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BACKGROUND: Plantar forefoot ulcers in individuals with diabetes often lead to deep infection and lower extremity amputation. Increasing evidence suggests that the process is initiated by increased passive stiffness within the gastrocnemius-soleus musculotendinous unit. The goal of this investigation was to perform ultrasound examination of the Achilles tendon in a consecutive group of asymptomatic diabetic individuals to identify any inherent structural pathology that might be associated with the increased stiffness that appears to be associated with the development of diabetic forefoot ulcers. MATERIALS AND METHODS: Seventy consecutive diabetic individuals with no past history of diabetic foot morbidity underwent ultrasonography of their Achilles tendons. Each patient was also tested for fasting glucose and glycosylated hemoglobin as a measure of diabetes control. Ten similarly aged non-diabetics with no history of Achilles tendinopathy served as controls. RESULTS: The ultrasonography revealed disorganized tendon fibers in 62 of 70 (89%) patients, and calcification within the Achilles tendon in 53 (76%) patients. The Achilles tendon thickness averaged 5.0 (range, 4 to 8) mm. There was no correlation between patient age and Achilles tendon thickness (r = 0.292, p = 0.014); however, there was a trend for duration of disease and Achilles tendon disorganization (p = 0.073). The oldest patients also appeared to demonstrate a trend for more ultrasound-measured Achilles tendinopathy. There was no correlation between fasting glucose or glycosylated hemoglobin and Achilles tendon alterations. CONCLUSION: This investigation confirms structural abnormalities within the Achilles tendon of diabetic individuals that might represent biologic changes affecting the inherent stiffness that leads to increased forefoot pressure and the development of plantar forefoot ulcers. This process appears to worsen with advanced age and does not appear to be related to diabetes control. [ABSTRACT FROM AUTHOR]

Infection is one of the main causes of orthopedic implants failure, with antibiotic-resistant bacteria playing a crucial role in this outcome. In this work, antimicrobial nanogels were developed to be applied in situ as implant coating to prevent orthopedic-device-related infections. To that regard, a broad-spectrum antimicrobial peptide, Dhvar5, was grafted onto chitosan via thiol-norbornene "photoclick" chemistry. Dhvar5-chitosan nanogels (Dhvar5-NG) were then produced using a microfluidic system. Dhvar5-NG (10 10 nanogels (NG)/mL) with a Dhvar5 concentration of 6 μg/mL reduced the burden of the most critical bacteria in orthopedic infections - methicillin-resistant Staphylococcus aureus (MRSA) - after 24 h in medium supplemented with human plasma proteins. Transmission electron microscopy showed that Dhvar5-NG killed bacteria by membrane disruption and cytoplasm release. No signs of cytotoxicity against a pre-osteoblast cell line were verified upon incubation with Dhvar5-NG. To further explore therapeutic alternatives, the potential synergistic effect of Dhvar5-NG with antibiotics was evaluated against MRSA. Dhvar5-NG at a sub-minimal inhibitory concentration (10 9 NG/mL) demonstrated synergistic effect with oxacillin (4-fold reduction: from 2 to 0.5 μg/mL) and piperacillin (2-fold reduction: from 2 to 1 μg/mL). This work supports the use of Dhvar5-NG as adjuvant of antibiotics to the prevention of orthopedic devices-related infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Tumefactive multiple sclerosis (TMS) is a rare variant of multiple sclerosis that presents with a large demyelinating lesion in the central nervous system, accompanied by peripheral ring-like enhancement, perilesional oedema and mass effect. We report a case of a 59-year-old woman who was admitted to the hospital with a four-day history of somnolence, muscle weakness in her left extremities and ultimately, loss of consciousness. Over the following 48 hours, the patient's condition worsened with progressive consciousness impairment. Although the results of the initial head computed tomography (CT) scan supported the diagnosis of a multifocal ischaemic stroke, toxoplasmosis was proposed as the most credible diagnostic hypothesis by brain magnetic resonance imaging (MRI). Due to the adverse clinical progression following the initiation of targeted therapy and inconclusive investigation, a brain biopsy was performed, which was indicative of active TMS in a subacute phase. The patient was started on plasmapheresis and natalizumab along with corticosteroids, with a very good response. In conclusion, we report a biopsy-proven TMS diagnosis in a patient that clinically mimicked an acute stroke and was radiographically confounded with intracranial toxoplasmosis. It highlights that TMS is an uncommon neurological demyelinating disease that is often misdiagnosed. It also emphasises the importance of establishing an accurate differential diagnosis to promptly initiate aggressive immunosuppressive treatment, which may result in a more favourable prognosis., Learning Points: Tumefactive multiple sclerosis is an uncommon variant of multiple sclerosis that presents a substantial diagnostic challenge due to its potential to resemble the clinical and radiological characteristics of other central nervous system (CNS) pathologies, including neoplasms, granulomatous diseases, abscesses and vasculitis.Despite the fact that multimodal imaging studies may help narrow the differential diagnosis, a biopsy is often required to reach a definitive diagnosis and should not be delayed.Awareness of this condition among non-neurologists is critical since a timely and accurate diagnosis prompts aggressive immunomodulatory treatments that may delay a second demyelinating event or progression to clinically definite multiple sclerosis., Competing Interests: Conflicts of Interests: The Authors declare that there are no competing interests., (© EFIM 2024.)

