Sequential Cascade Research Articles

Conceptualization of the species problem

The species problem is understood as a result of the contradiction between aspiration and inability to reduce diversity of species conceptions (SCs) to a single one. Any SC represents the natural spe-cies phenomenon in a certain cognitive situation and serves as a heuristic model of this phenomenon in the latter. SCs of various levels of generality emerge as a result of sequential multiple reduction cascade; the more reduction steps lead to a particular SC, the less it is adequate to the natural species phenomenon. The entire array of SCs can be represented by a conceptual pyramid, within which various SCs occur as particular interpretations of more general (inclusive) concepts and have no sense without contexts imposed by them. It is suggested that, in order to define natural “species in general,” a certain concept of biota should be fixed at the top of conceptual pyramid allowing to dis-tinguish between species and non-species (such as life form, syntaxa, guilds) phenomena. The on-tology of the natural species phenomenon is presumably determined by its essence, viz. species-hood. The latter is a part of the entire natural history of organisms, so its manifestations are group-specific and evolve with the evolutionary development of the structure of biota.

Paratransgenic manipulation of a tsetse microRNA alters the physiological homeostasis of the fly's midgut environment.

Tsetse flies are vectors of parasitic African trypanosomes, the etiological agents of human and animal African trypanosomoses. Current disease control methods include fly-repelling pesticides, fly trapping, and chemotherapeutic treatment of infected people and animals. Inhibiting tsetse's ability to transmit trypanosomes by strengthening the fly's natural barriers can serve as an alternative approach to reduce disease. The peritrophic matrix (PM) is a chitinous and proteinaceous barrier that lines the insect midgut and serves as a protective barrier that inhibits infection with pathogens. African trypanosomes must cross tsetse's PM in order to establish an infection in the fly, and PM structural integrity negatively correlates with trypanosome infection outcomes. Bloodstream form trypanosomes shed variant surface glycoproteins (VSG) into tsetse's gut lumen early during the infection establishment, and free VSG molecules are internalized by the fly's PM-producing cardia. This process results in a reduction in the expression of a tsetse microRNA (miR275) and a sequential molecular cascade that compromises PM integrity. miRNAs are small non-coding RNAs that are critical in regulating many physiological processes. In the present study, we investigated the role(s) of tsetse miR275 by developing a paratransgenic expression system that employs tsetse's facultative bacterial endosymbiont, Sodalis glossinidius, to express tandem antagomir-275 repeats (or miR275 sponges). This system induces a constitutive, 40% reduction in miR275 transcript abundance in the fly's midgut and results in obstructed blood digestion (gut weights increased by 52%), a significant increase (p-value < 0.0001) in fly survival following infection with an entomopathogenic bacteria, and a 78% increase in trypanosome infection prevalence. RNA sequencing of cardia and midgut tissues from paratransgenic tsetse confirmed that miR275 regulates processes related to the expression of PM-associated proteins and digestive enzymes as well as genes that encode abundant secretory proteins. Our study demonstrates that paratransgenesis can be employed to study microRNA regulated pathways in arthropods that house symbiotic bacteria.

A Nitric Oxide-Responsive Transcriptional Regulator NsrR Cooperates With Lrp and CRP to Tightly Control the hmpA Gene in Vibrio vulnificus.

Nitric oxide (NO) is an important antimicrobial effector produced by the host innate immune system to counteract invading pathogens. To survive and establish a successful infection, a fulminating human pathogen Vibrio vulnificus expresses the hmpA gene encoding an NO dioxygenase in an NO-responsive manner. In this study, we identified an Rrf2-family transcriptional regulator NsrR that is predicted to contain the Fe-S cluster coordinated by three cysteine residues. Transcriptome analysis showed that NsrR controls the expression of multiple genes potentially involved in nitrosative stress responses. Particularly, NsrR acts as a strong repressor of hmpA transcription and relieves the repression of hmpA upon exposure to NO. Notably, nsrR and hmpA are transcribed divergently, and their promoter regions overlap with each other. Molecular biological analyses revealed that NsrR directly binds to this overlapping promoter region, which is alleviated by loss of the Fe-S cluster, leading to the subsequent derepression of hmpA under nitrosative stress. We further found that a leucine-responsive regulatory protein (Lrp) negatively regulates hmpA in an NsrR-dependent manner by directly binding to the promoter region, presumably resulting in a DNA conformation change to support the repression by NsrR. Meanwhile, a cyclic AMP receptor protein (CRP) positively regulates hmpA probably through repression of nsrR and lrp by directly binding to each promoter region in a sequential cascade. Altogether, this collaborative regulation of NsrR along with Lrp and CRP enables an elaborate control of hmpA transcription, contributing to survival under host-derived nitrosative stress and thereby the pathogenesis of V. vulnificus.

