A tough bioadhesive hydrogel supports sutureless sealing of the dural membrane in porcine and ex vivo human tissue.

Complete sequestration of central nervous system tissue and cerebrospinal fluid by the dural membrane is fundamental to maintaining homeostasis and proper organ function, making reconstruction of this layer an essential step during neurosurgery. Primary closure of the dura by suture repair is the current standard, despite facing technical, microenvironmental, and anatomic challenges. Here, we apply a mechanically tough hydrogel paired with a bioadhesive for intraoperative sealing of the dural membrane in rodent, porcine, and human central nervous system tissue. Tensile testing demonstrated that this dural tough adhesive (DTA) exhibited greater toughness with higher maximum stress and stretch compared with commercial sealants in aqueous environments. To evaluate the performance of DTA in the range of intracranial pressure typical of healthy and disease states, ex vivo burst pressure testing was conducted until failure after DTA or commercial sealant application on ex vivo porcine dura with a punch biopsy injury. In contrast to commercial sealants, DTA remained adhered to the porcine dura through increasing pressure up to 300 millimeters of mercury and achieved a greater maximum burst pressure. Feasibility of DTA to repair cerebrospinal fluid leak in a simulated surgical context was evaluated in postmortem human dural tissue. DTA supported effective sutureless repair of the porcine thecal sac in vivo. Biocompatibility and adhesion of DTA was maintained for up to 4 weeks in rodents after implantation. The findings suggest the potential of DTA to augment or perhaps even supplant suture repair and warrant further exploration.

A Drosophila model relevant to chemotherapy-related cognitive impairment.

Chemotherapy-related cognitive impairment (CRCI) is a common adverse effect of treatment and is characterized by deficits involving multiple cognitive domains including memory. Despite the significant morbidity of CRCI and the expected increase in cancer survivors over the coming decades, the pathophysiology of CRCI remains incompletely understood, highlighting the need for new model systems to study CRCI. Given the powerful array of genetic approaches and facile high throughput screening ability in Drosophila, our goal was to validate a Drosophila model relevant to CRCI. We administered the chemotherapeutic agents cisplatin, cyclophosphamide, and doxorubicin to adult Drosophila. Neurologic deficits were observed with all tested chemotherapies, with doxorubicin and in particular cisplatin also resulting in memory deficits. We then performed histologic and immunohistochemical analysis of cisplatin-treated Drosophila tissue, demonstrating neuropathologic evidence of increased neurodegeneration, DNA damage, and oxidative stress. Thus, our Drosophila model relevant to CRCI recapitulates clinical, radiologic, and histologic alterations reported in chemotherapy patients. Our new Drosophila model can be used for mechanistic dissection of pathways contributing to CRCI (and chemotherapy-induced neurotoxicity more generally) and pharmacologic screens to identify disease-modifying therapies.

Methyl-Branched Liposomes as a Depot for Sustained Drug Delivery.

Inadequate drug loading and control of payload leakage limit the duration of the effect of liposomal drug carriers and may cause toxicity. Here, we report a liposome system as a depot for sustained drug delivery whose design is inspired by the low permeability of Archaeal membranes to protons and solutes. Incorporating methyl-branched phospholipids into lipid bilayers decreased payload diffusion across liposomal membranes, thereby enhancing the drug load capacity by 10-16% and reducing the release of small molecules in the first 24 h by 40-48%. The in vivo impact of this approach was demonstrated by injection at the sciatic nerve. Methyl-branched liposomes encapsulating the anesthetic tetrodotoxin (TTX) achieved markedly prolonged local anesthesia lasting up to 70 h, in comparison to the 16 h achieved with liposomes containing conventional lipids. The present work demonstrates the usefulness of methyl-branched liposomes to enhance liposomal depot systems for sustained drug delivery.

Aromatized liposomes for sustained drug delivery.

Insufficient drug loading and leakage of payload remain major challenges in designing liposome-based drug delivery systems. These phenomena can limit duration of effect and cause toxicity. Targeting the rate-limiting step in drug release from liposomes, we modify (aromatized) them to have aromatic groups within their lipid bilayers. Aromatized liposomes are designed with synthetic phospholipids with aromatic groups covalently conjugated onto acyl chains. The optimized aromatized liposome increases drug loading and significantly decreases the burst release of a broad range of payloads (small molecules and macromolecules, different degrees of hydrophilicity) and extends their duration of release. Aromatized liposomes encapsulating the anesthetic tetrodotoxin (TTX) achieve markedly prolonged effect and decreased toxicity in an application where liposomes are used clinically: local anesthesia, even though TTX is a hydrophilic small molecule which is typically difficult to encapsulate. Aromatization of lipid bilayers can improve the performance of liposomal drug delivery systems.

