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PURPOSE: Lumbar discectomy is among the most common spine procedures in the US, with 300,000 procedures performed each year. Like other surgical procedures, this procedure is not excluded from potential complications. This paper presents a video annotation methodology for microdiscectomy including the development of a surgical workflow. In future work, this methodology could be combined with computer vision and machine learning models to predict potential adverse events. These systems would monitor the intraoperative activities and possibly anticipate the outcomes. METHODS: A necessary step in supervised machine learning methods is video annotation, which involves labeling objects frame-by-frame to make them recognizable for machine learning applications. Microdiscectomy video recordings of spine surgeries were collected from a multi-center research collaborative. These videos were anonymized and stored in a cloud-based platform. Videos were uploaded to an online annotation platform. An annotation framework was developed based on literature review and surgical observations to ensure proper understanding of the instruments, anatomy, and steps. RESULTS: An annotated video of microdiscectomy was produced by a single surgeon. Multiple iterations allowed for the creation of an annotated video complete with labeled surgical tools, anatomy, and phases. In addition, a workflow was developed for the training of novice annotators, which provides information about the annotation software to assist in the production of standardized annotations. CONCLUSIONS: A standardized workflow for managing surgical video data is essential for surgical video annotation and machine learning applications. We developed a standard workflow for annotating surgical videos for microdiscectomy that may facilitate the quantitative analysis of videos using supervised machine learning applications. Future work will demonstrate the clinical relevance and impact of this workflow by developing process modeling and outcome predictors.
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OBJECTIVE: Perinatal mood and anxiety disorders (PMADs) affect many neonatal intensive care unit (NICU) parents and are a significant risk factor for maternal suicide. Lack of screening and treatment interferes with infant development and bonding, compounding risks in fragile infants. We aim to describe PMAD screening in level IV NICUs across the Children's Hospitals Neonatal Consortium (CHNC) and to determine the relationship between standardized screening and mental health professionals (MHPs) presence. STUDY DESIGN: We surveyed experts at 44 CHNC NICUs about PMADs screening practices and presence of NICU-specific MHPs. Kruskal-Wallis test was used to examine relationships. RESULTS: Of 44 centers, 34 (77%) responded. Fourteen centers (41%) perform screening with validated tools. Thirteen (38%) centers have NICU-dedicated psychologists. Formally screening centers tend to have higher cumulative MHPs (p = 0.089) than informally screening centers. Repeat screening practices were highly variable with no difference in the number of cumulative MHPs. CONCLUSION: Screening practices for PMADs vary across CHNC centers; less than half have additional MHPs beyond social workers. Creating a sustainable model to detect PMADs likely requires more MHPs in NICUs. KEY POINTS: · PMADs affect many NICU parents.. · Systematic screening for PMADs is limited in level IV children's hospital NICUs.. · Additional MHPs may contribute to enhanced screening and support of parents.. · Hospitals should prioritize funds for NICU-specific MHPs to optimize outcomes..
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Scanning electron microscopy (SEM) remains distinct in its ability to allow topographical visualization of structures. Key elements to consider for successful examination of biological specimens include appropriate preparative and imaging techniques. Chemical processing induces structural artifacts during specimen preparation, and several factors need to be considered when selecting fixation protocols to reduce these effects while retaining structures of interest. Particular care for proper dehydration of specimens is essential to minimize shrinkage and is necessary for placement under the high-vacuum environment required for routine operation of standard SEMs. Choice of substrate for mounting and coating specimens can reduce artifacts known as charging, and a basic understanding of microscope settings can optimize parameters to achieve desired results. This article describes fundamental techniques and tips for routine specimen preparation for a variety of biological specimens, preservation of labile or fragile structures, immune-labeling strategies, and microscope imaging parameters for optimal examination by SEM. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Chemical preparative techniques for preservation of biological specimens for examination by SEM Alternate Protocol 1: Practical considerations for the preparation of soft tissues Alternate Protocol 2: Removal of debris from the exoskeleton of invertebrates Alternate Protocol 3: Fixation of colonies grown on agar plates Alternate Protocol 4: Stabilization of polysaccharide structures with alcian blue and lysine Alternate Protocol 5: Preparation of non-adherent particulates in solution for SEM Support Protocol 1: Application of thin layer of adhesive on substrate to improve adherence Support Protocol 2: Poly-L-lysine coating specimen substrates for improved adherence Support Protocol 3: Microwave processing of biological specimens for examination by SEM Basic Protocol 2: Critical point drying of specimens Alternate Protocol 6: Chemical alternative to critical point drying Basic Protocol 3: Sputter coating Alternate Protocol 7: Improved bulk conductivity through "OTOTO" Basic Protocol 4: Immune-labeling strategies Alternate Protocol 8: Immune-labeling internal antigens with small gold probes Alternate protocol 9: Quantum dot or fluoronanogold preparations for correlative techniques Basic Protocol 5: Exposure of internal structures by mechanical fracturing Basic Protocol 6: Exposure of internal structures of tissues by fracturing with liquid nitrogen Basic Protocol 7: Anaglyph production from stereo pairs to produce 3D images.
