A Call for Better, Not Faster, Research Ethics Committee Reviews in the Covid-19 Era.

With the rapid spread of SARS-CoV2 has come a rapid proliferation of clinical research studies, resulting in considerable strain on research ethics committees (RECS), which need to review study proposals. RECs are pressured to move through the review process quickly so that studies can get underway to address the pandemic. These committees are also asked to increase efficiency without relaxing the standards for ethical review. RECs are accustomed to external pressure for approval from investigators; however, in the Covid-19 era, this pressure is coming from not only the sponsors and investigators but also many other stakeholders, including world leaders, the community, the media, and professional organizations. Drawing on the authors' experiences on a central REC reviewing complex multicenter Covid-19 studies, this commentary describes challenges that are inherent to Covid-19 research studies, such as the difficulty of obtaining informed consent from patients ill with the highly infectious virus. The commentary recommends several steps that RECs can take to ensure ethical review of research studies during the Covid-19 pandemic and future infectious disease outbreaks.

Three Dimensional Human Neuro-Spheroid Model of Alzheimer's Disease Based on Differentiated Induced Pluripotent Stem Cells.

The testing of candidate drugs to slow progression of Alzheimer's disease (AD) requires clinical trials that are lengthy and expensive. Efforts to model the biochemical milieu of the AD brain may be greatly facilitated by combining two cutting edge technologies to generate three-dimensional (3D) human neuro-spheroid from induced pluripotent stem cells (iPSC) derived from AD subjects. We created iPSC from blood cells of five AD patients and differentiated them into 3D human neuronal culture. We characterized neuronal markers of our 3D neurons by immunocytochemical staining to validate the differentiation status. To block the generation of pathologic amyloid ß peptides (Aß), the 3D-differentiated AD neurons were treated with inhibitors targeting ß-secretase (BACE1) and γ-secretases. As predicted, both BACE1 and γ-secretase inhibitors dramatically decreased Aß generation in iPSC-derived neural cells derived from all five AD patients, under standard two-dimensional (2D) differentiation conditions. However, BACE1 and γ-secretase inhibitors showed less potency in decreasing Aß levels in neural cells differentiated under 3D culture conditions. Interestingly, in a single subject AD1, we found that BACE1 inhibitor treatment was not able to significantly reduce Aß42 levels. To investigate underlying molecular mechanisms, we performed proteomic analysis of 3D AD human neuronal cultures including AD1. Proteomic analysis revealed specific reduction of several proteins that might contribute to a poor inhibition of BACE1 in subject AD1. To our knowledge, this is the first iPSC-differentiated 3D neuro-spheroid model derived from AD patients' blood. Our results demonstrate that our 3D human neuro-spheroid model can be a physiologically relevant and valid model for testing efficacy of AD drug.

Retromer disruption promotes amyloidogenic APP processing.

Retromer deficiency has been implicated in sporadic AD and animals deficient in retromer components exhibit pronounced neurodegeneration. Because retromer performs retrograde transport from the endosome to the Golgi apparatus and neuronal Aß is found in late endosomal compartments, we speculated that retromer malfunction might enhance amyloidogenic APP processing by promoting interactions between APP and secretase enzymes in late endosomes. We have evaluated changes in amyloid precursor protein (APP) processing and trafficking as a result of disrupted retromer activity by knockdown of Vps35, a vacuolar sorting protein that is an essential component of the retromer complex. Knocking down retromer activity produced no change in the quantity or cellular distribution of total cellular APP and had no affect on internalization of cell-surface APP. Retromer deficiency did, however, increase the ratio of secreted Aß42:Aß40 in HEK-293 cells over-expressing APP695, due primarily to a decrease in Aß40 secretion. Recent studies suggest that the retromer-trafficked protein, Wntless, is secreted at the synapse in exosome vesicles and that these same vesicles contain Aß. We therefore hypothesized that retromer deficiency may be associated with altered exosomal secretion of APP and/or secretase fragments. Holo-APP, Presenilin and APP C-terminal fragments were detected in exosomal vesicles secreted from HEK-293 cells. Levels of total APP C-terminal fragments were significantly increased in exosomes secreted by retromer deficient cells. These data suggest that reduced retromer activity can mimic the effects of familial AD Presenilin mutations on APP processing and promote export of amyloidogenic APP derivatives.

