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Abstract Short-hairpin RNAs (shRNA), targeting knockdown of specific genes, hold enormous promise for precision-based therapeutics to treat numerous neurodegenerative disorders. However, whether shRNA constructed molecules can modify neuronal circuits underlying certain behaviors has not been explored. We designed shRNA to knockdown the human HTR2A gene in vitro using iPSC-differentiated neurons. Multi-electrode array (MEA) results showed that the knockdown of the 5HT-2A mRNA and receptor protein led to a decrease in spontaneous electrical activity. In vivo, intranasal delivery of AAV9 vectors containing shRNA resulted in a decrease in anxiety-like behavior in mice and a significant improvement in memory in both mice (104%) and rats (92%) compared to vehicle-treated animals. Our demonstration of a non-invasive shRNA delivery platform that can bypass the blood–brain barrier has broad implications for treating numerous neurological mental disorders. Specifically, targeting the HTR2A gene presents a novel therapeutic approach for treating chronic anxiety and age-related cognitive decline.

Many of the current synthetic methodologies utilized within academic and industrial laboratories require knowledge of how to safely handle air- and moisture-sensitive reagents and work under inert atmospheres. As a result, the ACS Committee on Professional Training recommends incorporating synthetic methods that make use of these inert atmospheres into curricula for the development of students with career aspirations in science. However, incorporation of these methods into the curricula at primarily undergraduate institutions is challenging due to access to limited resources and infrastructure. This article reports a semester long, multistep laboratory synthesis of the tripodal bis[2-(2,3-dihydroxyphenyl)-6-pyridylmethyl](2-pyridylmethyl)amine (BCATTPA) compound involving metal halogen exchange, chlorine transfer, nucleophilic substitution, Suzuki cross-coupling, and Lewis acid dealkylation steps that was successfully carried out by sophomore or junior undergraduate researchers enrolled in a Mentored Research in Chemistry course at Fairleigh Dickinson University. Using routine laboratory characterization techniques such as 1H NMR and FT-IR spectroscopies as well as GC-MS, students were able to identify and assess the purities of their resulting products. The disclosed laboratory synthesis enabled students to get formal training in air- and moisture-free Schlenk-line techniques such as syringe and cannula transfers in a controlled learning environment that they could use in future research activities in organic, inorganic, analytical, physical, or biochemistry laboratories.

Bispecific antibodies (BsAbs) form an exciting class of bio-therapeutics owing to their multispecificity. Although numerous formats have been developed, generation of hetero-tetrameric IgG1-like BsAbs having acceptable safety and pharmacokinetics profiles from a single cell culture system remains challenging due to the heterogeneous pairing between the four chains. Herein, we employed a structure-guided approach to engineer mutations in the constant domain interfaces (CH1-CL and CH3-CH3) of heavy and κ light chains to prevent heavy-light mispairing in the antigen binding fragment (Fab) region and heavy-heavy homodimerization in the Fc region. Transient co-transfection of mammalian cells with heavy and light chains of pre-existing antibodies carrying the engineered constant domains generates BsAbs with percentage purity ranging from 78% to 85%. The engineered BsAbs demonstrate simultaneous binding of both antigens, while retaining the thermal stability, Fc-mediated effector properties and FcRn binding properties of the parental antibodies. Importantly, since the variable domains were not modified, the mutations may enable BsAb formation from antibodies belonging to different germline origins and isotypes. The rationally designed mutations reported in this work could serve as a starting point for generating optimized solutions required for large scale production.

Down syndrome (DS) is one of the most common genetic causes of intellectual disability and is characterized by a number of behavioral as well as cognitive symptoms. Many of the neuropathological features of early-onset Alzheimer's disease (AD) including senile plaques and neurofibrillary tangles (NFTs) are also present in people with DS as a result of triplication of the amyloid precursor gene on chromosome 21. Evidence suggests that harboring one or both apolipoprotein E4 (APOE4) alleles may increase the risk for AD due to the proteolytic cleavage of apoE4 and a subsequent loss of function. To investigate a role for the apoE proteolysis in vivo, we compared three autopsy groups; 7 DS with AD neuropathology cases over 40 years, 5 young DS cases without AD pathology under 40 years (YDS) and 5 age-matched control cases over 40 years by immunohistochemistry utilizing an antibody that detects the amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE fragment antibody (nApoECF) revealed labeling of pyramidal neurons in the frontal cortex of YDS cases, whereas in the DS-AD group, labeling with nApoECF was prominent within NFTs. NFT labeling with nApoECF was significantly greater in the hippocampus versus the frontal cortex in the same DS-AD cases, suggesting a regional distribution of truncated apoE. Colocalization immunofluorescence experiments indicated that 52.5% and 53.2% of AT8- and PHF-1-positive NFTs, respectively, also contained nApoECF. Collectively, these data support a role for the proteolytic cleavage of apoE in DS and suggest that apoE fragmentation is closely associated with NFTs.

