Divergent directed evolution of a TetR-type repressor towards aromatic molecules.

Reprogramming cellular behaviour is one of the hallmarks of synthetic biology. To this end, prokaryotic allosteric transcription factors (aTF) have been repurposed as versatile tools for processing small molecule signals into cellular responses. Expanding the toolbox of aTFs that recognize new inducer molecules is of considerable interest in many applications. Here, we first establish a resorcinol responsive aTF-based biosensor in Escherichia coli using the TetR-family repressor RolR from Corynebacterium glutamicum. We then perform an iterative walk along the fitness landscape of RolR to identify new inducer specificities, namely catechol, methyl catechol, caffeic acid, protocatechuate, L-DOPA, and the tumour biomarker homovanillic acid. Finally, we demonstrate the versatility of these engineered aTFs by transplanting them into the model eukaryote Saccharomyces cerevisiae. This work provides a framework for efficient aTF engineering to expand ligand specificity towards novel molecules on laboratory timescales, which, more broadly, is invaluable across a wide range of applications such as protein and metabolic engineering, as well as point-of-care diagnostics.

A New Case Series Suggests That SCA48 (ATX/STUB1) Is Primarily a Monogenic Disorder.

BACKGROUND: Monoallelic, pathogenic STUB1 variants cause autosomal dominant cerebellar ataxia (ATX-STUB1/SCA48). Recently, a genetic interaction between STUB1 variants and intermediate or high-normal CAG/CAA repeats in TBP was suggested, indicating digenic inheritance or a disease-modifying role for TBP expansions. OBJECTIVE: To determine the presence and impact of intermediate or high-normal TBP expansions in ataxic patients with heterozygous STUB1 variants. METHODS: We describe 21 patients with ataxia carrying a heterozygous STUB1 variant and determined TBP repeat length. RESULTS: A total of 15 of 21 patients (71%) carried a normal TBP<40 allele, 4 (19%) carried an intermediate TBP41-42 allele, and two carried a high-normal TBP40 allele (9.5%). Five of six carriers (83%) of both STUB1 variants and TBP40-42 alleles showed marked cognitive impairment. CONCLUSIONS: SCA48 is predominantly a monogenic disorder, because most patients carried an isolated, heterozygous STUB1 variant and presented with the typical combined phenotype of ataxia and cognitive dysfunction. Still, co-occurrence of TBP41-42 or high-normal TBP40 alleles was relatively frequent and associated with marked cognitive defects (28.5%), suggesting a modifying effect on clinical expression in some cases. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Immediate implant placement with immediate or delayed provisionalization in the maxillary aesthetic zone: A 10-year randomized trial.

AIM: To compare the marginal bone level of immediately placed implants, with either immediate or delayed provisionalization (IP or DP), in the maxillary aesthetic zone after 10 years of function. MATERIALS AND METHODS: Participants with a failing tooth in the maxillary aesthetic zone were randomly assigned to immediate implant placement with either IP (n = 20) or DP (n = 20) after primary wound closure with a free gingival graft. The final restoration was placed 3 months after provisionalization. The primary outcome was change in marginal bone level. In addition, implant survival, restoration survival and success, peri-implant tissue health, mucosa levels, aesthetic indices, buccal bone thickness and patient satisfaction were evaluated. RESULTS: After 10 years, the mean mesial and distal changes in marginal bone level were -0.47 ± 0.45 mm and -0.49 ± 0.52 mm in the IP group and -0.58 ± 0.76 mm and -0.41 ± 0.72 mm in the DP group (p = .61; p = .71). The survival rate was 100% for the implants; for the restorations, it was 88.9% in the IP group and 87.5% in the DP group. Restoration success, according to modified USPHS criteria, was 77.8% in the IP group and 75.0% in the DP group. The prevalence of peri-implant mucositis was 38.9% and 35.7% and of peri-implantitis 0.0% and 6.3%, respectively, in the IP group and DP group (p = 1.0; p = .40). The Pink Esthetic Score and White Esthetic Score was 15.28 ± 2.32 in the IP group and 14.64 ± 2.74 in the DP group, both clinically acceptable (p = .48). The buccal bone thickness was lower in the DP group. Patient satisfaction was similar in both groups (p = .75). CONCLUSIONS: The mean marginal bone level changes after immediate implant placement with IP were similar to those after immediate placement with DP. CLINICAL TRIAL REGISTRATION: Registered in the National Trial Register (NL9340).

