RESUMEN

Maternal opioid use poses a significant health concern not just to the expectant mother but also to the fetus. Notably, increasing numbers of children born suffering from neonatal opioid withdrawal syndrome (NOWS) further compounds the crisis. While epidemiological research has shown the heightened risk factors associated with NOWS, little research has investigated what molecular mechanisms underly the vulnerabilities these children carry throughout development and into later life. To understand the implications of in utero and post-natal opioid exposure on the developing brain, we sought to assess the response to one of the most common pediatric injuries: minor traumatic brain injury (mTBI). Using a rat model of in utero and post-natal oxycodone (IUO) exposure and a low force weight drop model of mTBI, we show that not only neonatal opioid exposure significantly affects neuroinflammation, brain metabolites, synaptic proteome, mitochondrial function, and altered behavior in juvenile rats, but also, in conjunction with mTBI these aberrations are further exacerbated. Specifically, we observed long term metabolic dysregulation, neuroinflammation, alterations in synaptic mitochondria, and impaired behavior were impacted severely by mTBI. Our research highlights the specific vulnerability caused by IUO exposure to a secondary stressor such as later life brain injury. In summary, we present a comprehensive study to highlight the damaging effects of prenatal opioid abuse in conjunction with mild brain injury on the developing brain.

RESUMEN

With the aim of developing the concept of pretargeted click chemistry for the diagnosis of Alzheimer's disease two antibodies specific for amyloid-ß were modified to incorporate trans-cyclooctene functional groups. Two bis(thiosemicarbazone) compounds with pendant 1,2,4,5-tetrazine functional groups were prepared and radiolabelled with positron emitting copper-64. The new copper-64 complexes rapidly react with the trans-cyclooctene functionalized antibodies in a bioorthogonal click reaction and cross the blood-brain barrier in mice.

Asunto(s)

RESUMEN

CD22 has been suggested to contribute to Alzheimer's disease (AD) pathogenesis by inhibiting microglial amyloid ß (Aß) phagocytosis. Soluble CD22 (sCD22) generated by cleavage from cell membranes may be a marker of inflammation and microglial dysfunction; but alterations of sCD22 levels in AD and their correlation with AD biomarkers remain unclear. Plasma sCD22 levels were measured in cognitively normal non-AD participants and patients with preclinical AD and AD dementia from a Chinese cohort and the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing. Plasma sCD22 levels were elevated in patients with preclinical and dementia AD. Plasma sCD22 levels were negatively correlated with cerebrospinal fluid (CSF) Aß42 levels and Aß42/Aß40, and positively correlated with CSF phosphorylated tau levels and brain Aß burden, but negatively correlated with cognitive function. Moreover, higher plasma sCD22 levels were associated with faster cognitive decline during follow-up. These findings suggest that CD22 plays important roles in AD development, and that sCD22 is a potential biomarker for AD.

Asunto(s)

RESUMEN

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterised by cognitive impairment, behavioural alteration, and functional decline. Over 130 AD-associated susceptibility loci have been identified by genome-wide association studies (GWAS), while whole genome sequencing (WGS) and whole exome sequencing (WES) studies have identified AD-associated rare variants. These variants are enriched in APOE, TREM2, CR1, CD33, CLU, BIN1, CD2AP, PILRA, SCIMP, PICALM, SORL1, SPI1, RIN3, and more genes. Given that aging is the single largest risk factor for late-onset AD (LOAD), the accumulation of somatic mutations in the brain and blood of AD patients have also been explored. Collectively, these genetic findings implicate the role of innate and adaptive immunity in LOAD pathogenesis and suggest that a systemic failure of cell-mediated amyloid-ß (Aß) clearance contributes to AD onset and progression. AD-associated variants are particularly enriched in myeloid-specific regulatory regions, implying that AD risk variants are likely to perturbate the expression of myeloid-specific AD-associated genes to interfere Aß clearance. Defective phagocytosis, endocytosis, and autophagy may drive Aß accumulation, which may be related to naturally-occurring antibodies to Aß (Nabs-Aß) produced by adaptive responses. Passive immunisation is providing efficiency in clearing Aß and slowing cognitive decline, such as aducanumab, donanemab, and lecanemab (ban2401). Causation of AD by impairment of the innate immunity and treatment using the tools of adaptive immunity is emerging as a new paradigm for AD, but immunotherapy that boosts the innate immune functions of myeloid cells is highly expected to modulate disease progression at asymptomatic stage.