Although the increased risk of developing sporadic Alzheimer's disease (AD) associated with the inheritance of the apolipoprotein E4 (APOE4) allele is well characterized, the molecular underpinnings of how ApoE4 imparts risk remains unknown. Enhanced proteolysis of the ApoE4 protein with a toxic-gain of function has been suggested and a 17 kDa amino-terminal ApoE4 fragment (nApoE41-151) has been identified in post-mortem human AD frontal cortex sections. Recently, we demonstrated in vitro, exogenous treatment of nApoE41-151 in BV2 microglial cells leads to uptake, trafficking to the nucleus and increased expression of genes associated with cell toxicity and inflammation. In the present study, we extend these findings to zebrafish (Danio rerio), an in vivo model system to assess the toxicity of nApoE41-151. Exogenous treatment of nApoE41-151 to 24-hour post-fertilization for 24 hours resulted in significant mortality. In addition, developmental abnormalities were observed following treatment with nApoE41-151 including improper folding of the hindbrain, delay in ear development, deformed yolk sac, enlarged cardiac cavity, and significantly lower heart rates. A similar nApoE31-151 fragment that differs by a single amino acid change (C>R) at position 112 had no effects on these parameters under identical treatment conditions. Decreased presence of pigmentation was noted for both nApoE31-151- and nApoE41-151-treated larvae compared with controls. Behaviorally, touch-evoked responses to stimulus were negatively impacted by treatment with nApoE41-151 but did not reach statistical significance. Additionally, triple-labeling confocal microscopy not only confirmed the nuclear localization of the nApoE41-151 fragment within neuronal populations following exogenous treatment, but also identified the presence of tau pathology, one of the hallmark features of AD. Collectively, these in vivo data demonstrating toxicity as well as sublethal effects on organ and tissue development support a novel pathophysiological function of this AD associated-risk factor.

Vascular dementia (VaD) is the second most common form of dementia and is currently defined as a cerebral vessel vascular disease leading to ischemic episodes. Apolipoprotein E (apoE) gene polymorphism has been proposed as a risk factor for VaD, however, to date there are few documented post-mortem studies on apoE pathology in the VaD brain. To investigate a potential role for the apoE protein, we analyzed seven confirmed cases of VaD by immunohistochemistry utilizing an antibody that specifically detects the amino-terminal fragment of apoE. Application of this antibody, termed N-terminal, apoE cleavage fragment (nApoECF) revealed consistent labeling within neurofibrillary tangles (NFTs), blood vessels, and reactive astrocytes. Labeling occurred in VaD cases that had confirmed APOE genotypes of 3/3, 3/4, and 4/4, with respect to NFTs, staining of the nApoECF co-localized with PHF-1 and was predominantly localized to large, stellate neurons in layer II of the entorhinal cortex. Quantitative analysis indicated that approximately 38.4% of all identified NFTs contained the amino-terminal fragment of apoE. Collectively, these data support a role for the proteolytic cleavage of apoE in the VaD and support previous reports that APOE polymorphism is significantly associated with susceptibility in this disease.

Although the cerebellum is generally thought of as an area spared of Alzheimer’s disease (AD) pathology, recent evidence suggests that balance and mobility dysfunction may be magnified in affected individuals. In the present study, we sought to determine the degree of pathological changes within the cerebellum utilizing an antibody that specifically detects caspase-cleaved GFAP within degenerating astrocytes. Compared to control subjects, application of this antibody, termed the GFAP caspase-cleavage product (GFAPccp) antibody, revealed widespread labeling in cerebellar white matter with little staining observed in grey matter. Staining was observed within damaged astrocytes, was often localized near blood vessels and co-localized with other markers of apoptosis including TUNEL and caspase-cleaved tau. Of interest was the association of beta-amyloid deposition in white matter together with GFAPccp in cerebellar AD sections. In contrast, utilizing the tangle marker, PHF-1, neuritic pathology was completely absent in AD cerebellar sections. It is suggested that the observed pathological changes found in the white matter of the cerebellum may contribute to the declined motor performance in AD.