A dedicated consultant-led vascular access team significantly reduces out-of-hours and emergency permanent central venous access insertions.

PURPOSE: The insertion of a permanent central venous access device (PCVAD i.e. Broviac or Hickman lines and vascuports) is often considered a low priority and performed as an emergency. In 2004, a vascular access team (VAT), responsible for all PCVAD insertions, was established in our institution to address this. METHODS: Data were collected retrospectively on all PCVAD insertions in 2 periods (January 2002-December 2003 and January 2005-December 2006). This included procedure, list type, surgeon grade, and operative time. RESULTS: During 2002 to 2003 and 2005 to 2006, 465 and 569 PCVADs were inserted, respectively (22% increase). After introduction of the VAT, the percentage of lines inserted during emergency lists fell from 24% (n = 112) to 13% (n = 72), and out-of-hours fell from 6% (n = 29) to 3% (n = 17; P < .05), respectively. Median time taken for PCVAD insertion in 2005 to 2006 was significantly less if using an elective list compared with insertions performed on an emergency list (elective, 67 [56-82] minutes vs emergency, 85 (65-110) minutes; P < .05). DISCUSSION: Introduction of a VAT has led to a significant decrease in emergency and out-of-hours PCVAD insertions, despite an increase in overall activity for the period. The median time taken for elective insertions is significantly less than emergency insertions, which has increased the efficiency of vascular access provision at our institution.

Insulin degrading enzyme is localized predominantly at the cell surface of polarized and unpolarized human cerebrovascular endothelial cell cultures.

Insulin degrading enzyme (IDE) is expressed in the brain and may play an important role there in the degradation of the amyloid beta peptide (Abeta). Our results show that cultured human cerebrovascular endothelial cells (HCECs), a primary component of the blood-brain barrier, express IDE and may respond to exposure to low levels of Abeta by upregulating its expression. When radiolabeled Abeta is introduced to the medium of cultured HCECs, it is rapidly degraded to smaller fragments. We believe that this degradation is largely the result of the action of IDE, as it can be substantially blocked by the presence of insulin in the medium, a competitive substrate of IDE. No inhibition is seen when an inhibitor of neprilysin, another protease that may degrade Abeta, is present in the medium. Our evidence suggests that the action of IDE occurs outside the cell, as inhibitors of internalization fail to affect the rate of the observed degradation. Further, our evidence suggests that degradation by IDE occurs on the plasma membrane, as much of the IDE present in HCECs was biotin-labeled by a plasma membrane impermeable reagent. This activity seems to be polarity dependent, as measurement of Abeta degradation by each surface of differentiated HCECs shows greater degradation on the basolateral (brain-facing) surface. Thus, IDE could be an important therapeutic target to decrease the amount of Abeta in the cerebrovasculature.

Upregulation of clusterin/apolipoprotein J in lactacystin-treated SH-SY5Y cells.

Clusterin (apolipoprotein J) is a highly conserved, multifunctional, vertebrate glycoprotein. Several isoforms of clusterin have been described including the predominant secreted isoform (sCLU) and several nuclear isoforms (nCLU) associated with cell death. sCLU has been shown to bind a variety of partly unfolded, stressed proteins including those associated with Lewy bodies (LBs) in patients with Parkinson's disease (PD). The development of familial and sporadic PD has been associated with the ubiquitin-proteasome system (UPS) dysfunction and aberrant protein degradation. This suggests that failure of the UPS to degrade abnormal proteins may underlie nigral degeneration and LB formation in PD. The effects of toxin-mediated proteasomal impairment on changes in gene expression and cell viability were studied in differentiated SH-SY5Y cells. Clusterin expression was increased in cells exposed for 24 hr to the proteasomal inhibitor lactacystin (10 microM) as determined by gene microarray analysis. RT-PCR showed that sCLU, not nCLU, was the major clusterin isoform expressed in both control and lactacystin-treated cells. Western blot analysis identified statistically significant increases in sCLU in total cell lysates after 24 hr of lactacystin exposure and showed that sCLU fractionates with the endoplasmic reticulum. Time-course studies demonstrated that maximal decreases in proteasome activity (4 hr) preceded maximal increases in clusterin expression (24 hr). Together these data suggest that proteasome impairment results in the upregulation of sCLU in SH-SY5Y cells, supporting the hypothesis that the association of clusterin with LBs in PD may be related to UPS failure.