The sugarcane industry generates byproducts that contribute to the proliferation of Stomoxys calcitrans . An analysis was carried out to verify the efficacy of Heterorhabditis bacteriophora HP88 and H. baujardi LPP7 at different vinasse temperatures to control S. calcitrans larvae. Ten fly larvae were deposited in plastic containers containing four mL of 50% vinasse. Each treatment consisted of 300 EPN/larvae of H. bacteriophora added to the containers and heated at temperatures of 25, 28, 31, 34, 37 and 40 °C. The same treatments were performed using H. baujardi . The treatments were carried out in a BOD incubator at 25 ± 1 °C and 70 ± 10% RH, and each treatment was replicated six times. The treated groups, controls and temperatures showed no statistical differences in terms of larval mortality rate (P=0.8573), percentage of dead pupae (P=0.1782) and adult emergence (P=0.4386). Larval mortality rates of 30% and 14.17% were achieved with H. bacteriophora and H. baujardi , respectively, while the control groups presented 3.89% with H. bacteriophora and 8.61% with H. baujardi . From the standpoint of temperatures, significant differences were found only at 37 and 40 °C for H. baujardi . The highest pupal mortality achieved with H. bacteriophora was 34.17% at 31 °C, while that reached with H. baujardi at 37 °C was 40%. The groups containing H. bacteriophora caused lower adult emergence rates at temperatures of 25, 28, 31 and 34 °C, while H. baujardi caused its lowest emergence rates at 37 and 40 °C. It is concluded that infection occurs in the immature stages of S. calcitrans by EPN when added to 50% vinasse solution at different temperatures and that nematodes caused negative effects on the emergence of fly larvae at varying temperatures., Competing Interests: Conflict of interests: ACMS, GCS, ACFS, DPS, JLLMN, MCMCC and AJB – No conflict of interest.

Breast cancer is the most prevalent cancer among women worldwide. Therapeutic strategies to control tumors and metastasis are still challenging. Three-dimensional (3D) spheroid-type systems more accurately replicate the features of tumors in vivo, working as a better platform for performing therapeutic response analysis. This work aimed to characterize the epithelial-mesenchymal transition and doxorubicin (dox) response in a mammary tumor spheroid (MTS) model. We evaluated the doxorubicin treatment effect on MCF-7 spheroid diameter, cell viability, death, migration and proteins involved in the epithelial-mesenchymal transition (EMT) process. Spheroids were also produced from tumors formed from 4T1 and 67NR cell lines. MTSs mimicked avascular tumor characteristics, exhibited adherens junction proteins and independently produced their own extracellular matrix. Our spheroid model supports the 3D culturing of cells isolated from mice mammary tumors. Through the migration assay, we verified a reduction in E-cadherin expression and an increase in vimentin expression as the cells became more distant from spheroids. Dox promoted cytotoxicity in MTSs and inhibited cell migration and the EMT process. These results suggest, for the first time, that this model reproduces aspects of the EMT process and describes the potential of dox in inhibiting the metastatic process, which can be further explored.

Acute myeloid leukaemia (AML) management remains a significant challenge in oncology due to its low survival rates and high post-treatment relapse rates, mainly attributed to treatment-resistant leukaemic stem cells (LSCs) residing in bone marrow (BM) niches. This review offers an in-depth analysis of AML progression, highlighting the pivotal role of extracellular vesicles (EVs) in the dynamic remodelling of BM niche intercellular communication. We explore recent advancements elucidating the mechanisms through which EVs facilitate complex crosstalk, effectively promoting AML hallmarks and drug resistance. Adopting a temporal view, we chart the evolving landscape of EV-mediated interactions within the AML niche, underscoring the transformative potential of these insights for therapeutic intervention. Furthermore, the review discusses the emerging understanding of endothelial cell subsets' impact across BM niches in shaping AML disease progression, adding another layer of complexity to the disease progression and treatment resistance. We highlight the potential of cutting-edge methodologies, such as organ-on-chip (OoC) and single-EV analysis technologies, to provide unprecedented insights into AML-niche interactions in a human setting. Leveraging accumulated insights into AML EV signalling to reconfigure BM niches and pioneer novel approaches to decipher the EV signalling networks that fuel AML within the human context could revolutionise the development of niche-targeted therapy for leukaemia eradication.

This study aimed to evaluate the virulence of Heterorhabditis amazonenses NEPT11 against larvae of Stomoxys calcitrans . Groups of 10 third-instar fly larvae were deposited in Petri dishes, to which were added 50, 100 and 200 EPNs/larva in 4ml of distilled water. The volume of the control group was the same as the treated group, but without EPNs. Larval mortality was observed daily, until larvae died or adults emerged. The Petri dishes were kept on laboratory shelves at 27 ± 1 °C and 70 ± 10% RH. The experiment was replicated six times. A regression analysis revealed quadratic behavior with increasing concentrations, indicating that the concentration of 200 EPNs/larva (48%) was the most efficient among the tested concentrations, while concentrations of 50 and 100 EPNs/larva killed 26.6 and 40% of larvae, respectively. In general, none of the treatments resulted in a mortality rate of more than 50%, but all the treated groups exhibited a higher mortality than that of the control group. It is concluded that the EPN H. amazonensis NEPT11 shows a promising potential to control third-instar larvae of S. calcitrans . However, further studies are needed in different situations to better understand the activity of this organism against the immature stages of the stable fly., Competing Interests: Conflict of interests: ACMS, ACFS, DPS, GPSA, VTS, JLLMN, MCMCC and AJB – No conflict of interest.

This narrative review explores the application of point-of-care ultrasound (POCUS) in palliative care and its feasibility in home care settings. POCUS has the potential to streamline diagnostic strategies without patient transfer to the hospital, expedite timely symptomatic relief, and reduce complications from specific palliative interventions. The advent of handheld ultrasound devices has made it an attractive diagnostic and interventional adjunct in acute palliative care. POCUS has gained widespread acceptance as part of routine care in emergency medicine and intensive care, guiding certain procedures and increasing their safety. The modernization and miniaturization of ultrasound equipment have made ultra-portable devices available, allowing for better-quality images at affordable prices. Handheld devices have the potential to revolutionize everyday clinical practice in home-based palliative care, contributing to important bedside clinical decisions. Palliative care patients often require diagnostic examinations in the last months of their lives, with CT being the most frequently performed imaging procedure. However, CT imaging is associated with high costs and burdens, leading to increased suffering and impaired quality of life. Clinical ultrasound, a dialogic imaging modality, offers a safer and more efficient approach to palliative care. POCUS applications, which are cost-effective, non-invasive, and well-tolerated, can be used to improve patient satisfaction and diagnostic understanding. POCUS is a valuable tool in palliative care, improving diagnostic accuracy and reducing the time to diagnosis for various pathologies. It is a standard of care for many procedures and improves patient safety. However, there are limitations to POCUS in palliative care, such as operator-dependent examination variability and limited availability of trained professionals. To overcome these limitations, palliative care physicians should receive mandatory training in POCUS, which can be incorporated into the core curriculum. Additionally, ultrasound teleconsulting can assist less experienced examiners in real-time examinations. The literature on POCUS in palliative care is limited, but research on patient-oriented outcomes is crucial. POCUS should be considered a supplement to good clinical reasoning and regulated radiological evaluations., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Monteiro et al.)

