Genetic and epigenetic instability as an underlying driver of progression and aggressive behavior in IDH-mutant astrocytoma.

In recent years, the classification of adult-type diffuse gliomas has undergone a revolution, wherein specific molecular features now represent defining diagnostic criteria of IDH-wild-type glioblastomas, IDH-mutant astrocytomas, and IDH-mutant 1p/19q-codeleted oligodendrogliomas. With the introduction of the 2021 WHO CNS classification, additional molecular alterations are now integrated into the grading of these tumors, given equal weight to traditional histologic features. However, there remains a great deal of heterogeneity in patient outcome even within these established tumor subclassifications that is unexplained by currently codified molecular alterations, particularly in the IDH-mutant astrocytoma category. There is also significant intercellular genetic and epigenetic heterogeneity and plasticity with resulting phenotypic heterogeneity, making these tumors remarkably adaptable and robust, and presenting a significant barrier to the design of effective therapeutics. Herein, we review the mechanisms and consequences of genetic and epigenetic instability, including chromosomal instability (CIN), microsatellite instability (MSI)/mismatch repair (MMR) deficits, and epigenetic instability, in the underlying biology, tumorigenesis, and progression of IDH-mutant astrocytomas. We also discuss the contribution of recent high-resolution transcriptomics studies toward defining tumor heterogeneity with single-cell resolution. While intratumoral heterogeneity is a well-known feature of diffuse gliomas, the contribution of these various processes has only recently been considered as a potential driver of tumor aggressiveness. CIN has an independent, adverse effect on patient survival, similar to the effect of histologic grade and homozygous CDKN2A deletion, while MMR mutation is only associated with poor overall survival in univariate analysis but is highly correlated with higher histologic/molecular grade and other aggressive features. These forms of genomic instability, which may significantly affect the natural progression of these tumors, response to therapy, and ultimately clinical outcome for patients, are potentially measurable features which could aid in diagnosis, grading, prognosis, and development of personalized therapeutics.

Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding.

Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here, we present an ExM variant, decrowding expansion pathology (dExPath), that can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and, perhaps, diagnosis.

CDKN2A mutations have equivalent prognostic significance to homozygous deletion in IDH-mutant astrocytoma.

Homozygous deletion of CDKN2A/B is currently considered a molecular signature for grade 4 in IDH-mutant astrocytomas, irrespective of tumor histomorphology. The 2021 WHO Classification of CNS Tumors does not currently include grading recommendations for histologically lower-grade (grade 2-3) IDH-mutant astrocytoma with CDKN2A mutation or other CDKN2A alterations, and little is currently known about the prognostic implications of these alternative CDKN2A inactivating mechanisms. To address this, we evaluated a cohort of institutional and publicly available IDH-mutant astrocytomas, 15 with pathogenic mutations in CDKN2A, 47 with homozygous CDKN2A deletion, and 401 with retained/wildtype CDKN2A. The IDH-mutant astrocytomas with mutant and deleted CDKN2A had significantly higher overall copy number variation compared to those with retained/wildtype CDKN2A, consistent with more aggressive behavior. Astrocytoma patients with CDKN2A mutation had significantly worse progression-free (p = 0.0025) and overall survival (p < 0.0001) compared to grade-matched patients with wildtype CDKN2A, but statistically equivalent progression-free survival and overall survival outcomes to patients with CDKN2A deletion. No significant survival difference was identified between CDKN2A mutant cases with or without loss of the second allele. These findings suggest that CDKN2A mutation has a detrimental effect on survival in otherwise lower-grade IDH-mutant astrocytomas, similar to homozygous CDKN2A deletion, and should be considered for future grading schemes.

Novel method for augmented reality guided endodontics: An in vitro study.

