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This paper discusses “Regulatory Alignment for Multi-Modal Infrastructure Corridors” in the context of the Canadian Northern Corridor Concept (CNC). In particular, the paper reviews whether there are any existing models “for regulatory oversight and approval for multi-modal corridors and the subsequent placement of infrastructure within” that might be appropriate in developing the CNC. The paper concludes that existing models do not include certain of the elements of the CNC Concept as it has evolved to date, nor could any of the models be applied within the framework of Canada’s jurisdictional realities that would govern the CNC. The paper identifies several questions that need to be addressed in order to advance the CNC Concept to a more fully-formed proposal, which would form the basis for further examination of the need for regulatory oversight and approval and the form that such oversight and approval might take. The paper also suggests a forum that might oversee this further work.

OBJECTIVES/SPECIFIC AIMS: To create a composite index, referred to as the Pediatric Hydrocephalus Severity Index (PHSI), to classify the severity of disease at baseline and predict outcomes among children treated for hydrocephalus. METHODS/STUDY POPULATION: The Hydrocephalus Outcome Questionnaire will be administered in person or online to the parents of 150 patients between the ages of 5 and 18 years who are followed at the Neurosurgery Clinic at St. Louis Children’s Hospital for hydrocephalus. Patients must have been diagnosed and treated for hydrocephalus at least 6 months prior to the survey date. Potential participants are excluded if their health status changed during the 4 weeks prior to survey date, as determined by the child’s parents. Potential risk factors (see anticipated results) will be identified on retrospective medical record review. We will create a clinical prediction rule, called the PHSI, to stratify patients on likelihood of experiencing a poor long-term outcome after surgical treatment. Participants will be classified as “good” or “poor” outcome based on thresholds set for questionnaire results. We will use a combination of bivariate analysis and clinical reasoning to restrict the number of factors for further analysis, and multivariate logistic regression to build a predictive model for poor outcome. Creation of the PHSI will involve assigning integer values to adjusted odds ratios for significant risk factors at a 95% confidence level. RESULTS/ANTICIPATED RESULTS: Risk factors that we anticipate will be predictive of long-term clinical outcome include signs and symptoms at onset (bulging fontanel, splayed sutures, papilledema, up-gaze palsy, headache, vomiting, lethargy), head circumference above the 97th percentile, frontal-occipital horn ratio greater than 0.4, etiology of meningitis or neonatal intraventricular hemorrhage, central nervous system comorbidities (seizures, Chiari malformation, scoliosis, periventricular leukomalacia), preoperative infection or sepsis, and frequent shunt revisions or infections. We hypothesize that a PHSI will be a valuable tool for stratifying patients in future research studies, as well as aiding prognosis in clinical situations. DISCUSSION/SIGNIFICANCE OF IMPACT: A validated composite PHSI would be a major advance in clinical hydrocephalus research and practice. A PHSI would allow investigators to stratify patients based on initial presentation for clinical research studies, which may in turn lead to the establishment of more standardized treatment guidelines. It would also facilitate studies investigating differential utilization of healthcare resources based on disease severity. Clinically, a PHSI would better equip physicians to counsel parents on what to expect for their child or future healthcare resource requirements.

Alzheimer's disease (AD) is characterised by the aggregation and deposition of amyloid-β (Aβ) peptides in the human brain. In age-related late-onset AD, deficient degradation and clearance, rather than enhanced production, of Aβ contributes to disease pathology. In the present study, we assessed the contribution of the two key Aβ-degrading zinc metalloproteases, insulin-degrading enzyme (IDE) and neprilysin (NEP), to Aβ degradation in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Using an Aβ fluorescence polarisation assay, inhibition of IDE but not of NEP, blocked the degradation of Aβ by human neurons. When the neurons were grown in a 3D extracellular matrix to visualise Aβ deposition, inhibition of IDE but not NEP, increased the number of Aβ deposits. The resulting Aβ deposits were stained with the conformation-dependent, anti-amyloid antibodies A11 and OC that recognise Aβ aggregates in the human AD brain. Inhibition of the Aβ-forming β-secretase prevented the formation of the IDE-inhibited Aβ deposits. These data indicate that inhibition of IDE in live human neurons grown in a 3D matrix increased the deposition of Aβ derived from the proteolytic cleavage of the amyloid precursor protein. This work has implications for strategies aimed at enhancing IDE activity to promote Aβ degradation in AD., Competing Interests: The authors declare that there are no competing interests associated with this manuscript., (© 2023 The Author(s).)

