Live-cell chromosome dynamics and outcome of X chromosome pairing events during ES cell differentiation.

Random X inactivation represents a paradigm for monoallelic gene regulation during early ES cell differentiation. In mice, the choice of X chromosome to inactivate in XX cells is ensured by monoallelic regulation of Xist RNA via its antisense transcription unit Tsix/Xite. Homologous pairing events have been proposed to underlie asymmetric Tsix expression, but direct evidence has been lacking owing to their dynamic and transient nature. Here we investigate the live-cell dynamics and outcome of Tsix pairing in differentiating mouse ES cells. We find an overall increase in genome dynamics including the Xics during early differentiation. During pairing, however, Xic loci show markedly reduced movements. Upon separation, Tsix expression becomes transiently monoallelic, providing a window of opportunity for monoallelic Xist upregulation. Our findings reveal the spatiotemporal choreography of the X chromosomes during early differentiation and indicate a direct role for pairing in facilitating symmetry-breaking and monoallelic regulation of Xist during random X inactivation.

Pattern of access determines influence of junk food diet on cue sensitivity and palatability.

AIMS: Like drug addiction, cues associated with palatable foods can trigger food-seeking, even when sated. However, whether susceptibility to the motivating influence of food-related cues is a predisposing factor in overeating or a consequence of poor diet is difficult to determine in humans. Using a rodent model, we explored whether a highly palatable 'junk food' diet impacts responses to reward-paired cues in a Pavlovian-to-instrumental transfer test, using sweetened condensed milk (SCM) as the reward. The hedonic impact of SCM consumption was also assessed by analyzing licking microstructure. METHODS: To probe the effects of pattern and duration of junk food exposure, we provided rats with either regular chow ad libitum (controls) or chow plus access to junk food for either 2 or 24 h per day for 1, 3, or 6 weeks. We also examined how individual susceptibility to weight gain related to these measures. RESULTS: Rats provided 24 h access to the junk food diet were insensitive to the motivational effects of a SCM-paired cue when tested sated even though their hedonic experience upon reward consumption was similar to controls. In contrast, rats provided restricted, 2 h access to junk food exhibited a cue generalization phenotype under sated conditions, lever-pressing with increased vigor in response to both a SCM-paired cue, and a cue not previously paired with reward. Hedonic response was also significantly higher in these animals relative to controls. CONCLUSIONS: These data demonstrate that the pattern of junk food exposure differentially alters the hedonic impact of palatable foods and susceptibility to the motivating influence of cues in the environment to promote food-seeking actions when sated, which may be consequential for understanding overeating and obesity.

Microglia disrupt mesolimbic reward circuitry in chronic pain.

Chronic pain attenuates midbrain dopamine (DA) transmission, as evidenced by a decrease in opioid-evoked DA release in the ventral striatum, suggesting that the occurrence of chronic pain impairs reward-related behaviors. However, mechanisms by which pain modifies DA transmission remain elusive. Using in vivo microdialysis and microinjection of drugs into the mesolimbic DA system, we demonstrate in mice and rats that microglial activation in the VTA compromises not only opioid-evoked release of DA, but also other DA-stimulating drugs, such as cocaine. Our data show that loss of stimulated extracellular DA is due to impaired chloride homeostasis in midbrain GABAergic interneurons. Treatment with minocycline or interfering with BDNF signaling restored chloride transport within these neurons and recovered DA-dependent reward behavior. Our findings demonstrate that a peripheral nerve injury causes activated microglia within reward circuitry that result in disruption of dopaminergic signaling and reward behavior. These results have broad implications that are not restricted to the problem of pain, but are also relevant to affective disorders associated with disruption of reward circuitry. Because chronic pain causes glial activation in areas of the CNS important for mood and affect, our findings may translate to other disorders, including anxiety and depression, that demonstrate high comorbidity with chronic pain.

Parsing the hedonic and motivational influences of nociceptin on feeding using licking microstructure analysis in mice.