OBJECTIVE: The aim of this study is to evaluate the accuracy in endodontics of a novel augmented reality (AR) method for guided access cavity preparation in 3D-printed jaws. METHODS: Two operators with different levels of experience in endodontics performed pre-planned virtually guided access cavities through a novel markerless AR system on three sets of 3D-printed jaw models (Objet Connex 350, Stratasys) mounted on a phantom. After the treatment, a post-operative high-resolution CBCT scan (NewTom VGI Evo, Cefla) was taken for each model and registered to the pre-operative model. All the access cavities were then digitally reconstructed by filling the cavity area using 3D medical software (3-Matic 15.0, materialize). For the anterior teeth and the premolars, the deviation at the coronal and apical entry points as well as the angular deviation of the access cavity were compared to the virtual plan. For the molars, the deviation at the coronal entry point was compared to the virtual plan. Additionally, the surface area of all access cavities at the entry point was measured and compared to the virtual plan. Descriptive statistics for each parameter were performed. A 95% confidence interval was calculated. RESULTS: A total of 90 access cavities were drilled up to a depth of 4 mm inside the tooth. The mean deviation in the frontal teeth and in the premolars at the entry point was 0.51 mm and 0.77 mm at the apical point, with a mean angular deviation of 8.5° and a mean surface overlap of 57%. The mean deviation for the molars at the entry point was 0.63 mm, with a mean surface overlap of 82%. CONCLUSION: The use of AR as a digital guide for endodontic access cavity drilling on different teeth showed promising results and might have potential for clinical use. However, further development and research might be needed before in vivo validation.

DNA methylation of the promoter region at the CREB1 binding site is a mechanism for the epigenetic regulation of brain-specific PKMζ.

Protein kinase M zeta, PKMζ, is a brain enriched kinase with a well characterized role in Long-Term Potentiation (LTP), the activity-dependent strengthening of synapses involved in long-term memory formation. However, little is known about the molecular mechanisms that maintain the tissue specificity of this kinase. Here, we characterized the epigenetic factors, mainly DNA methylation, regulating PKMζ expression in the human brain. The PRKCZ gene has an upstream promoter regulating Protein kinase C ζ (PKCζ), and an internal promoter driving PKMζ expression. A demethylated region, including a canonical CREB binding site, situated at the internal promoter was only observed in human CNS tissues. The induction of site-specific hypermethylation of this region resulted in decreased CREB1 binding and downregulation of PKMζ expression. Noteworthy, CREB binding sites were absent in the upstream promoter of PRKCZ locus, suggesting a specific mechanism for regulating PKMζ expression. These observations were validated using a system of human neuronal differentiation from induced pluripotent stem cells (iPSCs). CREB1 binding at the internal promoter was detected only in differentiated neurons, where PKMζ is expressed. The same epigenetic mechanism in the context of CREB binding site was identified in other genes involved in neuronal differentiation and LTP. Additionally, aberrant DNA hypermethylation at the internal promoter was observed in cases of Alzheimer's disease, correlating with decreased expression of PKMζ in patient brains. Altogether, we present a conserved epigenetic mechanism regulating PKMζ expression and other genes enhanced in the CNS with possible implications in neuronal differentiation and Alzheimer's disease.

Regeneration of the Pulp Tissue: Cell Homing versus Cell Transplantation Approach: A Systematic Review.

BACKGROUND: The main objective of this systematic review was to compare the apical healing, root maturation and histological characteristics of teeth treated with cell-based versus cell-free techniques. METHODS: The methodology of this review was based on the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. A literature search strategy was carried out on PubMed, EMBASE and the Web of Science databases. The last search was done on 1 August 2021. Articles written in languages other than English were excluded. Two researchers independently selected the studies and extracted the data. As no randomized clinical trials were available, animal studies were included. RESULTS: In total, 26 studies were included in the systematic review: 22 articles only researched the cell-free technique, 3 articles compared the cell-based to the cell-free technique, and 1 article compared the cell-based technique to apexification. In terms of apical healing, qualitative analysis of the data suggested that there seems to be no significant difference between cell-free and cell-based techniques. The results regarding tooth maturation are contradictory. The main difference between the cell-free and the cell-based techniques seems to be the histology of the treated tooth. The cell-free technique seems to result in cementum-like, bone-like or periodontal ligament-like tissue. One study, on the other hand, found that the cell-based technique resulted in regeneration of the whole pulp with an odontoblast layer, connective tissue, blood vessels and neuronal tissue. CONCLUSIONS: Currently, the number of randomized clinical trials on this topic are very scarce. This is probably due to the limited infrastructure and lack of resources to apply the cell-based technique. Even though both techniques seem to be promising for clinical application, long-term data need to be provided regarding the healing and reparative patterns.