Mechanical performance of patient-specific prefabricated temporary shell versus laboratory-fabricated CAD/CAM provisional implant-supported single-tooth restorations: A laboratory study.

OBJECTIVE: To evaluate the mechanical performance of patient-specific prefabricated temporary shell versus laboratory-fabricated CAD/CAM provisional restorations on titanium temporary abutments, with and without thermo-mechanical ageing. MATERIALS AND METHODS: Implants with a conical connection were divided into four groups (n = 24) and restored with temporary shell or laboratory-fabricated central or lateral incisor PMMA restorations that were relined or bonded on titanium temporary abutments. The diameter of the central and lateral incisor groups' implants was regular (ϕ 4.3 mm) or narrow (ϕ 3.5 mm), respectively. Half of each group's specimens were subjected to ageing, simultaneous thermocycling (5-55°C) and chewing simulation (120,000 cycles, 50 N, 1.7 Hz) resulting in eight groups in total (n = 12). The aged specimens were evaluated with optical microscopy, and survival and complication rates were determined according to modified USPHS criteria. The non-aged specimens and those that had survived ageing were loaded until failure, whereupon bending moments were calculated. RESULTS: Survival rates after ageing were 100% for all groups. Apart from wear facets (ϕ 2-3 mm) on the palatal restoration surface, no complications were observed. The mean fracture load and bending moments ranged between 597.6-847.1 N and 433.3-550.6 Ncm, respectively, with no significant differences between the eight groups (p = .25; p = .20). CONCLUSIONS: As patient-specific temporary shell central and lateral incisor provisional implant-supported restorations are mechanically stable enough to withstand clinical bite forces, even after thermo-mechanical ageing, they may serve as an alternative to laboratory-fabricated provisional restorations.

CRISPR-Cas tools to study gene function in cytokinesis.

Cytokinesis is the process that separates a cell into two daughter cells at the end of mitosis. Most of our knowledge of cytokinesis comes from overexpression studies, which affects our interpretation of protein function. Gene editing can circumvent this issue by introducing functional mutations or fluorescent probes directly into a gene locus. However, despite its potential, gene editing is just starting to be used in the field of cytokinesis. Here, we discuss the benefits of using gene editing tools for the study of cytokinesis and highlight recent studies that successfully used CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) technology to answer critical questions regarding the function of cytokinesis proteins. We also present methodologies for editing essential genes and discuss how CRISPR interference (CRISPRi) and activation (CRISPRa) can enable precise control of gene expression to answer important questions in the field. Finally, we address the need for gene editing to study cytokinesis in more physiologically relevant contexts. Therefore, this Review provides a roadmap for gene editing to be used in the study of cytokinesis and other cellular processes.

Pathway elucidation and microbial synthesis of proaporphine and bis-benzylisoquinoline alkaloids from sacred lotus (Nelumbo nucifera).