A Drosophila model of chemotherapy-related cognitive impairment.

Chemotherapy-related cognitive impairment (CRCI) is a common adverse effect of treatment and is characterized by deficits involving multiple cognitive domains including memory. Despite the significant morbidity of CRCI and the expected increase in cancer survivors over the coming decades, the pathophysiology of CRCI remains incompletely understood, highlighting the need for new model systems to study CRCI. Given the powerful array of genetic approaches and facile high throughput screening ability in Drosophila, our goal was to validate a Drosophila model of CRCI. We administered the chemotherapeutic agents cisplatin, cyclophosphamide, and doxorubicin to adult Drosophila. Neurocognitive deficits were observed with all tested chemotherapies, especially cisplatin. We then performed histologic and immunohistochemical analysis of cisplatin-treated Drosophila tissue, demonstrating neuropathologic evidence of increased neurodegeneration, DNA damage, and oxidative stress. Thus, our Drosophila model of CRCI recapitulates clinical, radiologic, and histologic alterations reported in chemotherapy patients. Our new Drosophila model can be used for mechanistic dissection of pathways contributing to CRCI and pharmacologic screens to identify novel therapies to ameliorate CRCI.

A Novel Approach to Improving Post-Operative Pain and Minimizing Opioid Consumption After a Hip Arthroscopy.

Background: There have been many different approaches to controlling pain in patients undergoing hip arthroscopy. These include medications, nerve blocks, and intra-articular injections among many others. We introduced a combination of a pre-operative pericapsular nerve group (PENG) block, and intra-operative pericapsular injection of BKK (bupivacaine, ketamine, and ketorolac). Methods: Patients undergoing primary hip arthroscopy were identified. There were three patient cohorts based on type of anesthesia: general anesthesia only (GA), general anesthesia and a pericapsular Marcaine injection (GA/Marcaine), or GA with pre-operative PENG block and an intraoperative BKK pericapsular injection (GA+PENG/BKK). Data collected included post-operative pain scores in the PACU (Post-Anesthesia Care Unit), time spent in the PACU, inpatient opioid consumption (both PACU and inpatient), and outpatient opioid prescriptions filled. Results: 20 patients received GA, 11 patients received GA/Marcaine, and 20 patients received GA+PENG/BKK. The GA+PENG/BKK group had average PACU pain score of 3.9 out of 10 compared to 7.7 in the GA group (p<.001) and 6.6 in the GA/Marcaine injection group (p=.048). The GA+PENG/BKK group had shorter mean PACU times than either other group (p<.001). The GA+PENG/BKK also consumed less opioids than the GA or GA/Marcaine groups in the PACU (p<.001), and in the total inpatient stay (p=.002, p=.003), as well as outpatient (p=.019, p=.040). Conclusion: In patients undergoing a hip arthroscopy, performing a pre-operative PENG block and intra-operative BKK pericapsular injection will result in decreased postoperative pain, PACU time, and inpatient and outpatient opioids compared to general anesthesia only and general anesthesia with intracapsular Marcaine.

An aptamer-based depot system for sustained release of small molecule therapeutics.

Delivery of hydrophilic small molecule therapeutics by traditional drug delivery systems is challenging. Herein, we have used the specific interaction between DNA aptamers and drugs to create simple and effective drug depot systems. The specific binding of a phosphorothioate-modified aptamer to drugs formed non-covalent aptamer/drug complexes, which created a sustained release system. We demonstrated the effectiveness of this system with small hydrophilic molecules, the site 1 sodium channel blockers tetrodotoxin and saxitoxin. The aptamer-based delivery system greatly prolonged the duration of local anesthesia and reduced systemic toxicity. The beneficial effects of the aptamers were restricted to the compounds they were specific to. These studies establish aptamers as a class of highly specific, modifiable drug delivery systems, and demonstrate potential usefulness in the management of postoperative pain.