The extracellular matrix protein fibulin-3/EFEMP1 promotes pleural mesothelioma growth by activation of PI3K/Akt signaling.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited therapeutic options. The extracellular matrix protein fibulin-3/EFEMP1 accumulates in the pleural effusions of MPM patients and has been proposed as a prognostic biomarker of these tumors. However, it is entirely unknown whether fibulin-3 plays a functional role on MPM growth and progression. Here, we demonstrate that fibulin-3 is upregulated in MPM tissue, promotes the malignant behavior of MPM cells, and can be targeted to reduce tumor progression. Overexpression of fibulin-3 increased the viability, clonogenic capacity and invasion of mesothelial cells, whereas fibulin-3 knockdown decreased these phenotypic traits as well as chemoresistance in MPM cells. At the molecular level, fibulin-3 activated PI3K/Akt signaling and increased the expression of a PI3K-dependent gene signature associated with cell adhesion, motility, and invasion. These pro-tumoral effects of fibulin-3 on MPM cells were disrupted by PI3K inhibition as well as by a novel, function-blocking, anti-fibulin-3 chimeric antibody. Anti-fibulin-3 antibody therapy tested in two orthotopic models of MPM inhibited fibulin-3 signaling, resulting in decreased tumor cell proliferation, reduced tumor growth, and extended animal survival. Taken together, these results demonstrate for the first time that fibulin-3 is not only a prognostic factor of MPM but also a relevant molecular target in these tumors. Further development of anti-fibulin-3 approaches are proposed to increase early detection and therapeutic impact against MPM.

Alpha2beta1 Integrin Polymorphism in Diffuse Astrocytoma Patients.

Integrins are heterodimeric transmembrane glycoproteins resulting from the non-covalent association of an α and ß chain. The major integrin receptor for collagen/laminin, α2ß1 is expressed on a wide variety of cell types and plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Integrin-triggered signaling pathways promote the invasion and survival of glioma cells by modifying the brain microenvironment. In this study, we investigated the association of a specific genetic polymorphism of integrin α2ß1 with the incidence of diffusely infiltrating astrocytoma and the progression of these tumors. Single-nucleotide polymorphism in intron 7 of the integrin ITGA2 gene was examined in 158 patients and 162 controls using polymerase chain reaction and restriction enzyme analysis. The ITGA2 genotype +/+ (with a BglII restriction site in both alleles) exhibited higher frequency in grade II astrocytoma compared to control (P = 0.02) whereas the genotype -/- (lacking the BglII site) correlated with the poorest survival rate (P = 0.04). In addition, in silico analyses of ITGA2 expression from low-grade gliomas (LGG, n = 515) and glioblastomas (GBM, n = 159) indicated that the higher expression of ITGA2 in LGG was associated with poor overall survival (P < 0.0001). However, the distribution of integrin ITGA2 BglII genotypes (+/+, +/-, -/-) was not significantly different between astrocytoma subgroups III and IV (P = 0.65, 0.24 and 0.33; 0.29, 0.48, 0.25, respectively) compared to control. These results suggest a narrow association between the presence of this SNP and indicate that further studies with larger samples are warranted to analyze the relation between tumor grade and overall survival, highlighting the importance of determining these polymorphisms for prognosis of astrocytomas.

Chromosomal instability in adult-type diffuse gliomas.

Chromosomal instability (CIN) is a fundamental property of cancer and a key underlying mechanism of tumorigenesis and malignant progression, and has been documented in a wide variety of cancers, including colorectal carcinoma with mutations in genes such as APC. Recent reports have demonstrated that CIN, driven in part by mutations in genes maintaining overall genomic stability, is found in subsets of adult-type diffusely infiltrating gliomas of all histologic and molecular grades, with resulting elevated overall copy number burden, chromothripsis, and poor clinical outcome. Still, relatively few studies have examined the effect of this process, due in part to the difficulty of routinely measuring CIN clinically. Herein, we review the underlying mechanisms of CIN, the relationship between chromosomal instability and malignancy, the prognostic significance and treatment potential in various cancers, systemic disease, and more specifically, in diffusely infiltrating glioma subtypes. While still in the early stages of discovery compared to other solid tumor types in which CIN is a known driver of malignancy, the presence of CIN as an early factor in gliomas may in part explain the ability of these tumors to develop resistance to standard therapy, while also providing a potential molecular target for future therapies.

Translating Neonatal Resuscitation Guidelines Into Practice in Brazil.

