Demonstration of a Modular Prototype End-to-End Simulator for Aquatic Remote Sensing Applications.

This study introduces a prototype end-to-end Simulator software tool for simulating two-dimensional satellite multispectral imagery for a variety of satellite instrument models in aquatic environments. Using case studies, the impact of variable sensor configurations on the performance of value-added products for challenging applications, such as coral reefs and cyanobacterial algal blooms, is assessed. This demonstrates how decisions regarding satellite sensor design, driven by cost constraints, directly influence the quality of value-added remote sensing products. Furthermore, the Simulator is used to identify situations where retrieval algorithms require further parameterization before application to unsimulated satellite data, where error sources cannot always be identified or isolated. The application of the Simulator can verify whether a given instrument design meets the performance requirements of end-users before build and launch, critically allowing for the justification of the cost and specifications for planned and future sensors. It is hoped that the Simulator will enable engineers and scientists to understand important design trade-offs in phase 0/A studies easily, quickly, reliably, and accurately in future Earth observation satellites and systems.

C. elegans germ granules sculpt both germline and somatic RNAome.

Germ granules are membrane-less organelles essential for small RNA biogenesis and germline development. Among the conserved properties of germ granules is their association with the nuclear membrane. Recent studies demonstrated that LOTUS domain proteins, EGGD-1 and EGGD-2 (also known as MIP-1 and MIP-2 respectively), promote the formation of perinuclear germ granules in C. elegans. This finding presents a unique opportunity to evaluate the significance of perinuclear localization of germ granules. Here we show that loss of eggd-1 causes the coalescence of germ granules and formation of abnormal cytoplasmic aggregates. Impairment of perinuclear granules affects certain germline classes of small RNAs including Piwi-interacting RNAs. Transcriptome profiling reveals overexpression of spermatogenic and cuticle-related genes in eggd-1 hermaphrodites. We further demonstrate that disruption of germ granules activates HLH-30-mediated transcriptional program in somatic tissues. Collectively, our findings underscore the essential role of EGGD-1 in germ granule organization and reveal an unexpected germ granule-to-soma communication.

Adult liver transplantation for the advanced clinical practitioner: an overview.

Liver transplantation (LT) is a major surgical undertaking but, in a carefully selected population, it provides excellent outcomes in terms of prolongation of life and improvements in quality of life. This article outlines the processes of referral, assessment, operative course and post-transplant complications of LT, in the UK context. Specific consideration is also given to immunosuppressive medications and considerations around their prescription. The role of the advanced clinical practitioner (ACP) in primary or secondary care may focus on identifying potential candidates for transplantation and ensuring timely discussion and referral. Thus, a familiarity with eligibility criteria, and where to access this information, is important for all ACPs. Additionally, the increasing numbers of transplants performed in the UK mean that there is a large population of post-transplant patients in the wider community. These patients may present to healthcare services with a variety of issues relating to their LT, where early recognition and treatment has the potential to have major impacts on patient, or graft, function and longevity. Due to this, early discussions with specialist transplant centres is advised.

Golgi stress induces SIRT2 to counteract Shigella infection via defatty-acylation.

Enzymes from pathogens often modulate host protein post-translational modifications (PTMs), facilitating survival and proliferation of pathogens. Shigella virulence factors IpaJ and IcsB induce proteolytic cleavage and lysine fatty acylation on host proteins, which cause Golgi stress and suppress innate immunity, respectively. However, it is unknown whether host enzymes could reverse such modifications introduced by pathogens' virulence factors to suppress pathogenesis. Herein, we report that SIRT2, a potent lysine defatty-acylase, is upregulated by the transcription factor CREB3 under Golgi stress induced by Shigella infection. SIRT2 in turn removes the lysine fatty acylation introduced by Shigella virulence factor IcsB to enhance host innate immunity. SIRT2 knockout mice are more susceptible to Shigella infection than wildtype mice, demonstrating the importance of SIRT2 to counteract Shigella infection.

Visualization and Purification of Caenorhabditis elegans Germ Granule Proteins Using Proximity Labeling.