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Microscopy, Electron, Scanning , Specimen Handling , Microscopy, Electron, Scanning/methods , Specimen Handling/methods , AnimalsABSTRACT
HIV-1 gp120 glycan binding to C-type lectin adhesion receptor L-selectin/CD62L on CD4 T cells facilitates viral attachment and entry. Paradoxically, the adhesion receptor impedes HIV-1 budding from infected T cells and the viral release requires the shedding of CD62L. To systematically investigate CD62L-shedding mediated viral release and its potential inhibition, we screened compounds specific for serine-, cysteine-, aspartyl-, and Zn-dependent proteases for CD62L shedding inhibition and found that a subclass of Zn-metalloproteinase inhibitors, including BB-94, TAPI, prinomastat, GM6001, and GI25423X, suppressed CD62L shedding. Their inhibition of HIV-1 infections correlated with enzymatic suppression of both ADAM10 and 17 activities and expressions of these ADAMs were transiently induced during the viral infection. These metalloproteinase inhibitors are distinct from the current antiretroviral drug compounds. Using immunogold labeling of CD62L, we observed association between budding HIV-1 virions and CD62L by transmission electron microscope, and the extent of CD62L-tethering of budding virions increased when the receptor shedding is inhibited. Finally, these CD62L shedding inhibitors suppressed the release of HIV-1 virions by CD4 T cells of infected individuals and their virion release inhibitions correlated with their CD62L shedding inhibitions. Our finding reveals a new therapeutic approach targeted at HIV-1 viral release.
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Centromeres are constricted chromosomal regions that are essential for cell division. In eukaryotes, centromeres display a remarkable architectural and genetic diversity. The basis of centromere-accelerated evolution remains elusive. Here, we focused on Pneumocystis species, a group of mammalian-specific fungal pathogens that form a sister taxon with that of the Schizosaccharomyces pombe, an important genetic model for centromere biology research. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of S. pombe. Using organisms from a short-term in vitro culture or infected animal models and chromatin immunoprecipitation (ChIP)-Seq, we identified CENP-A bound regions in two Pneumocystis species that diverged ~35 million years ago. Each species has a unique short regional centromere (<10 kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. These features suggest an epigenetic specification of centromere function. Analysis of centromeric DNA across multiple Pneumocystis species suggests a vertical transmission at least 100 million years ago. The common ancestry of Pneumocystis and S. pombe centromeres is untraceable at the DNA level, but the overall architectural similarity could be the result of functional constraint for successful chromosomal segregation.IMPORTANCEPneumocystis species offer a suitable genetic system to study centromere evolution in pathogens because of their phylogenetic proximity with the non-pathogenic yeast S. pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after the divergence of the two clades ~ 460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-Seq to characterize centromeres in multiple Pneumocystis species. We show that Pneumocystis have short epigenetic centromeres that function differently from those in S. pombe.
Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Centromere Protein A/genetics , Phylogeny , Chromosomal Proteins, Non-Histone/genetics , Centromere/metabolism , Schizosaccharomyces/genetics , DNA/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
Following several days of blood feeding by larval and nymphal ixodid (hard) ticks, the salivary glands degenerate and are completely replaced in the next life stage. Yet, what happens during the molt of immature argasid (soft) ticks after their rapid and small bloodmeal has remained a mystery. Multiple studies of nymphal Ornithodoros hermsi Wheeler (Acari: Argasidae) ticks infected with the relapsing fever spirochete Borrelia hermsii suggested the salivary glands in these ticks may not disintegrate after feeding. Therefore, cohorts of second-stage O. hermsi nymphs were fed and examined daily after the bloodmeal by fresh dissections and weekly by histological cross-sections of the entire tick. The composition of the salivary glands was typical for argasid ticks in having agranular (Type I) and granular (Type II) acini, the latter being surrounded by a myo-epithelial sheath. In all 197 ticks examined from 1 to 63 days after feeding, morphologically intact salivary glands were present. During apolysis, 5 ticks had extralimital clusters of granular acini adhering to otherwise intact glands. Our observations demonstrate that the salivary glands of nymphal O. hermsi do not disintegrate after feeding and new acini are produced during the molt for incorporation into the existing glands. Cumulatively, these findings suggest a fundamental difference in the transstadial development of argasid and ixodid ticks.
Subject(s)
Nymph , Ornithodoros , Salivary Glands , Animals , Ornithodoros/growth & development , Ornithodoros/physiology , Nymph/growth & development , Nymph/physiologyABSTRACT
PURPOSE: Congenital heart defects are the most common birth defects in the USA and in 25% of cases need to be treated with cardiovascular interventions. One of such interventions is the postoperative use of an extracorporeal membrane oxygenation (ECMO) machine for the treatment of cardiorespiratory failure. The process of placing the patient on the ECMO is extremely time-critical and requires the use of cardiac cannulation. For the first time, our team developed and evaluated a new quick-connect cannulation system that allows for rapid, easy, and safe ECMO cannulation in the pediatric population. The design should eliminate the need for purse-string sutures that are currently used to secure cannulas, as the cannulas will be inserted through a port that is glued to the cardiovascular tissue. METHODS: The rapid cannulation assistance device was designed on the SolidWorks computer-aided design software using the dimensions of the commercially available arterial and venous catheters. These designs were then 3D printed, and tensile testing was performed. Then, a flow loop was developed, and cannulation was performed and analyzed on both 3D-printed hearts and porcine hearts. RESULTS: The rapid cannulation assistance device was designed and 3D printed. Tensile testing found that the parts were strong enough to withstand forces that may be introduced in studies. 3D-printed and porcine heart tests with a flow loop found no leakage with the 3D-printed hearts but minimal leaking with the porcine hearts. However, this leakage was observed at the junction between the device and the heart, leading us to believe that a glue better suited to attach the device to the heart would prevent leakage in the future. CONCLUSIONS: This project successfully demonstrated how a rapid cannulation assistance device could be developed and tested. Future studies will be conducted that address device adhesion to the cardiovascular tissue so that accurate pressure and flow rates can be measured. Future studies will also include testing the device in a fluid environment to more effectively analyze the device success and comparing the time required to cannulate using our device compared to the standard of care.
Subject(s)
Extracorporeal Membrane Oxygenation , Heart Failure , Humans , Child , Animals , Swine , Catheterization/methods , Heart Failure/surgery , Lung , Arteries , Extracorporeal Membrane Oxygenation/methodsABSTRACT
PURPOSE: Bone biopsies are currently conducted under computed tomography (CT) guidance using a battery-powered drill to obtain tissue samples for diagnosis of suspicious bone lesions. However, this procedure is suboptimal as images produced under CT lack soft tissue discrimination and involve ionizing radiation. Therefore, our team developed an MRI-safe pneumatic drill to translate this clinical workflow into the MR environment, which can improve target visualization and eliminate radiation exposure. We compare drill times and quality of samples between the 2 drills using animal bones. METHODS: Five porcine spare rib bones were obtained from a butcher shop. Each bone was drilled twice using the Arrow OnControl battery-powered drill and twice using our pneumatically actuated drill. For this study, we used an 11-gauge bone biopsy needle set with an internal core capturing thread. A stopwatch recorded the overall time of drilling for each specimen obtained. RESULTS: All 20 samples collected contained a high-quality inner core and cortex. The total average time for drilling with the pneumatic drill was 8.5 s (+ / - 2.5 s) and 7.1 s (+ / - 1.4 s) with the standard battery-powered drill. CONCLUSION: Both drills worked well and were able to obtain comparable specimens. The pneumatic drill took slightly longer, 1.39 s on average, but this extra time would not be significant in clinical practice. We plan to use the pneumatic drill to enable MRI-safe bone biopsy for musculoskeletal lesions. Biopsy under MRI would provide excellent lesion visualization with no ionizing radiation.