Asunto(s)

RESUMEN

INTRODUCTION: Anesthesia residents are deemed competent based on subjective and objective metrics. Knowledge acquisition and procedural skill is often difficult to accurately measure. Inspecting tangible metrics of perioperative efficiency may provide a source for reliable evaluation. METHODS: Retrospective case-log database review yielded 3072 surgical cases involving residents over 5 years. Primary variable investigated was the time from surgery completion to exit from operating room. Other variables recorded included day of week, attending anesthesiologist name, attending surgeon name, patient age, sex, American Society of Anesthesiologists physical status (ASA PS) classification, and inpatient versus day surgery status. RESULTS: After controlling for procedure duration time, inpatient status, ASA PS, surgeon, and attending anesthesiologist, resident training time had high statistical significance. In the fully adjusted model, 1 year of resident training was associated with a reduction in emergence time by 28 seconds. A 1-hour increase in procedure time was associated with an increase in emergence time of 34 seconds. CONCLUSIONS: Although a statistically significant correlation between anesthesia resident training time and emergence time was demonstrated, the clinical significance is likely low given the relatively small amount of actual time saved. We caution the value of using perioperative metrics (e.g., emergence time) for evaluating anesthesia resident competency, until such metrics have undergone significant validation.

RESUMEN

Polymorphism in the microglial receptor CD33 gene has been linked to late-onset Alzheimer disease (AD), and reduced expression of the CD33 sialic acid-binding domain confers protection. Thus, CD33 inhibition might be an effective therapy against disease progression. Progress toward discovery of selective CD33 inhibitors has been hampered by the absence of an atomic resolution structure. We report here the crystal structures of CD33 alone and bound to a subtype-selective sialic acid mimetic called P22 and use them to identify key binding residues by site-directed mutagenesis and binding assays to reveal the molecular basis for its selectivity toward sialylated glycoproteins and glycolipids. We show that P22, when presented on microparticles, increases uptake of the toxic AD peptide, amyloid-ß (Aß), into microglial cells. Thus, the sialic acid-binding site on CD33 is a promising pharmacophore for developing therapeutics that promote clearance of the Aß peptide that is thought to cause AD.

RESUMEN

In the five-term, transitive inference task used with animals, pigeons are trained on four simultaneous discrimination premise pairs: A + B -, B + C -, C + D -, D + E -. Typically, when tested with the BD pair, most pigeons show a transitive inference effect, choosing B over D. Two non-inferential hypotheses have been proposed to account for this effect but neither has been reliably supported by research. Here we test a third non-inferential hypothesis that the preference for B arises because the animals have not had as much experience with B - in the A + B - discrimination as they have had with the D - in the C + D - discrimination. To test this hypothesis we trained the Experimental Group with the A + B - discrimination in which, over trials, there were four possible A + stimuli that could appear. This was done to encourage the pigeons to learn to reject the B - stimulus. For the Control Group there was only one A + stimulus over trials, as is typically the case. We also varied the nature of the stimuli between groups, such that colors served as the stimuli for half of the pigeons, whereas flags of different counties served as stimuli for the remaining pigeons. In both stimulus conditions, for the Experiment Group, we found little preference for stimulus B over stimulus D, whereas for the Control Group we found the typical preference for stimulus B. Thus, we propose that it is not necessary to attribute the transitive inference effect to an inferential process.

Asunto(s)

RESUMEN

The rules of engagement between zinc finger transcription factors and DNA have been partly defined by in vitro DNA-binding and structural studies, but less is known about how these rules apply in vivo. Here, we demonstrate how a missense mutation in the second zinc finger of Krüppel-like factor-1 (KLF1) leads to degenerate DNA-binding specificity in vivo, resulting in ectopic transcription and anemia in the Nan mouse model. We employed ChIP-seq and 4sU-RNA-seq to identify aberrant DNA-binding events genome wide and ectopic transcriptional consequences of this binding. We confirmed novel sequence specificity of the mutant recombinant zinc finger domain by performing biophysical measurements of in vitro DNA-binding affinity. Together, these results shed new light on the mechanisms by which missense mutations in DNA-binding domains of transcription factors can lead to autosomal dominant diseases.

Asunto(s)

RESUMEN

BACKGROUND: Proteolytic degradation of amyloid ß (Aß) peptides has been intensely studied due to the central role of Aß in Alzheimer's disease (AD) pathogenesis. While several enzymes have been shown to degrade Aß peptides, the main pathway of Aß degradation in vivo is unknown. Cerebrospinal fluid (CSF) Aß42 is reduced in AD, reflecting aggregation and deposition in the brain, but low CSF Aß42 is, for unknown reasons, also found in some inflammatory brain disorders such as bacterial meningitis. METHOD: Using 18O-labeling mass spectrometry and immune-affinity purification, we examined endogenous proteolytic processing of Aß in human CSF. RESULTS: The Aß peptide profile was stable in CSF samples from healthy controls but in CSF samples from patients with bacterial meningitis, showing increased leukocyte cell count, 18O-labeling mass spectrometry identified proteolytic activities degrading Aß into several short fragments, including abundant Aß1-19 and 1-20. After antibiotic treatment, no degradation of Aß was detected. In vitro experiments located the source of the proteolytic activity to blood components, including leukocytes and erythrocytes, with insulin-degrading enzyme as the likely protease. A recombinant version of the mid-domain anti-Aß antibody solanezumab was found to inhibit insulin-degrading enzyme-mediated Aß degradation. CONCLUSION: 18O labeling-mass spectrometry can be used to detect endogenous proteolytic activity in human CSF. Using this technique, we found an enzymatic activity that was identified as insulin-degrading enzyme that cleaves Aß in the mid-domain of the peptide, and could be inhibited by a recombinant version of the mid-domain anti-Aß antibody solanezumab.

