The regulatory feedback cycle of the insulin-degrading enzyme and the amyloid precursor protein intracellular domain: Implications for Alzheimer’s disease
One of the major pathological hallmarks of Alzheimer´s disease (AD) is an accumulation of amyloid-β (Aβ) in brain tissue leading to formation of toxic oligomers and senile plaques.
Under physiological conditions, a tightly balanced equilibrium between Aβ-production and -degradation is necessary to prevent pathological Aβ-accumulation. Here, we investigate the molecular mechanism how insulin-degrading enzyme (IDE), one of the major Aβ-degrading enzymes, is regulated and how amyloid precursor protein (APP) processing and Aβ-degradation is linked in a regulatory cycle to achieve this balance.
In absence of Aβ-production caused by APP or Presenilin deficiency, IDE-mediated Aβ-degradation was decreased, accompanied by a decreased IDE activity, protein level, and expression. Similar results were obtained in cells only expressing a truncated APP, lacking the APP intracellular domain (AICD) suggesting that AICD promotes IDE expression.
In return, APP overexpression mediated an increased IDE expression, comparable results were obtained with cells overexpressing C50, a truncated APP representing AICD. Beside these genetic approaches, also AICD peptide incubation and pharmacological inhibition of the γ-secretase preventing AICD production regulated IDE expression and promoter activity.
By utilizing CRISPR/Cas9 APP and Presenilin knockout SH-SY5Y cells results were confirmed in a second cell line in addition to mouse embryonic fibroblasts. In vivo, IDE expression was decreased in mouse brains devoid of APP or AICD, which was in line with a significant correlation of APP expression level and IDE expression in human postmortem AD brains.
Our results show a tight link between Aβ-production and Aβ-degradation forming a regulatory cycle in which AICD promotes Aβ-degradation via IDE and IDE itself limits its own production by degrading AICD.
Children discharged from an emergency department with bacteraemia had lower C-reactive protein and better outcomes than admissions
Aim: To investigate whether there are common clinical findings in bacteremic children that were discharged from the emergency department (ED) and to follow their clinical outcome.
Methods: A retrospective chart review of children above one month old with positive blood cultures obtained in Shamir Medical Center’s ED between January 2011 and December 2019 was conducted.
Results: 250 cases were analysed, of which 68 discharged after first evaluation. Streptococcus pneumonia was the most commonly isolated pathogen.
Compared to children that were admitted when first evaluated in the ED, discharged children had lower C-reactive protein (mean 50.5 ±62.8 vs 121.7 ±113.2 mg/L, p<0.001).
Dyspnoea and being ill-looking were less prevalent among the latter (6.7% versus 35.1%, p=<0.001, 3.0% versus 22.2% p<0.001, respectively), as were presence of Kingella kingae and other gram-negative bacteria.
Description: Intact Genomics T4 DNA Polymerase has both a DNA-dependent DNA polymerase activity and a potent 3´→5´ exonuclease activity. Product Includes:T4 DNA Polymerase10x T4 DNA Polymerase Buffer
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer10mM dNTP Mix
Of the children hospitalised in our institution, the duration of hospitalization was significantly lower than in those admitted during the first visit (6.3± 4.3 vs 9.0±7.4 days, p=0.002). None of the discharged children were admitted to paediatric intensive care unit.
Conclusion: Children with bacteraemia who were discharged home before knowing their positive blood cultures results had lower C-reactive protein and better outcome compared to those admitted on first evaluation in emergency department.
A high-throughput genetically directed protein crosslinking analysis reveals the physiological relevance of the ATP synthase “inserted” state
ATP synthase, a highly conserved protein complex that has a subunit composition of α3 β3 γδεab2 c8-15 for the bacterial enzyme, is a key player in supplying energy to living organisms. This protein complex consists of a peripheral F1 sector (α3 β3 γδε) and a membrane-integrated Fo sector (ab2 c8-15 ).
Structural analyses of the isolated protein components revealed that, remarkably, the C-terminal domain of its ε-subunit seems to adopt two dramatically different structures, but the physiological relevance of this conformational change remains largely unknown.
In an attempt to decipher this, we developed a high-throughput in vivo protein photocrosslinking analysis pipeline based on the introduction of the unnatural amino acid into the target protein via the scarless genome-targeted site-directed mutagenesis technique, and probing the crosslinked products via the high-throughput polyacrylamide gel electrophoresis technique.
Employing this pipeline, we examined the interactions involving the C-terminal helix of the ε-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the bacterial ATP synthase exists as an equilibrium between the “inserted” and “non-inserted” state in cells, maintaining a moderate but significant level of net ATP synthesis when shifting to the former upon exposing to unfavorable energetically stressful conditions.
Such a mechanism allows the bacterial ATP synthases to proportionally and instantly switch between two reversible functional states in responding to changing environmental conditions. Importantly, this high-throughput approach could allow us to decipher the physiological relevance of protein-protein interactions identified under in vitro conditions or to unveil novel physiological context-dependent protein-protein interactions that are unknown before.