Subject(s)
Bone Diseases , Bone and Bones , Humans , Bone and Bones/diagnostic imaging , Bone and Bones/surgery , Bone and Bones/pathology , Biopsy/methods , Tomography, X-Ray Computed , Magnetic Resonance ImagingABSTRACT
Severe COVID-associated lung injury is a major confounding factor of hospitalizations and death with no effective treatments. Here, we describe a non-classical fibrin clotting mechanism mediated by SARS-CoV-2 infected primary lung but not other susceptible epithelial cells. This infection-induced fibrin formation is observed in all variants of SARS-CoV-2 infections, and requires thrombin but is independent of tissue factor and other classical plasma coagulation factors. While prothrombin and fibrinogen levels are elevated in acute COVID BALF samples, fibrin clotting occurs only with the presence of viral infected but not uninfected lung epithelial cells. We suggest a viral-induced coagulation mechanism, in which prothrombin is activated by infection-induced transmembrane serine proteases, such as ST14 and TMPRSS11D, on NHBE cells. Our finding reveals the inefficiency of current plasma targeted anticoagulation therapy and suggests the need to develop a viral-induced ARDS animal model for treating respiratory airways with thrombin inhibitors.
Subject(s)
COVID-19 , Animals , Humans , SARS-CoV-2 , Thrombin , Prothrombin , Lung , Epithelial Cells , FibrinABSTRACT
Centromeres are genomic regions that coordinate accurate chromosomal segregation during mitosis and meiosis. Yet, despite their essential function, centromeres evolve rapidly across eukaryotes. Centromeres are often the sites of chromosomal breaks which contribute to genome shuffling and promote speciation by inhibiting gene flow. How centromeres form in strongly host-adapted fungal pathogens has yet to be investigated. Here, we characterized the centromere structures in closely related species of mammalian-specific pathogens of the fungal phylum of Ascomycota. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of Schizosaccharomyces pombe. Using organisms from a short-term in vitro culture or infected animal models and ChIP-seq, we identified centromeres in three Pneumocystis species that diverged ~100 million years ago. Each species has a unique short regional centromere (< 10kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. CENP-C, a scaffold protein that links the inner centromere to the kinetochore appears dispensable in one species, suggesting a kinetochore rewiring. Despite the loss of DNA methyltransferases, 5-methylcytosine DNA methylation occurs in these species, though not related to centromere function. These features suggest an epigenetic specification of centromere function.
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Mosquito salivary proteins play a crucial role in regulating hemostatic responses at the bite site during blood feeding. In this study, we investigate the function of Anopheles gambiae salivary apyrase (AgApyrase) in Plasmodium transmission. Our results demonstrate that salivary apyrase interacts with and activates tissue plasminogen activator, facilitating the conversion of plasminogen to plasmin, a human protein previously shown to be required for Plasmodium transmission. Microscopy imaging shows that mosquitoes ingest a substantial amount of apyrase during blood feeding which reduces coagulation in the blood meal by enhancing fibrin degradation and inhibiting platelet aggregation. Supplementation of Plasmodium infected blood with apyrase significantly enhanced Plasmodium infection in the mosquito midgut. In contrast, AgApyrase immunization inhibited Plasmodium mosquito infection and sporozoite transmission. This study highlights a pivotal role for mosquito salivary apyrase for regulation of hemostasis in the mosquito blood meal and for Plasmodium transmission to mosquitoes and to the mammal host, underscoring the potential for new strategies to prevent malaria transmission.
