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Abstract Laser microsurgery has long been an important means of assessing the functions of specific cells and tissues. Most laser ablation systems use short, highly focused laser pulses to create plasma-mediated lesions with dimensions on the order of the wavelength of light. While the small size of the lesion enables ablation with high spatial resolution, it also makes it difficult to ablate larger structures. We developed an infrared laser ablation system capable of thermally lesioning tissues with spot sizes tunable by the duration and amplitude of laser pulses. We used our laser system in the roundworm C. elegans to kill single neurons and to sever the dorsal and ventral nerve cords, structures that are difficult to lesion using a plasma-based ablation system. We used these ablations to investigate the source of convulsions in a gain-of-function mutant for the acetylcholine receptor ACR-2. Severing the ventral nerve cord caused convulsions to occur independently anterior and posterior to the lesion, suggesting that convulsions can arise independently from distinct subsets of the motor circuit.

Neural circuits coordinate with muscles and sensory feedback to generate motor behaviors appropriate to an animal’s environment. In C. elegans, the mechanisms by which the motor circuit generates undulations and modulates them based on the environment are largely unclear. We quantitatively analyzed C. elegans locomotion during free movement and during transient optogenetic muscle inhibition. Undulatory movements were highly asymmetrical with respect to the duration of bending and unbending during each cycle. Phase response curves induced by brief optogenetic inhibition of head muscles showed gradual increases and rapid decreases as a function of phase at which the perturbation was applied. A relaxation oscillator model based on proprioceptive thresholds that switch the active muscle moment was developed and is shown to quantitatively agree with data from free movement, phase responses, and previous results for gait adaptation to mechanical loadings. Our results suggest a neuromuscular mechanism underlying C. elegans motor pattern generation within a compact circuit.

Many cell types display the remarkable ability to alter their cellular phenotype in response to specific external or internal signals. Such phenotypic plasticity is apparent in the nematode Caenorhabditis elegans when adverse environmental conditions trigger entry into the dauer diapause stage. This entry is accompanied by structural, molecular, and functional remodeling of a number of distinct tissue types of the animal, including its nervous system. The transcription factor (TF) effectors of 3 different hormonal signaling systems, the insulin-responsive DAF-16/FoxO TF, the TGFβ-responsive DAF-3/SMAD TF, and the steroid nuclear hormone receptor, DAF-12/VDR, a homolog of the vitamin D receptor (VDR), were previously shown to be required for entering the dauer arrest stage, but their cellular and temporal focus of action for the underlying cellular remodeling processes remained incompletely understood. Through the generation of conditional alleles that allowed us to spatially and temporally control gene activity, we show here that all 3 TFs are not only required to initiate tissue remodeling upon entry into the dauer stage, as shown before, but are also continuously required to maintain the remodeled state. We show that DAF-3/SMAD is required in sensory neurons to promote and then maintain animal-wide tissue remodeling events. In contrast, DAF-16/FoxO or DAF-12/VDR act cell-autonomously to control anatomical, molecular, and behavioral remodeling events in specific cell types. Intriguingly, we also uncover non-cell autonomous function of DAF-16/FoxO and DAF-12/VDR in nervous system remodeling, indicating the presence of several insulin-dependent interorgan signaling axes. Our findings provide novel perspectives into how hormonal systems control tissue remodeling.

Introduction. Expectations for limb length differences after TKA are important for patient perception and outcomes. Limb length discrepancies may occur due to postoperative leg length increases, which can lead to decreased patient functionality and satisfaction and even possible litigation. The purpose of this study is to examine the frequency and extent of limb lengthening among various preoperative deformities and between two different implant systems. Methods. Preoperative and postoperative full-length standing radiographs were obtained between August 2018 and August 2019 to measure mechanical axis and limb length of operative limbs. Demographic information such as age, sex, and BMI was also collected. Patients were grouped into categories for pre- and postoperative subgroup analysis: valgus, varus, customized implant, and conventional implant. Regression analysis was performed to evaluate significant relationships. Results. Of the 121 primary TKAs analyzed, 62% of the knees showed an increase in limb length after TKA, with an average lengthening of 5.32 mm. Preoperative varus alignment was associated with a mean lengthening of 3.14 mm, while preoperative valgus alignment was associated with a mean lengthening of 16.2 mm. Overall, there were no statistically significant differences in limb lengths pre- and postoperatively (p = 0.23) and no significant changes in limb length for any subgroup. Further, no variables were associated with limb length changes (p = 0.49), including the use of customized implants (p = 0.2). Conclusions. Limb lengthening after TKA is common and, on average, occurs more significantly in valgus knees. No significant difference in limb lengthening could be demonstrated using customized over conventional implants. Preoperative counseling is important to manage patient expectations.