Opioid peptides are implicated in processes related to reward and aversion; however, how specific opioid peptides are involved remains unclear. We investigated the role of nociceptin (NOC) in voluntary licking for palatable and aversive tastants by studying the effect of intracerebroventricularly administered NOC on licking microstructure in wild-type and NOC receptor knockout (NOP KO) mice. Compared with the wild-type mice, NOP KO mice emitted fewer bouts of licking when training to lick for a 20% sucrose solution. Correspondingly, intracerebroventricular administration of NOC increased the number of licking bouts for sucrose and sucralose in wild-type, but not in NOP KO mice. The ability of NOC to initiate new bouts of licking for sweet solutions suggests that NOC may drive motivational aspects of feeding behavior. Conversely, adulterating a sucrose solution with the aversive tastant quinine reduced licking bout lengths in wild-type and NOP KOs, suggesting that NOC signaling is not involved in driving voluntary consumption of semiaversive tastants. Interestingly, when consuming sucrose following 20 h of food deprivation, NOP KO mice emitted longer bouts of licking than wild types, suggesting that under hungry conditions, NOC may also contribute toward hedonic aspects of feeding. Together, these results suggest differential roles for NOC in the motivational and hedonic aspects of feeding.

Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease.

Parkinson disease (PD) is a neurodegenerative disorder particularly characterized by the loss of dopaminergic neurons in the substantia nigra. Pesticide exposure has been associated with PD occurrence, and we previously reported that the fungicide benomyl interferes with several cellular processes potentially relevant to PD pathogenesis. Here we propose that benomyl, via its bioactivated thiocarbamate sulfoxide metabolite, inhibits aldehyde dehydrogenase (ALDH), leading to accumulation of the reactive dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), preferential degeneration of dopaminergic neurons, and development of PD. This hypothesis is supported by multiple lines of evidence. (i) We previously showed in mice the metabolism of benomyl to S-methyl N-butylthiocarbamate sulfoxide, which inhibits ALDH at nanomolar levels. We report here that benomyl exposure in primary mesencephalic neurons (ii) inhibits ALDH and (iii) alters dopamine homeostasis. It induces selective dopaminergic neuronal damage (iv) in vitro in primary mesencephalic cultures and (v) in vivo in a zebrafish system. (vi) In vitro cell loss was attenuated by reducing DOPAL formation. (vii) In our epidemiology study, higher exposure to benomyl was associated with increased PD risk. This ALDH model for PD etiology may help explain the selective vulnerability of dopaminergic neurons in PD and provide a potential mechanism through which environmental toxicants contribute to PD pathogenesis.

The redistribution of Drosophila vesicular monoamine transporter mutants from synaptic vesicles to large dense-core vesicles impairs amine-dependent behaviors.

Monoamine neurotransmitters are stored in both synaptic vesicles (SVs), which are required for release at the synapse, and large dense-core vesicles (LDCVs), which mediate extrasynaptic release. The contributions of each type of vesicular release to specific behaviors are not known. To address this issue, we generated mutations in the C-terminal trafficking domain of the Drosophila vesicular monoamine transporter (DVMAT), which is required for the vesicular storage of monoamines in both SVs and LDCVs. Deletion of the terminal 23 aa (DVMAT-Δ3) reduced the rate of endocytosis and localization of DVMAT to SVs, but supported localization to LDCVs. An alanine substitution mutation in a tyrosine-based motif (DVMAT-Y600A) also reduced sorting to SVs and showed an endocytic deficit specific to aminergic nerve terminals. Redistribution of DVMAT-Y600A from SV to LDCV fractions was also enhanced in aminergic neurons. To determine how these changes might affect behavior, we expressed DVMAT-Δ3 and DVMAT-Y600A in a dVMAT null genetic background that lacks endogenous dVMAT activity. When expressed ubiquitously, DVMAT-Δ3 showed a specific deficit in female fertility, whereas DVMAT-Y600A rescued behavior similarly to DVMAT-wt. In contrast, when expressed more specifically in octopaminergic neurons, both DVMAT-Δ3 and DVMAT-Y600A failed to rescue female fertility, and DVMAT-Y600A showed deficits in larval locomotion. DVMAT-Y600A also showed more severe dominant effects than either DVMAT-wt or DVMAT-Δ3. We propose that these behavioral deficits result from the redistribution of DVMAT from SVs to LDCVs. By extension, our data suggest that the balance of amine release from SVs versus that from LDCVs is critical for the function of some aminergic circuits.

Rines E3 ubiquitin ligase regulates MAO-A levels and emotional responses.