Immune cells educated by the primary breast tumor and their secreted factors support the formation of bone pre-metastatic niche. Indeed, we showed that RANKL + CD3 + T cells, specific for the 4T1 mammary carcinoma cell line, arrive at the bone marrow before metastatic cells and set the pre-metastatic niche. In the absence of RANKL expressed by T cells, there is no pre-metastatic osteolytic disease and bone metastases are completely blocked. Adding to the role of T cells, we have recently demonstrated that dendritic cells assist RANKL + T cell activities at bone pre-metastatic niche, by differentiating into potent bone resorbing osteoclast-like cells, keeping their antigen-presenting cell properties, providing a positive feedback loop to the osteolytic profile. Here we are showing that bone marrow-derived CD19 + B cells, from 4T1 tumor-bearing mice, also express the pro-osteoclastogenic cytokine receptor activator of NFκB ligand (RANKL). Analysis of trabecular bone mineral density by conventional histomorphometry and X-ray microtomography (micro-CT) demonstrated that B cells expressing RANKL cooperate with 4T1-primed CD3 + T cells to induce bone loss. Moreover, RANKL expression by B cells depends on T cells activity, since experiments performed with B cells derived from 4T1 tumor-bearing nude BALB/c mice resulted in the maintenance of trabecular bone mass instead of bone loss. Altogether, we believe that 4T1-primed RANKL + B cells alone are not central mediators of bone loss in vivo but when associated with T cells induce a strong decrease in bone mass, accelerating both breast cancer progression and bone metastases establishment. Although several studies performed in different pathological settings, showed that B cells, positively and negatively impact on osteoclastogenesis, due to their capacity to secret pro or anti-osteoclastogenic cytokines, as far as we know, this is the first report showing the role of RANKL expression by B cells on breast cancer-derived bone metastases scenario., Competing Interests: Declaration of competing interest The authors state that they have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Competing Interests: Declarations of Competing Interest None.

We describe a strategy for the development of a rational approach of neoplastic disease therapy based on the demonstration that scale-free networks are susceptible to specific attacks directed against its connective hubs. This strategy involves the (i) selection of up-regulated hubs of connectivity in the tumors interactome, (ii) drug repurposing of these hubs, (iii) RNA silencing of non-druggable hubs, (iv) in vitro hub validation, (v) tumor-on-a-chip, (vi) in vivo validation, and (vii) clinical trial. Hubs are protein targets that are assessed as targets for rational therapy of cancer in the context of personalized oncology. We confirmed the existence of a negative correlation between malignant cell aggressivity and the target number needed for specific drugs or RNA interference (RNAi) to maximize the benefit to the patient's overall survival. Interestingly, we found that some additional proteins not generally targeted by drug treatments might justify the addition of inhibitors designed against them in order to improve therapeutic outcomes. However, many proteins are not druggable, or the available pharmacopeia for these targets is limited, which justifies a therapy based on encapsulated RNAi.

The purpose of this study was to evaluate the effect of the EPN Heterorhabditis bacteriophora HP88 on Stmoxys. calcitrans larvae in sugarcane bagasse ash. Groups of 10 stable fly larvae were placed in Petri dishes containing filter paper and bagasse ash. Concentrations of 50, 150 and 250 EPNs/larva of S. calcitrans in four milliliters of distilled water were added to each plate. In the control group contained only distilled water, without EPNs. The bioassay had three replications and was maintained at 27 ± 1°C and 70-80% relative humidity. It was observed that mortality rate in all treated groups was significantly higher than in the control group (26,6%). The mortality rate in the presence of 50 EPNs/larva (46,6%) was lower than in 150 EPNs/larva (76,3%), which in turn was lower than 250 EPNs/larva group (93,3%). It was verified by analysis of variance and regression that there was a linear pattern of mortality, that is, the higher the EPNs/larva concentration, the higher the larval mortality. It was concluded that EPN H. bacteriophora HP88 was capable of infecting and causing mortality of stable fly larvae in sugarcane bagasse ash., Competing Interests: Conflict of interests: ACMS, ACFS, DPS, GCS, ILAC, JLLMN, MCMCC and AJB - No conflict of interest.

Introduction: In Krabbe disease (KD), mutations in β-galactosylceramidase (GALC), a lysosomal enzyme responsible for the catabolism of galactolipids, leads to the accumulation of its substrates galactocerebroside and psychosine. This neurologic condition is characterized by a severe and progressive demyelination together with neuron-autonomous defects and degeneration. Twitcher mice mimic the infantile form of KD, which is the most common form of the human disease. The Twitcher CNS and PNS present demyelination, axonal loss and neuronal defects including decreased levels of acetylated tubulin, decreased microtubule stability and impaired axonal transport., Methods: We tested whether inhibiting the α-tubulin deacetylase HDAC6 with a specific inhibitor, ACY-738, was able to counteract the early neuropathology and neuronal defects of Twitcher mice., Results: Our data show that delivery of ACY-738 corrects the low levels of acetylated tubulin in the Twitcher nervous system. Furthermore, it reverts the loss myelinated axons in the sciatic nerve and in the optic nerve when administered from birth to postnatal day 9, suggesting that the drug holds neuroprotective properties. The extended delivery of ACY-738 to Twitcher mice delayed axonal degeneration in the CNS and ameliorated the general presentation of the disease. ACY-738 was effective in rescuing neuronal defects of Twitcher neurons, stabilizing microtubule dynamics and increasing the axonal transport of mitochondria., Discussion: Overall, our results support that ACY-738 has a neuroprotective effect in KD and should be considered as an add-on therapy combined with strategies targeting metabolic correction., Competing Interests: OG and MJ were employed by Acetylon Pharmaceuticals Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Braz, Morgado, Pereira, Monteiro, Golonzhka, Jarpe, Brites, Sousa and Nogueira-Rodrigues.)