Summary: People tend to fall asleep when gently rocked or vibrated. Experimental studies have shown that rocking promotes sleep in humans and mice. However, the mechanisms underlying the phenomenon are not well understood. A habituation model proposes that habituation, a form of non-associative learning, mediates sleep induction by monotonous stimulation. Here, we show that gentle vibration promotes sleep in Drosophila in part through habituation. Vibration-induced sleep (VIS) leads to increased homeostatic sleep credit and reduced arousability, and can be suppressed by heightened arousal or reduced GABA signaling. Multiple mechanosensory organs mediate VIS, and the magnitude of VIS depends on vibration frequency and genetic background. Sleep induction improves over successive blocks of vibration. Furthermore, training with continuous vibration does not generalize to intermittent vibration, demonstrating stimulus specificity, a characteristic of habituation. Our findings suggest that habituation plays a significant role in sleep induction by vibration.

Getting a grip on how we may age healthily is a central interest of biogerontological research. To this end, a number of academic teams developed platforms for life- and healthspan assessment in Caenorhabditis elegans. These are very appealing for medium- to high throughput screens, but a broader implementation is lacking due to many systems relying on custom scripts for data analysis that others struggle to adopt. Hence, user-friendly recommendations would help to translate raw data into interpretable results. The aim of this communication is to streamline the analysis of data obtained by the WorMotel, an economically and practically appealing screening platform, in order to facilitate the use of this system by interested researchers. We here detail recommendations for the stepwise conversion of raw image data into activity values and explain criteria for assessment of health in C. elegans based on locomotion. Our analysis protocol can easily be adopted by researchers, and all needed scripts and a tutorial are available in S1 and S2 Files.

The roundworm Caenorhabditis elegans is widely used as a model for studying conserved pathways for fat storage, aging, and metabolism. The most broadly used methods for imaging fat in C. elegans require fixing and staining the animal. Here, we show that dark field images acquired through an ordinary light microscope can be used to estimate fat levels in worms. We define a metric based on the amount of light scattered per area, and show that this light scattering metric is strongly correlated with worm fat levels as measured by Oil Red O (ORO) staining across a wide variety of genetic backgrounds and feeding conditions. Dark field imaging requires no exogenous agents or chemical fixation, making it compatible with live worm imaging. Using our method, we track fat storage with high temporal resolution in developing larvae, and show that fat storage in the intestine increases in at least one burst during development.

The demand for revision total joint arthroplasties (rTJAs) is expected to increase as the age of the population continues to rise. Accurate cost data regarding hospital expenses for differing age groups are needed to deliver optimal care within value-based healthcare (VBHC) models. The aim of this study was to compare the total in-hospital costs by decadal groups following rTJA and to determine the primary drivers of the costs for these procedures. Time-driven activity-based costing (TDABC) was used to capture granular hospital costs. A total of 551 rTJAs were included in the study, with 294 sexagenarians, 198 septuagenarians, and 59 octogenarians and older. Sexagenarians had a lower ASA classification (2.3 vs. 2.4 and 2.7; p < 0.0001) and were more often privately insured (66.7% vs. 24.2% and 33.9%; p < 0.0001) as compared to septuagenarians and octogenarians and older, respectively. Sexagenarians were discharged to home at a higher rate (85.3% vs. 68.3% and 34.3%; p < 0.0001), experienced a longer operating room (OR) time (199.8 min vs. 189.7 min and 172.3 min; p = 0.0195), and had a differing overall hospital length of stay (2.8 days vs. 2.7 days and 3.6 days; p = 0.0086) compared to septuagenarians and octogenarians and older, respectively. Sexagenarians had 7% and 23% less expensive personnel costs from post-anesthesia care unit (PACU) to discharge (p < 0.0001), and 1% and 24% more expensive implant costs (p = 0.077) compared to septuagenarians and octogenarians and older, respectively. Sexagenarians had a lower total in-hospital cost for rTJAs by 0.9% compared to septuagenarians but 12% more expensive total in-hospital costs compared to octogenarians and older (p = 0.185). Multivariate linear regression showed that the implant cost (0.88389; p < 0.0001), OR time (0.12140; p < 0.0001), personnel cost from PACU through to discharge (0.11472; p = 0.0007), and rTHAs (−0.03058; p < 0.0001) to be the strongest associations with overall costs. Focusing on the implant costs and OR times to reduce costs for all age groups for rTJAs is important to provide cost-effective VBHC.