Sacred lotus (Nelumbo nucifera) has been utilized as a food, medicine, and spiritual symbol for nearly 3000 years. The medicinal properties of lotus are largely attributed to its unique profile of benzylisoquinoline alkaloids (BIAs), which includes potential anti-cancer, anti-malarial and anti-arrhythmic compounds. BIA biosynthesis in sacred lotus differs markedly from that of opium poppy and other members of the Ranunculales, most notably in an abundance of BIAs possessing the (R)-stereochemical configuration and the absence of reticuline, a major branchpoint intermediate in most BIA producers. Owing to these unique metabolic features and the pharmacological potential of lotus, we set out to elucidate the BIA biosynthesis network in N. nucifera. Here we show that lotus CYP80G (NnCYP80G) and a superior ortholog from Peruvian nutmeg (Laurelia sempervirens; LsCYP80G) stereospecifically convert (R)-N-methylcoclaurine to the proaporphine alkaloid glaziovine, which is subsequently methylated to pronuciferine, the presumed precursor to nuciferine. While sacred lotus employs a dedicated (R)-route to aporphine alkaloids from (R)-norcoclaurine, we implemented an artificial stereochemical inversion approach to flip the stereochemistry of the core BIA pathway. Exploiting the unique substrate specificity of dehydroreticuline synthase from common poppy (Papaver rhoeas) and pairing it with dehydroreticuline reductase enabled de novo synthesis of (R)-N-methylcoclaurine from (S)-norcoclaurine and its subsequent conversion to pronuciferine. We leveraged our stereochemical inversion approach to also elucidate the role of NnCYP80A in sacred lotus metabolism, which we show catalyzes the stereospecific formation of the bis-BIA nelumboferine. Screening our collection of 66 plant O-methyltransferases enabled conversion of nelumboferine to liensinine, a potential anti-cancer bis-BIA from sacred lotus. Our work highlights the unique benzylisoquinoline metabolism of N. nucifera and enables the targeted overproduction of potential lotus pharmaceuticals using engineered microbial systems.

Directional versus ring-mode deep brain stimulation for Parkinson's disease: protocol of a multi-centre double-blind randomised crossover trial.

BACKGROUND: The effectiveness of Deep Brain Stimulation (DBS) therapy for Parkinson's disease can be limited by side-effects caused by electrical current spillover into structures adjacent to the target area. The objective of the STEEred versus RING-mode DBS for Parkinson's disease (STEERING) study is to investigate if directional DBS for Parkinson's disease results in a better clinical outcome when compared to ring-mode DBS. METHODS: The STEERING study is a prospective multi-centre double-blind randomised crossover trial. Inclusion criteria are Parkinson's disease, subthalamic nucleus DBS in a 'classic' ring-mode setting for a minimum of six months, and optimal ring-mode settings have been established. Participants are categorised into one of two subgroups according to their clinical response to the ring-mode settings as 'responders' (i.e., patient with a satisfactory effect of ring-mode DBS) or 'non-responder' (i.e., patient with a non-satisfactory effect of ring-mode DBS). A total of 64 responders and 38 non-responders will be included (total 102 patients). After an optimisation period in which an optimal directional setting is found, participants are randomised to first receive ring-mode DBS for 56 days (range 28-66) followed by directional DBS for 56 days (28-66) or vice-versa. The primary outcome is the difference between ring-mode DBS and directional DBS settings on the Movement Disorders Society Unified Parkinson's Disease Rating Scale - Motor Evaluation (MDS-UPDRS-ME) in the off-medication state. Secondary outcome measures consist of MDS-UPDRS-ME in the on-medication state, MDS-UPDRS Activities of Daily Living, MDS-UPDRS Motor Complications-Dyskinesia, disease related quality of life measured with the Parkinson's Disease Questionnaire 39, stimulation-induced side-effects, antiparkinsonian medication use, and DBS-parameters. Participants' therapy preference is measured at the end of the study. Outcomes will be analysed for both responder and non-responder groups, as well as for both groups pooled together. DISCUSSION: The STEERING trial will provide insights into whether or not directional DBS should be standardly used in all Parkinson's disease DBS patients or if directional DBS should only be used in a case-based approach. TRIAL REGISTRATION: This trial was registered on the Netherlands Trial Register, as trial NL6508 ( NTR6696 ) on June 23, 2017.

Subthalamic and pallidal deep brain stimulation: are we modulating the same network?

