Mitochondrial Acyl Carrier Protein of Leishmania major Displays Features Distinct from the Canonical Type II ACP.

Prokaryotes synthesize fatty acids using a type II synthesis pathway (FAS). In this process, the central player, i.e., the acyl carrier protein (ACP), sequesters the growing acyl chain in its internal hydrophobic cavity. As the acyl chain length increases, the cavity expands in size, which is reflected in the NMR chemical shift perturbations and crystal structures of the acyl-ACP intermediates. A few eukaryotic organelles, such as plastids and mitochondria, also harbor type II fatty acid synthesis machinery. Plastid FAS from spinach and Plasmodium falciparum has been characterized at the molecular level, but the mitochondrial pathway remains unexplored. Here, we report NMR studies of the mitochondrial acyl-acyl carrier protein intermediates of Leishmania major (acyl-LmACP). Our studies show that LmACP experiences remarkably small conformational changes upon acylation, with perturbations confined to helices II and III only. CastP determined that the cavity size of apo-LmACP (PDB entry 5ZWT) is less than that of Escherichia coli ACP (PDB 1T8K). Thus, the small chemical shift perturbations observed in the LmACP intermediates, coupled with CastP results, suggest an unusually small cavity when fully expanded. The faster rate of C8-LmACP chain hydrolysis compared to E. coli ACP (EcACP) also supports these convictions. Structure comparison of LmACP with other type II ACP disclosed unique differences in the helix I and loop I conformations, as well as several residues present there. Numerous hydrophobic residues in helix I and loop I (conserved in all mitochondrial ACPs) are substituted with hydrophilic residues in the bacterial/plastid type II ACP. For instance, Phe and leucine at positions 14 and 34 in LmACP are substituted with a hydrophilic residue and Ala in bacterial/plastid type II ACP. Mutation of Leu 34 to Ala (corresponding residue in EcACP) resulted in a complete loss of structure, underscoring its importance in maintaining the ACP fold. Thus, our NMR studies, combined with insights from the crystal structure, highlight several unique features of LmACP, distinct from the prokaryote and plastid type II ACP. Given the high sequence identity, the features might be conserved in all mitochondrial ACPs.

A Cut/cohesin axis alters the chromatin landscape to facilitate neuroblast death.

Precise control of cell death in the nervous system is essential for development. Spatial and temporal factors activate the death of Drosophila neural stem cells (neuroblasts) by controlling the transcription of multiple cell death genes through a shared enhancer. The activity of this enhancer is controlled by abdominal A and Notch, but additional inputs are needed for proper specificity. Here, we show that the Cut DNA binding protein is required for neuroblast death, regulating reaper and grim downstream of the shared enhancer and of abdominal A expression. The loss of cut accelerates the temporal progression of neuroblasts from a state of low overall levels of H3K27me3 to a higher H3K27me3 state. This is reflected in an increase in H3K27me3 modifications in the cell death gene locus in the CNS on Cut knockdown. We also show that cut regulates the expression of the cohesin subunit Stromalin. Stromalin and the cohesin regulatory subunit Nipped-B are required for neuroblast death, and knockdown of Stromalin increases H3K27me3 levels in neuroblasts. Thus, Cut and cohesin regulate apoptosis in the developing nervous system by altering the chromatin landscape.

A systematic re-examination of processing of MHCI-bound antigenic peptide precursors by endoplasmic reticulum aminopeptidase 1.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mature antigenic peptides for presentation by major histocompatibility complex class I (MHCI) molecules and regulates adaptive immune responses. ERAP1 has been proposed to trim peptide precursors both in solution and in preformed MHCI-peptide complexes, but which mode is more relevant to its biological function remains controversial. Here, we compared ERAP1-mediated trimming of antigenic peptide precursors in solution or when bound to three MHCI alleles, HLA-B*58, HLA-B*08, and HLA-A*02. For all MHCI-peptide combinations, peptide binding onto MHCI protected against ERAP1-mediated trimming. In only a single MHCI-peptide combination, trimming of an HLA-B*08-bound 12-mer progressed at a considerable rate, albeit still slower than in solution. Results from thermodynamic, kinetic, and computational analyses suggested that this 12-mer is highly labile and that apparent on-MHC trimming rates are always slower than that of MHCI-peptide dissociation. Both ERAP2 and leucine aminopeptidase, an enzyme unrelated to antigen processing, could trim this labile peptide from preformed MHCI complexes as efficiently as ERAP1. A pseudopeptide analogue with high affinity for both HLA-B*08 and the ERAP1 active site could not promote the formation of a ternary ERAP1/MHCI/peptide complex. Similarly, no interactions between ERAP1 and purified peptide-loading complex were detected in the absence or presence of a pseudopeptide trap. We conclude that MHCI binding protects peptides from ERAP1 degradation and that trimming in solution along with the dynamic nature of peptide binding to MHCI are sufficient to explain ERAP1 processing of antigenic peptide precursors.