Despite the fact that harboring the apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer’s disease (AD), the exact mechanism by which ApoE4 contributes to disease progression remains unknown. Recently, we demonstrated that a 151 amino-terminal fragment of ApoE4 (nApoE41–151) localizes within the nucleus of microglia in the human AD brain and traffics to the nucleus causing toxicity in BV2 microglia cells. In the present study, we examined in detail what genes may be affected following treatment by nApoE41–151. Transcriptome analyses in BV2 microglial cells following sublethal treatment with nApoE41–151 revealed the upregulation of almost 4,000 genes, with 20 of these genes upregulated 182- to 715-fold compared to untreated control cells. The majority of these 20 genes play a role in the immune response and polarization toward microglial M1 activation. As a control, an identical nApoE31–151 fragment that differed by a single amino acid at position 112 (Cys→Arg) was tested and produced a similar albeit lower level of upregulation of an identical set of genes. In this manner, enriched pathways upregulated by nApoE31–151 and nApoE41–151 following exogenous treatment included Toll receptor signaling, chemokine/cytokine signaling and apoptosis signaling. There were unique genes differentially expressed by at least two-fold for either fragment. For nApoE31–151, these included 16 times as many genes, many of which are involved in physiological functions within microglia. For nApoE41–151, on the other hand the number genes uniquely upregulated was significantly lower, with many of the top upregulated genes having unknown functions. Taken together, our results suggest that while nApoE31–151 may serve a more physiological role in microglia, nApoE41–151 may activate genes that contribute to disease inflammation associated with AD. These data support the hypothesis that the link between harboring the APOE4 allele and dementia risk could be enhanced inflammation through activation of microglia.

Human clinical trials seek to ameliorate the disease states and symptomatic progression of illnesses that, as of yet, are largely untreatable according to clinical standards. Ideally, clinical trials test “disease-modifying drugs,” i.e., therapeutic agents that specifically modify pathological features or molecular bases of the disease and would presumably have a large impact on disease progression. In the case of Alzheimer’s disease (AD), however, this approach appears to have stalled progress in the successful development of clinically useful therapies. For the last 25 years, clinical trials involving AD have centered on beta-amyloid (Aβ) and the Aβ hypothesis of AD progression and pathology. According to this hypothesis, the progression of AD begins following an accumulation of Aβ peptide, leading to eventual synapse loss and neuronal cell death: the true overriding pathological feature of AD. Clinical trials arising from the Aβ hypothesis target causal steps in the pathway in order to reduce the formation of Aβ or enhance clearance, and though agents have been successful in this aim, they remain unsuccessful in rescuing cognitive function or slowing cognitive decline. As such, further use of resources in the development of treatment options for AD that target Aβ, its precursors, or its products should be reevaluated. The purpose of this review was to give an overview of how human clinical trials are conducted in the USA and to assess the results of recent failed trials involving AD, the majority of which were based on the Aβ hypothesis. Based on these current findings, it is suggested that lowering Aβ is an unproven strategy, and it may be time to refocus on other targets for the treatment of this disease including pathological forms of tau.

Despite the wealth of evidence supporting the activation of caspases in Alzheimer's disease (AD), chronic administration of a caspase inhibitor has never been tested in any animal model system. The purpose of the current report was to identify a suitable animal model that displays caspase activation and cleavage of critical proteins associated with AD, and secondly, to undertake a pilot study utilizing the novel caspase inhibitor, quinolyl-valyl-O-methylaspartyl-[-2, 6-difluorophenoxy]-methyl ketone (Q-VD-OPh). Analysis of 12 month-old TgCRND8 mice, which represent an early-onset animal model for AD, indicated the activation of caspase-7 as well as the cleavage of tau and the amyloid precursor protein (APP). Having established that TgCRND8 mice represent a suitable model system to target caspases therapeutically, a prophylactic study was initiated utilizing Q-VD-OPh. Three month-old TgCRND8 mice were injected intraperitoneally three times a week for three months with 10 mg/kg Q-VD-OPh and compared to control mice injected with vehicle. Although there was no apparent effect on extracellular Aβ deposition, chronic treatment with Q-VD-OPh did prevent caspase-7 activation and limited the pathological changes associated with tau, including caspase cleavage. These preliminary findings suggest that further studies examining the utility of Q-VD-OPh as a potential therapeutic compound for the treatment of AD are warranted.