Monoamine oxidase A (MAO-A), the catabolic enzyme of norepinephrine and serotonin, plays a critical role in emotional and social behavior. However, the control and impact of endogenous MAO-A levels in the brain remains unknown. Here we show that the RING finger-type E3 ubiquitin ligase Rines/RNF180 regulates brain MAO-A subset, monoamine levels, and emotional behavior. Rines interacted with MAO-A and promoted its ubiquitination and degradation. Rines knock-out mice displayed impaired stress responses, enhanced anxiety, and affiliative behavior. Norepinephrine and serotonin levels were altered in the locus ceruleus, prefrontal cortex, and amygdala in either stressed or resting conditions, and MAO-A enzymatic activity was enhanced in the locus ceruleus in Rines knock-out mice. Treatment of Rines knock-out mice with MAO inhibitors showed genotype-specific effects on some of the abnormal affective behaviors. These results indicated that the control of emotional behavior by Rines is partly due to the regulation of MAO-A levels. These findings verify that Rines is a critical regulator of the monoaminergic system and emotional behavior and identify a promising candidate drug target for treating diseases associated with emotion.

Targeted deletion of the mouse α2 nicotinic acetylcholine receptor subunit gene (Chrna2) potentiates nicotine-modulated behaviors.

Baseline and nicotine-modulated behaviors were assessed in mice harboring a null mutant allele of the nicotinic acetylcholine receptor (nAChR) subunit gene α2 (Chrna2). Homozygous Chrna2(-/-) mice are viable, show expected sex and Mendelian genotype ratios, and exhibit no gross neuroanatomical abnormalities. A broad range of behavioral tests designed to assess genotype-dependent effects on anxiety (elevated plus maze and light/dark box), motor coordination (narrow bean traverse and gait), and locomotor activity revealed no significant differences between mutant mice and age-matched wild-type littermates. Furthermore, a panel of tests measuring traits, such as body position, spontaneous activity, respiration, tremors, body tone, and startle response, revealed normal responses for Chrna2-null mutant mice. However, Chrna2(-/-) mice do exhibit a mild motor or coordination phenotype (a decreased latency to fall during the accelerating rotarod test) and possess an increased sensitivity to nicotine-induced analgesia in the hotplate assay. Relative to wild-type, Chrna2(-/-) mice show potentiated nicotine self-administration and withdrawal behaviors and exhibit a sex-dependent enhancement of nicotine-facilitated cued, but not trace or contextual, fear conditioning. Overall, our results suggest that loss of the mouse nAChR α2 subunit has very limited effects on baseline behavior but does lead to the potentiation of several nicotine-modulated behaviors.

Involvement of NMDAR2A tyrosine phosphorylation in depression-related behaviour.

Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression-related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression-related behaviour. The NR2A subunit of the NMDA receptor is tyrosine-phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr-1325-Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock-in mice show antidepressant-like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock-in mice, DARPP-32 phosphorylation at Thr 34, which is important for the regulation of depression-related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src-induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor-mediated downstream signalling to modulate depression-related behaviour.

Voluntary alcohol intake alters the motivation to seek intravenous oxycodone and neuronal activation during the reinstatement of oxycodone and sucrose seeking.

Opioid-alcohol polysubstance use is prevalent and worsens treatment outcomes. Here we assessed whether co-consumption of oxycodone and alcohol influence the intake of one another, demand for oxycodone, and the neurocircuitry underlying cue-primed reinstatement of oxycodone-seeking. Male and female rats underwent oxycodone intravenous self-administration (IVSA) with homecage access to alcohol (20% v/v) and/or water immediately after the IVSA session. Next, economic demand for intravenous oxycodone was assessed while access to alcohol and/or water continued. Control rats self-administered sucrose followed by access to alcohol and/or water. Rats underwent a cue-primed reinstatement test and brains were processed for c-fos mRNA expression. While both sexes decreased oxycodone intake if they had access to alcohol, and decreased alcohol intake if they had access to oxycodone, only female oxycodone + alcohol rats exhibited decreased demand elasticity and increased cue-primed reinstatement. Alcohol consumption increased the number of basolateral and central amygdala neurons activated during sucrose and oxycodone reinstatement and the number of ventral and dorsal striatum neurons engaged by sucrose reinstatement. Nucleus accumbens shell dopamine 1 receptor expressing neurons displayed activation patterns consistent with oxycodone reinstatement. Thus, alcohol alters the motivation to seek oxycodone in a sex-dependent manner and the neural circuitry engaged by cue-primed reinstatement of sucrose and oxycodone-seeking.

Voluntary alcohol intake alters the motivation to seek intravenous oxycodone and neuronal activation during the reinstatement of oxycodone and sucrose seeking.