BACKGROUND AND OBJECTIVES: The Brazilian Neonatal Resuscitation Program releases guidelines based on local interpretation of international consensus on science and treatment recommendations. We aimed to analyze whether guidelines for preterm newborns were applied to practice in the 20 Brazilian Network on Neonatal Research centers of this middle-income country. METHODS: Prospectively collected data from 2014 to 2020 were analyzed for 8514 infants born at 230/7 to 316/7 weeks' gestation. The frequency of procedures was evaluated by gestational age (GA) category, including use of a thermal care bundle, positive pressure ventilation (PPV), PPV with a T-piece resuscitator, maximum fraction of inspired oxygen (Fio2) concentration during PPV, tracheal intubation, chest compressions and medications, and use of continuous positive airway pressure in the delivery room. Logistic regression, adjusted by center and year, was used to estimate the probability of receiving recommended treatment. RESULTS: For 3644 infants 23 to 27 weeks' GA and 4870 infants 28 to 31 weeks' GA, respectively, the probability of receiving care consistent with guidelines per year increased, including thermal care (odds ratio [OR], 1.52 [95% confidence interval (CI) 1.44-1.61] and 1.45 [1.38-1.52]) and PPV with a T-piece (OR, 1.45 [95% CI 1.37-1.55] and 1.41 [1.32-1.51]). The probability of receiving PPV with Fio2 1.00 decreased equally in both GA groups (OR, 0.89; 95% CI, 0.86-0.93). CONCLUSIONS: Between 2014 and 2020, the resuscitation guidelines for newborns <32 weeks' GA on thermal care, PPV with a T-piece resuscitator, and decreased use of Fio2 1.00 were translated into clinical practice.

Global DNA methylation profiling reveals chromosomal instability in IDH-mutant astrocytomas.

Diffusely infiltrating gliomas are among the most common central nervous system tumors in adults. Over the past decade, the subcategorization of these tumors has changed to include both traditional histologic features and more recently identified molecular factors. However, one molecular feature that has yet to be integrated is the presence/absence of chromosomal instability (CIN). Herein, we use global methylation profiling to evaluate a reference cohort of IDH-mutant astrocytomas with and without prior evidence of CIN (n = 42), and apply the resulting methylation-based characteristics to a larger test cohort of publicly-available IDH-mutant astrocytomas (n = 245). We demonstrate that IDH-mutant astrocytomas with evidence of CIN cluster separately from their chromosomally-stable counterparts. CIN cases were associated with higher initial histologic grade, altered expression patterns of genes related to CIN in other cancers, elevated initial total copy number burden, and significantly worse progression-free and overall survival. In addition, in a grade-for-grade analysis, patients with CIN-positive WHO grade 2 and 3 tumors had significantly worse survival. These results suggest that global methylation profiling can be used to discriminate between chromosomally stable and unstable IDH-mutant astrocytomas, and may therefore provide a reliable and cost-effective method for identifying gliomas with chromosomal instability and resultant poor clinical outcome.

A re-appraisal of the ENSO response to volcanism with paleoclimate data assimilation.

The potential for explosive volcanism to affect the El Niño-Southern Oscillation (ENSO) has been debated since the 1980s. Several observational studies, based largely on tree-ring proxies, have since found support for a positive ENSO phase in the year following large eruptions. In contrast, recent coral data from the heart of the tropical Pacific suggest no uniform ENSO response to explosive volcanism over the last millennium. Here we leverage paleoclimate data assimilation to integrate both tree-ring and coral proxies into a reconstruction of ENSO state, and re-appraise this relationship. We find only a weak statistical association between volcanism and ENSO, and identify the selection of volcanic events as a key variable to the conclusion. We discuss the difficulties of conclusively establishing a volcanic influence on ENSO by empirical means, given the myriad factors affecting the response, including the spatiotemporal details of the forcing and ENSO phase preconditioning.

The scaffolding protein DLG5 promotes glioblastoma growth by controlling Sonic Hedgehog signaling in tumor stem cells.