Introduction: Neural induction of human induced pluripotent stem cells represents a critical switch in cell state during which pluripotency is lost and commitment to a neural lineage is initiated. Although many of the key transcription factors involved in neural induction are known, we know little of the temporal and causal relationships that are required for this state transition., Methods: Here, we have carried out a longitudinal analysis of the transcriptome of human iPSCs undergoing neural induction. Using the temporal relationships between the changing profile of key transcription factors and subsequent changes in their target gene expression profiles, we have identified distinct functional modules operative throughout neural induction., Results: In addition to modules that govern loss of pluripotency and gain of neural ectoderm identity, we discover other modules governing cell cycle and metabolism. Strikingly, some of these functional modules are retained throughout neural induction, even though the gene membership of the module changes. Systems analysis identifies other modules associated with cell fate commitment, genome integrity, stress response and lineage specification. We then focussed on OTX2, one of the most precociously activated transcription factors during neural induction. Our temporal analysis of OTX2 target gene expression identified several OTX2 regulated gene modules representing protein remodelling, RNA splicing and RNA processing. Further CRISPRi inhibition of OTX2 prior to neural induction promotes an accelerated loss of pluripotency and a precocious and aberrant neural induction disrupting some of the previously identified modules., Discussion: We infer that OTX2 has a diverse role during neural induction and regulates many of the biological processes that are required for loss of pluripotency and gain of neural identity. This dynamical analysis of transcriptional changes provides a unique perspective of the widespread remodelling of the cell machinery that occurs during neural induction of human iPSCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gupta, Polit, Fitzgerald, Rowland, Murali, Buckley and Subramaniam.)

Establishing preclinical models of Alzheimer's disease that predict clinical outcomes remains a critically important, yet to date not fully realized, goal. Models derived from human cells offer considerable advantages over non-human models, including the potential to reflect some of the inter-individual differences that are apparent in patients. Here we report an approach using induced pluripotent stem cell-derived cortical neurons from people with early symptomatic Alzheimer's disease where we sought a match between individual disease characteristics in the cells with analogous characteristics in the people from whom they were derived. We show that the response to amyloid-β burden in life, as measured by cognitive decline and brain activity levels, varies between individuals and this vulnerability rating correlates with the individual cellular vulnerability to extrinsic amyloid-β in vitro as measured by synapse loss and function. Our findings indicate that patient-induced pluripotent stem cell-derived cortical neurons not only present key aspects of Alzheimer's disease pathology but also reflect key aspects of the clinical phenotypes of the same patients. Cellular models that reflect an individual's in-life clinical vulnerability thus represent a tractable method of Alzheimer's disease modelling using clinical data in combination with cellular phenotypes., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

The cellular prion protein (PrP C ) is a key neuronal receptor for β-amyloid oligomers (AβO), mediating their neurotoxicity, which contributes to the neurodegeneration in Alzheimer's disease (AD). Similarly to the amyloid precursor protein (APP), PrP C is proteolytically cleaved from the cell surface by a disintegrin and metalloprotease, ADAM10. We hypothesized that ADAM10-modulated PrP C shedding would alter the cellular binding and cytotoxicity of AβO. Here, we found that in human neuroblastoma cells, activation of ADAM10 with the muscarinic agonist carbachol promotes PrP C shedding and reduces the binding of AβO to the cell surface, which could be blocked with an ADAM10 inhibitor. Conversely, siRNA-mediated ADAM10 knockdown reduced PrP C shedding and increased AβO binding, which was blocked by the PrP C -specific antibody 6D11. The retinoic acid receptor analog acitretin, which up-regulates ADAM10, also promoted PrP C shedding and decreased AβO binding in the neuroblastoma cells and in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Pretreatment with acitretin abolished activation of Fyn kinase and prevented an increase in reactive oxygen species caused by AβO binding to PrP C Besides blocking AβO binding and toxicity, acitretin also increased the nonamyloidogenic processing of APP. However, in the iPSC-derived neurons, Aβ and other amyloidogenic processing products did not exhibit a reciprocal decrease upon acitretin treatment. These results indicate that by promoting the shedding of PrP C in human neurons, ADAM10 activation prevents the binding and cytotoxicity of AβO, revealing a potential therapeutic benefit of ADAM10 activation in AD., (© 2019 Jarosz-Griffiths et al.)

Developing cellular models of sporadic Alzheimer's disease (sAD) is challenging due to the unknown initiator of disease onset and the slow disease progression that takes many years to develop in vivo. The use of human induced pluripotent stem cells (iPSCs) has revolutionised the opportunities to model AD pathology, investigate disease mechanisms and screen potential drugs. The majority of this work has, however, used cells derived from patients with familial AD (fAD) where specific genetic mutations drive disease onset. While these provide excellent models to investigate the downstream pathways involved in neuronal toxicity and ultimately neuronal death that leads to AD, they provide little insight into the causes and mechanisms driving the development of sAD. In this review we compare the data obtained from fAD and sAD iPSC-derived cell lines, identify the inconsistencies that exist in sAD models and highlight the potential role of Aβ clearance mechanisms, a relatively under-investigated area in iPSC-derived models, in the study of AD. We discuss the development of more physiologically relevant models using co-culture and three-dimensional culture of iPSC-derived neurons with glial cells. Finally, we evaluate whether we can develop better, more consistent models for sAD research using genetic stratification of iPSCs and identification of genetic and environmental risk factors that could be used to initiate disease onset for modelling sAD. These considerations provide exciting opportunities to develop more relevant iPSC models of sAD which can help drive our understanding of disease mechanisms and identify new therapeutic targets.