Membraneless organelles, such as germ granules and stress granules, are liquid-like condensates formed by phase transition. Recently, we and others have adopted proximity-based labeling methods to determine the composition of these membraneless compartments. Here, we describe the use of TurboID-an engineered promiscuous biotin ligase-to label and purify proteins localizing to Caenorhabditis elegans germ granules, known as P granules. We provide a detailed protocol for visualization of the subcellular localization of biotinylated proteins from dissected gonads, assessment of TurboID enrichment using streptavidin blots, and enrichment of biotinylated proteins under stringent conditions. Altogether, this protocol provides a workflow to unravel the proteome of C. elegans germ granules. Importantly, the assays described here can be applied to interrogate many membraneless organelles, in a diversity of living multicellular organisms. Graphical abstract.

Long-chain fatty acyl coenzyme A inhibits NME1/2 and regulates cancer metastasis.

SignificanceThe study provided a long-sought molecular mechanism that could explain the link between fatty acid metabolism and cancer metastasis. Further understanding may lead to new strategies to inhibit cancer metastasis. The chemical proteomic approach developed here will be useful for discovering other regulatory mechanisms of protein function by small molecule metabolites.

Chronic kidney disease and renal replacement therapy: an overview for the advanced clinical practitioner.

Chronic kidney disease (CKD), also known as chronic renal failure (CRF), is a common, globally significant condition, with associated significant mortality and morbidity. Due to the disease prevalence, advanced clinical practitioners (ACPs) in a variety of primary and secondary care settings will encounter patients with this diagnosis who will require care and treatment. This article examines definitions of CKD, describes a clinical approach to a patient with CKD, including key history and examination findings, and provides a brief overview of renal replacement strategies for the patient with end-stage renal failure. ACPs require a thorough understanding of this condition and the key elements of treatment and care in this population.

Binding Affinity Determines Substrate Specificity and Enables Discovery of Substrates for N-Myristoyltransferases.

Kinetic parameters (k cat and K m) derived from the Michaelis-Menten equation are widely used to characterize enzymes. k cat/K m is considered the catalytic efficiency or substrate specificity of an enzyme toward its substrate. N-Myristoyltransferases (NMTs) catalyze the N-terminal glycine myristoylation of numerous eukaryotic proteins. Surprisingly, we find that in vitro human NMT1 can accept acetyl-CoA and catalyze acetylation with k cat and K m values similar to that of myristoylation. However, when both acetyl-CoA and myristoyl-CoA are present in the reaction, NMT1 catalyzes almost exclusively myristoylation. This phenomenon is caused by the dramatically different binding affinities of NMT1 for myristoyl-CoA and acetyl-CoA (estimated K d of 14.7 nM and 10.1 µM, respectively). When both are present, NMT1 is essentially entirely bound by myristoyl-CoA and thus catalyzes myristoylation exclusively. The NMT1 example highlights the crucial role of binding affinity in determining the substrate specificity of enzymes, which in contrast to the traditionally held view in enzymology that the substrate specificity is defined by k cat/K m values. This understanding readily explains the vast biological literature showing the coimmunoprecipitation of enzyme-substrate pairs for enzymes that catalyzes protein post-translational modifications (PTM), including phosphorylation, acetylation, and ubiquitination. Furthermore, this understanding allows the discovery of substrate proteins by identifying the interacting proteins of PTM enzymes, which we demonstrate by identifying three previously unknown substrate proteins (LRATD1, LRATD2, and ERICH5) of human NMT1/2 by mining available interactome data.

Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans.