Asunto(s)

RESUMEN

C-reactive protein (CRP) concentrations rise in response to tissue injury or infection. Circulating pentameric CRP (pCRP) localizes to damaged tissue where it leads to complement activation and further tissue damage. In-depth knowledge of the pCRP activation mechanism is essential to develop therapeutic strategies to minimize tissue injury. Here we demonstrate that pCRP by binding to cell-derived microvesicles undergoes a structural change without disrupting the pentameric symmetry (pCRP*). pCRP* constitutes the major CRP species in human-inflamed tissue and allows binding of complement factor 1q (C1q) and activation of the classical complement pathway. pCRP*-microvesicle complexes lead to enhanced recruitment of leukocytes to inflamed tissue. A small-molecule inhibitor of pCRP (1,6-bis(phosphocholine)-hexane), which blocks the pCRP-microvesicle interactions, abrogates these proinflammatory effects. Reducing inflammation-mediated tissue injury by therapeutic inhibition might improve the outcome of myocardial infarction, stroke and other inflammatory conditions.

Asunto(s)

RESUMEN

PURPOSE: We sought to evaluate the feasibility, safety, and difficulty of performing the per-oral endoscopic myotomy (POEM) procedure in the setting of a prior Heller myotomy using a survival porcine model. METHODS: Four pigs underwent laparoscopic Heller myotomy with Dor partial anterior fundoplication followed by the POEM performed 4 weeks later. Two additional pigs served as controls, undergoing only the POEM. RESULTS: All procedures were completed without complications. The revisional POEM was not significantly more difficult than POEM controls based on procedure time, POEM procedure components, or procedure difficulty scores. Revisional POEM had a longer mean operative time when compared with Heller myotomy (126.0 vs. 83.8 min; P<0.01) but had a lower total difficulty score (28.6 vs. 52.1; P≪0.01). CONCLUSIONS: A POEM after previous Heller myotomy is safe and feasible in the porcine model and has potential as an option for patients suffering from recurrent or persistent symptoms after failed surgical myotomy.

Asunto(s)

RESUMEN

Solanezumab and Crenezumab are two humanized antibodies targeting Amyloid-ß (Aß) which are currently tested in multiple clinical trials for the prevention of Alzheimer's disease. However, there is a scientific discussion ongoing about the target engagement of these antibodies. Here, we report the immunohistochemical staining profiles of biosimilar antibodies of Solanezumab, Crenezumab and Bapineuzumab in human formalin-fixed, paraffin-embedded tissue and human fresh frozen tissue. Furthermore, we performed a direct comparative immunohistochemistry analysis of the biosimilar versions of the humanized antibodies in different mouse models including 5XFAD, Tg4-42, TBA42, APP/PS1KI, 3xTg. The staining pattern with these humanized antibodies revealed a surprisingly similar profile. All three antibodies detected plaques, cerebral amyloid angiopathy and intraneuronal Aß in a similar fashion. Remarkably, Solanezumab showed a strong binding affinity to plaques. We also reaffirmed that Bapineuzumab does not recognize N-truncated or modified Aß, while Solanezumab and Crenezumab do detect N-terminally modified Aß peptides Aß4-42 and pyroglutamate Aß3-42. In addition, we compared the results with the staining pattern of the mouse NT4X antibody that recognizes specifically Aß4-42 and pyroglutamate Aß3-42, but not full-length Aß1-42. In contrast to the biosimilar antibodies of Solanezumab, Crenezumab and Bapineuzumab, the murine NT4X antibody shows a unique target engagement. NT4X does barely cross-react with amyloid plaques in human tissue. It does, however, detect cerebral amyloid angiopathy in human tissue. In Alzheimer mouse models, NT4X detects intraneuronal Aß and plaques comparable to the humanized antibodies. In conclusion, the biosimilar antibodies Solanezumab, Crenezumab and Bapineuzumab strongly react with amyloid plaques, which are in contrast to the NT4X antibody that hardly recognizes plaques in human tissue. Therefore, NT4X is the first of a new class of therapeutic antibodies.