The predictive value of partial MGMT promoter methylation for IDH-wild-type glioblastoma patients.

Background: Glioblastoma patients with hypermethylation of the O6-methylguanine-methyltransferase (MGMT) gene promoter have significantly improved survival when treated with temozolomide compared to patients with unmethylation of the MGMT promoter. However, the prognostic and predictive significance of partial MGMT promoter methylation is unclear. Methods: The National Cancer Database was queried for patients newly diagnosed in 2018 with histopathologically confirmed isocitrate dehydrogenase (IDH)-wildtype glioblastoma. The overall survival (OS) associated with MGMT promoter methylation status was assessed using multivariable Cox regression with Bonferroni correction for multiple testing (P < .008 was significant). Results: Three thousand eight hundred twenty-five newly diagnosed IDH-wildtype glioblastoma patients were identified. The MGMT promoter was unmethylated in 58.7% (n = 2245), partially methylated in 4.8% (n = 183), hypermethylated in 3.5% (n = 133), and methylated not otherwise specified (NOS; likely consisting predominantly of hypermethylated cases) in 33.0% (n = 1264) of cases. Among patients that received first-line single-agent chemotherapy (ie likely temozolomide), compared to partial methylation (referent), MGMT promoter unmethylation was associated with worse OS (hazard ratio [HR] 1.94; 95% confidence interval [95 CI]: 1.54-2.44; P < .001) in multivariable Cox regression adjusted for major prognostic confounders. In contrast, a significant OS difference was not observed between partially methylated promoters and either hypermethylated (HR 1.02; 95 CI: 0.72-1.46; P = .90) or methylated NOS (HR 0.99; 95 CI: 0.78-1.26; P = .93) promoters. Among IDH-wildtype glioblastoma patients who did not receive first-line chemotherapy, MGMT promoter methylation status was not associated with significant differences in OS (P = 0.39-0.83). Conclusions: Compared to MGMT promoter unmethylation, partial methylation was predictive of improved OS among IDH-wildtype glioblastoma patients treated with first-line single-agent chemotherapy-supporting the use of temozolomide therapy in these patients.

Revisiting a Telencephalic Extent of the Ascending Reticular Activating System.

Is the cerebrum involved in its own activation to states of attention or arousal? "Telencephalon" is a term borrowed from embryology to identify not only the cerebral hemispheres of the forebrain, but also the basal forebrain. We review a generally undercited literature that describes nucleus basalis of Meynert, located within the substantia innominata of the ventrobasal forebrain, as a telencephalic extension of the ascending reticular activating formation. Although that formation's precise anatomical definition and localization have proven elusive over more than 70 years, a careful reading of sources reveals that there are histological features common to certain brainstem neurons and those of the nucleus basalis, and that a largely common dendritic architecture may be a morphological aspect that helps to define non-telencephalic structures of the ascending reticular activating formation (e.g., in brainstem) as well as those parts of the formation that are telencephalic and themselves responsible for cortical activation. We draw attention to a pattern of dendritic arborization described as "isodendritic," a uniform (isos-) branching in which distal dendrite branches are significantly longer than proximal ones. Isodendritic neurons also differ from other morphological types based on their heterogeneous, rather than specific afferentation. References reviewed here are consistent in their descriptions of histology, particularly in studies of locales rich in cholinergic neurons. We discuss the therapeutic implications of a basal forebrain site that may activate cortex. Interventions that specifically target nucleus basalis and, especially, the survival of its constituent neurons may benefit afflictions in which higher cortical function is compromised due to disturbed arousal or attentiveness, including not only coma and related syndromes, but also conditions colloquially described as states of cognitive "fog" or of "long-haul" mental compromise.

Focused Ultrasound Thalamotomy: Correlation of Postoperative Imaging with Neuropathological Findings.

Magnetic resonance-guided high-intensity focused ultrasound (MRgFUS) is a rapidly developing technique used for tremor relief in tremor-predominant Parkinson's disease (PD) and essential tremor that has demonstrated successful results. Here, we describe the neuropathological findings in a woman who died from a fall 10 days after successful MRgFUS for tremor-predominant PD. Histological analysis demonstrates the characteristic early postoperative MRI findings including 3 distinct zones on T2-weighted imaging: (1) a hypointense core, (2) a hyperintense region with hypointense rim, and (3) a slightly hyperintense, poorly marginated surrounding area. Histopathological analyses also demonstrate the suspected cellular processes composing each of these regions including central hemorrhagic necrosis with surrounding cytotoxic edema and a rim of mostly unaffected vasogenic edema with some reactive and reparative processes. Overall, this case demonstrates the correlation of postoperative imaging findings with the subacute neuropathological findings after MRgFUS for PD.