The proportion of patients over the age of 90 years continues to grow, and the anticipated demand for total joint arthroplasty (TJA) in this population is expected to rise concomitantly. As the country shifts to alternative reimbursement models, data regarding hospital expenses is needed for accurate risk-adjusted stratification. The aim of this study was to compare total in-hospital costs following primary TJA in octogenarians and nonagenarians, and to determine the primary drivers of cost. This was a retrospective analysis from a single institution in the U.S. We used time-drive activity-based costing (TDABC) to capture granular total hospital costs for each patient. 889 TJA’s were included in the study, with 841 octogenarians and 48 nonagenarians. Nonagenarians were more likely to undergo total hip arthroplasty (THA) (70.8% vs. 42.4%; p < 0.0001), had higher ASA classification (2.6 vs. 2.4; p = 0.049), and were more often privately insured (35.4% vs. 27.8%; p = 0.0001) as compared to octogenarians. Nonagenarians were more often discharged to skilled nursing facilities (56.2% vs. 37.5%; p = 0.0011), experienced longer operating room (OR) time (142 vs. 133; p = 0.0201) and length of stay (3.7 vs. 3.1; p = 0.0003), and had higher implant and total in-hospital costs (p < 0.0001 and 0.0001). Multivariate linear regression showed implant cost (0.700; p < 0.0001), length of stay (0.546; p < 0.0001), and OR time (0.288; p < 0.0001) to be the strongest associations with overall costs. Primary TJA for nonagenarians was more expensive than octogenarians. Targeting implant costs, length of stay, and OR time can reduce costs for nonagenarians in order to provide cost-effective value-based care.

Patients who are discharged home following primary hip and knee arthroplasty have lower associated costs and better outcomes than patients who are discharged to skilled nursing facilities (SNFs). However, patients who live alone are more likely to be discharged to an SNF. We studied the factors that determine the discharge destination for patients who live alone after total joint arthroplasty (TJA) at an urban tertiary care academic hospital between April 2016 and April 2017. We identified 127 patients who lived alone: 79 (62.2%) were sent home, and 48 (37.8%) were sent to an SNF after surgery. Patients who went home versus to an SNF differed in age, employment status, exercise/active status, patient expectation of discharge to an SNF, ASA score, and the length of stay. After controlling for expectations of discharge to an SNF (OR: 28.98), patients who were younger (OR: 0.03) and employed (OR: 6.91) were more likely to be discharged home. In conclusion, the expectation of discharge location was the strongest predictor of discharge to an SNF even after controlling for age and employment. Future research should include a multi-hospital approach to strengthen the validity of our findings and investigate additional factors that impact discharge destination.

Sleep during development is involved in refining brain circuitry, but a role for sleep in the earliest periods of nervous system elaboration, when neurons are first being born, has not been explored. Here we identify a sleep state in Drosophila larvae that coincides with a major wave of neurogenesis. Mechanisms controlling larval sleep are partially distinct from adult sleep: octopamine, the Drosophila analog of mammalian norepinephrine, is the major arousal neuromodulator in larvae, but dopamine is not required. Using real-time behavioral monitoring in a closed-loop sleep deprivation system, we find that sleep loss in larvae impairs cell division of neural progenitors. This work establishes a system uniquely suited for studying sleep during nascent periods, and demonstrates that sleep in early life regulates neural stem cell proliferation.

Coordinated rhythmic movements are ubiquitous in animal behavior. In many organisms, chains of neural oscillators underlie the generation of these rhythms. In C. elegans, locomotor wave generation has been poorly understood; in particular, it is unclear where in the circuit rhythms are generated, and whether there exists more than one such generator. We used optogenetic and ablation experiments to probe the nature of rhythm generation in the locomotor circuit. We found that multiple sections of forward locomotor circuitry are capable of independently generating rhythms. By perturbing different components of the motor circuit, we localize the source of secondary rhythms to cholinergic motor neurons in the midbody. Using rhythmic optogenetic perturbation, we demonstrate bidirectional entrainment of oscillations between different body regions. These results show that, as in many other vertebrates and invertebrates, the C. elegans motor circuit contains multiple oscillators that coordinate activity to generate behavior.