Opioid-alcohol polysubstance use is prevalent and worsens treatment outcomes. Here we assessed whether co-consumption of oxycodone and alcohol would influence intake of one another, demand for oxycodone, and the neurocircuitry underlying cue-primed reinstatement of oxycodone-seeking. Male and female rats underwent oxycodone intravenous self-administration (IVSA) with access to either alcohol (20% v/v) and water or only water immediately after the IVSA session. Next, economic demand for intravenous oxycodone was assessed while access to alcohol and/or water continued. Control rats self-administered sucrose followed by access to alcohol and/or water. Rats underwent extinction training and brains were processed for c-fos mRNA expression immediately following a cue-primed reinstatement test. While both sexes decreased oxycodone intake if they had access to alcohol, and decreased alcohol intake if they had access to oxycodone, female oxycodone+alcohol rats exhibited decreased demand elasticity for intravenous oxycodone and increased cue-primed reinstatement while male rats did not. Spontaneous withdrawal signs were correlated with oxycodone intake while alcohol intake was correlated with anxiety-like behavior. Alcohol consumption increased the number of basolateral and central amygdala neurons activated during sucrose and oxycodone reinstatement and the number of ventral and dorsal striatum neurons engaged by sucrose reinstatement. Nucleus accumbens shell dopamine 1 receptor containing neurons displayed activation patterns consistent with oxycodone reinstatement. Thus, alcohol alters the motivation to seek oxycodone in a sex-dependent manner and alters the neural circuitry engaged by cue-primed reinstatement of sucrose and oxycodone-seeking.

An ethogram analysis of cutaneous thermal pain sensitivity and oxycodone reward-related behaviors in rats.

Inter-relationships between pain sensitivity, drug reward, and drug misuse are of considerable interest given that many analgesics exhibit misuse potential. Here we studied rats as they underwent a series of pain- and reward-related tests: cutaneous thermal reflex pain, induction and extinction of conditioned place preference to oxycodone (0.56 mg/kg), and finally the impact of neuropathic pain on reflex pain and reinstatement of conditioned place preference. Oxycodone induced a significant conditioned place preference that extinguished throughout repeated testing. Correlations identified of particular interest included an association between reflex pain and oxycodone-induced behavioral sensitization, and between rates of behavioral sensitization and extinction of conditioned place preference. Multidimensional scaling analysis followed by k-clustering identified three clusters: (1) reflex pain, rate of behavioral sensitization and rate of extinction of conditioned place preference (2) basal locomotion, locomotor habituation, acute oxycodone-stimulated locomotion and rate of change in reflex pain during repeated testing, and (3) magnitude of conditioned place preference. Nerve constriction injury markedly enhanced reflex pain but did not reinstate conditioned place preference. These results suggest that high rates of behavioral sensitization predicts faster rates of extinction of oxycodone seeking/reward, and suggest that cutaneous thermal reflex pain may be predictive of both.

An ethogram analysis of cutaneous thermal pain sensitivity and oxycodone reward-related behaviors in rats.

Inter-relationships between pain sensitivity, drug reward, and drug misuse are of considerable interest given that many analgesics exhibit misuse potential. Here we studied rats as they underwent a series of pain- and reward-related tests: cutaneous thermal reflex pain, induction and extinction of conditioned place preference to oxycodone (0.56 mg/kg), and finally the impact of neuropathic pain on reflex pain and reinstatement of conditioned place preference. Oxycodone induced a significant conditioned place preference that was extinguished throughout repeated testing. Correlations identified of particular interest included an association between reflex pain and oxycodone-induced behavioral sensitization, and between rates of behavioral sensitization and extinction of conditioned place preference. Multidimensional scaling analysis followed by k-clustering identified three clusters: (1) reflex pain and the rate of change in reflex pain response throughout repeated testing, (2) basal locomotion, locomotor habituation, and acute oxycodone-stimulated locomotion, and (3) behavioral sensitization, strength of conditioned place preference, and rate of extinction. Nerve constriction injury markedly enhanced reflex pain but did not reinstate conditioned place preference. These results support the notion that behavioral sensitization relates to the acquisition and extinction of oxycodone seeking/reward, but suggest that generally cutaneous thermal reflex pain poorly predicts oxycodone reward-related behaviors except for behavioral sensitization.

Extracellular dopamine levels in striatal subregions track shifts in motivation and response cost during instrumental conditioning.