Benefits of Implementing an Enhanced Recovery After Surgery Protocol in Ambulatory Surgery.

Background: Enhanced recovery after surgery (ERAS) protocols in orthopaedic surgery have garnered significant focus due to their ability to control pain adequately in the immediate postoperative window, allowing for earlier mobilization, shorter hospital stays, and fewer complications. Virginia Commonwealth University created a multimodal pain management approach in which patients receive a preoperative femoral nerve block followed by periarticular intraoperative local injection anesthesia consisting of bupivacaine, ketamine, and ketorolac. Hypothesis: We hypothesized that implementation of the ERAS protocol will decrease postoperative pain scores, decrease recovery time in the postanesthesia care unit (PACU), and decrease opioid use in anterior cruciate ligament (ACL) reconstruction. Study Design: Cohort study; Level of evidence, 3. Methods: Two patient cohorts were involved: before ERAS implementation (pre-ERAS) and after (post-ERAS). Patients with ACL reconstruction only and patients with ACL reconstruction with meniscal repair were analyzed separately. Post-ERAS patients received an intraoperative periarticular injection of bupivacaine, ketamine, and ketorolac and a postoperative multimodal pain regimen. Outcomes included time spent in the PACU, short-term and long-term opioid consumption, and pain score at discharge from the PACU. Results: Compared with pre-ERAS patients, post-ERAS patients had decreased pain (2.1 vs 0.84 out of 10, respectively), spent less time in the PACU (79.4 vs 62.8 minutes, respectively), and had less opioid consumption in the immediate postoperative period (4.55 vs 2.26 total morphine milligram equivalents [MMEs], respectively) (P < .001 for all). After ERAS implementation, long-term MME use decreased from 410 to 321 between 0 and 2 weeks postoperatively, 92.6 to 1.69 between 2 and 6 weeks, and 494.5 to 323 between 0 and 6 weeks (P < .001 for all). All domains showed significant improvements for both the ACL and the ACL plus meniscal repair cohorts, with the exception of pain at discharge in the ACL plus meniscal repair group. Conclusion: The study findings suggest that an enhanced recovery pathways protocol that includes a standardized intraoperative periarticular bupivacaine, ketamine, and ketorolac injection improves pain scores in the immediate postoperative window, decreases opioid consumption, and reduces recovery time in the PACU for patients undergoing ACL reconstruction.

The epidemiology of primary and metastatic brain tumors in infancy through childhood.

PURPOSE: To evaluate the epidemiology of primary and metastatic pediatric brain tumors in the United States according to the WHO CNS 4th and 5th editions classifications. METHODS: Pediatric patients (age ≤ 14) presenting between 2004 and 2017 with a brain tumor were identified in the National Cancer Database and categorized by NICHD age stages. Patients' age, sex, race/ethnicity, overall survival, and tumor characteristics were evaluated according to WHO CNS 4th and 5th editions. RESULTS: 23,978 pediatric brain tumor patients were identified. Overall, other (i.e. circumscribed) astrocytic gliomas (21%), diffuse astrocytic/oligodendroglial gliomas (21%; 64% of which were midline), and embryonal tumors (16%) predominated. A minority of brain tumors were of ependymal (6%), glioneuronal & neuronal (6%), germ cell tumor (GCT; 4%), mesenchymal non-meningothelial (2%), cranial nerve (2%), choroid plexus (2%), meningioma (2%), pineal (1%), and hematolymphoid (0.4%) types. GCTs were more likely in patients of Asian/Pacific Islander race/ethnicity. Brain metastases were exceedingly rare, accounting for 1.4% overall, with the most common primary tumor being neuroblastoma (61%) and non-CNS sarcoma (16%). Brain metastatic, choroid plexus, and embryonal tumors peaked during infancy and toddlerhood; whereas diffuse gliomas peaked in middle-late childhood. GCTs and glioneuronal & neuronal tumors uniquely displayed bimodal distributions, with elevated prevalence in both infancy and middle-to-late childhood. CONCLUSION: We systematically described the epidemiology of pediatric brain tumors in the context of contemporary classification schema, thereby validating our current understanding and providing key insights.

