Biology subjects

Murphy, P.

Publications and source records attributed to Murphy, P..

22 records · Page 2Linked to original sources

bioRxiv2021

Joint development recovery on resumption of embryonic movement following paralysis

Fetal activity in utero is a normal part of pregnancy and reduced or absent movement can lead to long-term skeletal defects such as Fetal Akinesia Deformation Sequence (FADS), joint dysplasia and arthrogryposis. A variety of animal models with decreased or absent embryonic movements show a consistent set of developmental defects providing insight into the aetiology of congenital skeletal abnormalities. At developing joints defects include reduced joint interzones with frequent fusion of cartilaginous skeletal rudiments across the joint. At the spine defects include shortening and a spectrum of curvature deformations. An important question, with relevance to possible therapeutic interventions for human conditions, is the capacity for recovery with resumption of movement following short term immobilisation. Here we use the well-established chick model to compare the effects of sustained immobilisation from embryonic day (E) 4-10 to two different recovery scenarios: (i) natural recovery from E6 until E10 and (ii) the addition of hyperactive movement stimulation during the recovery period. We demonstrate partial recovery of movement and partial recovery of joint development under both recovery conditions, but no improvement in spine defects. The joints examined (elbow, hip and knee) showed better recovery in hindlimb than forelimb, with hyperactive mobility leading to greater recovery in the knee and hip. The hip joint showed the best recovery with improved rudiment separation, tissue organisation and commencement of cavitation. This work demonstrates that movement post paralysis can partially-recover specific aspects of joint development which could inform therapeutic approaches to ameliorate the effects of human fetal immobility. Summary StatementThe study reveals that embryonic movement post paralysis can partially-recover specific aspects of joint development, which could inform therapeutic approaches to ameliorate the effects of restricted fetal movement in utero.

developmental biology↗

bioRxiv2020

Quorum sensing N-Acyl homoserine lactones are a new class of anti-schistosomal.

BackgroundSchistosomiasis is a prevalent neglected tropical disease that affects approximately 300 million people worldwide. Its treatment is through a single class chemotherapy, praziquantel. Concerns surrounding the emergence of praziquantel insensitivity have led to a need for developing novel anthelmintics. Methodology/Principle findingsThrough evaluating and screening fourteen compounds (initially developed for anti-cancer and anti-viral projects) against Schistosoma mansoni, one of three species responsible for most cases of human schistosomiasis, a racemic N-acyl homoserine (1) demonstrated good efficacy against all intra mammalian lifecycle stages including schistosomula (EC50 = 4.7 {micro}M), juvenile worms (EC50 = 4.3 {micro}M) and adult worms (EC50 = 8.3 {micro}M). To begin exploring structural activity relationships, a further 8 analogues of this compound were generated, including individual (R)- and (S)- enantiomers. Upon anti-schistosomal screening of these analogues, the (R)- enantiomer retained activity, whereas the (S)- lost activity. Furthermore, modification of the lactone ring to a thiolactone ring (3) improved potency against schistosomula (EC50 = 2.1 {micro}M), juvenile worms (EC50 = 0.5 {micro}M) and adult worms (EC50 = 4.8 {micro}M). As the active racemic parent compound is structurally similar to quorum sensing signaling peptides used by bacteria, further evaluation of its effect (along with its stereoisomers and the thiolactone analogues) against Gram+ (Staphylococcus aureus) and Gram- (Escherichia coli) species was conducted. While some activity was observed against both Gram+ and Gram- bacteria species for the racemic compound 1 (MIC 125 mg/L), the (R) stereoisomer had better activity (125 mg/L) than the (S) (>125mg/L). However, the greatest antimicrobial activity (MIC 31.25 mg/L against S. aureus) was observed for the thiolactone containing analogue (3). Conclusion/SignificanceTo the best of our knowledge, this is the first demonstration that N-Acyl homoserines exhibit anthelmintic activities. Furthermore, their additional action on Gram+ bacteria opens a new avenue for exploring these molecules more broadly as part of future anti-infective initiatives. Author SummarySchistosomiasis, caused by infection with blood fluke schistosomes, is a neglected tropical disease that negatively impacts the lives of approximately 300 million people worldwide. In the absence of a vaccine, it is currently controlled by a single drug, Praziquantel (PZQ). Although incredibly valuable in controlling disease burden, PZQ-mediated chemotherapy is ineffective against juvenile worms and may not be sustainable should resistance develop. The need to identify an alternative or combinatorial drug is, therefore, a priority in contributing to the control of this parasitic disease into the 21st century. In this study, we have identified a new class of anthelmintic, N-acyl homoserine lactones, which are normally used by bacteria for quorum sensing and population density control. The tested N-acyl homoserine lactones were active against all intra-human schistosome lifecycle stages, in particular, when a thiolactone modification to the core N-acyl homoserine ring was made. Interestingly, these N-acyl homoserine lactones also displayed antimicrobial activities against Gram+ Staphylococcus aureus. By demonstrating broad activities against schistosomes and bacteria exemplars, this study identified a potential route for the further development of a new anti-infective class.