Mounting evidence suggests the involvement of caspases in the disease process associated with Alzheimer’s disease (AD). The activation of caspases may be responsible for the neurodegeneration associated with AD and several recent studies have suggested that caspases may also play a role in promoting pathogenic mechanisms underlying this disease. Thus, caspase activation and cleavage of the amyloid precursor protein (APP) and tau may facilitate both the production of beta-amyloid (Aβ) as well as the formation of neurofibrillary tangles (NFTs). Because the activation of caspases in AD may be a proximal event that is not just associated with neurodegeneration, caspases are potential therapeutic targets for the treatment of this disorder. In this review, studies documenting the role of caspases in the AD brain will be discussed. In this context, a discussion of the therapeutic value of targeting caspase inhibition in the treatment of AD will be evaluated including drug targets, delivery and selectivity.

Multidentate ligands involving tethered pyridyl groups coordinated to transition metal ions have been frequently used to mimic the 3-histidine (3H), 2-histidine-1-carboxylate (2H1C) brace motifs or other combinations of histidine and carboxylate endogenous ligating residues found in bioinorganic metalloenzymes. It is of interest to immobilize these ligand chelates onto heterogeneous supports. This, however, requires the use of bromine-substituted (chloromethyl)pyridines, whose current synthetic routes involve the use of extremely pyrophoric chemicals, such as n-butyllithium that require cryogenic reaction conditions, and toxic chemicals, such as thionyl chloride, that are challenging to handle and require extensive hazard controls. Herein, we report alternative methodologies towards the syntheses of 2-bromo-6-hydroxymethylpyridine and 2-bromo-6-chloromethylpyridine from inexpensive commercially available 2,6-dibromopyridine using isopropylmagnesium chloride lithium chloride complex (Turbo Grignard) and cyanuric chloride which are easier to handle and require milder reaction conditions than the conventional reagents. Gas chromatography-mass spectrometry (GC-MS) methods were developed and simple 1H- and 13C- nuclear magnetic resonance (NMR) and Fourier-transform infrared (FT-IR) spectroscopies were also used to monitor the conversion of both reaction steps and showed that products could be obtained and isolated through simple workups without the presence of unreacted starting material or undesired overchlorinated 2-chloro-6-chloromethylpyridine side product.

The Center of Biomedical Research Excellence in Matrix Biology strives to improve our understanding of extracellular matrix at molecular, cellular, tissue, and organismal levels to generate new knowledge about pathophysiology, normal development, and regenerative medicine. The primary goals of the Center are to i) support junior investigators, ii) enhance the productivity of established scientists, iii) facilitate collaboration between both junior and established researchers, and iv) build biomedical research infrastructure that will support research relevant to cell−matrix interactions in disease progression, tissue repair and regeneration, and v) provide access to instrumentation and technical support. A Pilot Project program provides funding to investigators who propose applying their expertise to matrix biology questions. Support from the National Institute of General Medical Sciences at the National Institutes of Health that established the Center of Biomedical Research Excellence in Matrix Biology has significantly enhanced the infrastructure and the capabilities of researchers at Boise State University, leading to new approaches that address disease diagnosis, prevention, and treatment. New multidisciplinary collaborations have been formed with investigators who may not have previously considered how their biomedical research programs addressed fundamental and applied questions involving the extracellular matrix. Collaborations with the broader matrix biology community are encouraged.

Samples were collected in 35 states as part of a national monitoring system to detect multiple diseases in feral swine (Sus scrofa). During March 2009 through December 2010, we collected serum samples from 4,479 feral swine from 13 states, and 159 animals tested were seropositive for brucellosis. No difference in likelihood of infection was found between males and females, but adults were more likely than sub-adults or juveniles to be exposed to brucellosis. Feral swine sampled during winter months also were more likely to be seropositive than animals sampled during other seasons. Apparent prevalence varied among states, and seropositive animals often were clustered in specific counties within a state. We recommend improved diagnostics and stricter regulations on movement of feral swine both intra- and inter-state to minimize further spread of the disease and to decrease the risk of reintroduction of brucellosis into livestock.

The utility of Orlando and Thissen's ("2000", "2003") S-X[squared] fit index was extended to the model-fit analysis of the graded response model (GRM). The performance of a modified S-X[squared] in assessing item-fit of the GRM was investigated in light of empirical Type I error rates and power with a simulation study having various conditions typically encountered in applied testing situations. The results show that the Type I error rates were controlled adequately around the nominal alpha by S-X[squared]. The power of the S-X[squared] statistic was much lower when the source of misfit was multidimensionality than when it was due to discrepancy from the true GRM curves. Once the data size increased sufficiently, however, appropriate power was obtained regardless of the source of the item-misfit. In summary, the generalized S-X[squared] appears to be a promising index for investigating item fit for polytomous items in educational and psychological assessments. (Contains 5 tables and 2 figures.)