BACKGROUND: Tumor invasion, a hallmark of malignant gliomas, involves reorganization of cell polarity and changes in the expression and distribution of scaffolding proteins associated with polarity complexes. The scaffolding proteins of the DLG family are usually downregulated in invasive tumors and regarded as tumor suppressors. Despite their important role in regulating neurodevelopmental signaling, the expression and functions of DLG proteins have remained almost entirely unexplored in malignant gliomas. METHODS: Western blot, immunohistochemistry, and analysis of gene expression were used to quantify DLG members in glioma specimens and cancer datasets. Over-expression and knockdown of DLG5, the highest-expressed DLG member in glioblastoma, were used to investigate its effects on tumor stem cells and tumor growth. qRT-PCR, Western blotting, and co-precipitation assays were used to investigate DLG5 signaling mechanisms. RESULTS: DLG5 was upregulated in malignant gliomas compared to other solid tumors, being the predominant DLG member in all glioblastoma molecular subtypes. DLG5 promoted glioblastoma stem cell invasion, viability, and self-renewal. Knockdown of this protein in vivo disrupted tumor formation and extended survival. At the molecular level, DLG5 regulated Sonic Hedgehog (Shh) signaling, making DLG5-deficient cells insensitive to Shh ligand. Loss of DLG5 increased the proteasomal degradation of Gli1, underlying the loss of Shh signaling and tumor stem cell sensitization. CONCLUSIONS: The high expression and pro-tumoral functions of DLG5 in glioblastoma, including its dominant regulation of Shh signaling in tumor stem cells, reveal a novel role for this protein that is strikingly different from its proposed tumor-suppressor role in other solid tumors.

Ko143 Reverses MDR in Glioblastoma via Deactivating P-Glycoprotein, Sensitizing a Resistant Phenotype to TMZ Treatment.

BACKGROUND/AIM: Over-expression of both P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) has been associated with multidrug-resistance in glioblastoma (GBM). Though previously studied broad-spectrum inhibitors of drug efflux pumps have failed to progress in clinical studies due to in vivo toxicity, research into clinically viable targeted inhibitors is needed. This study evaluated the effects of Ko143, a non-toxic analog of fumitremorgin C, on temozolomide (TMZ) efficacy in resistant glioblastoma stem cells. MATERIALS AND METHODS: We used ATP-Glo assay to determine cell viabilities and flow cytometry to perform cell cycle analysis. Comparative gene expression was analysed through RT-qPCR. RESULTS: TMZ IC50 decreased 41.07% (p<0.01) in the resistant phenotype when delivered in combination with Ko143. Additionally, the TMZ-resistant phenotype (GBM146) displayed 44-fold greater P-gp expression than the TMZ-sensitive phenotype (GBM9) (p<0.01), yet a 0.6-fold lower BCRP expression. Ko143 potentiates TMZ efficacy and likely inhibits P-glycoprotein more potently than previously indicated. CONCLUSION: Further development of non-toxic, targeted inhibitors of drug efflux pumps for use in combinatorial chemotherapy may improve glioblastoma patient prognosis.

Indisulam Reduces Viability and Regulates Apoptotic Gene Expression in Pediatric High-Grade Glioma Cells.

Pediatric high-grade glioma (pHGG) is one of the most aggressive brain tumors. Treatment includes surgery, radiotherapy, chemotherapy, or combination therapy in children older than 3−5 years of age. These devastating tumors are influenced by the hypoxic microenvironment that coordinatively increases the expression of carbonic anhydrases (CA9 and CA12) that are involved in pH regulation, metabolism, cell invasion, and resistance to therapy. The synthetic sulphonamide Indisulam is a potent inhibitor of CAs. The aim of this study was to evaluate the effects of Indisulam on CA9 and CA12 enzymes in pHGG cell lines. Our results indicated that, under hypoxia, the gene and protein expression of CA9 and CA12 are increased in pHGG cells. The functional effects of Indisulam on cell proliferation, clonogenic capacity, and apoptosis were measured in vitro. CA9 and CA12 gene and protein expression were analyzed by RT-PCR and western blot. The treatment with Indisulam significantly reduced cell proliferation (dose-time-dependent) and clonogenic capacity (p < 0.05) and potentiated the effect of apoptosis (p < 0.01). Indisulam promoted an imbalance in the anti-apoptotic BCL2 and pro-apoptotic BAX protein expression. Our results demonstrate that Indisulam contributes to apoptosis via imbalance of apoptotic proteins (BAX/BCL2) and suggests a potential to overcome chemotherapy resistance caused by the regulation these proteins.