Cell- or network-driven oscillators underlie motor rhythmicity. The identity of C. elegans oscillators remains unknown. Through cell ablation, electrophysiology, and calcium imaging, we show: (1) forward and backward locomotion is driven by different oscillators; (2) the cholinergic and excitatory A-class motor neurons exhibit intrinsic and oscillatory activity that is sufficient to drive backward locomotion in the absence of premotor interneurons; (3) the UNC-2 P/Q/N high-voltage-activated calcium current underlies A motor neuron’s oscillation; (4) descending premotor interneurons AVA, via an evolutionarily conserved, mixed gap junction and chemical synapse configuration, exert state-dependent inhibition and potentiation of A motor neuron’s intrinsic activity to regulate backward locomotion. Thus, motor neurons themselves derive rhythms, which are dually regulated by the descending interneurons to control the reversal motor state. These and previous findings exemplify compression: essential circuit properties are conserved but executed by fewer numbers and layers of neurons in a small locomotor network.

BackgroundCerebral venous thrombosis is a rare cause of stroke that poses diagnostic, therapeutic, and prognostic challenges. Mainstay treatment is systemic anticoagulation, but endovascular treatment is increasingly advocated. Our objectives were to describe the epidemiology, treatment, and prognosis of 152 patients with cerebral venous thrombosis. Methods and ResultsThis was a retrospective study of consecutive cerebral venous thrombosis cases from 2006 to 2013 at a comprehensive stroke center through hospital discharge. Predictors of full recovery (modified Rankin Scale scores 0–1) were analyzed with multiple logistic regression and presented as adjusted odds ratios (AORs) with 95% confidence intervals (CIs). The population was young (average age: 42 years), majority female (69%), and commonly presenting with cerebral edema (63%), and 72% were transferred in. All patients received systemic anticoagulation; 49% (n=73) required endovascular treatment. Reasons for requiring endovascular treatment included cerebral edema, herniation, or hemorrhagic infarct (n=38); neurologic decline (n=17); rethrombosis, persistent occlusion, or clot propagation (n=10); extensive clot burden (n=7); and persistent headache despite anticoagulation (n=1). There were 7 (10%) procedural complications. Recanalization was successful (61%), partial (30%), and unsuccessful (9%). Overall, 60% fully recovered. Positive predictors of full recovery included hormonal etiology, particularly for patients who were transferred in (AOR: 7.06 [95% CI, 2.27–21.96], interaction P=0.03) and who had migraine history (AOR: 4.87 [95% CI, 1.01–23.50], P=0.05), whereas negative predictors of full recovery were cerebral edema (AOR: 0.11 [95% CI, 0.04–0.34], P

Background: Proximal humeral fractures are common in the elderly and can present with multiple fracture configurations. Our case series describes a three-part valgus-impacted fracture pattern of the proximal humerus that underwent spontaneous reduction by nonsurgical management with good functional outcomes. Methods: Seven patients who sustained low-energy three-part valgus-impacted fractures of the proximal humerus were studied. We retrospectively reviewed serial anteroposterior radiographs of each patient taken as part of routine follow-up. For clinical outcomes, patients were assessed by a physical examination of both shoulders. A Disability of Arm, Shoulder, and Hand (DASH) questionnaire assessed the functional outcomes. Results: In all seven patients, fractures reduced spontaneously without any complications. Radiologically, the neck–shaft angle decreased from an average of 169° to 141 ° . In the six patients examined, range of motion was decreased in all planes in the affected shoulder relative to the contralateral shoulder. Two patients exhibited no loss of power in the affected arm, while the rest exhibited Medical Research Council grade 4 of 5 power in the affected arm. The mean DASH score obtained in our patients was 18.17. None of our patients reported any significant limitations in their activities of daily living and all achieved premorbid functional levels. Conclusion: Low-energy three-part valgus-impacted fracture pattern of the proximal humerus has the ability to spontaneously reduce by nonsurgical management with good functional outcomes.

The roundworm C. elegans is a mainstay of aging research due to its short lifespan and easily manipulable genetics. Current, widely used methods for long-term measurement of C. elegans are limited by low throughput and the difficulty of performing longitudinal monitoring of aging phenotypes. Here we describe the WorMotel, a microfabricated device for long-term cultivation and automated longitudinal imaging of large numbers of C. elegans confined to individual wells. Using the WorMotel, we find that short-lived and long-lived strains exhibit patterns of behavioral decline that do not temporally scale between individuals or populations, but rather resemble the shortest and longest lived individuals in a wild type population. We also find that behavioral trajectories of worms subject to oxidative stress resemble trajectories observed during aging. Our method is a powerful and scalable tool for analysis of C. elegans behavior and aging.