Introduction: Visceral adipose tissue (VAT) is related to cardiometabolic risk. Estimating it using the visceral adiposity index (VAI) could identify this risk in the Brazilian Air Force (BAF) aviator population. The aim here is to verify the predictive capacity of the VAI for identifying visceral fat areas compared to traditional obesity indicators in BAF pilots., Materials and Methods: Forty male BAF pilots were recruited. The study was conducted in two stages: the first applied a structured questionnaire to characterize the sample and identify sedentary behavior and the second obtained nutritional, anthropometric, and body composition data, carrying out biochemical and magnetic resonance imaging tests and investigating physical activity level in this stage. The comparison of the predictive capacity of the VAI with that of other adiposity indicators (body mass index [BMI], waist circumference, waist-height ratio, waist-hip ratio, and neck circumference) for detecting increased VAT and the determination of the optimal cutoff points for the different adiposity indicators were carried out using receiving operating characteristic (ROC) curves. An association was verified between the adiposity indicators and excess visceral fat using Poisson regression analysis with robust variance., Results: The VAI presented a better predictive capacity for VAT (area under the ROC curve = 0.941), while the BMI did not present diagnostic accuracy (95% CI < 0.5). The strength of the association with high visceral fat was also greater for the VAI than for the other indicators evaluated., Conclusions: The VAI was shown to be a better predictor of excess VAT in relation to the other indicators studied. As it is a more easy-access and lower-cost technique than resonance, it enables greater applicability in tracing and monitoring the visceral obesity of a large contingent of military personnel., (© The Association of Military Surgeons of the United States 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A 35-Year-Old Man with Chronic Cough and DyspneaA 35-year-old man presented for evaluation of chronic cough and worsening dyspnea. How do you approach the evaluation, and what is the diagnosis?

Background: Different pathologies, affecting the skeletal system, were reported to display altered bone and/or cartilage innervation profiles leading to the deregulation of the tissue homeostasis. The patterning of peripheral innervation is achieved through the tissue-specific expression of attractive or repulsive axonal guidance cues in specific space and time frames. During the last decade, emerging findings attributed to the extracellular vesicles (EV) trading a central role in peripheral tissue innervation. However, to date, the contribution of EV in controlling bone innervation is totally unknown., Results: Here we show that sensory neurons outgrowth induced by the bone resorbing cells-osteoclasts-is promoted by osteoclast-derived EV. The EV induced axonal growth is achieved by targeting epidermal growth factor receptor (EGFR)/ErbB2 signaling/protein kinase C phosphorylation in sensory neurons. In addition, our data also indicate that osteoclasts promote sensory neurons electrophysiological activity reflecting a possible pathway in nerve sensitization in the bone microenvironment, however this effect is EV independent., Conclusions: Overall, these results identify a new mechanism of sensory bone innervation regulation and shed the light on the role of osteoclast-derived EV in shaping/guiding bone sensory innervation. These findings provide opportunities for exploitation of osteoclast-derived EV based strategies to prevent and/or mitigate pathological uncontrolled bone innervation., (© 2022. The Author(s).)