Community‐based mental health project in Davao Region

@#Mental health has profound effects on an individual’s quality of life, and it can also affect the families and the communities of persons with mental illness. In early 2020, the World Health Organization (WHO) Special Initiative for Mental Health in the Philippines recorded at least 3.6 million Filipinos suffering from a mental, neurological, or substance abuse disorder.1 In 2015, schizophrenia was the top mental disorder in the Philippines,2 and it is estimated that 1 million Filipinos (1% of the population) suffer from schizophrenia.3 Schizophrenia, a debilitating mental health condition, is characterized by both positive (e.g., hallucinations, delusions, confused thoughts, etc.) and negative (e.g., lack of pleasure, flattening, withdrawal, etc.) symptoms.4 5 6 The condition can affect the individual’s personal and social aspects of daily life, such as self-care, interpersonal relationships, education, and employment.7 8 9 10 Hospitalization is generally indicated for patients who are actively experiencing delusions/hallucinations, those who pose a serious threat of harm to themselves or others, or those who are unable to care for themselves and need constant supervision and support. Other possible indications for hospitalization include the presence of general medical or psychiatric problems that may render outpatient treatment unsafe or ineffective.11 12 Recent therapeutic advances, especially the introduction of atypical antipsychotic medications that have demonstrated better efficacy rates compared to older generation oral antipsychotic drugs, have allowed the reintegration of persons with mental health disorders (e.g., schizophrenia, other psychotic disorders, major depression, mood disorder, or bipolar disorder) into the society.13 14 15 Yet, despite the availability of such medications, there still exists substantial gaps in the delivery of mental health services. In the Philippines, mental health has remained poorly-resourced, with only 3-5% of the total health budget spent on mental health care.16 Mental health specialists have been in shortage, and a large proportion of these specialists work in urban for-profit services or private practices. 16 Mental health care services are delivered largely in hospital and private clinic settings,16 18 while community-based services remain underdeveloped.17 19 Prohibitive economic conditions and stigma on mental illness20 21 are some of the factors that contribute to low diagnosis and treatment rates. These factors, as well as funding issues that limit patient access, especially to newer innovative drugs, have rendered mental health care relatively inaccessible, leaving many patients undiagnosed and untreated or undertreated.17 In 2015, the Davao Center for Development (DCHD) Mental Health Program facilitated the establishment of Community-Based Mental Health Programs (CBMHPs) and the implementation of the WHO Mental Health Gap Action Programme Intervention Guide (mhGAP-IG) in several municipalities in the region. Since their launching, CBMHPs in Davao Region documented annual increases ranging from 10 to 13% in the number of patients availing mental health services in primary and tertiary care facilities from 2016 to 2019. 22 During the program implementation review conducted by the National Mental Health Program in 2019, two Centers for Health Development (CHD)—those in Davao and in CaLaBaRZon—planned to implement a common project for patients with schizophrenia in some areas with CBMHPs in their respective regions.22 This is in accordance with the Republic Act 11036, also known as the Philippine Mental Health Act, which mandates that basic mental health services be provided in community settings.23 The Schizophrenia Project was designed to make mental health services accessible and antipsychotic medications readily available in municipalities with the highest burden of schizophrenia, the most common mental health condition in the region. The project involves community-level assessment, management, and follow up of patients with schizophrenia through the process described in the mhGAP-IG. In Davao, the ongoing Schizophrenia Project implementation has been made possible by the collaboration of primary care providers in rural health units (RHU) with psychiatrist consultants. Johnson & Johnson Philippines, the marketing authorization holder of paliperidone palmitate in the country, conducts training of health care workers involved in the project. DCHD finances the project and provides technical assistance to the RHUs. A pilot phase of the project was planned to run for one year in four implementation sites—Boston in Davao Oriental, Santo Tomas in Davao del Norte, Sta. Cruz in Davao del Sur, and Jose Abad Santos in Davao Occidental—which have been identified by DOH DCHD as having the highest numbers of patients diagnosed with schizophrenia in Davao Region. DCHD purchased paliperidone palmitate needed for project implementation in December 2019. In March 2020, Johnson & Johnson conducted the first training for health workers. Patient enrollment into the pilot phase started in July 2020 and was completed in October 2020. RHUs in the four implementation sites identified patients with probable schizophrenia through a community-based case-finding and referral strategy patterned after the mhGAP-IG. Among the four municipalities, a total of 49 patients—9 from Boston, 10 from Santo Tomas, 11 from Sta. Cruz, and 19 from Jose Abad Santos—were enrolled into the program. The enrolled patients were diagnosed, treated, and monitored at least every six months by the collaborating psychiatrists and RHU physicians. The RHUs were also tasked with setting-up communication lines for emergency consultations with the collaborating psychiatrists during crisis, providing counseling to the patients’ families, and conducting health promotion events to raise awareness on schizophrenia. After the one-year implementation of the pilot phase, health care providers were highly satisfied with the project. After initiation of treatment, most of the patients demonstrated improved symptoms, and some of them were able to perform household chores or return to work within a few months. None of the patients experienced hospitalization or relapse during the pilot phase. Health workers involved in drug dispensing and patient monitoring observed that the intravenous preparation of paliperidone palmitate used in this project facilitates easier supply inventory and patient tracking. DCHD also noted cost savings in expenditure on antipsychotic drugs when paliperidone palmitate was used in this project instead of the oral and conventional depot antipsychotic drugs used in the past. Health workers involved in the project also encountered some challenges during implementation. Many caregivers and families of patients in the project were not very cooperative in complying with the demands of the treatment sessions and social reintegration. After initiation of antipsychotic treatment, many patients could not comply with the regular follow up sessions, mostly because they live very far from the RHUs and could not afford the transportation costs of the visits. The Schizophrenia Project did not have a structured reintegration program, so many patients who were already in remission after a few months of treatment could not be properly reintegrated into their respective families and communities. During the project review after the pilot phase, stakeholders pointed out several good practices of individual municipalities that can possibly be scaled up or replicated by other CBMHPs implementing similar projects in the future. In Boston, the municipal health officer conducted a series of lectures on the social dimensions of mental health disorders to the caregivers and families of patients in the project. Those who attended the lectures have expressed an increased understanding of the nature of their patients’ condition. In Sta. Cruz, the RHU provided food (rice and snacks) and fare reimbursements to the families of patients who came during follow up checkups. The RHUs of Santo Tomas and Sta. Cruz involved the social welfare service units of their respective municipalities in the process of family and community reintegration of patients on remission. The pilot phase implementation of the Schizophrenia Project in the four municipalities in Davao has demonstrated that it is highly possible to integrate mental health services at the primary care and community settings, and achieve positive outcomes for patients, caregivers, health care providers, and the health system.