Orlando and Thissen's S-X[superscript 2] item fit index has performed better than traditional item fit statistics such as Yen' s Q[subscript 1] and McKinley and Mill' s G[superscript 2] for dichotomous item response theory (IRT) models. This study extends the utility of S-X[superscript 2] to polytomous IRT models, including the generalized partial credit model, partial credit model, and rating scale model. The performance of the generalized S-X[superscript 2] in assessing item model fit was studied in terms of empirical Type I error rates and power and compared to G[superscript 2]. The results suggest that the generalized S-X[superscript 2] is promising for polytomous items in educational and psychological testing programs. (Contains 2 figures, 3 tables, and 52 formulas.)

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by microscopic lesions consisting of beta-amyloid plaques and neurofibrillary tangles (NFTs). The majority of cases are defined as sporadic and are likely caused by a combination of both genetic and environmental factors. Of the genetic risk factors identified, the 34 kDa protein, apolipoprotein (apo) E4, is of significant importance as APOE4 carriers account for 65%–80% of all AD cases. Although apoE4 plays a normal role in lipoprotein transport, how it contributes to AD pathogenesis is currently unknown. One potential mechanism by which apoE4 contributes to disease risk is its propensity to undergo proteolytic cleavage generating N- and C-terminal fragments. The purpose of this review will be to examine the mechanisms by which apoE4 contributes to AD pathogenesis focusing on the potential loss or gain of function that may occur following cleavage of the full-length protein. In this context, a discussion of whether targeting apoE4 therapeutically is a rationale approach to treating this disease will be assessed.

The Beclin-1 protein is essential for the initiation of autophagy, and recent studies suggest this function may be compromised in Alzheimer's disease (AD). In addition, in vitro studies have supported a loss of function of Beclin-1 due to proteolytic modification by caspases. In the present study, we examined whether caspase-cleavage of Beclin-1 occurs in the AD brain by designing a site-directed caspase-cleavage antibody based upon a known cleavage site within the protein at position D149. We confirmed that Beclin-1 is an excellent substrate for caspase-3 and demonstrates cleavage led to the formation of a 35-kDa C-terminal fragment labeled by our novel antibody following Western blot analysis. Application of this antibody termed Beclin-1 caspase-cleavage product antibody or BeclinCCP in frontal cortex tissue sections revealed strong immunolabeling within astrocytes that localized with plaque regions and along blood vessels in all AD cases examined. In addition, weaker, more variable BeclinCCP labeling was also observed within neurofibrillary tangles that colocalized with the early tau conformational marker, MC-1 as well as the late tangle marker, PHF-1. Collectively, these data support a depletion of Beclin-1 in AD following caspase-cleavage. This article is part of a Special Issue entitled “Autophagy and protein degradation in neurological diseases.”

Vascular dementia (VaD) is the second most common form of dementia in the United States and is characterized as a cerebral vessel vascular disease that leads to ischemic episodes. Whereas the relationship between caspase-cleaved tau and neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) has been previously described, whether caspase activation and cleavage of tau occurs in VaD is presently unknown. To investigate a potential role for caspase-cleaved tau in VaD, we analyzed seven confirmed cases of VaD by immunohistochemistry utilizing a well-characterized antibody that specifically detects caspase-cleaved tau truncated at Asp421. Application of this antibody (TauC3) revealed consistent labeling within NFTs, dystrophic neurites within plaque-rich regions and corpora amylacea (CA) in the human VaD brain. Labeling of CA by the TauC3 antibody was widespread throughout the hippocampus proper, was significantly higher compared to age matched controls, and co-localized with ubiquitin. Staining of the TauC3 antibody co-localized with MC-1, AT8, and PHF-1 within NFTs. Quantitative analysis indicated that roughly 90% of PHF-1-labeled NFTs contained caspase-cleaved tau. In addition, we documented the presence of active caspase-3 within plaques, blood vessels and pretangle neurons that co-localized with TauC3. Collectively, these data support a role for the activation of caspase-3 and proteolytic cleavage of TauC3 in VaD providing further support for the involvement of this family of proteases in NFT pathology.