The phenomenon of clasmatodendrosis.

Clasmatodendrosis derives from the Greek for fragment (klasma), tree (dendron), and condition (- osis). Cajal first used the term in 1913: he observed disintegration of the distal cell processes of astrocytes, along with a fragmentation or beading of proximal processes closer to the astrocyte cell body. In contemporary clinical and experimental reports, clasmatodendrosis has been observed in models of cerebral ischemia and seizures (including status epilepticus), in elderly brains, in white matter disease, in hippocampal models and cell cultures associated with amyloid plaques, in head trauma, toxic exposures, demyelinating diseases, encephalitides and infection-associated encephalopathies, and in the treatment of cancer using immune effector cells. We examine evidence to support a claim that clasmatodendrotic astrocyte cell processes overtly bead (truncate) as a morphological sign of ongoing damage premortem. In grey and white matter and often in relationship to vascular lumina, beading becomes apparent with immunohistochemical staining of glial fibrillary acidic protein when specimens are examined at reasonably high magnification, but demonstration of distal astrocytic loss of processes may require additional marker study and imaging. Proposed mechanisms for clasmatodendrotic change have examined hypoxic-ischemic, osmotic-demyelinating, and autophagic models. In these models as well as in neuropathological reports, parenchymal swelling, vessel-wall leakage, or disturbed clearance of toxins can occur in association with clasmatodendrosis. Clasmatodendrotic features may serve as a marker for gliovascular dysregulation either acutely or chronically. We review correlative evidence for blood-brain barrier (BBB) dysfunction associated with astrocytic structural change, with attention to interactions between endothelial cells, pericytes, and astrocytic endfeet.

Elevated Oxidative Stress and DNA Damage in Cortical Neurons of Chemotherapy Patients.

The unintended neurologic sequelae of chemotherapy contribute to significant patient morbidity. Chemotherapy-related cognitive impairment (CRCI) is observed in up to 80% of cancer patients treated with chemotherapy and involves multiple cognitive domains including executive functioning. The pathophysiology underlying CRCI and the neurotoxicity of chemotherapy is incompletely understood, but oxidative stress and DNA damage are highly plausible mechanisms based on preclinical data. Unfortunately, validating pathways relevant to CRCI in humans is limited by an absence of relevant neuropathologic studies of patient brain tissue. In the present study, we stained sections of frontal lobe autopsy tissue from cancer patients treated with chemotherapy (n = 15), cancer patients not treated with chemotherapy (n = 10), and patients without history of cancer (n = 10) for markers of oxidative stress (nitrotyrosine, 4-hydroxynonenal) and DNA damage (pH2AX, pATM). Cancer patients treated with chemotherapy had increased staining for markers of oxidative stress and DNA damage in frontal lobe cortical neurons compared to controls. We detected no statistically significant difference in oxidative stress and DNA damage by the duration between last administration of chemotherapy and death. The study highlights the potential relevance of oxidative stress and DNA damage in the pathophysiology of CRCI and the neurotoxicity of chemotherapy.

IDH-mutant gliomas with additional class-defining molecular events.

The 2016 WHO classifies IDH-mutant gliomas into oligodendroglioma or diffuse astrocytoma based on co-occurring genetic events. Recent literature addresses the concept of stratifying IDH-mutant gliomas based on prognostically significant molecular events. However, the presence of a second class-defining driver alteration in IDH-mutant gliomas has not been systematically described. We searched the sequencing database at our institutions as well as The Cancer Genome Atlas (TCGA) and cBioPortal for IDH-mutant gliomas with other potentially significant alterations. For each case, we reviewed the clinical information, histology and genetic profile. Of 1702 gliomas tested on our targeted exome sequencing panel, we identified 364 IDH-mutated gliomas, four of which had pathogenic FGFR alterations and one with BRAF V600E mutation. Five additional IDH-mutant gliomas with NTRK fusions were identified through collaboration with an outside institution. Also, a search in the glioma database in cBioPortal (5379 total glioma samples, 1515 cases [28.1%] with IDH1/2 mutation) revealed eight IDH-mutated gliomas with FGFR, NTRK or BRAF pathogenic alterations. All IDH-mutant gliomas with dual mutations identified were hemispheric and had a mean age at diagnosis of 36.2 years (range 16-55 years old). Co-occurring genetic events involved MYCN, RB and PTEN. Notable outcomes included a patient with an IDH1/FGFR1-mutated anaplastic oligodendroglioma who has survived 20 years after diagnosis. We describe a series of 18 IDH-mutant gliomas with co-occurring genetic events that have been described as independent class-defining drivers in other gliomas. While these tumors are rare and the significance of these alterations needs further exploration, alterations in FGFR, NTRK, and BRAF could have potential therapeutic implications and affect clinical trial design and results in IDH-mutant studies. Our data highlights that single gene testing for IDH1 in diffuse gliomas may be insufficient for detection of targets with potential important prognostic and treatment value.