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PURPOSE: Surgical data science is an emerging field focused on quantitative analysis of pre-, intra-, and postoperative patient data (Maier-Hein et al. in Med Image Anal 76: 102306, 2022). Data science approaches can decompose complex procedures, train surgical novices, assess outcomes of actions, and create predictive models of surgical outcomes (Marcus et al. in Pituitary 24: 839-853, 2021; Røadsch et al. in Nat Mach Intell, 2022). Surgical videos contain powerful signals of events that may impact patient outcomes. A necessary step before the deployment of supervised machine learning methods is the development of labels for objects and anatomy. We describe a complete method for annotating videos of transsphenoidal surgery. METHODS: Endoscopic video recordings of transsphenoidal pituitary tumor removal surgeries were collected from a multicenter research collaborative. These videos were anonymized and stored in a cloud-based platform. Videos were uploaded to an online annotation platform. Annotation framework was developed based on a literature review and surgical observations to ensure proper understanding of the tools, anatomy, and steps present. A user guide was developed to trained annotators to ensure standardization. RESULTS: A fully annotated video of a transsphenoidal pituitary tumor removal surgery was produced. This annotated video included over 129,826 frames. To prevent any missing annotations, all frames were later reviewed by highly experienced annotators and a surgeon reviewer. Iterations to annotated videos allowed for the creation of an annotated video complete with labeled surgical tools, anatomy, and phases. In addition, a user guide was developed for the training of novice annotators, which provides information about the annotation software to ensure the production of standardized annotations. CONCLUSIONS: A standardized and reproducible workflow for managing surgical video data is a necessary prerequisite to surgical data science applications. We developed a standard methodology for annotating surgical videos that may facilitate the quantitative analysis of videos using machine learning applications. Future work will demonstrate the clinical relevance and impact of this workflow by developing process modeling and outcome predictors.
Subject(s)
Algorithms , Pituitary Neoplasms , Humans , Supervised Machine Learning , Endoscopy , Machine Learning , Multicenter Studies as TopicABSTRACT
OBJECTIVE: The natural extension of inpatient-focused neonatal neurocritical care (NNCC) programs is the evaluation of long-term neurodevelopmental outcomes in the same patient population. CLINICAL DESIGN: A dedicated and collaborative team of neonatologists, neonatal neurologists, neuropsychologists, neurosurgeons, physical medicine and rehabilitation physicians, and psychologists are necessary to provide personalized medicine, developmental assessments, and parental education for NNCC graduates. To achieve this goal, we devised a two-clinic follow-up model at Children's Wisconsin: HOPE (Healthy Outcomes Post-ICU Engagement) and DREAM: Developmentally Ready: Engagement for Achievement of Milestones) clinics. Those infants with significant neurologic diagnoses attend DREAM clinic, while all other high-risk neonatal intensive care unit (NICU) infants are seen in the HOPE clinic. CONCLUSION: These clinic models allow for a targeted approach to post-NICU care, which has improved family engagement and perceptions of value. KEY POINTS: · Infants with neurologic compromise are a specialized population with increasing survival.. · Interdisciplinary NICU follow-up brings together previously separated outpatient service lines.. · Our novel clinic model allows for specialized developmental assessments..
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HIV infection remains incurable to date and there are no compounds targeted at the viral release. We show here HIV viral release is not spontaneous, rather requires caspases activation and shedding of its adhesion receptor, CD62L. Blocking the caspases activation caused virion tethering by CD62L and the release of deficient viruses. Not only productive experimental HIV infections require caspases activation for viral release, HIV release from both viremic and aviremic patient-derived CD4 T cells also require caspase activation, suggesting HIV release from cellular viral reservoirs depends on apoptotic shedding of the adhesion receptor. Further transcriptomic analysis of HIV infected CD4 T cells showed a direct contribution of HIV accessory gene Nef to apoptotic caspases activation. Current HIV cure focuses on the elimination of latent cellular HIV reservoirs that are resistant to infection-induced cell death. This has led to therapeutic strategies to stimulate T cell apoptosis in a "kick and kill" approach. Our current work has shifted the paradigm on HIV-induced apoptosis and suggests such approach would risk to induce HIV release and thus be counter-productive. Instead, our study supports targeting of viral reservoir release by inhibiting of caspases activation.