Insulin degrading enzyme is expressed in the human cerebrovascular endothelium and in cultured human cerebrovascular endothelial cells.

Insulin degrading enzyme (IDE) is found in the cytosol, peroxisomes and plasma membrane of many cells. Although it preferentially cleaves insulin it can also cleave many other small proteins with diverse sequences including the monomeric form of the amyloid beta peptide (A beta). In the brain, IDE has been reported to be expressed predominantly in neurons. In this study, IDE expression was detected in cultured human cerebrovascular endothelial cells. Using laser capture microdissection followed by PCR analysis, it was found that IDE mRNA is expressed in human brain blood vessels. Using immunofluorescence and multiphoton microscopy IDE was localized to the endothelium of the cerebrovascular blood vessels in human.

Identification of the protein disulfide isomerase family member PDIp in experimental Parkinson's disease and Lewy body pathology.

Parkinson's disease (PD) is a slowly progressing neurodegenerative disorder with no clear etiology. Pathological hallmarks of the disease include the loss of dopaminergic neurons from the substantia nigra (SN) and the presence of Lewy bodies (LBs) (alpha-synuclein and ubiquitin-positive, eosinophilic, cytoplasmic inclusions) in many of the surviving neurons. Experimental modeling of PD neurodegeneration using the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenyl-pyridinium (MPP(+)) has identified changes in gene expression of different endoplasmic reticulum (ER) stress proteins associated with MPTP- and PD-related neurodegeneration. We show that the protein disulfide isomerase (PDI) family member pancreatic protein disulfide isomerase (PDIp), previously considered exclusively expressed in pancreatic tissue, is uniquely upregulated among PDI family members within 24 h following exposure of retinoic acid (RA)-differentiated SH-SY5Y human neuroblastoma cells to either 1 mM MPP(+) or 10 microM of the highly specific proteasome inhibitor lactacystin. RT-PCR confirms PDIp expression in brain of post-mortem human PD subjects and immunohistochemical studies demonstrate PDIp immunoreactivity in LBs. Collectively, these findings suggest that increased PDIp expression in dopaminergic (DA) neurons might contribute to LB formation and neurodegeneration, and that this increased PDIp expression may be the result of proteasome impairment.

Escherichia coli Shiga toxin 1 and TNF-alpha induce cytokine release by human cerebral microvascular endothelial cells.

Infection with Shiga toxin (Stx)-producing Escherichia coli can lead to development of hemolytic uremic syndrome (HUS). Patients with severe HUS often exhibit central nervous system (CNS) pathology, which is thought to involve damage to brain endothelium, a component of the blood-brain barrier. We hypothesized that this neuropathology occurs when cerebral endothelial cells of the blood-brain barrier, sensitized by exogenous TNF-alpha and stimulated by Stx1, produce and release proinflammatory cytokines. This was tested by measuring changes in cytokine mRNA and protein expression in human brain endothelial cells (hBEC) in vitro when challenged by TNF-alpha and/or Stx. High doses of Stx1 alone were somewhat cytotoxic to hBEC; Stx1-treated cells produced increased amounts of IL-6 mRNA and secreted this cytokine. IL-1beta and TNF-alpha mRNA, but not protein, were increased, and IL-8 secretion increased without an observed increase in mRNA. Cells pretreated with TNF-alpha were more sensitive to Stx1, displaying greater Stx1-induction of mRNA for TNF-alpha, IL-1beta, and IL-6, and secretion of IL-6 and IL-8. These observations suggest that in the pathogenesis of HUS, Stx can induce cytokine release from hBEC, which may contribute toward the characteristic CNS neuropathology.