Accumulating evidence supports a role for the activation of proteolytic enzymes, caspases, in the Alzheimer's disease (AD) brain. Neurons committed to apoptosis may do so through a mitochondrial pathway employing caspase-9 or through an alternative, receptor-mediated pathway involving caspase-8. Considering the role of mitochondrial dysfunction in AD, we examined the possible activation of caspase-9 in the AD brain using an antibody that recognizes the active fragments of caspase-9, but not the full-length proform of the enzyme. In vivo immunohistochemical analysis demonstrated little caspase-9 activation in the majority of hippocampal brain sections from control brains. However, labeling of neurons as well as dystrophic neurites within plaque regions was observed in all AD hippocampal brain sections examined. In addition, active caspase-9 was colocalized with active caspase-8 and the accumulation of caspase-3-cleavage products of fodrin. The activation of caspase-9 was also observed in neurons positive for oxidative damage to DNA/RNA. A quantitative analysis indicates that as the number of neurons containing neurofibrillary tangles (NFTs) increases, the extent of caspase-9 activation decreases, supporting the idea that caspase-9 activation may precede NFT formation. In addition, a site-directed caspase-cleavage antibody was designed to the amino-terminal caspase-3 consensus cleavage site located in tau, and shown to be an effective marker for caspase-cleaved fragments of tau in vitro. Analysis with this antibody using age-matched control or AD brain sections demonstrated no staining in control brains while widespread labeling of NFTs, neuropil threads, and dystrophic neurites was observed in AD sections. Taken together, these results demonstrate the activation of caspases and cleavage of tau in the AD brain, events which may precede and lead to the formation of NFTs.

Recent studies support the activation of apoptotic pathways in the Alzheimer's disease (AD) brain. Neurons committed to apoptosis may do so by either activation of a receptor-mediated pathway employing caspase-8 or through an alternative mitochondrial pathway involving oxidative stress. In the present study, the role of caspase-8 in the AD brain was examined by designing a caspase-cleavage site-directed antibody to one of the active fragments of caspase-8. In vitro analysis with this antibody, termed CASP-8p18, demonstrated that it recognized the active 18-kDa fragment of caspase-8 but not the precursor protein. In vivo immunohistochemical analysis using hippocampal tissue sections from AD or aged-matched control brains demonstrated CASP-8p18 immunolabeling of neurons in all AD cases, whereas little staining was observed in controls. These results were confirmed using a commercially available antibody that, like the CASP-8p18 antibody reacts only with the 18-kDa fragment of caspase-8 and not full-length caspase-8. As with CASP-8p18 antibody, the commercial antibody-labeled neurons in all AD cases, while showing a relative paucity of staining in representative control cases. Labeling of CASP-8p18 within tangle-bearing neurons was observed in double-labeling studies with AT8 or PHF-1, both markers for neurofibrillary tangles (NFTs). In addition, using a caspase-cleavage site-directed antibody that recognizes cleavage products of caspase-3 showed colocalization of this antibody with the CASP-8p18 antibody within NFTs. These results suggest a role for caspase-8 and the receptor-mediated apoptotic pathway as a mechanism leading to the activation of caspase-3 within neurons of the AD brain.

The Alzheimer disease-associated β-amyloid peptide has been shown to induce apoptotic neuronal death. In the present study, we test the hypothesis that the apoptotic pathway activated by β-amyloid is similar to the pathway activated by the Fas/TNFR family of death receptors, which requires caspase-8 activity and adaptor proteins such as FADD. We demonstrate that the selective caspase-8 inhibitor IETD-fmk blocks neuronal death induced by β-amyloid. Furthermore, using viral-mediated gene delivery, we show that neurons expressing dominant-negative FADD are protected from apoptosis induced by β-amyloid. Together these results indicate that the apoptotic pathway activated by β-amyloid requires both caspase-8 activity and FADD. These findings further support the hypothesis that β-amyloid might initiate apoptosis by cross-linking death receptors of the Fas/TNFR family.

Although the risk factor for apolipoprotein E (apoE) polymorphism in Alzheimer's disease (AD) has been well described, the role that apoE plays in other neurodegenerative diseases, including Pick's disease, is not well established. To examine a possible role of apoE in Pick's disease, an immunohistochemical analysis was performed utilizing a novel site-directed antibody that is specific for an amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE cleavage fragment (nApoECF) antibody, consistently labeled Pick bodies within area CA1 of the hippocampus in 4 of the 5 cases examined. Co-localization of the nApoECF antibody with PHF-1, a general marker for Pick bodies, as well as with an antibody to caspase-cleaved tau (TauC3) was evident within the hippocampus. While staining of the nApoECF antibody was robust in area CA1, little co-localization with PHF-1 in Pick bodies within the dentate gyrus was observed. A quantitative analysis indicated that approximately 86% of the Pick bodies identified in area CA1 labeled with the nApoECF antibody. The presence of truncated apoE within Pick bodies suggests a broader role of apoE beyond AD and raises the question as to whether this protein contributes to pathogenesis associated with Pick's disease.

Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are common causes of motor and cognitive deficits and are associated with the abnormal accumulation of alpha-synuclein (α-syn). This study investigated whether passive immunization with a novel monoclonal α-syn antibody (9E4) against the C-terminus (CT) of α-syn was able to cross into the CNS and ameliorate the deficits associated with α-syn accumulation. In this study we demonstrate that 9E4 was effective at reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved α-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4 traffics into the CNS, binds to cells that display α-syn accumulation and promotes α-syn clearance via the lysosomal pathway. These results suggest that passive immunization with monoclonal antibodies against the CT of α-syn may be of therapeutic relevance in patients with PD and DLB.

Although much is known regarding the molecular mechanisms leading to neuronal cell loss in Parkinson's disease (PD), the initiating event has not been identified. Prevailing theories including a chemical insult or infectious agent have been postulated as possible triggers, leading to neuroinflammation. We present immunohistochemical data indicating the presence of influenza A virus within the substantia nigra pars compacta (SNpc) from postmortem PD brain sections. Influenza A virus labeling was identified within neuromelanin granules as well as on tissue macrophages in the SNpc. Further supporting a role for neuroinflammation in PD was the identification of T-lymphocytes that colocalized with an antibody to caspase-cleaved Beclin-1 within the SNpc. The presence of influenza A virus together with macrophages and T-lymphocytes may contribute to the neuroinflammation associated with this disease.

Hands-on continuous process improvement and ability to train others are highly desired industry job skills for industrial and systems engineering (ISyE) graduates. It is important for higher education institutions to continuously strive for innovative curriculum and prepare their students adequately for industry. Traditional ISyE curricula typically involve fewer equipment and instrumentations compared to other engineering disciplines. As a result, ISyE students are often trained by using software, simulations and conceptual projects. To bridge the hands-on skill gap in our graduates, a new integrated manufacturing processes lab instructional pedagogy was introduced to ISyE juniors. In the new curriculum model, students were provided with the opportunity to apply continuous process improvement concept to traditional manufacturing processes while learning basic manufacturing operations and machine tools. Students cither implemented their own continuous process improvement ideas on self-designed products, or trained other peers to execute their improvement proposals. A survey was conducted at the end of the lab course to assess students' experiences with the new lab curriculum and their perception of industry job skills. Results show that students highly valued the new learning experiences in the manufacturing processes lab course and wished to have more similar opportunities in their ISyE courses. Data also reveals that student perception of hands-on continuous process improvement skill and ability to train others were consistent with industry expectations with respect to the level of importance. However, students perceived the ability to train others to be more highly desired by their potential employers compared to the hands-on continuous process improvement skill, whereas industry shared the opposite expectation. The outcome of this study encourages the implementation of an integrated curriculum and instructional pedagogy model in which hands-on training of crosscutting concepts is incorporated into traditional courses. Several areas of course improvement was also identified at the end of the study to further increase the effectiveness in preparing ISyE graduates for careers in industry. [ABSTRACT FROM AUTHOR]

Background: Many survivors of lung injury, including bacterial pneumonia and COVID‐19, suffer from incident dementia. Patients who have had pneumonia and other infections are at a higher risk for developing Alzheimer's disease and related dementias (ADRD) (Chu et al., BBI, 2022, Sipila et al., Lancet Infec Dis, 2021, Girard et al., J Gen Intern Med, 2018). There is growing appreciation and strong evidence that neurovascular uncoupling, cerebral blood flow reductions and dysregulation, and breakdown of the blood‐brain barrier (BBB), including the loss of pericytes, are early events in the ADRD pathophysiological cascade. Furthermore, astrocytes and microglia are activated in the ADRD brain and can also disrupt cerebrovascular health and impact cognition. Because these are key events in ADRD, we aimed to determine the effects of bacterial pneumonia on the cerebrovascular system. Method: C57BL/6J mice were intratracheally infected with P. aeruginosa PA103 (ExoU and ExoT competent, 2.5×105 CFU) was introduced as a virulent bacterial strain for 24 or 48 hours. In addition, ΔPcrV (PA103ΔPcrV, 2.5×107 CFU), a mutant lacking a functional Type III secretion system was used for control. Following transcardial perfusion, brains were snap frozen in OCT and serially sectioned. Next, we performed immunofluorescence staining for CD13 (pericytes), lectin (endothelial cells), IgG (BBB leakage), Gfap (reactive astrocytes), and Iba1 (microglia). Result: Significant BBB leakage of IgG was detected in both cortex and hippocampus 48 hours post‐infection with PA103 compared to ΔPcrV. Significant activation of astrocytes and microglia was detected 48 hours post‐PA103 infection compared to ΔPcrV. Pericyte coverage was unchanged in cortex and hippocampus both 24‐ and 48‐hours post‐infection with PA103 compared to ΔPcrV. Conclusion: Here we have shown that bacterial pneumonia causes BBB breakdown and gliosis in young mice. Because BBB breakdown and gliosis are early events in many neurodegenerative diseases leading to cognitive dysfunction, understanding how pneumonia can accelerate these events is extremely timely and important. Future studies should focus on the mechanism(s) by which pneumonia causes cerebrovascular dysfunction and how this may increase ADRD. [ABSTRACT FROM AUTHOR]