The "Recognition Helix" of the Type II Acyl Carrier Protein (ACP) Utilizes a "Ubiquitin Interacting Motif (UIM)"-like Surface To Bind Its Partners.

Interaction interfaces comprise a finite number of sequence/structural motifs, which are often repeated in nature. Here we show how a helical motif present in the acyl carrier protein (ACP), involved in multiprotein interactions, displays a binding interface similar to that of the "ubiquitin interacting motif (UIM)". Analysis of the crystal structures of the ACP-enzyme complexes gave the first hint that helix II of the ACP ("universal recognition helix") utilizes UIM-like noncovalent interactions to associate with the type II fatty acid biosynthesis (FAS) pathway enzymes. The ACP interacting functional surface of the FAS enzymes comprises positively charged residues, flanking a central hydrophobic patch, akin to ubiquitin. Our nuclear magnetic resonance chemical shift perturbation studies, relaxation studies, and surface plasmon resonance measurements unequivocally show that helix II of ACP behaves like a UIM motif in its interactions with ubiquitin, by binding the Ile 44 functional surface of the latter. A synthetic peptide with the ACP helix II sequence showed equivalent binding to ubiquitin. The evolution of similar interaction motifs in the two systems has probably been driven by functional constraints, as both ACP and UIM participate in multiprotein interactions.

Cell death in development: Signaling pathways and core mechanisms.

Programmed cell death eliminates unneeded and dangerous cells in a timely and effective manner during development. In this review, we examine the role cell death plays during development in worms, flies and mammals. We discuss signaling pathways that regulate developmental cell death, and describe how they communicate with the core cell death pathways. In most organisms, the majority of developmental cell death is seen in the nervous system. Therefore we focus on what is known about the regulation of developmental cell death in this tissue. Understanding how the cell death is regulated during development may provide insight into how this process can be manipulated in the treatment of disease.

Cell death regulates muscle fiber number.

Cell death can have both cell autonomous and non-autonomous roles in normal development. Previous studies have shown that the central cell death regulators grim and reaper are required for the developmentally important elimination of stem cells and neurons in the developing central nervous system (CNS). Here we show that cell death in the nervous system is also required for normal muscle development. In the absence of grim and reaper, there is an increase in the number of fibers in the ventral abdominal muscles in the Drosophila adult. This phenotype can be partially recapitulated by inhibition of cell death specifically in the CNS, indicating a non-autonomous role for neuronal death in limiting muscle fiber number. We also show that FGFs produced in the cell death defective nervous system are required for the increase in muscle fiber number. Cell death in the muscle lineage during pupal stages also plays a role in specifying fiber number. Our work suggests that FGFs from the CNS act as a survival signal for muscle founder cells. Thus, proper muscle fiber specification requires cell death in both the nervous system and in the developing muscle itself.

Structural and Functional Significance of the N- and C-Terminal Appendages in Arabidopsis Truncated Hemoglobin.