Organ-on-a-chip in vitro platforms accurately mimic complex microenvironments offering the ability to recapitulate and dissect mechanisms of physiological and pathological settings, revealing their major importance to develop new therapeutic targets. Bone diseases, such as osteoarthritis, are extremely complex, comprising of the action of inflammatory mediators leading to unbalanced bone homeostasis and de-regulation of sensory innervation and angiogenesis. Although there are models to mimic bone vascularization or innervation, in vitro platforms merging the complexity of bone, vasculature, innervation, and inflammation are missing. Therefore, in this study a microfluidic-based neuro-vascularized bone chip (NVB chip) is proposed to 1) model the mechanistic interactions between innervation and angiogenesis in the inflammatory bone niche, and 2) explore, as a screening tool, novel strategies targeting inflammatory diseases, using a nano-based drug delivery system. It is possible to set the design of the platform and achieve the optimized conditions to address the neurovascular network under inflammation. Moreover, this system is validated by delivering anti-inflammatory drug-loaded nanoparticles to counteract the neuronal growth associated with pain perception. This reliable in vitro tool will allow understanding the bone neurovascular system, enlightening novel mechanisms behind the inflammatory bone diseases, bone destruction, and pain opening new avenues for new therapies discovery., (© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Regeneration of adult mammalian central nervous system (CNS) axons is abortive, resulting in inability to recover function after CNS lesion, including spinal cord injury (SCI). Here, we show that the spiny mouse (Acomys) is an exception to other mammals, being capable of spontaneous and fast restoration of function after severe SCI, re-establishing hind limb coordination. Remarkably, Acomys assembles a scarless pro-regenerative tissue at the injury site, providing a unique structural continuity of the initial spinal cord geometry. The Acomys SCI site shows robust axon regeneration of multiple tracts, synapse formation, and electrophysiological signal propagation. Transcriptomic analysis of the spinal cord following transcriptome reconstruction revealed that Acomys rewires glycosylation biosynthetic pathways, culminating in a specific pro-regenerative proteoglycan signature at SCI site. Our work uncovers that a glycosylation switch is critical for axon regeneration after SCI and identifies β3gnt7, a crucial enzyme of keratan sulfate biosynthesis, as an enhancer of axon growth., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Before bone colonization, immune cells primed by breast primary tumor cells actively modify the bone microenvironment, disturbing the complex and tightly homeostatic signaling network regulated by osteoblasts and osteoclasts. Indeed, we have shown that RANKL + CD4 + T cells specific for the 4T1 mammary carcinoma cell line, arrive at the bone marrow (BM) before metastatic cells and set the pre-metastatic niche. In the absence of RANKL expressed by T cells, there is no pre-metastatic osteolytic disease and bone metastases are blocked. Adding to the role of T cells, we have recently demonstrated that dendritic cells (DCs) provide a positive feedback loop to the osteolytic profile induced by the metastatic tumor. In this setting, DCs are able to differentiate into potent bone resorbing osteoclast-like cells keeping their antigen-presenting cell (APC) properties to maintain RANKL + CD4 + Th17 T cells activities, via IL-23 expression. Here we show that 67NR non-metastatic tumor cells, a sibling of 4T1 tumor cell line, induce an increase in trabecular bone mass on day 11 post-tumor implant. This observation was associated with an expansion of the osteoblastic lineage cells accompanied by a reduction of osteoclasts numbers. Moreover, BM derived CD8 + T cells from 67NR tumor-bearing mice, express an anti-osteoclastogenic cytokine milieu enriched by IFN-γ, IL-10 and producing low levels of RANKL. The frequency of BM derived CD8 + FoxP3 + regulatory T cells, known as potent suppressors of osteoclastogenesis both in vitro and in vivo, was also increased in such animals. This milieu was capable to suppress 4T1 tumor-specific CD4 + T cells phenotype in vivo and in vitro and strongly inhibited bone metastases establishment, restoring trabecular bone mass volume. We concluded that the 67NR + tumor derived CD8 + T cells phenotypes, either contributing to bone homeostasis and/or control of 4T1 breast tumor pre-metastatic disease, interfere with osteoclasts and osteoblasts activities inside BM. Our study highlights the opposing roles of subverted tumor CD4 + and CD8 + T cell subtypes in directing breast cancer progression and bone metastases establishment. For non-metastatic tumors, the role of T cells regarding bone remodeling has never been addressed before. As far as we know, this is the first description that an in situ carcinoma can modify distant sites. In the case showed here, modification of the distant bone site disfavors pre-metastatic bone niche formation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction: We have previously reported on neurogenic bladder dysfunction among Congenital Zika Vírus Syndrome (CZS) patients, but it is unknown how they will respond to treatment., Objective: To assess whether children with neurological lower urinary tract dysfunction and CZS will respond to Standard therapies., Methodology: A prospective observational cohort study of children with CZS referred for urological assessment between 2016 and 2020 to our quaternary center in Brazil. Urological protocol included clinical history, urinalysis and culture, renal and bladder ultrasonography and urodynamic study. Patients were treated based on findings from the first evaluation, with oxybutynin chloride for overactive bladder and low bladder compliance, clean intermittent catheterization for ineffective bladder emptying, or dual therapy when both were observed. Urological outcomes were evaluated between the first and second visits considering patient's adherence. Outcomes measured included clinical, imaging, and urodynamic variables. Data was analyzed using the IBM SPSS 22 software., Results: From the cohort of 90 patients, 56 completed the second urodynamic assessment and were included. One presented underactive bladder and 55 overactive bladder. Among these 55, 39 were adherent and 16 non-adherents to the prescribed treatment. Among the 39 adherents, 8 adhered regularly to oxybutynin and clean intermittent catheterization (CIC), 29 to oxybutynin alone, and two to catheterization alone. During follow-up, the number of patients with urinary tract infection and postvoid residual increased, but all other parameters had improved. Renal and bladder ultrasonography improved in 10, maximum bladder pressure decreased in 22 and maximum cystometric capacity and compliance increased in 14 patients. Sixteen patients did not adhere regularly to the prescribed treatment and although the number of patients with urinary tract infection reduced with antibiotic therapy, their bladder capacity and compliance did not improve during follow-up., Discussion: Ultrasonographic and urodynamic improvements were observed after 10.8 ± 7.5 months of treatment, including one patient with ureterohydronephrosis that resolved. Adherence to CIC remains a challenge and reflected in the number of patients presenting urinary tract infection and postvoid residual. The immediate clinical relevance is the major study strength, given the previously uncharacterized therapy options for this patient population. The number of patients remains one of the study limitations, reducing our ability to perform more advanced statistical analyses., Conclusion: Patients with Zika-related neurological lower urinary tract dysfunction may benefit from conventional therapies. Results confirmed ultrasonographic and urodynamic improvements after treatment, although not statically significant. Adherence to treatment, specifically to CIC, remains a challenge., Competing Interests: Conflicts of interest None., (Copyright © 2021 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Deregulation of miRNAs contributes to the development of distinct cancer types, including melanoma, an aggressive form of skin cancer characterized by high metastatic potential and poor prognosis. The expression of a set of 580 miRNAs was investigated in a model of murine melanoma progression, comprising non-metastatic (4C11-) and metastatic melanoma (4C11+) cells. A significant increase in miR-138-5p expression was found in the metastatic 4C11+ melanoma cells compared to 4C11-, which prompted us to investigate its role in melanoma aggressiveness. Functional assays, including anoikis resistance, colony formation, collective migration, serum-deprived growth capacity, as well as in vivo tumor growth and experimental metastasis were performed in 4C11- cells stably overexpressing miR-138-5p. miR-138-5p induced an aggressive phenotype in mouse melanoma cell lines leading to increased proliferation, migration and cell viability under stress conditions. Moreover, by overexpressing miR-138-5p, low-growing and non-metastatic 4C11- cells became highly proliferative and metastatic in vivo, similar to the metastatic 4C11+ cells. Luciferase reporter analysis identified the tumor suppressor Trp53 as a direct target of miR-138-5p. Using data sets from independent melanoma cohorts, miR-138-5p and P53 expression were also found deregulated in human melanoma samples, with their levels negatively and positively correlated with prognosis, respectively. Our data shows that the overexpression of miR-138-5p contributes to melanoma metastasis through the direct suppression of Trp53., (Copyright © 2021. Published by Elsevier Inc.)

Bone marrow (BM) is a preferential metastatic site for solid cancers, contributing to higher morbidity and mortality among millions of oncologic patients worldwide. There are no current efficient therapies to minimize this health burden. Microfluidic based in vitro models emerge as powerful alternatives to animal testing, as well as promising tools for the development of personalized medicine solutions. The complexity associated with the BM metastatic niche originated a wide variety of microfluidic platforms designed to mimic this microenvironment. This review gathers the essential parameters to design an accurate in vitro microfluidic device, based on a comparative analysis of existing models created to address the different steps of the metastatic cascade., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Immune system dysfunction is paramount in coronavirus disease 2019 (COVID-19) severity and fatality rate. Mucosal-associated invariant T (MAIT) cells are innate-like T cells involved in mucosal immunity and protection against viral infections. Here, we studied the immune cell landscape, with emphasis on MAIT cells, in cohorts totaling 208 patients with various stages of disease. MAIT cell frequency is strongly reduced in blood. They display a strong activated and cytotoxic phenotype that is more pronounced in lungs. Blood MAIT cell alterations positively correlate with the activation of other innate cells, proinflammatory cytokines, notably interleukin (IL)-18, and with the severity and mortality of severe acute respiratory syndrome coronavirus 2 infection. We also identified a monocyte/macrophage interferon (IFN)-α-IL-18 cytokine shift and the ability of infected macrophages to induce the cytotoxicity of MAIT cells in an MR1-dependent manner. Together, our results suggest that altered MAIT cell functions due to IFN-α-IL-18 imbalance contribute to disease severity, and their therapeutic manipulation may prevent deleterious inflammation in COVID-19 aggravation.