Development of Continuous Flow Systems to Access Secondary Amines Through Previously Incompatible Biocatalytic Cascades*.

A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof‐of‐principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed‐bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross‐reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O‐methylnorbelladine.

Molecular mechanism of Mad1 kinetochore targeting by phosphorylated Bub1.

During metaphase, in response to improper kinetochore‐microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase‐promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation‐dependent signalling cascade. The Mad1‐Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C‐terminal domain of Mad1 (Mad1CTD) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg‐Leu‐Lys) motif, but also directly acts as an N‐terminal cap to the CD1 α‐helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled‐coil of Mad1CTD and has implications for how the Mad1‐Bub1 complex at kinetochores promotes efficient MCC assembly.

Development of Continuous Flow Systems to Access Secondary Amines Through Previously Incompatible Biocatalytic Cascades.

A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof-of-principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed-bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross-reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O-methylnorbelladine.

In Vitro Analysis of E3 Ubiquitin Ligase Function.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

Gaps in TB preventive therapy for persons initiating antiretroviral therapy in Uganda: an explanatory sequential cascade analysis

BACKGROUND: The WHO recommends TB symptom screening and TB preventive therapy (TPT) for latent TB infection (LTBI) in persons living with HIV (PLWH). However, TPT uptake remains limited. We aimed to characterize and contextualize gaps in the TPT care cascade among persons enrolling for antiretroviral therapy (ART).SETTING: Four PEPFAR-supported facilities in Uganda.METHODS: We studied a proportionate stratified random sample of persons registering for ART when TPT was available. Patient-level data on eligibility, initiation, and completion were obtained from registers to determine proportion of eligible patients completing each cascade step. We interviewed providers and administrators and used content analysis to identify barriers to guideline-concordant TPT practices.RESULTS: Of 399 study persons, 309 (77%) were women. Median age was 29 (IQR 25-34), CD4 count 405 cells/µL (IQR 222-573), and body mass 23 kg/m² (IQR 21-25). Of 390 (98%) screened, 372 (93%) were TPT-eligible. Only 62 (17%) eligible PLWH initiated and 36 (58%) of 62 completed TPT. Providers reported hesitating to prescribe TPT because they lacked confidence excluding TB by symptom screening alone and feared promoting drug resistance. Although isoniazid was available, past experience of irregular supply discouraged TPT initiation. Providers pointed to insufficient TB-dedicated staff, speculated that patients discounted TB risk, and worried TPT pill burden and side effects depressed ART adherence.CONCLUSIONS: While screening was nearly universal, most eligible PLWH did not initiate TPT. Only about half of those who initiated completed treatment. Providers feared promoting drug resistance, harbored uncertainty about continued availability, and worried TPT could antagonize ART adherence. Our findings suggest urgent need for stakeholder engagement in TPT provision.

Enhanced ductility in Cu64Zr36 metallic glasses induced by prolonged low-energy ion irradiation: A molecular dynamics study

The irradiation induced variations in microstructure and mechanical properties of the Cu64Zr36 metallic glasses (MGs) are investigated via large-scale molecular dynamics simulations. The irradiation condition is modeled by sequential and prolonged collision cascades. The simulation results show a simultaneous decrease of the density and the fraction of Cu-centered full icosahedron (CCFI) clusters in the irradiated samples. This microstructural evolution induced by the prolonged irradiation is proposed to be responsible for the change of deformation mode of tested samples under uniaxial tensile loading. When increasing the irradiation dose, the ultimate tensile strength of MG samples decreases gradually, while their deformation mechanism switches from localized shear banding to ductile necking and finally to ideally plastic flow, enhancing the MG ductility. Careful atomic-scale examinations on the initiation and development of shear transformation zones (STZs) are carried out for a better understanding of the irradiation-induced ductility enhancement in MGs. Two related effects of the irradiation are unveiled. On the one hand, the irradiation releases the stored strain energy by promoting the STZ activation within the MG matrix. On the other hand, the irradiation damages restrain the propagation of shear band through the innovatively proposed two-unit “STZ-vortex” mechanism. Under the combined effect of these two mechanisms, the low-temperature ductility of irradiated MGs is improved and the brittle-like failure driven by single shear banding is prevented.