cDNA microarray analysis of changes in gene expression associated with MPP+ toxicity in SH-SY5Y cells.

cDNA microarray analysis of 1-methyl-4-phenyl-pyridinium (MPP+) toxicity (1 mM, 72 h) in undifferentiated SH-SY5Y cells identified 48 genes that displayed a signal intensity greater than the mean of all differentially expressed genes and a two-fold or greater difference in normalized expression. RT-PCR analysis of a subset of genes showed that c-Myc and RNA-binding protein 3 (RMB3) expression decreased by approximately 50% after 72 h of exposure to MPP+ (1 mM) but did not change after 72 h of exposure to 6-hydroxydopamine (25 microM), rotenone (50 nM), and hydrogen peroxide (600 microM). Exposure of retinoic acid (RA)-differentiated SH-SY5Y cells to MPP+ (1 mM, 72 h) also resulted in a decrease in RMB3 expression and an increase in GADD153 expression. In contrast, c-Myc expression was slightly increased in RA-differentiated cells. Collectively, these data provide new insights into the molecular mechanisms of MPP+ toxicity and show that MPP+ can elicit distinct patterns of gene expression in undifferentiated and RA-differentiated SH-SY5Y cells.

Specific up-regulation of GADD153/CHOP in 1-methyl-4-phenyl-pyridinium-treated SH-SY5Y cells.

Growth arrest DNA damage-inducible 153 (GADD153) expression was increased in 1-methyl-4-phenyl-pyridinium (MPP(+))-treated human SH-SY5Y neuroblastoma cells as determined by gene microarray analysis. GADD153 expression increased after 24 hr of MPP(+) (1 mM) exposure and preceded activation of caspase 3. Comparison of GADD153 expression among cultures treated with other toxins whose primary mode of action is either via mitochondrial impairment (rotenone) or via oxidative stress (6-hydroxydopamine or hydrogen peroxide) showed that GADD153 was uniquely up-regulated by MPP(+). Together these data suggest that a cellular mechanism distinct from mitochondrial impairment or oxidative stress contributes significantly to the up-regulation of GADD153 by MPP(+) and that GADD153 may function as an inducer of apoptosis following MPP(+) exposure. Published 2002 Wiley-Liss, Inc.

In vitro Inhibition of Soft-Rotting Bacteria by EDTA and Nisin and in vivo Response on Inoculated Fresh Cut Carrots.

EDTA and the antibiotic nisin, in combination with heat, were tested for inhibition of growth of six pectolytic, soft-rotting bacteria in 80% trypticase soy broth (TSB). Fifty percent reduction of growth by EDTA at 25°C in TSB occurred at 3.24 mM for Erwinia chrysanthemi, 2.57 mM for Pseudomonas fluorescens, 0.96 mM for E. carotovora (subsp. carotovora), 0.48 mM for P. viridiflava, 0.17 mM for Xanthomonas campestris (pv. campestris), and 0.16 mM for Cytophaga johnsonae. Nisin at 50 ug/ml was effective against X. campestris and C. johnsonae (over 90% inhibition of growth) but not against the other four bacteria (less than 20% inhibition), which are the more economically important soft-rotters. Combinations of EDTA and nisin were synergistic. A combination of 0.3 mM EDTA + nisin at 50 µg/ml inhibited growth of E. carotovora, E. chrysanthemi, and P. viridiflava by over 70%, and growth of P. fluorescens by 37%. Hot water treatments for 0.3 min at 37 or 49°C reduced survival of bacteria in the presence of EDTA + nisin, but not of EDTA, nisin, or water alone. EDTA + nisin at 37°C reduced CFU/ml of E. carotovora, E. chrysanthemi, P. fluorescens, and P. viridiflava by 2 log units, and at 49°C by 3 log units, compared with the 25°C treatment. Decay of carrot disks inoculated at two inoculum levels (103 and 104 CFU per disk) with E. carotovora, P. fluorescens, or P. viridiflava was reduced by a 1.5-min immersion in 45°C water, with or without EDTA and nisin additives. Immersion in 0.3 mM EDTA + nisin at 15 to 50 µg/ml at 45°C reduced decay due to E. carotovora and to P. fluorescens at the lower inoculum level by an average of about 50% compared with water alone at 45°C, but differences were statistically significant only at the 90% level of confidence and no different than a standard chlorine dip, current commercial practice for cut carrot slices.