Bone metastases occur in 70% of patients with advanced breast cancer, causing severe morbidity and increased mortality due to osteolytic lesions driven by osteoclasts (OCs) inside the bone marrow (BM) microenvironment. A reciprocal vicious cycle between bone remodeling system and the tumor itself is established by the release of growth factors stored in the mineralized matrix, which in turn feed the tumor, changing tumor behavior and growth. However, BM is not a passive host microenvironment for circulating tumor cells, but instead can be actively modified by the primary tumor before metastatic spread occurs. Indeed, we have shown that T cells specific for the 4T1 mammary carcinoma cell line, are characteristically RANKL + IL-17F + CD4 + T cells. Those cells arrive in the BM before metastatic cells and set the pre-metastatic niche. In the absence of T cell derived RANKL, there is no pre-metastatic osteolytic disease and bone metastases do not take place. Recently, dendritic cells (DCs), the main T cell partner at the beginning of the immune response, came into the spotlight as a potential source of OCs progenitors under inflammatory conditions. Regarding bone metastasis, nothing is currently known about DCs plasticity or even its partnership with tumor induced T cells for BM pre-metastatic niche formation. Here, we show that splenic CD11c + DCs stimulated with 4T1 conditioned media (CM) efficiently differentiated into mature and activated multinucleated giant cells (DC-OC) expressing TRAP and IL-23 cytokine. More important, 4T1 CM derived DC-OCs build a positive loop which amplifies the osteolytic phenomena by maintaining the RANKL + Th17 T cells and by its own osteoclastic activity. In conclusion, our results indicate that differentiation of OCs from DCs may be achievable in the bone pre osteolytic disease context representing an alternative OC differentiation pathway. Besides being induced by high levels of T cells pro osteoclastogenic cytokines, especially by RANKL, DC-OC keep a positive feedback loop towards osteolysis, maintaining the pro-osteoclastogenic T cell phenotype in the BM., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: To describe the trajectory of respiratory failure in COVID-19 and explore factors associated with risk of invasive mechanical ventilation (IMV)., Materials and Methods: A retrospective, observational cohort study of 112 inpatient adults diagnosed with COVID-19 between March 12 and April 16, 2020. Data were manually extracted from electronic medical records. Multivariable and Univariable regression were used to evaluate association between baseline characteristics, initial serum markers and the outcome of IMV., Results: Our cohort had median age of 61 (IQR 45-74) and was 66% male. In-hospital mortality was 6% (7/112). ICU mortality was 12.8% (6/47), and 18% (5/28) for those requiring IMV. Obesity (OR 5.82, CI 1.74-19.48), former (OR 8.06, CI 1.51-43.06) and current smoking status (OR 10.33, CI 1.43-74.67) were associated with IMV after adjusting for age, sex, and high prevalence comorbidities by multivariable analysis. Initial absolute lymphocyte count (OR 0.33, CI 0.11-0.96), procalcitonin (OR 1.27, CI 1.02-1.57), IL-6 (OR 1.17, CI 1.03-1.33), ferritin (OR 1.05, CI 1.005-1.11), LDH (OR 1.57, 95% CI 1.13-2.17) and CRP (OR 1.13, CI 1.06-1.21), were associated with IMV by univariate analysis., Conclusions: Obesity, smoking history, and elevated inflammatory markers were associated with increased need for IMV in patients with COVID-19., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: SYC consults for PureTech on their deupirfenidone in COVID study. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

In osteoarthritis (OA), pain is the dominant clinical symptom, yet the therapeutic approaches remain inadequate. The knowledge of the nociceptive mechanisms in OA, which will allow to develop effective therapies for OA pain, is of utmost need. In this study, we investigated the nociceptive mechanisms involved in post-traumatic OA pain, using the destabilization of the medial meniscus (DMM) mouse model. Our results revealed the development of peripheral pain sensitization, reflected by augmented mechanical allodynia. Along with the development of pain behaviour, we observed an increase in the expression of calcitonin gene-related peptide (CGRP) in both the sensory nerve fibers of the periosteum and the dorsal root ganglia. Interestingly, we also observed that other nociceptive mechanisms commonly described in non-traumatic OA phenotypes, such as infiltration of the synovium by immune cells, neuropathic mechanisms and also central sensitization were not present. Overall, our results suggest that CGRP in the sensory nervous system is underlying the peripheral sensitization observed after traumatic knee injury in the DMM model, highlighting the CGRP as a putative therapeutic target to treat pain in post-traumatic OA. Moreover, our findings suggest that the nociceptive mechanisms involved in driving pain in post-traumatic OA are considerably different from those in non-traumatic OA.

The association of ipsilateral talar and calcaneal fractures is an uncommon combination and is typically the result of a high-energy trauma. It is often associated with comminution, marked fracture displacement, and soft-tissue compromise. Obtaining satisfactory clinical and radiographic results is very challenging. Residual deformities, multiple procedures, and limitations are usually the norm. Therefore, studies have suggested that primary arthrodesis may represent the best option of surgical treatment. In this study, we report a case of a 30-year-old male patient with a rare combination of a highly comminuted transcalcaneal fracture-dislocation associated with a talar neck fracture successfully treated with open reduction and internal fixation (ORIF) with an 18-month follow-up. This case demonstrates that even when there are associated fractures of the talus and calcaneus with severe bone loss, ORIF may provide satisfactory outcomes in the short-term postoperative period. Levels of Evidence : Level V: Case report.