Asunto(s)

RESUMEN

Solanezumab (Eli Lilly) and crenezumab (Genentech) are the leading clinical antibodies targeting Amyloid-ß (Aß) to be tested in multiple Phase III clinical trials for the prevention of Alzheimer's disease in at-risk individuals. Aß capture by these clinical antibodies is explained here with the first reported mid-region Aß-anti-Aß complex crystal structure. Solanezumab accommodates a large Aß epitope (960 Å(2) buried interface over residues 16 to 26) that forms extensive contacts and hydrogen bonds to the antibody, largely via main-chain Aß atoms and a deeply buried Phe19-Phe20 dipeptide core. The conformation of Aß captured is an intermediate between observed sheet and helical forms with intramolecular hydrogen bonds stabilising residues 20-26 in a helical conformation. Remarkably, Aß-binding residues are almost perfectly conserved in crenezumab. The structure explains the observed shared cross reactivity of solanezumab and crenezumab with proteins abundant in plasma that exhibit this Phe-Phe dipeptide.

Asunto(s)

Asunto(s)

RESUMEN

Reducing amyloid-ß peptide (Aß) burden at the pre-symptomatic stages of Alzheimer's disease (AD) is currently the advocated clinical strategy for treating this disease. The most developed method for targeting Aß is the use of monoclonal antibodies including bapineuzumab, solanezumab and crenezumab. We have synthesized these antibodies and used surface plasmon resonance (SPR) and mass spectrometry to characterize and compare the ability of these antibodies to target Aß in transgenic mouse tissue as well as human AD tissue. SPR analysis showed that the antibodies were able to bind Aß with high affinity. All of the antibodies were able to bind Aß in mouse tissue. However, significant differences were observed in human brain tissue. While bapineuzumab was able to capture a variety of N-terminally truncated Aß species, the Aß detected using solanezumab was barely above detection limits while crenezumab did not detect any Aß. None of the antibodies were able to detect any Aß species in human blood. Immunoprecipitation experiments using plasma from AD subjects showed that both solanezumab and crenezumab have extensive cross-reactivity with non-Aß related proteins. Bapineuzumab demonstrated target engagement with brain Aß, consistent with published clinical data. Solanezumab and crenezumab did not, most likely as a result of a lack of specificity due to cross-reactivity with other proteins containing epitope overlap. This lack of target engagement raises questions as to whether solanezumab and crenezumab are suitable drug candidates for the preventative clinical trials for AD.

Asunto(s)

RESUMEN

Bapineuzumab (AAB-001) and its derivative (AAB-003) are humanized versions of the anti-Aß murine antibody 3D6 and are immunotherapy candidates in Alzheimer's disease. The common Fab fragment of these immunotherapies has been expressed, purified and crystallized in complex with ß-amyloid peptides (residues 1-8 and 1-28). Diffraction data at high resolution were acquired from crystals of Fab-Aß8 (2.0 Å) and Fab-Aß28 (2.2 Å) complexes at the Australian Synchrotron. Both crystal forms belonged to the primitive orthorhombic space group P21221.

Asunto(s)

RESUMEN

Bapineuzumab is a humanized antibody developed by Pfizer and Johnson & Johnson targeting the amyloid (Aß) plaques that underlie Alzheimer's disease neuropathology. Here we report the crystal structure of a Fab-Aß peptide complex that reveals Bapineuzumab surprisingly captures Aß in a monomeric helical conformation at the N-terminus. Microscale thermophoresis suggests that the Fab binds soluble Aß(1-40) with a K(D) of 89 (±9) nM. The structure explains the antibody's exquisite selectivity for particular Aß species and why it cannot recognize N-terminally modified or truncated Aß peptides.

Asunto(s)

RESUMEN

In this mini-review we focus on metal interactions with proteins with a particular emphasis on the evident synergism between different biophysical approaches toward understanding metallobiology. We highlight three recent examples from our own laboratory. Firstly, we describe metallodrug interactions with glutathione S-transferases, an enzyme family known to attack commonly used anti-cancer drugs. We then describe a protein target for memory enhancing drugs called insulin-regulated aminopeptidase in which zinc plays a role in catalysis and regulation. Finally we describe our studies on a protein, amyloid precursor protein, that appears to play a central role in Alzheimer's disease. Copper ions have been implicated in playing both beneficial and detrimental roles in the disease by binding to different regions of this protein.

Asunto(s)

RESUMEN

In vitro translation systems derived from a wide range of organisms have been described in the literature and are widely used in biomedical research laboratories. Perhaps the most robust and efficient of these cell-free systems is that derived from Escherichia coli. Over the past decade or so, experimental strategies have been developed which have enhanced the efficiency and stability of E. coli cell-free systems such that we can now prepare recombinant proteins on a scale suitable for purification and analysis by biophysical and structural biology techniques, which commonly require relatively large quantities of protein. This chapter describes in detail the protocols employed in our laboratory to prepare translationally active E. coli extracts and to synthesise proteins on a milligram scale from these extracts.

Asunto(s)