The germ line produces gametes that transmit genetic and epigenetic information to the next generation. Maintenance of germ cells and development of gametes require germ granules-well-conserved membraneless and RNA-rich organelles. The composition of germ granules is elusive owing to their dynamic nature and their exclusive expression in the germ line. Using Caenorhabditis elegans germ granule, called P granule, as a model system, we employed a proximity-based labeling method in combination with mass spectrometry to comprehensively define its protein components. This set of experiments identified over 200 proteins, many of which contain intrinsically disordered regions (IDRs). An RNA interference-based screen identified factors that are essential for P granule assembly, notably EGGD-1 and EGGD-2, two putative LOTUS-domain proteins. Loss of eggd-1 and eggd-2 results in separation of P granules from the nuclear envelope, germline atrophy, and reduced fertility. We show that IDRs of EGGD-1 are required to anchor EGGD-1 to the nuclear periphery while its LOTUS domains are required to promote the perinuclear localization of P granules. Taken together, our work expands the repertoire of P granule constituents and provides new insights into the role of LOTUS-domain proteins in germ granule organization.

pre-piRNA trimming and 2'-O-methylation protect piRNAs from 3' tailing and degradation in C. elegans.

The Piwi-interacting RNA (piRNA) pathway suppresses transposable elements and promotes fertility in diverse organisms. Maturation of piRNAs involves pre-piRNA trimming followed by 2'-O-methylation at their 3' termini. Here, we report that the 3' termini of Caenorhabditis elegans piRNAs are subject to nontemplated nucleotide addition, and piRNAs with 3' addition exhibit extensive base-pairing interaction with their target RNAs. Animals deficient for PARN-1 (pre-piRNA trimmer) and HENN-1 (2'-O-methyltransferase) accumulate piRNAs with 3' nontemplated nucleotides. In henn-1 mutants, piRNAs are shortened prior to 3' addition, whereas long isoforms of untrimmed piRNAs are preferentially modified in parn-1 mutant animals. Loss of either PARN-1 or HENN-1 results in modest reduction in steady-state levels of piRNAs. Deletion of both enzymes leads to depletion of piRNAs, desilenced piRNA targets, and impaired fecundity. Together, our findings suggest that pre-piRNA trimming and 2'-O-methylation act collaboratively to protect piRNAs from tailing and degradation.

Simultaneous Inhibition of SIRT2 Deacetylase and Defatty-Acylase Activities via a PROTAC Strategy.

As a member of the sirtuin family of enzymes, SIRT2 promotes tumor growth and regulates various biological pathways through lysine deacetylation and defatty-acylation. In the past few years, many SIRT2-selective small molecule inhibitors have been developed, but none have demonstrated simultaneous inhibition of both SIRT2 activities in cells. To further scrutinize the physiological importance and significance of SIRT2 deacetylase and defatty-acylase activities, small molecules that can selectively inhibit both activities of SIRT2 in living cells are needed. Here, we have applied the Proteolysis Targeting Chimera (PROTAC) strategy and synthesized a new SIRT2 inhibitor (TM-P4-Thal) to degrade SIRT2 selectively, which led to simultaneous inhibition of its deacetylase and defatty-acylase activities in living cells. Additionally, this compound exemplifies the advantage of the PROTAC strategy that allows complete eradication of an enzyme and its activity in biological settings.

NMT1 and NMT2 are lysine myristoyltransferases regulating the ARF6 GTPase cycle.

Lysine fatty acylation in mammalian cells was discovered nearly three decades ago, yet the enzymes catalyzing it remain unknown. Unexpectedly, we find that human N-terminal glycine myristoyltransferases (NMT) 1 and 2 can efficiently myristoylate specific lysine residues. They modify ADP-ribosylation factor 6 (ARF6) on lysine 3 allowing it to remain on membranes during the GTPase cycle. We demonstrate that the NAD+-dependent deacylase SIRT2 removes the myristoyl group, and our evidence suggests that NMT prefers the GTP-bound while SIRT2 prefers the GDP-bound ARF6. This allows the lysine myrisotylation-demyristoylation cycle to couple to and promote the GTPase cycle of ARF6. Our study provides an explanation for the puzzling dissimilarity of ARF6 to other ARFs and suggests the existence of other substrates regulated by this previously unknown function of NMT. Furthermore, we identified a NMT/SIRT2-ARF6 regulatory axis, which may offer new ways to treat human diseases.

