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2022 ◽  
Francesco Limone ◽  
Jana M. Mitchell ◽  
Irune Guerra San Juan ◽  
Janell L.M. Smith ◽  
Kavya Raghunathan ◽  

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modelling and drug discovery, especially when access to primary tissue is limited, such as in the brain. Current neuronal differentiation approaches use either small molecules for directed differentiation or transcription-factor-mediated programming. In this study we coupled the overexpression of the neuralising transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes). We showed that this approach induced activation of the motor neuron (MN) specific transcription factor Hb9/MNX1, using an Hb9::GFP-reporter line, with up to 95% of cells becoming Hb9::GFP+. These cells acquired and maintained expression of canonical early and mature MN markers. Molecular and functional profiling revealed that liMoNes resembled bona fide hPSC-derived MN differentiated by conventional small molecule patterning. liMoNes exhibited spontaneous electrical activity, expressed synaptic markers and formed contacts with muscle cells in vitro. Pooled, multiplex single-cell RNA sequencing on 50 cell lines revealed multiple anatomically distinct MN subtypes of cervical and brachial, limb-innervating MNs in reproducible quantities. We conclude that combining small molecule patterning with Ngn2 can facilitate the high-yield, robust and reproducible production of multiple disease-relevant MN subtypes, which is fundamental in the path to propel forward our knowledge of motoneuron biology and its disruption in disease.

Jorge Ripoll-Rozada ◽  
Joshua W. C. Maxwell ◽  
Richard J. Payne ◽  
Pedro José Barbosa Pereira

Tyrosine-O-sulfation is a common post-translational modification (PTM) of proteins following the cellular secretory pathway. First described in human fibrinogen, tyrosine-O-sulfation has long been associated with the modulation of protein–protein interactions in several physiological processes. A number of relevant interactions for hemostasis are largely dictated by this PTM, many of which involving the serine proteinase thrombin (FIIa), a central player in the blood-clotting cascade. Tyrosine sulfation is not limited to endogenous FIIa ligands and has also been found in hirudin, a well-known and potent thrombin inhibitor from the medicinal leech, Hirudo medicinalis. The discovery of hirudin led to successful clinical application of analogs of leech-inspired molecules, but also unveiled several other natural thrombin-directed anticoagulant molecules, many of which undergo tyrosine-O-sulfation. The presence of this PTM has been shown to enhance the anticoagulant properties of these peptides from a range of blood-feeding organisms, including ticks, mosquitos and flies. Interestingly, some of these molecules display mechanisms of action that mimic those of thrombin's bona fide substrates.

2022 ◽  
Vol 12 (1) ◽  
Genevieve E. Melling ◽  
Ross Conlon ◽  
Paschalia Pantazi ◽  
Elizabeth R. Dellar ◽  
Priya Samuel ◽  

AbstractAssessing genuine extracellular vesicle (EV) uptake is crucial for understanding the functional roles of EVs. This study measured the bona fide labelling of EVs utilising two commonly used fluorescent dyes, PKH26 and C5-maleimide-Alexa633. MCF7 EVs tagged with mEmerald-CD81 were isolated from conditioned media by size exclusion chromatography (SEC) and characterised using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), MACsPlex immunocapture assay and immunoblots. These fluorescently tagged EVs were subsequently stained with C5-maleimide-Alexa633 or PKH26, according to published protocols. Colocalisation of dual-labelled EVs was assessed by confocal microscopy and quantified using the Rank-Weighted Colocalisation (RWC) algorithm. We observed strikingly poor colocalisation between mEmerald-CD81-tagged EVs and C5-Maleimide-Alexa633 (5.4% ± 1.8) or PKH26 (4.6% ± 1.6), that remained low even when serum was removed from preparations. Our data confirms previous work showing that some dyes form contaminating aggregates. Furthermore, uptake studies showed that maleimide and mEmerald-CD81-tagged EVs can be often located into non-overlapping subcellular locations. By using common methods to isolate and stain EVs we observed that most EVs remained unstained and most dye signal does not appear to be EV associated. Our work shows that there is an urgent need for optimisation and standardisation in how EV researchers use these tools to assess genuine EV signals.