Background: CNS neurons and microglia participate in neuroimmune pathways linked to Alzheimer's Disease (AD) and AD Related Dementias (ADRD). However, CNS neurons and microglia cannot be isolated from living people, and primary microglia harvested post‐mortem rapidly de‐differentiate in culture. hiPSC‐neurons and hiPSC‐MG represent a practical alternative for in vitro research, as they are human‐derived, upscalable, and can be made from people with AD/ADRD‐sensitive or ‐resilient genotypes. We are developing high‐throughput assays utilizing hiPSC‐neurons/‐MG, to explore interactions between these cell types and pathways relevant to AD, and to test compounds for beneficial effects on neuronal health. Method: hiPSC‐neurons (excitatory) were purchased from Fujifilm CDI or differentiated in‐house (with kits from Elixirgen). hiPSC‐MG were differentiated in‐house from hiPSCs via a protocol in which yolk sac‐like structures are produced as an intermediary step. Mono‐ or cocultures were prepared in 96‐ or 384‐well dishes. Results were obtained via automated digital microscopy, with cellular structures and biomarkers analyzed via high content analysis (HCA) and cell activity (calcium or voltage transients) analyzed via Kinetic Image Cytometry; in certain experiments, LDH release, a general measure of cytotoxicity, was also assayed. Result: Coculture of hiPSC‐MG with hiPSC‐neurons led to a 2‐fold increase in neurites. hiPSC‐MG also elicited calcium transients in the hiPSC‐neurons via close contact. The APOE‐e4 gene variant confers the greatest risk for late‐onset AD, and proteolysis of ApoE4 may produce neurotoxic fragments. In our hands, a fragment of ApoE4 (ApoECFp17, aa 1‐151) induced LDH release in both hiPSC‐neurons and hiPSC‐MG, indicating cytotoxicity, whereas the corresponding fragment of ApoE3 had no effect; additionally, the toxic effect on hiPSC‐MG was partially blocked by PH002 (an ApoE4 "structure corrector") and by RAP (a protein that blocks ApoE4 binding to cell‐surface receptors). Conclusion: Our data demonstrate that hiPSC‐MG alter neurite expression and calcium transient activity in cocultured hiPSC‐neurons, and that the aa 1‐151 fragment of ApoE4 is toxic to human neurons and microglia, which could potentially play a role in AD etiology. Future research will incorporate hiPSC‐astrocytes into the cultures to further model CNS neuronal‐glial interactions and elucidate pathways relevant to AD/ADRD. [ABSTRACT FROM AUTHOR]

Background The annual incidence of Clostridium difficile infection (CDI) in the United States is estimated to be 330,000 cases. We evaluated the impact of using a launderable mattress and bed deck cover on the incidence of hospital-onset CDI in 2 long-term acute care hospitals (LTACHs). Methods Two LTACHs began using a launderable mattress and bed deck cover on beds starting in May 2013. One facility had 74 beds, and the other had 30 beds. Covers were changed after every patient. The covers were laundered using hot water, detergent, and chlorine. Rates for CDIs were compared using Poisson regression between the 16 months before use of the launderable cover and the 14 months after the cover started being used. Results At hospital A, the use of bedcovers reduced the rate of infection by 47.8% (95% confidence interval [CI], 47.1-48.6), controlling for the rate of handwashing compliance and length of stay in days. At hospital B, the use of bedcovers reduced the rate of infection by 50% (95% CI, 47.5-52.7), controlling for the rate of handwashing compliance and length of stay in days. Conclusion The use of a launderable cover for mattresses and bed decks of hospital beds was associated with a decreased rate of health care–associated CDI in 2 LTACHs. [ABSTRACT FROM AUTHOR]