Subject(s)
HIV Infections , HIV Seropositivity , HIV-1 , nef Gene Products, Human Immunodeficiency Virus , Humans , Caspases/metabolism , CD4-Positive T-Lymphocytes/metabolism , Cell Death , HIV Infections/drug therapy , HIV-1/genetics , nef Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Allergic diseases are a global health challenge. Individuals harboring loss-of-function variants in transforming growth factor-ß receptor (TGFßR) genes have an increased prevalence of allergic disorders, including eosinophilic esophagitis. Allergic diseases typically localize to mucosal barriers, implicating epithelial dysfunction as a cardinal feature of allergic disease. Here, we describe an essential role for TGFß in the control of tissue-specific immune homeostasis that provides mechanistic insight into these clinical associations. Mice expressing a TGFßR1 loss-of-function variant identified in atopic patients spontaneously develop disease that clinically, immunologically, histologically, and transcriptionally recapitulates eosinophilic esophagitis. In vivo and in vitro, TGFßR1 variant-expressing epithelial cells are hyperproliferative, fail to differentiate properly, and overexpress innate proinflammatory mediators, which persist in the absence of lymphocytes or external allergens. Together, our results support the concept that TGFß plays a fundamental, nonredundant, epithelial cell-intrinsic role in controlling tissue-specific allergic inflammation that is independent of its role in adaptive immunity.
Subject(s)
Eosinophilic Esophagitis , Hypersensitivity, Immediate , Animals , Mice , Eosinophilic Esophagitis/genetics , Receptors, Transforming Growth Factor beta/genetics , InflammationABSTRACT
OBJECTIVES: Research and clinical expertise have emphasized the mental health needs of parents and caregivers of medically complex children. Evidence-based interventions are available for adult mental health, including those designed specifically for caregivers caring for children with a variety of health-care needs. This paper describes practical and legal considerations of 3 possible pathways for psychologists to address the needs of caregivers within pediatric hospital settings. METHODS: Literature regarding the mental health needs of caregivers of children with medical conditions, evidence-based interventions, and pediatric subspecialty psychosocial guidelines was reviewed. Relevant legal and ethical obligations for psychologists were also summarized. RESULTS: The mental health needs of caregivers of medically complex children are often high, yet programmatic, institutional, legal, and ethical barriers can limit access to appropriate care. SIGNIFICANCE OF THE RESULTS: Integration of screening and treatment of caregivers' mental health within the pediatric hospital setting is one pathway to addressing caregivers' needs. The development of programs for caregiver mental health screening and treatment within pediatric hospital settings will enhance the well-being of children and families and reduce legal and ethical risks for pediatric psychologists. Consultation with institutional compliance, legal/risk, and medical records departments and the creation of electronic medical records for the caregiver may be useful and practical opportunities for integration.
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BACKGROUND: The gold standard workflow for targeting structures in the brain involves manual path planning. This preoperative manual path planning is very time-intensive and laborious, especially when some outcome measures such as maximum ablation and penetration depth has to be optimised. METHODS: Our novel path planner generates an optimal path which maximises the hippocampus penetration and distance from critical structures using a precomputed cost map and a reward map. RESULTS: The average penetration ratio for 12 cases is 88.13 ± 23.23% for a resolution of 1° and a safety margin of 1 mm. Average run time for the path planner based on 1° resolution was 1.99 ± 0.68 min. CONCLUSIONS: Results show that the algorithm can generate safe and clinically relevant paths with a quantitative representation of the penetration depth and is faster than the average reported time for manual path planning.