2022 ◽  
Vol 5 (4) ◽  
pp. 135-147
A. V. Krasyukov

The subject. The article is devoted to the study of the mechanism of tax obligation fulfillment. The author established that there are several points of view in understanding the legal essence of the fulfillment of an obligation in Russia and abroad: 1) contractual theories (the general contractual theory, the theory of a real contract, the limited contractual theory); 2) the theory of target impact; 3) the theory of real impact.The purpose of the article is to determine the legal essence of tax obligation fulfillment, to study the concept of the tax obligation fulfillment, the mechanism for exercising subjective rights and obligations, and the criteria for the proper fulfillment of a tax obligation.The methodology. The author uses general and specific scientific methods of scientific research: observation, systemic-structural, dialectical, analysis, comparative jurisprudence and others.The main results, scope of application. There are two sides of the tax obligation fulfillment: legal and factual. From an actual point of view, the tax obligation fulfillment is a set of operations by its parties with the object of the obligation. As a result, the object of the obligation must pass from one owner to another. From a legal point of view, the tax obligation fulfillment always represents the realization of its content through the exercise of rights and the fulfillment of obligations.The author believes that the tax obligation fulfillment should not be equated with the fulfillment of the obligation to pay tax, since not every obligation is executed through the payment of tax. In this regard, a situation may arise when the obligation is properly fulfilled by the debtor, and the creditor does not receive the property grant (for example, if a loss is received at the end of the tax period).The mechanism of the implementation of subjective tax rights and the fulfillment of obligations is a certain system of legal means and algorithms of behavior that allow to determine the appropriate size of the claim of a public law entity on the taxpayer's property and ensuring the transfer of the monetary is equivalent to this economic benefit to the budget system. This mechanism is a set of algorithms for the behavior of subjects of tax liability, defined by law, using specific legal means. As such means, depending on the party of the tax obligation, the law provides, for example, a tax return, a requirement to pay tax, etc. The mechanism of exercising subjective rights and fulfilling obligations includes the following stages: 1) pre-implementation; 2) procedural implementation; 3) actual implementation; 4) protection of the violated right.The author identifies five criteria for the proper fulfillment of a tax obligation: 1) the proper subjects of execution; 2) the proper place; 3) the proper time; 4) the proper object; 5) the proper way.Conclusions. The legal essence of the tax obligation fulfillment can be characterized as a transaction between its parties, aimed to mutual termination and the emergence of the rights of its parties. In order to protect the rights of bona fide taxpayers the author proposes to enshrine in tax legislation a ban on contradictory behavior in the process of fulfilling a tax obligation.

2022 ◽  
Vol 7 (1) ◽  
Samir Chowdhury ◽  
Steve Huntsman ◽  
Matvey Yutin

AbstractPath homology is a powerful method for attaching algebraic invariants to digraphs. While there have been growing theoretical developments on the algebro-topological framework surrounding path homology, bona fide applications to the study of complex networks have remained stagnant. We address this gap by presenting an algorithm for path homology that combines efficient pruning and indexing techniques and using it to topologically analyze a variety of real-world complex temporal networks. A crucial step in our analysis is the complete characterization of path homologies of certain families of small digraphs that appear as subgraphs in these complex networks. These families include all digraphs, directed acyclic graphs, and undirected graphs up to certain numbers of vertices, as well as some specially constructed cases. Using information from this analysis, we identify small digraphs contributing to path homology in dimension two for three temporal networks in an aggregated representation and relate these digraphs to network behavior. We then investigate alternative temporal network representations and identify complementary subgraphs as well as behavior that is preserved across representations. We conclude that path homology provides insight into temporal network structure, and in turn, emergent structures in temporal networks provide us with new subgraphs having interesting path homology.

2022 ◽  
Ana C. Alcalá ◽  
José L. Maravillas ◽  
David Meza ◽  
Octavio T. Ramirez ◽  
Juan E. Ludert ◽  

The dengue virus NS1 is a multifunctional protein that forms part of replication complexes. NS1 is also secreted, as a hexamer, to the extracellular milieu. Circulating NS1 has been associated with dengue pathogenesis by several mechanisms. Cell binding and internalization of soluble NS1 result in endothelial hyperpermeability and in the downregulation of the innate immune response. In this work, we report that the HDL scavenger receptor B1 (SRB1) in human hepatic cells and a scavenger receptor B1-like in mosquito C6/36 cells act as cell surface binding receptors for dengue virus NS1. The presence of the SRB1 on the plasma membrane of C6/36 cells, as well as in Huh7 cells, was demonstrated by confocal microscopy. The internalization of NS1 can be efficiently blocked by anti-SRB1 antibodies and previous incubation of the cells with HDL significantly reduces NS1 internalization. Significant reduction in NS1 internalization was observed in C6/36 cells transfected with siRNAs specific for SRB1. In addition, the transient expression of SRB1 in Vero cells, which lacks the receptor, allows NS1 internalization in these cells. Direct interaction between soluble NS1 and the SRB1 in Huh7 and C6/36 cells was demonstrated in situ by proximity ligation assays and in vitro by surface plasmon resonance. Finally, results are presented indicating that the SRB1 also acts as a cell receptor for Zika virus NS1. These results demonstrate that dengue virus NS1, a bona fide lipoprotein, usurps the HDL receptor for cell entry and offers explanations for the altered serum lipoprotein homeostasis observed in dengue patients. Importance Dengue is the most common viral disease transmitted to humans by mosquitoes. The dengue virus NS1 is a multifunctional glycoprotein necessary for viral replication. NS1 is also secreted as a hexameric lipoprotein and circulates in high concentrations in the sera of patients. Circulating NS1 has been associated with dengue pathogenesis by several mechanisms, including favoring of virus replication in hepatocytes and dendritic cells and disruption of the endothelial glycocalyx leading to hyperpermeability. Those last actions require NS1 internalization. Here, we identify the scavenger cell receptor B1, as the cell-binding receptor for dengue and Zika virus NS1, in cultured liver and in mosquito cells. The results indicate that flavivirus NS1, a bona fide lipoprotein, usurps the human HDL receptor and may offer explanations for the alterations in serum lipoprotein homeostasis observed in dengue patients.