Plant hemoglobins constitute three distinct groups: symbiotic, nonsymbiotic, and truncated hemoglobins. Structural investigation of symbiotic and nonsymbiotic (class I) hemoglobins revealed the presence of a vertebrate-like 3/3 globin fold in these proteins. In contrast, plant truncated hemoglobins are similar to bacterial truncated hemoglobins with a putative 2/2 α-helical globin fold. While multiple structures have been reported for plant hemoglobins of the first two categories, for plant truncated globins only one structure has been reported of late. Here, we report yet another crystal structure of the truncated hemoglobin from Arabidopsis thaliana (AHb3) with two water molecules in the heme pocket, of which one is distinctly coordinated to the heme iron, unlike the only available crystal structure of AHb3 with a hydroxyl ligand. AHb3 was monomeric in its crystallographic asymmetric unit; however, dimer was evident in the crystallographic symmetry, and the globin indeed existed as a stable dimer in solution. The tertiary structure of the protein exhibited a bacterial-like 2/2 α-helical globin fold with an additional N-terminal α-helical extension and disordered C-termini. To address the role of these extended termini in AHb3, which is yet unknown, N- and C-terminal deletion mutants were created and characterized and molecular dynamics simulations performed. The C-terminal deletion had an insignificant effect on most properties but perturbed the dimeric equilibrium of AHb3 and significantly influenced azide binding kinetics in the ferric state. These results along with the disordered nature of the C-terminus indicated its putative role in intramolecular or intermolecular interactions probably regulating protein-ligand and protein-protein interactions. While the N-terminal deletion did not change the overall globin fold, stability, or ligand binding kinetics, it seemed to have influenced coordination at the heme iron, the hydration status of the active site, and the quaternary structure of AHb3. Evidence indicated that the N-terminus is the predominant factor regulating the quaternary interaction appropriate to physiological requirements, dynamics of the side chains in the heme pocket, and tunnel organization in the protein matrix.

Phe28B10 Induces Channel-Forming Cytotoxic Amyloid Fibrillation in Human Neuroglobin, the Brain-Specific Hemoglobin.

Since its discovery, neuroglobin (Ngb), a neuron-specific oxygen binding hemoglobin, distinct from the classical myoglobin and blood hemoglobin, has attracted attention as an endogenous neuroprotectant. Recent reports suggest that Ngb protects neurons from brain stroke, ischemic stress-induced degeneration, and other brain disorders. Proteins with a specific role in neuroprotection are often associated with neurodegeneration, as well, depending on the cellular environment or specific cellular triggers that tilt the balance one way or the other. This investigation explored the potential role of Ngb in amyloid fibril-related neuronal disorder. Ngb was capable of amyloid formation in vitro at neutral pH and ambient temperature, in both apo and holo forms, albeit at a slower rate in the holo form, unlike other hemoglobins that exhibit such behavior exclusively in the apo states. Elevated temperature enhanced the rate of fibril formation significantly. The B-helix, which is known to play a major role in Ngb ligand binding kinetics, was found to be amyloidogenic with the Phe28B10 amino acid side chain as the key inducer of fibrillation. The Ngb amyloid fibril was also significantly cytotoxic to neuroblastoma cell lines, compared to those obtained from reference hemoglobins. The Ngb fibril probably promoted toxicity by inducing channel formation in the cell membrane, as investigated here using synthetic lipid bilayer membranes and the propidium iodide uptake assay. These findings imply that Ngb plays a role in neurodegenerative disorders in vivo, for which there seems to be indirect evidence by association. Ngb thus presents a novel prospect for understanding amyloid-related brain disorders beyond the limited set of proteins currently investigated for such diseases.

The Structure of the Holo-Acyl Carrier Protein of Leishmania major Displays a Remarkably Different Phosphopantetheinyl Transferase Binding Interface.

The genome of Leishmania major encodes a type II fatty acid biosynthesis pathway for which no structural or biochemical information exists. Here, for the first time, we have characterized the central player of the pathway, the acyl carrier protein (LmACP), using nuclear magnetic resonance (NMR). Structurally, the LmACP molecule is similar to other type II ACPs, comprising a four-helix bundle, enclosing a hydrophobic core. Dissimilarities in sequence, however, exist in helix II (recognition helix) of the protein. The enzymatic conversion of apo-LmACP into the holo form using type I (Escherichia coli AcpS) and type II (Sfp type) phosphopantetheinyl transferases (PPTs) is relatively slow. Mutagenesis studies underscore the importance of the residues present at the protein-protein interaction interface of LmACP in modulating the activity of PPTs. Interestingly, the cognate PPT for this ACP, the L. major 4'-phosphopantetheinyl transferase (LmPPT), does not show any enzymatic activity toward it, though it readily converts other type I and type II ACPs into their holo forms. NMR chemical shift perturbation studies suggest a moderately tight complex between LmACP and its cognate PPT, suggesting inhibition. We surmise that the unique surface of LmACP might have evolved to complement its cognate enzyme (LmPPT), possibly for the purpose of regulation.