Metal-Free C-C/C-N/C-C Bond Formation Cascade for the Synthesis of (Trifluoromethyl)sulfonylated Cyclopenta[b]indolines.

A bis(triflyl)ethylation [triflyl = (trifluoromethyl)sulfonyl] inserted into a sequential cyclization cascade resulted in the direct formation of gem-bis(triflyl)ated cyclopenta[b]indolines from anilide-derived allenols and alkenols. This catalyst- and irradiation-free sequence facilitated the efficient preparation of functionalized tricyclic indoline cores bearing two contiguous stereocenters. The formed cyclopenta[b]indolines can be easily transformed into a wide variety of triflylated indolines, including the tetracycle ring system found in polyveoline.

Responsive Emulsions for Sequential Multienzyme Cascades.

Multienzyme cascade biocatalysis is an efficient synthetic process, avoiding the isolation/purification of intermediates and shifting the reaction equilibrium to the product side.. However, multienzyme systems are often limited by their incompatibility and cross‐reactivity. Herein, we report a multi‐responsive emulsion to proceed multienzyme reactions sequentially for high reactivity. The emulsion is achieved using a CO2, pH, and thermo‐responsive block copolymer as a stabilizer, allowing the on‐demand control of emulsion morphology and phase composition. Applying this system to a three‐step cascade reaction enables the individual optimal condition for each enzyme, and a high overall conversion (ca. 97 % of the calculated limit) is thereby obtained. Moreover, the multi‐responsiveness of the emulsion allows the facile and separate yielding/recycling of products, polymers and active enzymes. Besides, the system could be scaled up with a good yield.

Responsive Emulsions for Sequential Multienzyme Cascades

AbstractMultienzyme cascade biocatalysis is an efficient synthetic process, avoiding the isolation/purification of intermediates and shifting the reaction equilibrium to the product side.. However, multienzyme systems are often limited by their incompatibility and cross‐reactivity. Herein, we report a multi‐responsive emulsion to proceed multienzyme reactions sequentially for high reactivity. The emulsion is achieved using a CO2, pH, and thermo‐responsive block copolymer as a stabilizer, allowing the on‐demand control of emulsion morphology and phase composition. Applying this system to a three‐step cascade reaction enables the individual optimal condition for each enzyme, and a high overall conversion (ca. 97 % of the calculated limit) is thereby obtained. Moreover, the multi‐responsiveness of the emulsion allows the facile and separate yielding/recycling of products, polymers and active enzymes. Besides, the system could be scaled up with a good yield.

Recent developments in multifunctional catalysts for fatty acid hydrodeoxygenation as a route towards biofuels

Catalytic hydrodeoxygenation (HDO) provides a promising route for upgrading biomass-derived fatty acids to alkanes, which are potential biofuels (e.g. jet fuel (C8–C16) and diesel (C12–C22)) that could reduce our reliance on unsustainable fossil fuels. Currently, catalytic HDO, conducted over catalysts such as molybdenum disulfide, necessitates harsh operating conditions (>300 °C) which is both environmentally and economically unsustainable and promotes unwanted side reactions, e.g. cracking, which compromises product selectivity. Accordingly, the development of novel catalysts, which enable efficient and sustainable HDO, under milder operating conditions, and their translation from lab bench to large-scale production are highly desired. This review discusses the recent development of heterogeneous catalysts for HDO (including reaction pathways, mechanisms, and side reactions) and explores design strategies for the development of new multifunctional catalysts with potential to enable future development of HDO processes under mild conditions. In particular, we consider the sequential cascade transformation of fatty acids into fatty alcohols (via hydrodeoxygenation) and then hydrocarbons (via dehydration and hydrogenation), which requires the coupling of different but complementary catalytic sites, as an attractive alternative mild HDO strategy.

Acoustohydrodynamic tweezers via spatial arrangement of streaming vortices.

Acoustics-based tweezers provide a unique toolset for contactless, label-free, and precise manipulation of bioparticles and bioanalytes. Most acoustic tweezers rely on acoustic radiation forces; however, the accompanying acoustic streaming often generates unpredictable effects due to its nonlinear nature and high sensitivity to the three-dimensional boundary conditions. Here, we demonstrate acoustohydrodynamic tweezers, which generate stable, symmetric pairs of vortices to create hydrodynamic traps for object manipulation. These stable vortices enable predictable control of a flow field, which translates into controlled motion of droplets or particles on the operating surface. We built a programmable droplet-handling platform to demonstrate the basic functions of planar-omnidirectional droplet transport, merging droplets, and in situ mixing via a sequential cascade of biochemical reactions. Our acoustohydrodynamic tweezers enables improved control of acoustic streaming and demonstrates a previously unidentified method for contact-free manipulation of bioanalytes and digitalized liquid handling based on a compact and scalable functional unit.