Expanded Clinical Phenotype, Oncological Associations, and Immunopathologic Insights of Paraneoplastic Kelch-like Protein-11 Encephalitis.

Importance: Recognizing the presenting and immunopathological features of Kelch-like protein-11 immunoglobulin G seropositive (KLHL11 IgG+) patients may aid in early diagnosis and management. Objective: To describe expanding neurologic phenotype, cancer associations, outcomes, and immunopathologic features of KLHL11 encephalitis. Design, Setting, and Participants: This retrospective tertiary care center study, conducted from October 15, 1998, to November 1, 2019, prospectively identified 31 KLHL11 IgG+ cases in the neuroimmunology laboratory. Eight were identified by retrospective testing of patients with rhomboencephalitis (confirmed by tissue-based-immunofluorescence and transfected-cell-based assays). Main Outcomes and Measures: Outcome variables included modified Rankin score and gait aid use. Results: All 39 KLHL11 IgG+ patients were men (median age, 46 years; range, 28-73 years). Initial clinical presentations were ataxia (n = 32; 82%), diplopia (n = 22; 56%), vertigo (n = 21; 54%), hearing loss (n = 15; 39%), tinnitus (n = 14; 36%), dysarthria (n = 11; 28%), and seizures (n = 9; 23%). Atypical neurologic presentations included neuropsychiatric dysfunction, myeloneuropathy, and cervical amyotrophy. Hearing loss or tinnitus preceded other neurologic deficits by 1 to 8 months in 10 patients (26%). Among patients screened for malignancy (n = 36), testicular germ-cell tumors (n = 23; 64%) or testicular microlithiasis and fibrosis concerning for regressed germ cell tumor (n = 7; 19%) were found in 83% of the patients (n = 30). In 2 patients, lymph node biopsy diagnosed metastatic lung adenocarcinoma in one and chronic lymphocytic leukemia in the other. Initial brain magnetic resonance imaging revealed T2 hyperintensities in the temporal lobe (n = 12), cerebellum (n = 9), brainstem (n = 3), or diencephalon (n = 3). Among KLHL11 IgG+ patients who underwent HLA class I and class II genotyping (n = 10), most were found to have HLA-DQB1*02:01 (n = 7; 70%) and HLA-DRB1*03:01 (n = 6; 60%) associations. A biopsied gadolinium-enhancing temporal lobe lesion demonstrated T cell-predominant inflammation and nonnecrotizing granulomas. Cerebellar biopsy (patient with chronic ataxia) and 2 autopsied brains demonstrated Purkinje neuronal loss and Bergmann gliosis, supporting early active inflammation and later extensive neuronal loss. Compared with nonautoimmune control peripheral blood mononuclear cells, cluster of differentiation (CD) 8+ and CD4+ T cells were significantly activated when patient peripheral blood mononuclear cells were cultured with KLHL11 protein. Most patients (58%) benefitted from immunotherapy and/or cancer treatment (neurological disability stabilized [n = 10] or improved [n = 9]). Kaplan-Meier curve demonstrated significantly higher probability of wheelchair dependence among patients without detectable testicular cancer. Long-term outcomes in KLHL11-IgG+ patients were similar to Ma2 encephalitis. Conclusions and Relevance: Kelch-like protein-11 IgG is a biomarker of testicular germ-cell tumor and paraneoplastic neurologic syndrome, often refractory to treatment. Described expanded neurologic phenotype and paraclinical findings may aid in its early diagnosis and treatment.