive exoskeletal devices could address. In these dangerous situations, independent mobility for the injured can preserve their vital capabilities so that they can safely evacuate and minimize the need for additional personnel to help. This expert view article discusses how different exoskeleton archetypes could provide independent mobility while satisfying the requisite needs for portability, maintainability, durability, and adaptability to be available and useful within austere environments. The authors also discuss areas of development that would enable exoskeletons to operate more effectively in these scenarios as well as preserve the health of the injured limb so that definitive treatment after evacuation will produce better outcomes.

Phase separation modulates the assembly and dynamics of a polarity-related scaffold-signaling hub.

Asymmetric cell division (ACD) produces morphologically and behaviorally distinct cells and is the primary way to generate cell diversity. In the model bacterium Caulobacter crescentus, the polarization of distinct scaffold-signaling hubs at the swarmer and stalked cell poles constitutes the basis of ACD. However, mechanisms involved in the formation of these hubs remain elusive. Here, we show that a swarmer-cell-pole scaffold, PodJ, forms biomolecular condensates both in vitro and in living cells via phase separation. The coiled-coil 4-6 and the intrinsically disordered regions are the primary domains that contribute to biomolecular condensate generation and signaling protein recruitment in PodJ. Moreover, a negative regulation of PodJ phase separation by the stalked-cell-pole scaffold protein SpmX is revealed. SpmX impedes PodJ cell-pole accumulation and affects its recruitment ability. Together, by modulating the assembly and dynamics of scaffold-signaling hubs, phase separation may serve as a general biophysical mechanism that underlies the regulation of ACD in bacteria and other organisms.

Repurposing Peptide Nanomaterials as Synthetic Biomolecular Condensates in Bacteria.

Peptide nanomaterials exhibit diverse applications in vitro, such as drug delivery. Here, we consider the utility of de novo peptide nanomaterials to organize biochemistry within the bacterial cytoplasm. Toward this goal, we discovered that ABC coiled-coil triblock peptides form gel-like biomolecular condensates with a csat of 10 µM in addition to their well-known hydrogel-forming capabilities. Expression of the coiled-coil triblock peptides in bacteria leads to cell pole accumulation via a nucleoid occlusion mechanism. We then provide a proof of principle that these synthetic biomolecular condensates could sequester clients at the cell pole. Finally, we demonstrate that triblock peptides and another biomolecular condensate, RNase E, phase-separate as distinct protein-rich assemblies in vitro and in vivo. These results reveal the potential of using peptide nanomaterials to divide the bacterial cytoplasm into distinct subcellular zones with future metabolic engineering and synthetic biology applications.

Protein engineering strategies to stimulate the functions of bacterial pseudokinases.

Enzymes orchestrate an array of concerted functions that often culminate in the chemical conversion of substrates into products. In the bacterial kingdom, histidine kinases autophosphorylate, then transfer that phosphate to a second protein called a response regulator. Bacterial genomes can encode large numbers of histidine kinases that provide surveillance of environmental and cytosolic stresses through signal stimulation of histidine kinase activity. Pseudokinases lack these hallmark catalytic functions but often retain binding interactions and allostery. Characterization of bacterial pseudokinases then takes a fundamentally different approach than their enzymatic counterparts. Here we discuss models for how bacterial pseudokinases can utilize protein-protein interactions and allostery to serve as crucial signaling pathway regulators. Then we describe a protein engineering strategy to interrogate these models, emphasizing how signals flow within bacterial pseudokinases. This description includes design considerations, cloning strategies, and the purification of leucine zippers fused to pseudokinases. We then describe two assays to interrogate this approach. First is a C. crescentus swarm plate assay to track motility phenotypes related to a bacterial pseudokinase. Second is an in vitro coupled-enzyme assay that can be applied to test if and how a pseudokinase regulates an active kinase. Together these approaches provide a blueprint for dissecting the mechanisms of cryptic bacterial pseudokinases.

Preoperative anemia is a risk factor for poor perioperative outcomes in ventral hernia repair.