Inhibition of deubiquitination by PR-619 induces apoptosis and autophagy via ubi-protein aggregation-activated ER stress in oesophageal squamous cell carcinoma
Objectives: Targeting the deubiquitinases (DUBs) has become a promising avenue for anti-cancer drug development. However, the effect and mechanism of pan-DUB inhibitor, PR-619, on oesophageal squamous cell carcinoma (ESCC) cells remain to be investigated.
Materials and methods: The effect of PR-619 on ESCC cell growth and cell cycle was evaluated by CCK-8 and PI staining.
Annexin V-FITC/PI double staining was performed to detect apoptosis. LC3 immunofluorescence and acridine orange staining were applied to examine autophagy. Intercellular Ca2+ concentration was monitored by Fluo-3AM fluorescence. The accumulation of ubi-proteins and the expression of the endoplasmic reticulum (ER) stress-related protein and CaMKKβ-AMPK signalling were determined by immunoblotting.
Results: PR-619 could inhibit ESCC cell growth and induce G2/M cell cycle arrest by downregulating cyclin B1 and upregulating p21. Meanwhile, PR-619 led to the accumulation of ubiquitylated proteins, induced ER stress and triggered apoptosis by the ATF4-Noxa axis. Moreover, the ER stress increased cytoplasmic Ca2+ and then stimulated autophagy through Ca2+ -CaMKKβ-AMPK signalling pathway.
Ubiquitin E1 inhibitor, PYR-41, could reduce the accumulation of ubi-proteins and alleviate ER stress, G2/M cell cycle arrest, apoptosis and autophagy in PR-619-treated ESCC cells. Furthermore, blocking autophagy by chloroquine or bafilomycin A1 enhanced the cell growth inhibition effect and apoptosis induced by PR-619.
Conclusions: Our findings reveal an unrecognized mechanism for the cytotoxic effects of general DUBs inhibitor (PR-619) and imply that targeting DUBs may be a potential anti-ESCC strategy.
Description: Creative Biogene Monkeypox Virus Real Time PCR Kit is used for the detection of monkeypox Virus in serum or lesion exudate samples by using real time PCR systems. Monkeypox virus (MPV) is a double-stranded DNA, zoonotic virus and a species of the genus Orthopoxvirus in the family Poxviridae. It is one of the human orthopoxviruses that includes variola (VARV), cowpox (CPX), and vaccinia (VACV) viruses. The kit contains a specific ready-to-use system for the detection of the monkeypox Virus. Fluorescence is emitted and measured by the real time systems' optical unit during the PCR.
Description: The Bioperfectus Monkeypox Virus Real Time PCR Kit is an in vitro diagnostic test, based on real-time PCR technology, for the detection of DNA from the Monkeypox virus. Specimens can be obtained from human serum, lesion exudate samples and scab. BSL-2 facilities with standard BSL-2 work practices may be used for the test of t he Monkeypox virus.
Description: Monkeypox virus is the virus that causes the disease monkeypox in both humans and animals. Monkeypox virus is an Orthopoxvirus, a genus of the family Poxviridae that contains other viral species that target mammals. The virus is mainly found in tropical rainforest regions of central and West Africa. The primary route of infection is thought to be contact with the infected animals or their bodily fluids. The genome is not segmented and contains a single molecule of linear double-stranded DNA, 185000 nucleotides long. The Monkeypox Virus real time PCR Kit contains a specific ready-to-use system for the detection of the Monkeypox Virusthrough polymerase chain reaction (PCR) in the real-time PCR system. The master contains reagents and enzymes for the specific amplification of the Monkeypox Virus DNA. Fluorescence is emitted and measured by the real time systems ́ optical unit during the PCR. The detection of amplified Monkeypox Virus DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1. DNA extraction buffer is available in the kit and serum or lesion exudate samples are used for the extraction of the DNA. In addition, the kit contains a system to identify possible PCR inhibition by measuring the 560nm fluorescence of the internal control (IC). An external positive control defined as 1×10^7 copies/ml is supplied which allow the determination of the gene load.
Description: Monkeypox virus is the virus that causes the disease monkeypox in both humans and animals. Monkeypox virus is an Orthopoxvirus, a genus of the family Poxviridae that contains other viral species that target mammals. The virus is mainly found in tropical rainforest regions of central and West Africa. The primary route of infection is thought to be contact with the infected animals or their bodily fluids.The genome is not segmented and contains a single molecule of linear double-stranded DNA, 185000 nucleotides long.The Monkeypox Virus real time PCR Kit contains a specific ready-to-use system for the detection of the Monkeypox Virusthrough polymerase chain reaction (PCR) in the real-time PCR system. The master contains reagents and enzymes for the specific amplification of theMonkeypox VirusDNA. Fluorescence is emitted and measured by the real time systems ́ optical unit during the PCR. The detection of amplified Monkeypox Virus DNA fragment is performed in fluorimeter channelFAM with the fluorescent quencher BHQ1. DNA extraction buffer is available in the kit and serum or lesion exudate samples are used for the extraction of the DNA. In addition, the kit contains a system to identify possible PCR inhibition by measuring the HEX/VIC/JOE fluorescence of the internal control (IC). An external positive control defined as 1×107copies/ml is supplied which allow the determination of the gene load.