Patients with upper limb amputation and brachial plexus injuries have high rates of prosthesis rejection. Study purpose is to describe experiences of subjects with transhumeral amputation and brachial plexus injury, who were fit with, and trained to use, a DEKA Arm.This was a mixed-methods study utilizing qualitative (e.g. interview, survey) and quantitative data (e.g. self-report and performance measures). Subject 1, a current prosthesis user, had a shoulder arthrodesis. Subject 2, not a prosthesis user, had a subluxed shoulder. Both were trained in laboratory and participated in a trial of home use. Descriptive analyses of processes and outcomes were conducted.Subject 1 was fitted with the transhumeral configuration (HC) DEKA Arm using a compression release stabilized socket. He had 12 hours of prosthetic training and participated in all home study activities. Subject 1 had improved dexterity and prosthetic satisfaction with the DEKA Arm and reported better quality of life (QOL) at the end of participation. Subject 2 was fit with the shoulder configuration (SC) DEKA Arm using a modified X-frame socket. He had 30 hours of training and participated in 3 weeks of home activities. He reported less functional disability at the end of training as compared to baseline, but encountered personal problems and exacerbation of PTSD symptoms and withdrew from home use portion at 3 weeks. Both subjects reported functional benefits from use, and expressed a desire to receive a DEKA Arm in the future.This paper reported on two different strategies for prosthetic fitting and their outcomes. The advantages and limitations of each approach were discussed.Use of both the HC and SC DEKA Arm for patients with TH amputation and brachial plexus injury was reported. Lessons learned may be instructive to clinicians considering prosthetic choices for future cases.

In response to environments that cause cellular stress, animals engage in sleep behavior that facilitates recovery from the stress. In Caenorhabditis elegans, stress-induced sleep(SIS) is regulated by cytokine activation of the ALA neuron, which releases FLP-13 neuropeptides characterized by an amidated arginine-phenylalanine (RFamide) C-terminus motif. By performing an unbiased genetic screen for mutants that impair the somnogenic effects of FLP-13 neuropeptides, we identified the gene dmsr-1, which encodes a G-protein coupled receptor similar to an insect RFamide receptor. DMSR-1 is activated by FLP-13 peptides in cell culture, is required for SIS in vivo, is expressed non-synaptically in several wake-promoting neurons, and likely couples to a Gi/o heterotrimeric G-protein. Our data expand our understanding of how a single neuroendocrine cell coordinates an organism-wide behavioral response, and suggest that similar signaling principles may function in other organisms to regulate sleep during sickness.

Schistosomiasis, caused by parasitic flatworms of the genus Schistosoma, is a neglected tropical disease affecting hundreds of millions globally. Praziquantel (PZQ), the only drug currently available for treatment and control, is largely ineffective against juvenile worms, and reports of PZQ resistance lend added urgency to the need for development of new therapeutics. Ion channels, which underlie electrical excitability in cells, are validated targets for many current anthelmintics. Transient receptor potential (TRP) channels are a large family of non-selective cation channels. TRP channels play key roles in sensory transduction and other critical functions, yet the properties of these channels have remained essentially unexplored in parasitic helminths. TRP channels fall into several (7-8) subfamilies, including TRPA and TRPV. Though schistosomes contain genes predicted to encode representatives of most of the TRP channel subfamilies, they do not appear to have genes for any TRPV channels. Nonetheless, we find that the TRPV1-selective activators capsaicin and resiniferatoxin (RTX) induce dramatic hyperactivity in adult worms; capsaicin also increases motility in schistosomula. SB 366719, a highly-selective TRPV1 antagonist, blocks the capsaicin-induced hyperactivity in adults. Mammalian TRPA1 is not activated by capsaicin, yet knockdown of the single predicted TRPA1-like gene (SmTRPA) in S. mansoni effectively abolishes capsaicin-induced responses in adult worms, suggesting that SmTRPA is required for capsaicin sensitivity in these parasites. Based on these results, we hypothesize that some schistosome TRP channels have novel pharmacological sensitivities that can be targeted to disrupt normal parasite neuromuscular function. These results also have implications for understanding the phylogeny of metazoan TRP channels and may help identify novel targets for new or repurposed therapeutics.

One advantage of the nematode Caenorhabditis elegans as a model organism is its suitability for in vivo optical microscopy. Imaging C. elegans often requires animals to be immobilized to avoid movement-related artifacts. Immobilization has been performed by application of anesthetics or by introducing physical constraints using glue or specialized microfluidic devices. Here we present a method for immobilizing C. elegans using polystyrene nanoparticles and agarose pads. Our technique is technically simple, does not expose the worm to toxic substances, and allows recovery of animals. We evaluate the method and show that the polystyrene beads increase friction between the worm and agarose pad. We use our method to quantify calcium transients and long-term regrowth in single neurons following axotomy by a femtosecond laser.