Detecting apoptosis in Drosophila tissues and cells.

In this chapter we discuss methods that can be used to study apoptotic cell death in the Drosophila embryo, ovary, as well as in cultured cell lines. These methods assay various aspects of the cell death process, from mitochondrial changes to caspase activation and DNA cleavage. The assays are useful for examining apoptosis in normal development and in response to developmental perturbations and external stresses. These techniques include Acridine Orange staining, TUNEL, cleaved caspase staining, caspase activity assays and assays for mitochondrial fission and permeabilization.

Coordinated expression of cell death genes regulates neuroblast apoptosis.

Properly regulated apoptosis in the developing central nervous system is crucial for normal morphogenesis and homeostasis. In Drosophila, a subset of neural stem cells, or neuroblasts, undergo apoptosis during embryogenesis. Of the 30 neuroblasts initially present in each abdominal hemisegment of the embryonic ventral nerve cord, only three survive into larval life, and these undergo apoptosis in the larvae. Here, we use loss-of-function analysis to demonstrate that neuroblast apoptosis during embryogenesis requires the coordinated expression of the cell death genes grim and reaper, and possibly sickle. These genes are clustered in a 140 kb region of the third chromosome and show overlapping patterns of expression. We show that expression of grim, reaper and sickle in embryonic neuroblasts is controlled by a common regulatory region located between reaper and grim. In the absence of grim and reaper, many neuroblasts survive the embryonic period of cell death and the ventral nerve cord becomes massively hypertrophic. Deletion of grim alone blocks the death of neuroblasts in the larvae. The overlapping activity of these multiple cell death genes suggests that the coordinated regulation of their expression provides flexibility in this crucial developmental process.

Cut homeodomain transcription factor is a novel regulator of growth and morphogenesis of cortex glia niche around neural cells.

Cortex glia in Drosophila central nervous system form a niche around neural cells for necessary signals to establish cross talk with their surroundings. These cells grow and expand their thin processes around neural cell bodies. Although essential for the development and function of the nervous system, how these cells make extensive and intricate connected networks remains largely unknown. In this study, we show that Cut, a homeodomain transcription factor, directly regulates the fate of the cortex glia, impacting neural stem cell (NSC) homeostasis. Focusing on the thoracic ventral nerve cord, we found that Cut is required for the normal growth and development of cortex glia and timely increase in DNA content through endocycle to later divide via acytokinetic mitosis. Knockdown of Cut in cortex glia significantly reduces the growth of cellular processes, the network around NSCs, and their progeny's cell bodies. Conversely, overexpression of Cut induces overall growth of the main processes at the expense of side ones. Whereas the Cut knockdown slows down the timely increase of DNA, the Cut overexpression results in a significant increase in nuclear size and volume and a 3-fold increase in DNA content of cortex glia. Further, we note that constitutively high Cut also interfered with nuclei separation during acytokinetic mitosis. Since the cortex glia form syncytial networks around neural cells, the finding identifies Cut as a novel regulator of glial growth and variant cell cycles to support a functional nervous system.

Clinical status of established MRONJ in oncology patients continuing bone-modifying agents.

The continuation of bone-modifying agents (BMAs) in patients with established medication-related osteonecrosis of the jaw (MRONJ) is a common concern among dentists and oncologists. There is little evidence supporting or refuting the continued use of BMAs or drug holidays and their impact on established MRONJ. This paper evaluates the outcome of continued BMAs use on the patient's MRONJ status. A retrospective review of 29 oncology patients undergoing active cancer care for either metastatic disease or multiple myeloma was conducted. Data on demographics, oncological status, BMA history and MRONJ status were collected. In total, 90% of patients were judged to have healed or stable MRONJ while continuing BMAs. Most patients (69%) continued the same BMA regime (three- or four-weekly) that they were on before developing MRONJ. The average number of BMAs doses received after an MRONJ diagnosis was 12 (range 1-48). Three patients (10.3%) were found to have MRONJ progression, with two patients developing new sites of necrosis. This real-world dataset suggests that the majority of MRONJ cases remain stable and will not worsen with the continuation of BMAs.