Spatial progression and molecular heterogeneity of IDH-mutant glioblastoma determined by DNA methylation-based mapping.

Glioblastoma (GBM) is the most common malignant primary central nervous system (CNS) neoplasm in adults, and has an almost universally poor prognosis. Recently, an emphasis on genetic and epigenetic profiling has revealed a number of molecular features useful in the diagnostic and prognostic classification of GBM, advancing our understanding of the underlying features that make these tumors so aggressive and providing the rationale for the creation of better targeted therapeutics. One such method, DNA methylation profiling, has recently emerged as an important technique for the classification of CNS tumors, with diagnostic accuracy in some cases surpassing traditional methods. However, how DNA methylation profiles change with the course of the disease remains less understood. Here, we present a case of a 30-year-old male with primary IDH-mutant GBM with widespread recurrence and death two years later. Using unsupervised hierarchical clustering of methylation probes, we created a phylogenetic map to trace the tumor path as it spread from the initial biopsy site throughout the right hemisphere, across the corpus callosum to the contralateral hemisphere, and into the brainstem. We identified molecular divergence between the right and left hemisphere GBM samples marked by distinct copy number profile alterations, alterations in specific methylation sites, and regional loss of MGMT promoter methylation, providing a potential mechanism for treatment resistance in this case. In summary, this case both highlights the molecular diversity in GBM, and illustrates a novel use for methylation profiling in establishing a phylogenetic profile to allow for spatial mapping of tumor progression.

The Carbonic Anhydrase Inhibitor E7070 Sensitizes Glioblastoma Cells to Radio- and Chemotherapy and Reduces Tumor Growth.

Glioblastomas (GBMs), the most common and lethal primary brain tumor, show inherent infiltrative nature and high molecular heterogeneity that make complete surgical resection unfeasible and unresponsive to conventional adjuvant therapy. Due to their fast growth rate even under hypoxic and acidic conditions, GBM cells can conserve the intracellular pH at physiological range by overexpressing membrane-bound carbonic anhydrases (CAs). The synthetic sulfonamide E7070 is a potent inhibitor of CAs that harbors putative anticancer properties; however, this drug has still not been tested in GBMs. The present study aimed to evaluate the effects of E7070 on CA9 and CA12 enzymes in GBM cells as well as in the tumor cell growth, migration, invasion, and resistance to radiotherapy and chemotherapy. We found that E7070 treatment significantly reduced tumor cell growth and increased radio- and chemotherapy efficacy against GBM cells under hypoxia. Our data suggests that E7070 has therapeutic potential as a radio-chemo-sensitizing in drug-resistant GBMs, representing an attractive strategy to improve the adjuvant therapy. We showed that CA9 and CA12 represent potentially valuable therapeutic targets that should be further investigated as useful diagnostic and prognostic biomarkers for GBM tailored therapy.