Subject(s)
Imaging, Three-Dimensional , Neurosurgical Procedures , Humans , Retrospective Studies , Neurosurgical Procedures/methods , Algorithms , Brain/surgeryABSTRACT
The intestinal mucosa protects the body from physical damage, pathogens, and antigens. However, inflammatory bowel diseases (IBDs) patients suffer from poor mucosal tissue function, including the lack of an effective cellular and/or mucus barrier. We investigated the mucus producing human colonic epithelial cell line HT29-MTX E12 to study its suitability as an in vitro model of cell/mucus barrier adaption during IBD. It was found that the proinflammatory cytokine interferon-gamma (IFN-γ), but not tumor necrosis factor-alpha (TNF-α), reduced cell viability. IFN-γ and TNF-α were found to synergize to decrease barrier function, as measured by trans-epithelial electric resistance (TER) and molecular flux assays. Cells cultured under an air-liquid interface produced an adherent mucus layer, and under these conditions reduced barrier function was found after cytokine exposure. Furthermore, IFN-γ, but not TNF-α treatment, upregulated the IFN-γ receptor 1 (IFNGR1) and TNF-α receptor super family 1A (TNFRSF1A) subunit mRNA in vitro. Co-stimulation resulted in increased mRNA expression of CLDN 2 and 5, two gene known to play a role in epithelial barrier integrity. Analysis of IBD patient samples revealed IFNGR1 and TNFRSF mRNA increased coincidently with guanylate binding protein 1 (GBP1) expression, an indicator of NFkB activity. Lastly, CLDN2 was found at higher levels in IBD patients while HNF4a was suppressed with disease. In conclusion, IFN-γ and TNF-α degrade epithelial/mucus barriers coincident with changes in CLDN gene and cytokine receptor subunit mRNA expression in HT29-MTX E12 cells. These changes largely reflect those observed in IBD patient samples.
Subject(s)
Inflammatory Bowel Diseases , Interferon-gamma , Cytokines/metabolism , HT29 Cells , Humans , Interferon-gamma/metabolism , Interferon-gamma/pharmacology , Intestinal Mucosa/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Cytokine/metabolism , Receptors, Interferon/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factor-alpha/metabolism , Interferon gamma ReceptorABSTRACT
Current vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are administered parenterally and appear to be more protective in the lower versus the upper respiratory tract. Vaccines are needed that directly stimulate immunity in the respiratory tract, as well as systemic immunity. We used avian paramyxovirus type 3 (APMV3) as an intranasal vaccine vector to express the SARS-CoV-2 spike (S) protein. A lack of pre-existing immunity in humans and attenuation by host-range restriction make APMV3 a vector of interest. The SARS-CoV-2 S protein was stabilized in its prefusion conformation by six proline substitutions (S-6P) rather than the two that are used in most vaccine candidates, providing increased stability. APMV3 expressing S-6P (APMV3/S-6P) replicated to high titers in embryonated chicken eggs and was genetically stable, whereas APMV3 expressing non-stabilized S or S-2P were unstable. In hamsters, a single intranasal dose of APMV3/S-6P induced strong serum IgG and IgA responses to the S protein and its receptor-binding domain, and strong serum neutralizing antibody responses to SARS-CoV-2 isolate WA1/2020 (lineage A). Sera from APMV3/S-6P-immunized hamsters also efficiently neutralized Alpha and Beta variants of concern. Immunized hamsters challenged with WA1/2020 did not exhibit the weight loss and lung inflammation observed in empty-vector-immunized controls; SARS-CoV-2 replication in the upper and lower respiratory tract of immunized animals was low or undetectable compared to the substantial replication in controls. Thus, a single intranasal dose of APMV3/S-6P was highly immunogenic and protective against SARS-CoV-2 challenge, suggesting that APMV3/S-6P is suitable for clinical development.
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OBJECTIVE: To describe an electronic parent support tool for the neonatal intensive care unit (NICU), and to assess whether support requests changed with staff availability. METHODS: We implemented secure text- or email-based parent support in the NICU and in the week after discharge. Questionnaires asked whether a parent would like psychology, social work, child life, chaplain, or post-discharge nurse support. Requested referrals were placed, and customized online resources and contacts were provided. We assessed whether requests changed based on in-person resource availability. RESULTS: Of 378 infants in our NICU from May to December, 202 parents agreed to participate. The proportion agreeing to participate increased over time (38-59%, p = 0.012). Post-discharge nurse requests decreased over time (90-45%, p = 0.033); other requests did not change significantly. CONCLUSIONS: An electronic tool increased parent support availability in the NICU and following discharge, even after staff were available at the bedside.