High levels of naturally occurring arsenic are found in the shallow reducing aquifers of West Bengal, Bangladesh, and other areas of Southeast Asia. These aquifers are used extensively for drinking water and irrigation by the local population. Mechanisms for its release are unclear, although increasing evidence points to a microbial control. The type of organic matter present is of vital importance because it has a direct impact on the rate of microbial activity and on the amount of arsenic released into the ground water. The discovery of naturally occurring hydrocarbons in an arsenic-rich aquifer from West Bengal provides a source of potential electron donors for this process. Using microcosm-based techniques, seven sediments from a site containing naturally occurring hydrocarbons in West Bengal were incubated with synthetic ground water for 28 d under anaerobic conditions without the addition of an external electron donor. Arsenic release and Fe(III) reduction appeared to be microbially mediated, with variable rates of arsenic mobilization in comparison to Fe(III) reduction, suggesting that multiple processes are involved. All sediments showed a preferential loss of petroleum-sourced n-alkanes over terrestrially sourced sedimentary hydrocarbons n-alkanes during the incubation, implying that the use of petroleum-sourced n-alkanes could support, directly or indirectly, microbial Fe(III) reduction. Samples undergoing maximal release of As(III) contained a significant population of Sulfurospirillum sp., a known As(V)-reducing bacterium, providing the first evidence that such organisms may mediate arsenic release from West Bengali aquifers.

The health implications of the consumption of high arsenic groundwater in Bangladesh and West Bengal are well-documented, however, little is known about the level of arsenic exposure elsewhere in Southeast Asia, where widespread exploitation of groundwater resources is less well established. We measured the arsenic concentrations of nail and hair samples collected from residents of Kandal province, Cambodia, an area recently identified to host arsenic-rich groundwaters, in order to evaluate the extent of arsenic exposure. Nail and hair arsenic concentrations ranged from 0.20 to 6.50 microg g(-1) (n=70) and 0.10 to 7.95 microg g(-1) (n=40), respectively, in many cases exceeding typical baseline levels. The arsenic content of the groundwater used for drinking water purposes (0.21-943 microg L(-1) (n=31)) was positively correlated with both nail (r=0.74, p<0.0001) and hair (r=0.86, p<0.0001) arsenic concentrations. In addition, the nail and hair samples collected from inhabitants using groundwater that exceeded the Cambodian drinking water legal limit of 50 microg L(-1) arsenic contained significantly more arsenic than those of individuals using groundwater containing <50 microg L(-1) arsenic. X-ray absorption near edge structure (XANES) spectroscopy suggested that sulfur-coordinated arsenic was the dominant species in the bulk of the samples analysed, with additional varying degrees of As(III)-O character. Tentative linear least squares fitting of the XANES data pointed towards differences in the pattern of arsenic speciation between the nail and hair samples analysed, however, mismatches in sample and standard absorption peak intensity prevented us from unambiguously determining the arsenic species distribution. The good correlation with the groundwater arsenic concentration, allied with the relative ease of sampling such tissues, indicate that the arsenic content of hair and nail samples may be used as an effective biomarker of arsenic intake in this relatively recently exposed population.

The bactericidal and opsonic activity of serum from 12 homozygous sicklers was compared with that of a single control pool of healthy nonsickler African serum. Factorially designed experiments were used to calculate 4 indices which were expressed as the ratio of test to control. Using S. enteritidis and normal human neutrophils viability indices (VI) were calculated for serum, for ingested organisms and for cells + serum, together with a phagocytic index (PI) for cells. Only 1 of the 12 sickler sera gave values equal to that for the control, the remainder showing marked deviation. The survival of organisms in sickler serum was 3 times that in control serum. Phagocytosis by neutrophils in the presence of test sera was only 10% of that in control sera and survival of ingested bacteria was 6 times greater. In the presence of neutrophils overall bacterial survival was 22 times greater for sickler than for control serum.

Thirty-eight homozygous sickler sera were compared with a large pool of serum from healthy African non-sicklers with regard to bactericidal and phagocytic indices. One third of the sera showed reduced bactericidal activity against Salmonella enteritidis which was restored by the addition of 4% control serum; control serum provided both heat-labile (HL) and absorbable (ABS) serum factors. 76% of test sera showed greatly defective opsonization as indicated by ingestion by normal human neutrophils. Activity was not readily restored by the addition of control serum which provided only HL factors. Intracellular survival was increased when bacteria were ingested from sickler serum; activity was readily restored by control serum which provided both HL and ABS factors. In the presence of both serum and neutrophils 84% of test sera permitted increased bacterial survival; the defect was not readily reversed by the addition of control serum which provided both HL and ABS factors.