PURPOSE: Ventral hernia repairs (VHR) are among the most commonly performed operations by general surgeons. Despite advances in technology there remains high complication and readmission rates. Preoperative anemia has been linked to poor outcomes and readmission across several surgical procedures, however the link to ventral hernia repair outcomes is limited. METHODS: Utilizing the American College of Surgeons National Safety and Quality Improvement Project (NSQIP) database for years 2016-2018, a total of 115,000 patients met inclusion criteria. Using propensity matching we matched two groups of patients who underwent VHR: (1) those with preoperative anemia and (2) those with normal hemoglobin levels. Anemia criteria was set forth by the World Health Organization (WHO). RESULTS: Univariate analysis did demonstrate statistical significance in post-operative outcomes percentage of serious surgical site infection, poor renal outcomes, transfusion, and unplanned remission in those with preoperative anemia who underwent VHR. In a multivariate analysis, patients who underwent ventral hernia repair with pre-operative anemia had significantly greater odds of unplanned readmission (odds ratio 1.35, 95% confidence interval 1.16-1.57) and serious surgical site infection (odds ratio 1.35, 95% confidence interval 1.04-1.74) independent of known risk factors such as smoking, diabetes and obesity. CONCLUSIONS: Preoperative anemia is a risk factor for poor postoperative outcomes in those undergoing ventral hernia repair and should be considered when evaluating a patient for repair.

Regulation of the activity of the bacterial histidine kinase PleC by the scaffolding protein PodJ.

Scaffolding proteins can customize the response of signaling networks to support cell development and behaviors. PleC is a bifunctional histidine kinase whose signaling activity coordinates asymmetric cell division to yield a motile swarmer cell and a stalked cell in the gram-negative bacterium Caulobacter crescentus. Past studies have shown that PleC's switch in activity from kinase to phosphatase correlates with a change in its subcellular localization pattern from diffuse to localized at the new cell pole. Here we investigated how the bacterial scaffolding protein PodJ regulates the subcellular positioning and activity of PleC. We reconstituted the PleC-PodJ signaling complex through both heterologous expressions in Escherichia coli and in vitro studies. In vitro, PodJ phase separates as a biomolecular condensate that recruits PleC and inhibits its kinase activity. We also constructed an in vivo PleC-CcaS chimeric histidine kinase reporter assay and demonstrated using this method that PodJ leverages its intrinsically disordered region to bind to PleC's PAS sensory domain and regulate PleC-CcaS signaling. Regulation of the PleC-CcaS was most robust when PodJ was concentrated at the cell poles and was dependent on the allosteric coupling between PleC-CcaS's PAS sensory domain and its downstream histidine kinase domain. In conclusion, our in vitro biochemical studies suggest that PodJ phase separation may be coupled to changes in PleC enzymatic function. We propose that this coupling of phase separation and allosteric regulation may be a generalizable phenomenon among enzymes associated with biomolecular condensates.

Altering the tuning parameter settings of a commercial powered prosthetic foot to increase power during push-off may not reduce collisional work in the intact limb during gait.

BACKGROUND: Increased knee osteoarthritis risk in patients with unilateral lower extremity limb loss is attributed to increased intact limb loading. Modulating powered ankle prosthesis push-off power may be an effective way to modulate intact limb loading. We examined how changes in the parameter settings of a commercial prosthetic ankle affect power delivery during push-off and the resulting collisional work experienced by the intact limb in persons with unilateral lower extremity limb loss. METHODS: Five subjects with unilateral transtibial amputation were fitted with a commercially available powered ankle prosthesis (Ottobock Empower). Subjects walked on a treadmill in seven conditions, where ankle power delivery settings were adjusted using methods accessible to clinicians. Kinetics and kinematics data were collected. RESULTS: Standard adjustment of parameter settings within the prosthetic foot did not alter timing of peak prosthesis power or intact limb collisional work but did have a significant effect on the magnitude of positive prosthesis ankle work. Increased prosthesis work did not decrease intact limb collisional work as predicted. CONCLUSIONS: Altering the parameter settings on a commercial powered ankle prosthesis affected the magnitude, but not the timing, of power delivered. Increased prosthesis push-off power did not decrease intact limb loading.

A Biosensor for Detection of Indole Metabolites.

Microbially produced indole metabolites serve as a diverse family of interspecies and interkingdom signaling molecules in the context of human health, crop production, and antibiotic resistance. We mined the protein database for sensors of indole metabolites and developed a biosensor for indole-3-aldehyde (I3A). Microbially produced I3A has been associated with reducing inflammation in diseases such as ulcerative colitis by stimulating the aryl hydrocarbon receptor pathway. We engineered an E. coli strain embedded with a single plasmid carrying a chimeric two-component system that detects I3A. Our I3A receptor characterization confirmed binding site residues that contribute to the sensor's I3A detection range of 0.1-10 µM. This new I3A biosensor opens the door to sensing indole metabolites produced at various host-microbe interfaces and provides new parts for synthetic biology applications.