Tonic dopamine (DA) signaling is widely regarded as playing a central role in effort-based decision making and in the motivational control of instrumental performance. The current study used microdialysis to monitor changes in extracellular DA levels across subregions of the nucleus accumbens and dorsal striatum of rats as they lever pressed for food reward on a probabilistic schedule of reinforcement, a procedure that ensured they would experience variation in the amount of effort needed to earn rewards across tests. Each rat was given three tests. Rats were hungry for the first and last test, but were sated on food before the middle test, allowing us to assess the effects of a downshift in motivational state on task performance and conditioning-induced DA efflux. During hungry tests, DA levels rose in both the shell and core of the accumbens and, to a lesser degree, in both the medial and lateral divisions of the dorsal striatum. Interestingly, changes in DA efflux across hungry tests in the accumbens core were negatively correlated with changes in the effort required to obtain rewards. We also found that--across regions--the DA response to instrumental conditioning was attenuated when rats were sated before testing. Furthermore, the effect of satiety on DA efflux in the accumbens shell was positively correlated with its effect on task performance. Together, the results indicate that tonic DA contributes to the control of instrumental performance by conveying information about the costs and benefits of responding to different striatal subregions.

Slitrk2 deficiency causes hyperactivity with altered vestibular function and serotonergic dysregulation.

SLITRK2 encodes a transmembrane protein that modulates neurite outgrowth and synaptic activities and is implicated in bipolar disorder. Here, we addressed its physiological roles in mice. In the brain, the Slitrk2 protein was strongly detected in the hippocampus, vestibulocerebellum, and precerebellar nuclei-the vestibular-cerebellar-brainstem neural network including pontine gray and tegmental reticular nucleus. Slitrk2 knockout (KO) mice exhibited increased locomotor activity in novel environments, antidepressant-like behaviors, enhanced vestibular function, and increased plasticity at mossy fiber-CA3 synapses with reduced sensitivity to serotonin. A serotonin metabolite was increased in the hippocampus and amygdala, and serotonergic neurons in the raphe nuclei were decreased in Slitrk2 KO mice. When KO mice were treated with methylphenidate, lithium, or fluoxetine, the mood stabilizer lithium showed a genotype-dependent effect. Taken together, Slitrk2 deficiency causes aberrant neural network activity, synaptic integrity, vestibular function, and serotonergic function, providing molecular-neurophysiological insight into the brain dysregulation in bipolar disorders.

Characterising the efficacy and bioavailability of bioactive peptides identified for attenuating muscle atrophy within a Vicia faba-derived functional ingredient.

Characterising key components within functional ingredients as well as assessing efficacy and bioavailability is an important step in validating nutritional interventions. Machine learning can assess large and complex data sets, such as proteomic data from plants sources, and so offers a prime opportunity to predict key bioactive components within a larger matrix. Using machine learning, we identified two potentially bioactive peptides within a Vicia faba derived hydrolysate, NPN_1, an ingredient which was previously identified for preventing muscle loss in a murine disuse model. We investigated the predicted efficacy of these peptides in vitro and observed that HLPSYSPSPQ and TIKIPAGT were capable of increasing protein synthesis and reducing TNF-α secretion, respectively. Following confirmation of efficacy, we assessed bioavailability and stability of these predicted peptides and found that as part of NPN_1, both HLPSYSPSPQ and TIKIPAGT survived upper gut digestion, were transported across the intestinal barrier and exhibited notable stability in human plasma. This work is a first step in utilising machine learning to untangle the complex nature of functional ingredients to predict active components, followed by subsequent assessment of their efficacy, bioavailability and human plasma stability in an effort to assist in the characterisation of nutritional interventions.

An Artificial-Intelligence-Discovered Functional Ingredient, NRT_N0G5IJ, Derived from Pisum sativum, Decreases HbA1c in a Prediabetic Population.