Objective: To describe the care of pediatric patients who had a gastrostomy and developed peristomal lesions and received a systematic single adapted crusting technique in a pediatric ICU in a tertiary Brazilian hospital., Methods: An analysis of six cases presenting traumatic, noninfectious peristomal lesions with ostium enlargement resulting in gastric residual leaks. All six patients received the same treatment over 7 to 15 days., Results: Lesion improvement was observed in all patients after 48 hours and considered attributable to the standard treatment recommended by ostomy professionals., Conclusions: The adapted crusting technique was effective in the treatment of children with peristomal lesions. This technique may be beneficial to other patient and organizational outcomes such as improving safety of care, decreasing pain and discomfort, reducing nursing workload and hospital costs, and improving quality of life.

Background: Zone 1 fractures of the proximal fifth metatarsal are usually treated nonsurgically using some type of immobilization. The aim of this study was to compare clinical and functional outcomes, time to return to prior activity levels, and rate of bone healing when using a hard-soled shoe (HSS) vs a controlled ankle motion (CAM)-walker boot (CWB)., Methods: Seventy-two consecutive patients with zone 1 fractures of the fifth metatarsal base were treated conservatively with either an HSS or CWB by 2 different providers. We included 57 women and 15 men, average age of 41.3 (range, 16-88) years. Radiographic findings, visual analog scale (VAS) for pain, and American Orthopaedic Ankle & Foot Society (AOFAS) midfoot score were assessed. Patients were followed at 4, 8, 10, 12, and 24 weeks or until asymptomatic and able to return to prior level of activities. Statistical analysis was performed using Mann-Whitney U , Fisher exact, and chi-square tests. P values <.05 were considered significant., Results: Age and gender distributions were similar in both groups ( P = .23 and P = .57). Patients had similar VAS and AOFAS scores after 8 ( P = .34 and P = .83) and 12 ( P = .87 and P = .79) weeks. Average time for bone healing was significantly faster using the CWB (7.2 weeks) when compared to the HSS (8.6 weeks) ( P < .001). The average time to return to prior level of activities was similar in both groups (8.3 weeks for CWB and 9.7 weeks for HSS) ( P = .11). Fracture displacement was equal in both groups, with a mean of 1.9 mm of displacement in patients using the HSS, and a mean of 1.6 mm in those using the CWB ( P = .26)., Conclusion: Zone 1 fractures of the proximal fifth metatarsal can be treated conservatively with either a hard-soled shoe or a CAM-walker boot. Even though patients treated in the CAM-walker boot demonstrated earlier signs of complete healing, similar clinical and functional results were achieved with both treatments., Level of Evidence: Level III, retrospective comparative series.

In contrast to the continuous increase in survival rates for many cancer entities, colorectal cancer (CRC) and pancreatic cancer are predicted to be ranked among the top 3 cancer-related deaths in the European Union by 2025. Especially, fighting metastasis still constitutes an obstacle to be overcome in CRC and pancreatic cancer. As described by Fearon and Vogelstein, the development of CRC is based on sequential mutations leading to the activation of proto-oncogenes and the inactivation of tumour suppressor genes. In pancreatic cancer, genetic alterations also attribute to tumour development and progression. Recent findings have identified new potentially important transcription factors in CRC, among those the activating transcription factor 2 (ATF2). ATF2 is a basic leucine zipper protein and is involved in physiological and developmental processes, as well as in tumorigenesis. The mutation burden of ATF2 in CRC and pancreatic cancer is rather negligible; however, previous studies in other tumours indicated that ATF2 expression level and subcellular localisation impact tumour progression and patient prognosis. In a tissue- and stimulus-dependent manner, ATF2 is activated by upstream kinases, dimerises and induces target gene expression. Dependent on its dimerisation partner, ATF2 homodimers or heterodimers bind to cAMP-response elements or activator protein 1 consensus motifs. Pioneering work has been performed in melanoma in which the dual role of ATF2 is best understood. Even though there is increasing interest in ATF2 recently, only little is known about its involvement in CRC and pancreatic cancer. In this review, we summarise the current understanding of the underestimated 'cancer gene chameleon' ATF2 in apoptosis, epithelial-to-mesenchymal transition and microRNA regulation and highlight its functions in CRC and pancreatic cancer. We further provide a novel ATF2 3D structure with key phosphorylation sites and an updated overview of all so-far available mouse models to study ATF2 in vivo., (© The Author(s) 2019. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.)

Introduction: Congenital Zika syndrome (CZS) is a recently discovered condition that affects central nervous system structures that control the lower urinary tract. The first cases of neurogenic bladder (NB) were recently reported as a sequalae of CZS in neurologically impaired children., Objective: Our goal is to further evaluate NB in the setting of CZS, identifying urological risk indicators in hopes that early diagnosis will mitigate the impact of the disease., Study Design: Urological assessment was performed in all patients with CZS and neurological impairment who were referred to our urodynamic clinic between June 2016 and May 2018. Neurogenic bladder was confirmed by urodynamic evaluation, and urological risk was based on urodynamic results., Results: Sixty-nine patients with CZS were tested. The majority (63 patients, 91.3%) presented with overactive bladder with increased pressures and reduced capacity for age (table 1). Different urodynamic patterns were observed, and the association of reduced bladder capacity for age, high bladder-filling pressure, and increased postvoid residual were frequently observed., Discussion: NB continues to be consistently diagnosed in our cohort of CZS, mostly with high-risk indicators for renal impairment. When not intervened upon in a timely manner, NB can cause progressive damage to the urinary tract, but the lack of knowledge that CZS causes NB delays investigation and treatment. Parents and health professionals will need to be sensitized to the risks that ZIKV can pose to the urinary tract so that appropriate therapies are initiated to prevent irreversible renal damage., Conclusion: NB is a common condition among our patients with CZS and microcephaly. This is a new cause of NB, unknown to urologists. While further investigation is necessary to understand long-term disease behavior and therapeutic response, increased knowledge among urologists may help to reduce morbidity related to untreated NB and to mitigate the disease burden for patients and families., (Copyright © 2019 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

p21 cip1/waf1 is a central regulator of cell cycle control and survival. While mutations are rare, it is commonly dysregulated in several human cancers due to epigenetic mechanisms influencing its transcriptional control. These mechanisms include promoter hypermethylation as well as additional pathways such as histone acetylation or methylation. The epigenetic regulators include writers, such as DNA methyltransferases (DNMTs); histone acetyltransferases (HATs) and histone lysine methyltransferases; erasers, such as histone deacetylases (HDACs); histone lysine demethylases [e.g., the Lysine Demethylase (KDM) family]; DNA hydroxylases; readers, such as the methyl-CpG-binding proteins (MBPs); and bromodomain-containing proteins, including the bromo- and extraterminal domain (BET) family. We further discuss the roles that long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play in the epigenetic control of p21 cip1/waf1 expression and its function in human cancers., Competing Interests: The authors declare no conflicts of interest.