Temporary phosphorus partitioning in mycelial systems of the cord-forming basidiomycete Phanerochaete velutina.

Mycelial cord systems, up to 50-cm diameter, of the basidiomycete Phanerochaete velutina (DC.: Pers.) Parmasto, a common woodland saprotroph, grown on non-sterile soil in model laboratory microcosms were baited, after 27 d, with pairs of fresh beech wood blocks (baits), placed at 10 d intervals behind the foraging colony margin. System development was quantified by image analysis. Mean radial extent and hyphal cover increased linearly with time until day 21, but declined before the mycelial systems reached the edges of the laboratory microcosms. The mass (DBM ) and border (DBS ) fractal dimensions of the mycelial systems changed with time but the ratio DBM ∶DBS became constant after 14 d. A separate central compartment containing the inoculum was supplied with 32 P orthophosphate and its translocation to wood baits monitored non-destructively for 73 d. Whilst total 32 P acquisition by wood baits increased linearly with time, the proportion of total allocated to baits varied significantly both temporally and according to the length of time that baits had been in contact with the mycelium. Most recently supplied wood baits were not the main sink for supplied phosphorus; rather, the rate of 32 P acquisition was initially greatest in baits from which egress of the fungus had already occurred. The rate of 32 P acquisition by the most recently added baits increased with time, supported by efflux from other wood baits, which had initially been the main sinks for translocated phosphorus. The results raise important questions about the ecological and functional significance of nutrient partitioning in cord systems and imply that 'observed' translocation, rather than being an absolute measure, indicates the degree to which phosphorus is loaded from a translocation stream in regions where it is being actively utilized and/or stored.

Patch formation and developmental polarity in mycelial cord systems of Phanerochaete velutina on a nutrient-depleted soil.

Development of mycelial cord systems of Phanerochaete velutina (DC.: Pers.) Parmasto from 4-cm3 inocula on a nutrient-depleted non-sterile soil was studied in laboratory microcosms using image analysis techniques. Cord systems were "baited" after 13d growth with either fresh, non-sterile 4-cm3 wood baits or control Perspex® blocks of the same contact area placed behind the foraging mycelial front. After 26 d growth, mycelial 'patches' arose by dedifferentiation of consolidated mycelial cords in both wood- and Perspex-baited cord systems. 'Patches' comprised fine, highly branched separate hyphae extending radially from points of aggregated hyphae in cords. 'Patches' and cords could be readily distinguished by image analysis and the areas covered by patches and cords could be measured and compared. Whilst the total hyphal cover of Perspex- and wood-baited systems did not differ significantly (P > 0.05), patch cover in wood-baited systems was up to 10 times greater than in Perspex-baited systems. Patches were temporary structures, regressing more rapidly with age than mycelial cords. Patch development ceased after application of a nutrient solution which replenished phosphate levels in the soil. Wood-baited mycelial systems displayed significant developmental polarity (P≤ 005) of both total hyphal cover (patches plus cords) and hyphae in patches towards the 'baited' sector of cord systems after 42 d, which corresponded with peak patch development. However, significant (P≤ 0.05) developmental polarity of the mycelial systems along the bait-inoculum line could be detected 8 d before patch formation when assessed by fractal geometry. Radiotracer studies showed that mycelial patches were not sinks for supplied 32 P, but that they were sites of increased nutrient uptake capacity compared with that of mycelial cords. We discuss the need for mycelial cord systems to balance allocation of mycelial biomass between the two essential processes of colonizing wood resource units, and the acquisition of soluble inorganic nutrients from soil.