The prevalence of prediabetes is rapidly increasing, and this can lead to an increased risk for individuals to develop type 2 diabetes and associated diseases. Therefore, it is necessary to develop nutritional strategies to maintain healthy glucose levels and prevent glucose metabolism dysregulation in the general population. Functional ingredients offer great potential for the prevention of various health conditions, including blood glucose regulation, in a cost-effective manner. Using an artificial intelligence (AI) approach, a functional ingredient, NRT_N0G5IJ, was predicted and produced from Pisum sativum (pea) protein by hydrolysis and then validated. Treatment of human skeletal muscle cells with NRT_N0G5IJ significantly increased glucose uptake, indicating efficacy of this ingredient in vitro. When db/db diabetic mice were treated with NRT_N0G5IJ, we observed a significant reduction in glycated haemoglobin (HbA1c) levels and a concomitant benefit on fasting glucose. A pilot double-blinded, placebo controlled human trial in a population of healthy individuals with elevated HbA1c (5.6% to 6.4%) showed that HbA1c percentage was significantly reduced when NRT_N0G5IJ was supplemented in the diet over a 12-week period. Here, we provide evidence of an AI approach to discovery and demonstrate that a functional ingredient identified using this technology could be used as a supplement to maintain healthy glucose regulation.

X11-like protein deficiency is associated with impaired conflict resolution in mice.

Understanding how emotion is generated, how conflicting emotions are regulated, and how emotional states relate to sophisticated behaviors is a crucial challenge in brain research. Model animals showing selective emotion-related phenotypes are highly useful for examining these issues. Here, we describe a novel mouse model that withdraws in approach-avoidance conflicts. X11-like (X11L)/Mint2 is a neuronal adapter protein with multiple protein-protein interaction domains that interacts with several proteins involved in modulating neuronal activity. X11L-knock-out (KO) mice were subordinate under competitive feeding conditions. X11L-KO mice lost significantly more weight than cohoused wild-type mice without signs of decreased motivation to eat or physical weakness. In a resident-intruder test, X11L-KO mice showed decreased intruder exploration behavior. Moreover, X11L-KO mice displayed decreased marble-burying, digging and burrowing behaviors, indicating aberrant ethological responses to attractive stimuli. In contrast, X11L-KO mice were indistinguishable from wild-type mice in the open field, elevated plus maze, and light/dark transition tests, which are often used to assess anxiety-like behavior. Neurochemical analysis revealed a monoamine imbalance in several forebrain regions. The defective ethological responses and social behaviors in X11L-KO mice were rescued by the expression of X11L under a Camk2a promoter using the Tet-OFF system during development. These findings suggest that X11L is involved in the development of neuronal circuits that contribute to conflict resolution.

The use of 3D printing technology in the creation of patient-specific facial prostheses.

BACKGROUND: Personalised medicine aims to optimise patient outcomes by tailoring treatments and interventions to the individual. While this approach can offer a number of benefits, it can be accompanied by significant overheads in terms of resources. Prostheses exist in order to restore and replicate the normal functions and appearance of the body but if these are not individually tailored to the patient's needs then a true restoration cannot be fully achieved. Traditionally a labour intensive process, the fabrication of craniofacial prostheses, involves taking a plaster cast of the area to be treated, hand carving wax models of the restoration and multiple meetings with the patient to alter this wax restoration before making a final prosthesis in silicone. AIMS: Utilising the patient's pre-existing computed tomography (CT) images and 3D printing technology, a patient-specific prosthesis can be created with improved efficiency and accuracy. METHODS: This study demonstrates methods used to create a patient-specific orbital prosthesis using CT images. These images were manipulated in a way which allowed for the intact orbit to be mirrored and used to develop a 3D printed model which acted as the starting point to create a silicone prosthesis. RESULTS: The benefits of using this method include reduced manufacturing time, decreased outpatient appointments, improved personalised outcomes and a repeatable process allowing multiple prostheses to be made. CONCLUSIONS: 3D printing is a valuable tool which can provide significant savings in time and improve patient outcomes by offering a tailored approach to each individual's treatment.

Stochastic pulsing of gene expression enables the generation of spatial patterns in Bacillus subtilis biofilms.

Stochastic pulsing of gene expression can generate phenotypic diversity in a genetically identical population of cells, but it is unclear whether it has a role in the development of multicellular systems. Here, we show how stochastic pulsing of gene expression enables spatial patterns to form in a model multicellular system, Bacillus subtilis bacterial biofilms. We use quantitative microscopy and time-lapse imaging to observe pulses in the activity of the general stress response sigma factor σB in individual cells during biofilm development. Both σB and sporulation activity increase in a gradient, peaking at the top of the biofilm, even though σB represses sporulation. As predicted by a simple mathematical model, increasing σB expression shifts the peak of sporulation to the middle of the biofilm. Our results demonstrate how stochastic pulsing of gene expression can play a key role in pattern formation during biofilm development.