ARL15, a GTPase implicated in rheumatoid arthritis, potentially repositions its truncated N-terminus as a function of guanine nucleotide binding.

The ADP ribosylation factor like protein 15 (ARL15) gene encodes for an uncharacterized GTPase associated with rheumatoid arthritis (RA) and other metabolic disorders. Investigation of the structural and functional attributes of ARL15 is important to position the protein as a potential drug target. Using spectroscopy, we demonstrated that ARL15 exhibits properties inherent of GTPases. The Km and Vmax of the enzyme were calculated to be 100 µM and 1.47 µmole/min/µL, respectively. The equilibrium dissociation constant (Kd) of GTP binding with ARL15 was estimated to be about eight-fold higher than that of GDP. Small Angle X-ray Scattering (SAXS) data indicated that in solution, the apo state of monomeric ARL15 adopts a shape characterized by a globe of maximum linear dimension (Dmax) of 6.1 nm, and upon binding to GTP or GDP, the vector distribution profile changes to peak-n-tail shoulder with Dmax extended to 7.6 and 7.7 nm, respectively. Structure restoration using a sequence-based template and experimental SAXS data provided the first visual insight revealing that the folded N-terminal in the unbound state of the protein may toggle open upon binding to guanine nucleotides. The conformational dynamics observed in the N-terminal region offer a scope to develop drugs that target this unique GTPase, potentially providing treatments for a range of metabolic disorders.

E-scooter-related facial injuries: a one-year review following implementation of a citywide trial.

Objective Birmingham City Council commenced electric scooter (e-scooter) trials in September 2020 as part of the wider UK effort to introduce e-scooters as an alternative method of transport. We aimed to review and evaluate maxillofacial injuries in the initial trial period of one year and comment on the safety implications.Method The Queen Elizabeth Hospital is a Level 1 Major Trauma Centre and the hub for maxillofacial services in Birmingham, UK. A single-centre retrospective study captured patients who sustained e-scooter-related facial injuries in the Birmingham e-scooter trial period from September 2020 to September 2021.Results A total of 29 patients were identified as having facial injuries. Of those patients: 59% (n = 17) were men and aged under 30; 43% (n = 18) of all injuries recorded involved hard tissue; and 41% (n = 12) were recorded to be under the influence of alcohol or cannabis. The non-use of helmets was recorded in 34% (n = 10) of patients. Additionally, 20 patients were managed operatively and 100% of patients (n = 12) that were under the influence of drugs or alcohol required operative management.Conclusion With the introduction of these trials, it is shown that facial injuries represent a sizeable proportion of all injuries. E-scooters have significant safety issues. Our study may influence legislation to account for improvements in users' compliance with safety measures and enforcement of those using e-scooters illegally. Legislation regarding the future of e-scooters is expected in the coming year as outlined in the 2022 Queen's Speech.

Survival of Inoculated Vibrio spp., Shigatoxigenic Escherichia coli, Listeria monocytogenes, and Salmonella spp. on Seaweed (Sugar Kelp) During Storage.

Bacteria including Vibrio spp. persist in coastal waters and can contaminate edible seaweeds. Pathogens such as Listeria monocytogenes, shigatoxigenic Escherichia coli (STEC), and Salmonella have been associated with and present serious health risks in minimally processed vegetables including seaweeds. This study evaluated the survival of four pathogens inoculated onto two product forms of sugar kelp subjected to different storage temperatures. The inoculation comprised of a cocktail of two Listeria monocytogenes and STEC strains, two Salmonella serovars, and two Vibrio species. STEC and Vibrio were grown and applied in salt-containing media to simulate preharvest contamination, whereas L. monocytogenes and Salmonella inocula were prepared to simulate postharvest contamination. Samples were stored at 4°C and 10°C for 7 days, and 22°C for 8 h. Microbiological analyses were performed periodically (1, 4, 8, 24 h, etc.) to evaluate the effects of storage temperature on pathogen survival. Pathogen populations decreased under all storage conditions, but survival was greatest for all species at 22°C, with STEC exhibiting significantly less reduction (1.8 log CFU/g) than Salmonella, L. monocytogenes, and Vibrio (3.1, 2.7, and 2.7 log CFU/g, respectively) after storage. The largest population reduction (5.3 log CFU/g) was observed in Vibrio stored at 4°C for 7 days. Regardless of storage temperature, all pathogens remained detectable at the end of the study duration. Results emphasize the need for strict adherence to temperature control for kelp as temperature abuse may support pathogen survival, especially STEC, during storage, and the need for prevention of postharvest contamination, particularly with Salmonella.