zoology↗

bioRxiv2019

Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer

Post-translational modification of proteins with ubiquitin represents a widely used mechanism for cellular regulation. Ubiquitin is activated by an E1 enzyme, transferred to an E2 conjugating enzyme and covalently linked to substrates by one of an estimated 600 E3 ligases (1). RING E3 ligases play a pivotal role in selecting substrates and priming the ubiquitin loaded E2 (E2~Ub) for catalysis (2,3). RING E3 RNF4 is a SUMO targeted ubiquitin ligase (4) with important roles in arsenic therapy for cancer (4,5) and in DNA damage responses (6,7). RNF4 has a RING domain and a substrate recognition domain containing multiple SUMO Interaction Motifs (SIMO_SCPLOWSC_SCPLOW) embedded in a region thought to be intrinsically disordered (8). While molecular details of SUMO recognition by the SIMs (8-10) and RING engagement of ubiquitin loaded E2 (3,11-15) have been determined, the mechanism by which SUMO substrate is delivered to the RING to facilitate ubiquitin transfer is an important question to be answered. Here, we show that the intrinsically disordered substrate-recognition domain of RNF4 maintains the SIMs in a compact global architecture that facilitates SUMO binding, while a highly-basic region positions substrate for nucleophilic attack on RING-bound ubiquitin loaded E2. Contrary to our expectation that the substrate recognition domain of RNF4 was completely disordered, distance measurements using single molecule Fluorescence Resonance Energy Transfer (smFRET) and NMR paramagnetic relaxation enhancement (PRE) revealed that it adopts a defined conformation primed for SUMO interaction. Mutational and biochemical analysis indicated that electrostatic interactions involving the highly basic region linking the substrate recognition and RING domains juxtaposed those regions and mediated substrate ubiquitination. Our results offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.

biophysics↗

bioRxiv2019

Effects of abnormal muscle forces on prenatal joint morphogenesis in mice

Fetal movements are essential for normal development of the human skeleton. When fetal movements are reduced or restricted, infants are at higher risk of developmental dysplasia of the hip and arthrogryposis (multiple joint contractures). Joint shape abnormalities have been reported in mouse models with abnormal or absent musculature, but the effects on joint shape in such models have not been quantified or characterised in detail. In this study, embryonic mouse forelimbs and hindlimbs at a single developmental stage (Theiler Stage 23) with normal, reduced or absent muscle were imaged in 3D. Skeletal rudiments were virtually segmented and rigid image registration was used to reliably align rudiments with each other, enabling repeatable assessment and measurement of joint shape differences between normal, reduced-muscle and absent muscle groups. We demonstrate qualitatively and quantitatively that joint shapes are differentially affected by a lack of, or reduction in, skeletal muscle, with the elbow joint being the most affected of the major limb joints. Surprisingly, the effects of reduced muscle were often more pronounced than those of absent skeletal muscle, indicating a complex relationship between muscle mass and joint morphogenesis. These findings have relevance for human developmental disorders of the skeleton in which abnormal fetal movements are implicated, particularly developmental dysplasia of the hip and arthrogryposis.

developmental biology↗