Anin vitro–in vivosequential cascade for the synthesis of iminosugars from aldoses

Here, we report a chemoenzymatic approach for the preparation of a small panel of biologically important iminosugars from readily available aldoses, employing a transaminase in combination withGluconobacter oxydanswhole cells.

PQQ-dependent Dehydrogenase Enables One-pot Bi-enzymatic Enantio-convergent Biocatalytic Amination of Racemic sec-Allylic Alcohols.

The asymmetric amination of secondary racemic allylic alcohols bears several challenges like the reactivity of the bi‐functional substrate/product as well as of the α,β‐unsaturated ketone intermediate in an oxidation‐reductive amination sequence. Heading for a biocatalytic amination cascade with a minimal number of enzymes, an oxidation step was implemented relying on a single PQQ‐dependent dehydrogenase with low enantioselectivity. This enzyme allowed the oxidation of both enantiomers at the expense of iron(III) as oxidant. The stereoselective amination of the α,β‐unsaturated ketone intermediate was achieved with transaminases using 1‐phenylethylamine as formal reducing agent as well as nitrogen source. Choosing an appropriate transaminase, either the (R)‐ or (S)‐enantiomer was obtained in optically pure form (>98 % ee). The enantio‐convergent amination of the racemic allylic alcohols to one single allylic amine enantiomer was achieved in one pot in a sequential cascade.

Continuous extension of maps between sequential cascades

The contour of a family of filters along a filter is a set-theoretic lower limit. Topologicity and regularity of convergences can be characterized with the aid of the contour operation. Contour inversion is studied, in particular, for iterated contours of sequential cascades. A related problem of continuous extension of maps between maximal elements of sequential cascades to full subcascades is solved in full generality.

Elucidation of a Sequential Iminium Ion Cascade Reaction Triggered by a Silica Gel-Promoted Aza-Peterson Reaction.

In a recent methodological study investigating the synthesis of N-alkoxyazomethine ylides, an unexpected aminal byproduct was generated during our attempt to isolate O-benzyl-N-((trimethylsilyl)methyl)hydroxylamine. After a strategic investigation, silica gel was discovered to be the cause of the byproduct formation. Through the mechanistic insight from control and trapping experiments, we propose the formation of a methaniminium ion via a novel aza-Peterson reaction, which ultimately triggers a sequential iminium ion cascade sequence. Herein, we discuss the elucidation of this cascade reaction mechanism and the constraints for the byproduct formation.

Chemo‐ and Stereoselective Synthesis of Fluorinated Amino Alcohols through One‐pot Reactions using Alcohol Dehydrogenases and Amine Transaminases

AbstractA series of amino alcohols have been prepared in a chemo‐, diastereo‐ and enantioselective fashion starting from the corresponding (het)aryl diketones, avoiding tedious chemical protection and deprotection steps. Different alcohol dehydrogenases have been able to selectively reduce the more reactive trifluoroacetyl groups under optimized conditions, while amine transaminases catalyzed the biotransamination of the less hindered acetyl groups. Based on the different reactivity of the acetyl and trifluoroacetyl groups, the design of sequential and concurrent cascades was investigated. The proper selection of the enzymes permits the synthesis of amino alcohol stereoisomers in high to excellent yields (86–&gt;99% conversion) and remarkable stereocontrol (up to &gt;99% de and &gt;99% ee) using an aqueous medium and mild reaction conditions.magnified image

Oxidative Stress, Neuroinflammation and Mitochondria in the Pathophysiology of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron (MN) disease. Its primary cause remains elusive, although a combination of different causal factors cannot be ruled out. There is no cure, and prognosis is poor. Most patients with ALS die due to disease-related complications, such as respiratory failure, within three years of diagnosis. While the underlying mechanisms are unclear, different cell types (microglia, astrocytes, macrophages and T cell subsets) appear to play key roles in the pathophysiology of the disease. Neuroinflammation and oxidative stress pave the way leading to neurodegeneration and MN death. ALS-associated mitochondrial dysfunction occurs at different levels, and these organelles are involved in the mechanism of MN death. Molecular and cellular interactions are presented here as a sequential cascade of events. Based on our present knowledge, the discussion leads to the idea that feasible therapeutic strategies should focus in interfering with the pathophysiology of the disease at different steps.