Local-to-global signal transduction at the core of a Mn2+ sensing riboswitch.

The widespread Mn2+-sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch aptamer from Xanthomonas oryzae at 2.96 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >100 µs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.

Identifying culturally acceptable cognitive tests for use in remote northern Australia.

BACKGROUND: A lack of culturally appropriate tests hampers accurate assessment of cognition in remote Australian Aboriginal communities. In Arnhem Land, this study employed a community consultation process to evaluate commonly used Western tests of executive function, memory, attention, and visuospatial function. METHODS: An initial consultation process and a follow-up pilot study resulted in the rejection of some common tests, the development of new tests, and culturally adapted versions of others. In the subsequent 30-person main trial, adult Aboriginal volunteers were examined on nine tests, plus the Kimberly Indigenous Cognitive Assessment screen, and a brief literacy test. RESULTS: Executive function, memory, and attention tests were found to group separately after an exploratory principal components analysis. Correlations between new tests and similar Kimberly screen items were not significant, but ceiling effects may be relevant. Six of 13 test scores were found to correlate with the literacy measure. CONCLUSIONS: A selection of cognitive tests were identified that Aboriginal people found culturally acceptable and engaging. In particular, Self-Ordered Pointing, Trail-Making, a verbal-switching task, and a new test "Which car?" show promise for further development. This work may contribute to the need for culturally appropriate cognitive testing in Aboriginal communities.

ATP/ADP modulates gp16-pRNA conformational change in the Phi29 DNA packaging motor.

Packaging of phage phi29 genome requires the ATPase gp16 and prohead RNA (pRNA). The highly conserved pRNA forms the interface between the connector complex and gp16. Understanding how pRNA interacts with gp16 under packaging conditions can shed light on the molecular mechanism of the packaging motor. Here, we present 3D models of the pRNA-gp16 complex and its conformation change in response to ATP or ADP binding. Using a combination of crystallography, small angle X-ray scattering and chemical probing, we find that the pRNA and gp16 forms a 'Z'-shaped complex, with gp16 specifically binds to pRNA domain II. The whole complex closes in the presence of ATP, and pRNA domain II rotates open as ATP hydrolyzes, before resetting after ADP is released. Our results suggest that pRNA domain II actively participates in the packaging process.

A Glycoconjugated SIRT2 Inhibitor with Aqueous Solubility Allows Structure-Based Design of SIRT2 Inhibitors.

Small molecule inhibitors for SIRT2, a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent protein lysine deacylases, have shown promise in treating cancer and neurodegenerative diseases. Developing SIRT2-selective inhibitors with better pharmacological properties is key to further realize the therapeutic potential of targeting SIRT2. One of the best SIRT2-selective inhibitors reported is a thiomyristoyl lysine compound called TM, which showed promising anticancer activity in mouse models without much toxicity to normal cells. The main limitations of TM, however, are the low aqueous solubility and lack of X-ray crystal structures to aid future drug design. Here, we designed and synthesized a glucose-conjugated TM (glucose-TM) analog with superior aqueous solubility. Although glucose-TM is not cell permeable, the excellent aqueous solubility allowed us to obtain a crystal structure of SIRT2 in complex with it. The structure enabled us to design several new TM analogs, one of which, NH4-6, showed superior water solubility and better anticancer activity in cell culture. The results of these studies provided important insights that will further fuel the future development of improved SIRT2 inhibitors as promising therapeutics for treating cancer and neurodegeneration.

Novel Lysine-Based Thioureas as Mechanism-Based Inhibitors of Sirtuin 2 (SIRT2) with Anticancer Activity in a Colorectal Cancer Murine Model.