Al-Duhaa ◽  
2021 ◽  
Vol 2 (02) ◽  
pp. 338-353
Muhammad Salman Mir Salman ◽  
Dr. Hafiz Muhammad Sajjad Khan

Atheism is a major topic of discussion in modern time. Critical study of Contemporary atheist literature is a bona-fide task for the emerging researchers for the decontamination of Muslim youth. On the basis of Rationality, Empiricism and critical thinking, brains are draining toward the realm of non-religions. Every person born on the nature of Islam but onward adopt the prevailing doctrines. In the past century an overwhelming critique has been put in academic field by the new atheists in favouring atheism in masquerading by science. Science has nothing to do with supernatural or metaphysical phenomena. Atheism is being attributed to modernity. Considering overwhelming scientific attacks by contemporary atheist, it is the need of the hour to tackle account seriously and use Islamic doctrine to cope the penetrating insurgency of atheist through in Islamic world. Stephen hawking (late) is a famous theoretical physicist and has imparted a valuable influential thoughts regarding origin of time, origin of universe and other hot cosmological and physical issues. Brief answers to the big questions are his last compilation. He argues that for interpretation of origin of Universe nothing has to be with personal God. Instead of attributing God, he attributes the laws of nature for the origin of universe. He argues that it was “time” that causes the universe to begin. He mixed up mythical accounts with the creation accounts as described by the revelation of different religions. In first chapter, he focused on the question: Is there a God? He deliberately asked the question and mentioned that science has the capacity to answer that very question. He argues that before creation of universe God does not have “time” to initiate or trigger and creation process and hence God does not exit. Hawking arguments regarding origin of Universe and existence of God has been critically analysed.

2021 ◽  
Vol 23 (2) ◽  
pp. 32-39
Iulia Crișan ◽  
Florin Alin Sava ◽  
Laurențiu Paul Maricuțoiu

Objective: Two experimental studies were conducted to compare the ability of immediate and delayed recall indicators to discriminate between performances of simulators and full-effort clinical and nonclinical participants. Methods: Three groups of simulators (uncoached, symptom-coached, and testcoached), one group of community controls, and one group of cognitively impaired patients were assessed with four experimental memory tests, in which the immediate and delayed recall tasks were separated by three other tasks. Results: Across both studies, delayed recall demonstrated higher accuracy than immediate recall in classifying simulated performances as invalid, as compared to performances of bona fide clinical participants. ROC curve results showed sensitivities below 50% for both indicators at specificities of ≥ 90%. Computing performance curves across recall trials revealed descending trends for all three simulator groups indicating a suppressed learning effect as a marker of noncredible performances. Among types of coaching, test-coaching proved to decrease differences between simulators and patients. Discussion: The effectiveness of such indicators in clinical evaluations and their vulnerability to information about test-taking strategies are discussed.

2021 ◽  
Vol 18 (2) ◽  
pp. 85-100
Mojca Krevel

In her 2019 novel Frankissstein: A Love Story, Jeanette Winterson weaves an intricate transtemporal and trans-spatial multiplicity, the coding of which is governed by Mary Shelley’s Frankenstein (1818). Through the double first-person narrative of Mary Shelley and her 21st-century reincarnation, Ry Shelley, Winterson approaches the literary phenomenon of Frankenstein in its entirety, seamlessly traversing and fusing the levels of the novel’s production, thematic and formal structuring, and reception. This paper argues that by employing the patchwork nature of Shelley’s monster as the principal metaphor for the creation of her own textual hybrid, Winterson upgrades the essentially Cartesian device of metafictional referencing into a bona fide world-building device that functions according to the governing principles of the post-Cartesian, i.e., postmodern, ontological order.

2021 ◽  
Vol 23 (1) ◽  
pp. 240
Abdullah Y. Hassan ◽  
Sairah Yousaf ◽  
Moran R. Levin ◽  
Osamah J. Saeedi ◽  
Saima Riazuddin ◽  

Congenital cataracts (CC) are responsible for approximately one-tenth of childhood blindness cases globally. Here, we report an African American family with a recessively inherited form of CC. The proband demonstrated decreased visual acuity and bilateral cataracts, with nuclear and cortical cataracts in the right and left eye, respectively. Exome sequencing revealed a novel homozygous variant (c.563A > G; p.(Asn188Ser)) in GJA3, which was predicted to be pathogenic by structural analysis. Dominantly inherited variants in GJA3 are known to cause numerous types of cataracts in various populations. Our study represents the second case of recessive GJA3 allele, and the first report in African Americans. These results validate GJA3 as a bona fide gene for recessively inherited CC in humans.

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