Experimental resin-modified calcium-silicate cement containing N-(2-hydroxyethyl) acrylamide monomer for pulp tissue engineering.

AIM: Our study aimed to measure (1) the flexural strength, (2) shear bond strength to dentin, (3) pH, and (4) calcium (Ca) release of a series of innovative resin-modified calcium-silicate pulp-capping cements (Rm-CSCs). Using an ex-vivo human vital tooth-culture model, we additionally assessed (5) their pulp-healing initiation when brought in direct contact with human dental pulp tissue. METHODOLOGY: Three experimental Rm-CSCs, being referred to 'Exp_HEAA', 'Exp_GDM' and 'Exp_HEAA/GDM', contained either 20 wt% N-(2-hydroxyethyl) acrylamide (HEAA), 20 wt% glycerol dimethacrylate (GDM) or 10 wt% HEAA plus 10 wt% GDM, added to a common base composition consisting of 25 wt% urethane dimethacrylate (UDMA), 10 wt% 4-methacryloxyethyl trimellitate anhydride (4-MET), and 5 wt% N,N'-{[(2-acrylamido-2-[(3-acrylamidopropoxy)methyl] propane-1,3-diyl)bis(oxy)]bis-(propane-1,3-diyl)}diacrylamide (FAM-401). As Ca source and radiopacifier, 37 wt% tricalcium silicate powder (TCS) and 3 wt% zirconium oxide (ZrO 2) were respectively added. RESULTS: All three experimental Rm-CSCs revealed a significantly higher flexural strength and shear bond strength to dentin (p < 0.05) than the commercial reference Rm-CSC TheraCal LC (Bisco). Exp_HEAA presented with a significantly higher Ca release and pH at 24 h compared with the other Rm-CSCs (p < 0.05). At 1 week, the Ca release and pH of Exp_HEAA and Exp_HEAA/GDM was significantly higher than those of Exp_GDM and TheraCal LC (p < 0.05). Using the ex-vivo human vital tooth culture model, Exp_HEAA revealed pulp-healing initiation capacity as documented by nestin and collagen-I expression. CONCLUSIONS: Depending on the formulation, the innovative Rm-CSCs performed favorably for primary properties of relevance regarding pulp capping, this more specifically in terms of flexural strength, bond strength to dentin, as well as alkaline pH and Ca release. However, only Exp_HEAA revealed pulp-healing initiation in direct contact with human dental pulp tissue in the ex-vivo human vital tooth-culture model. This promising outcome for Exp_HEAA should be attributed to the combined use of (1) a novel hydrophilic acrylamide monomer, enabling sufficient polymerization while maintaining adequate hydrophilicity, with (2) the functional monomer 4-MET, possessing chemical bonding potential to dentin, and (3) tricalcium silicate powder to achieve an alkaline pH and to release Ca in a sufficient and controlled way.

Targeting glioblastoma using a novel peptide specific to a deglycosylated isoform of brevican.

Glioblastoma multiforme (GBM) is the most common and deadliest form of brain tumor and remains amongst the most difficult cancers to treat. Brevican (Bcan), a central nervous system (CNS)-specific extracellular matrix protein, is upregulated in high-grade glioma cells, including GBM. A Bcan isoform lacking most glycosylation, dg-Bcan, is found only in GBM tissues. Here, dg-Bcan is explored as a molecular target for GBM. In this study, we screened a d-peptide library to identify a small 8-amino acid dg-Bcan-Targeting Peptide (BTP) candidate, called BTP-7 that binds dg-Bcan with high affinity and specificity. BTP-7 is preferentially internalized by dg-Bcan-expressing patient-derived GBM cells. To demonstrate GBM targeting, we radiolabeled BTP-7 with 18F, a radioisotope of fluorine, and found increased radiotracer accumulation in intracranial GBM established in mice using positron emission tomography (PET) imaging. dg-Bcan is an attractive molecular target for GBM, and BTP-7 represents a promising lead candidate for further development into novel imaging agents and targeted therapeutics.