The high mortality rate of melanoma is broadly associated with its metastatic potential. Tumor cell dissemination is strictly dependent on vascularization; therefore, angiogenesis and lymphangiogenesis play an essential role in metastasis. Hence, a better understanding of the players of tumor vascularization and establishing them as new molecular biomarkers might help to overcome the poor prognosis of melanoma patients. Here, we further characterized a linear murine model of melanoma progression and showed that the aggressiveness of melanoma cells is closely associated with high expression of angiogenic factors, such as Vegfc, Angpt2, and Six1, and that blockade of the vascular endothelial growth factor pathway by the inhibitor axitinib abrogates their tumorigenic potential in vitro and in the in vivo chicken chorioallantoic membrane assay. Furthermore, analysis of The Cancer Genome Atlas data revealed that the expression of the angiogenic factor ANGPT2 (P-value = 0.044) and the lymphangiogenic receptor VEGFR-3 (P-value = 0.002) were independent prognostic factors of overall survival in melanoma patients. Enhanced reduced representation bisulfite sequencing-based methylome profiling revealed for the first time a link between abnormal VEGFC, ANGPT2, and SIX1 gene expression and promoter hypomethylation in melanoma cells. In patients, VEGFC (P-value = 0.031), ANGPT2 (P-value < 0.001), and SIX1 (P-value = 0.009) promoter hypomethylation were independent prognostic factors of shorter overall survival. Hence, our data suggest that these angio- and lymphangiogenesis factors are potential biomarkers of melanoma prognosis. Moreover, these findings strongly support the applicability of our melanoma progression model to unravel new biomarkers for this aggressive human disease., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

The ability to perceive the emotions of others is crucial for everyday social interactions. Important aspects of visual socioemotional processing, such as the recognition of facial expressions, are known to depend on largely automatic mechanisms. However, whether and how properties of automaticity extend to the auditory domain remains poorly understood. Here we ask if nonverbal auditory emotion recognition is a controlled deliberate or an automatic efficient process, using vocalizations such as laughter, crying, and screams. In a between-subjects design (N = 112), and covering eight emotions (four positive), we determined whether emotion recognition accuracy (a) is improved when participants actively deliberate about their responses (compared with when they respond as fast as possible) and (b) is impaired when they respond under low and high levels of cognitive load (concurrent task involving memorizing sequences of six or eight digits, respectively). Response latencies were also measured. Mixed-effects models revealed that recognition accuracy was high across emotions, and only minimally affected by deliberation and cognitive load; the benefits of deliberation and costs of cognitive load were significant mostly for positive emotions, notably amusement/laughter, and smaller or absent for negative ones; response latencies did not suffer under low or high cognitive load; and high recognition accuracy (approximately 90%) could be reached within 500 ms after the stimulus onset, with performance exceeding chance-level already between 300 and 360 ms. These findings indicate that key features of automaticity, namely fast and efficient/effortless processing, might be a modality-independent component of emotion recognition. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Amblyomma parvum Aragão (Acari: Ixodidae) is a tick species found with wide distribution in the Neotropical region. Even though it is a wildlife-related tick, it is also a frequent parasite of domestic animals, is aggressive to human beings and may harbor pathogenic microorganisms. Therefore, it is a target species for control on domestic animals, particularly those at the rural-wildlife interface. Herein, the efficacy of two isolates (E9 and IBCB 425) of an entomopathogenic fungus, Metarhizium anisopliae sensu lato, already evaluated for ticks that parasitize domestic animals, was tested against unfed A. parvum adults. Both isolates displayed high acaricidal efficacy after immersion in fungal conidial suspensions for 5 min. Isolate E9 killed all ticks by the 7th day post-treatment, and isolate IBCB 425 did so by the 11th day. Tick mortality of 80 and 90% was achieved as early as the 3rd and 4th days, respectively, with both treatments. Thus, if a commercial M. anisopliae s.l. acaricide against domestic animal ticks is developed, it would also be effective against A. parvum.

Introduction: Congenital Zika Syndrome (CZS) has been associated with microcephaly and other central nervous system abnormalities including areas that have been implicated in the control of the lower urinary tract. As such, this descriptive case series has aimed to investigate whether CZS is linked with neurogenic bladder. Identifying such an association is paramount in the effort to recognize CZS complications that have putative treatment options that could mitigate the impact of CZS in infected children., Methods: Following IRB approval, urological assessment was performed in all patients referred to our clinic between June 2016 and May 2017 who presented with confirmed CZS-associated microcephaly. The research protocol consisted of obtaining clinical history, laboratory tests, lower and upper urinary tract ultrasounds, as well as a diagnostic urodynamic evaluation. ZIKA virus infection was previously confirmed by maternal history and positive PCR in babies and mothers. Microcephaly and other central nervous system abnormalities were established based on neurological assessment and associated imaging of the central nervous system (CT head and/or Brain MRI)., Results: Twenty-two consecutive CZS patients were tested and confirmed to have neurogenic bladder. Of the 22 patients assessed, 21 presented with an overactive bladder combined with reduced bladder capacity and elevated detrusor filling pressures. Clinically significant increases in postvoid residual (PVR) were confirmed in 40% of cases while a urinary tract infection (UTI) was identified in 23% of cases., Conclusion: Neurogenic bladder, a known treatable health condition, was confirmed in 100% of patients tested in this study, most presenting with high-risk urodynamic patterns known to lead to renal damage when left untreated. Follow up studies are necessary to provide further insight onto long-term disease progression and to investigate the response to standard therapies for neurogenic bladder. Nonetheless, we emphasize the importance of proactive management of neurogenic bladder and prompt referral so as to help mitigate CZS disease burden for patients and their families.