Identification of a polycomb responsive region in human HoxA cluster and its long-range interaction with polycomb enriched genomic regions.

Polycomb and Trithorax group proteins (PcG, TrxG) epigenetically regulate developmental genes. These proteins bind with specific DNA elements, the Polycomb Response Element (PRE). Apart from mutations in polycomb/ trithorax proteins, altered cis-elements like PRE underlie the modified function and thus disease etiology. PREs are well studied in Drosophila, while only a few human PREs have been reported. We have identified a polycomb responsive DNA element, hPRE-HoxA3, in the intron of the HoxA3 gene. The hPRE-HoxA3 represses luciferase reporter activity in a PcG-dependent manner. The endogenous hPRE-HoxA3 element recruits PcG proteins and is enriched with repressive H3K27me3 marks, demonstrating that hPRE-HoxA3 is a part of the PcG-dependent gene regulatory network. Furthermore, it interacts with D11-12, the well-known PRE in the human Hox cluster. hPRE-Hox3 is a part of the 3-dimensional chromosomal domain organization as it is involved in the long-range interaction with other PcG enriched regions of Hox A, B, C, and D clusters.

Record-keeping in intravenous sedation: Are we meeting the standards?

As primary care dental services continue to offer conscious sedation in practice, this article presents the findings from a record-keeping audit carried out at a dental teaching hospital in the UK. National guidance was used to set out the gold standards for record-keeping. Various shortcomings in terms of documentation were identified. This article enables dental practitioners involved in dental sedation to review their own sedation record-keeping to meet current national standards and ultimately improve clinical practice and the quality of patient care.

An evaluation of patient informed consent for dental extractions.

Dental practitioners are well versed in informing patients of the risks and benefits associated with dental extractions. The purpose of this service evaluation was to determine whether patients understood and recalled information relevant to their planned oral surgery procedure, prior to second stage consent.A questionnaire was distributed to patients who were attending for their elective treatment appointment. This explored their ability to recall the planned intervention, the modality of treatment (local anaesthetic, intravenous sedation, or general anaesthetic), understanding of alternative treatment options and the risks associated with the procedure. Completed responses were received from 29 of the distributed questionnaires (response rate=58%). The majority of patients were not aware of the following risks with their procedure: pain, bleeding, bruising, swelling, infection, damage to adjacent structures.Despite a well-documented consent form and comprehensive discussion, we identified that patients may not comprehend or recollect the risks associated with their dental extraction. As dental professionals we have a duty to seek ways to facilitate patient understanding and maximise their autonomy.

Vocal Fatigue in School Teachers and It's Relation to the Acoustic analysis of Voice.

Voice disorders are thought to be one of the major occupational hazards of school teaching. There is a need to study the prevalence of vocal fatigue in school teachers as it is unknown in Indian population and its awareness is at a very basic level. We aim to investigate percentage of school teachers reporting vocal fatigue and to find if there is any relationship between vocal fatigue and acoustic voice parameters. A total of 100 subjects (50-males and 50-females) in the age range of 25-35 years participated in the study. Voice Sample was obtained from the subject using a digital tape recorder and was analyzed in Visi pitch and Dr.Speech softwares. The sample was taken twice in a day, that is, before teaching and after teaching. The mean values obtained on all the questions of the vocal fatigue questionnaire show a significant increase in mean values on all the questions at post teaching ratings. Results revealed a statistically significant difference in pre and post teaching values of F0 and shimmer in males. In females, statistical significant difference was obtained in Noise to Harmonic Ratio in /u/ production. No significant correlation between acoustic parameters and overall vocal fatigue was found except for Noise to Harmonic ratio in females for /u/.