Sirtuin 2 (SIRT2) is a protein lysine deacylase that has been indicated as a therapeutic target for cancer. To further establish the role of SIRT2 in cancers, it is necessary to develop selective and potent inhibitors. Here, we report the facile synthesis of novel lysine-derived thioureas as mechanism-based SIRT2 inhibitors with anticancer activity. Compounds AF8, AF10, and AF12 selectively inhibited SIRT2 with IC50 values of 0.06, 0.15, and 0.08 µM, respectively. Compounds AF8 and AF10 demonstrated broad cytotoxicity amongst cancer cell lines, but minimal toxicity in noncancerous cells. AF8 and AF10 inhibited the anchorage-independent growth of human colorectal cancer cell line HCT116 with GI50 values of ∼7 µM. Furthermore, AF8 potently inhibited tumor growth in a HCT116 xenograft murine model, supporting that SIRT2 is a viable therapeutic target for colorectal cancer.

A Small-Molecule SIRT2 Inhibitor That Promotes K-Ras4a Lysine Fatty-Acylation.

SIRT2, a member of the sirtuin family of protein lysine deacylases, has been identified as a promising therapeutic target for treating cancer. In addition to catalyzing deacetylation, SIRT2 has recently been shown to remove fatty acyl groups from K-Ras4a and promote its transforming activity. Among the SIRT2-specific inhibitors, only the thiomyristoyl lysine compound TM can weakly inhibit the demyristoylation activity of SIRT2. Therefore, more potent small-molecule SIRT2 inhibitors are needed to further evaluate the therapeutic potential of SIRT2 inhibition, and to understand the function of protein lysine defatty-acylation. Herein we report a SIRT2 inhibitor, JH-T4, which can increase K-Ras4a lysine fatty acylation. This is the first small-molecule inhibitor that can modulate the lysine fatty acylation levels of K-Ras4a. JH-T4 also inhibits SIRT1 and SIRT3 in vitro. The increased potency of JH-T4 is likely due to the formation of hydrogen bonding between the hydroxy group and SIRT1, SIRT2, and SIRT3. This is further supported by in vitro studies with another small-molecule inhibitor, NH-TM. These studies provide useful insight for future SIRT2 inhibitor development.

Direct Comparison of SIRT2 Inhibitors: Potency, Specificity, Activity-Dependent Inhibition, and On-Target Anticancer Activities.

Sirtuin inhibitors have attracted much interest due to the involvement of sirtuins in various biological processes. Several SIRT2-selective inhibitors have been developed, and some exhibit anticancer activities. To facilitate the choice of inhibitors in future studies and the development of better inhibitors, we directly compared several reported SIRT2-selective inhibitors: AGK2, SirReal2, Tenovin-6, and TM. In vitro, TM is the most potent and selective inhibitor, and only TM could inhibit the demyristoylation activity of SIRT2. SirReal2, Tenovin-6, and TM all showed cytotoxicity in cancer cell lines, with Tenovin-6 being the most potent, but only TM showed cancer-cell-specific toxicity. All four compounds inhibited the anchorage-independent growth of HCT116 cells, but the effect of TM was most significantly affected by SIRT2 overexpression, suggesting that the anticancer effect of TM depends more on SIRT2 inhibition. These results not only provide useful guidance about choosing the right SIRT2 inhibitor in future studies, but also suggest general practices that should be followed for small-molecule inhibitor development activities.

Functional characterization and crystal structure of thermostable amylase from Thermotoga petrophila, reveals high thermostability and an unusual form of dimerization.

Thermostable α-amylases have many industrial applications and are therefore continuously explored from novel sources. We present the characterization of a novel putative α-amylase gene product (Tp-AmyS) cloned from Thermotoga petrophila. The purified recombinant enzyme is highly thermostable and able to hydrolyze starch into dextrin between 90 and 100°C, with optimum activity at 98°C and pH8.5. The activity increased in the presence of Rb1+, K1+ and Ca2+ ions, whereas other ions inhibited activity. The crystal structure of Tp-AmyS at 1.7Å resolution showed common features of the GH-13 family, however was apparently found to be a dimer. Several residues from one monomer interacted with a docked acarbose, an inhibitor of Tp-AmyS, in the other monomer, suggesting catalytic cooperativity within the dimer. The most striking feature of the dimer was that it resembled the dimerization of salivary amylase from a previous crystal structure, and thus could be a functional feature of some amylases.