The subthalamic nucleus and internal pallidum are main target sites for deep brain stimulation in Parkinson's disease. Multiple trials that investigated subthalamic versus pallidal stimulation were unable to settle on a definitive optimal target between the two. One reason could be that the effect is mediated via a common functional network. To test this hypothesis, we calculated connectivity profiles seeding from deep brain stimulation electrodes in 94 patients that underwent subthalamic and 28 patients with pallidal treatment based on a normative connectome atlas calculated from 1000 healthy subjects. In each cohort, we calculated connectivity profiles that were associated with optimal clinical improvements. The two maps showed striking similarity and were able to cross-predict outcomes in the respective other cohort (R = 0.37 at P < 0.001; R = 0.34 at P = 0.032). Next, we calculated an agreement map, which retained regions common to both target sites. Crucially, this map was able to explain an additional amount of variance in clinical improvements of either cohort when compared to the maps calculated on each cohort alone. Finally, we tested profiles and predictive utility of connectivity maps calculated from different motor symptom subscores with a specific focus on bradykinesia and rigidity. While our study is based on retrospective data and indirect connectivity metrics, it may deliver empirical data to support the hypothesis of a largely overlapping network associated with effective deep brain stimulation in Parkinson's disease irrespective of the specific target.

The Fast Gray Matter Acquisition T1 Inversion Recovery Sequence in Deep Brain Stimulation: Introducing the Rubral Wing for Dentato-Rubro-Thalamic Tract Depiction and Tremor Control.

BACKGROUND: The dentato-rubro-thalamic tract (DRT) is currently considered as a potential target in deep brain stimulation (DBS) for various types of tremor. However, tractography depiction can vary depending on the included brain regions. The fast gray matter acquisition T1 inversion recovery (FGATIR) sequence, with excellent delineation of gray and white matter, possibly provides anatomical identification of rubro-thalamic DRT fibers. OBJECTIVE: This study aimed to evaluate the FGATIR sequence by comparison with DRT depiction, electrode localization, and effectiveness of DBS therapy. MATERIALS AND METHODS: In patients with DBS therapy because of medication-refractory tremor, the FGATIR sequence was evaluated for depiction of the thalamus, red nucleus (RN), and rubro-thalamic connections. Deterministic tractography of the DRT, electrode localization, and tremor control were compared. The essential tremor rating scale was used to assess (hand) tremor. Tremor control was considered successful when complete tremor suppression (grade 0) or almost complete suppression (grade 1) was observed. RESULTS: In the postoperative phase, we evaluated 14 patients who underwent DRT-guided DBS: 12 patients with essential tremor, one with tremor-dominant Parkinson disease, and one with multiple sclerosis, representing 24 trajectories. Mean follow-up was 11.3 months (range 6-19 months). The FGATIR sequence provided a clear delineation of a hypointense white matter tract within the hyperintense thalamus. In coronal plane, this tract was most readily recognizable as a "rubral wing," with the round RN as base and lateral triangular convergence. The deterministic DRT depiction was consistently situated within the rubral wing. The number of active contacts located within the DRT (and rubral wing) was 22 (92%), of which 16 (73%) showed successful tremor control. CONCLUSIONS: The FGATIR sequence offers visualization of the rubro-thalamic connections that form the DRT, most readily recognizable as a "rubral wing" in coronal plane. This sequence contributes to tractographic depiction of DRT and provides a direct anatomical DBS target area for tremor control.

Preventing contamination of PCR-based multiplex assays including the use of a dedicated biosafety cabinet.

We retrospectively investigated cases of false-positive diagnoses using the BIOFIRE® FilmArray® meningitis/encephalitis (ME) panel to measure the impact of using a dedicated biosafety cabinet combined with preventive measures to reduce the prevalence of false-positive diagnoses due to pre-analytical in-laboratory contamination. False-positive results were identified by reviewing clinical data, biological parameters and cytology results of cerebrospinal fluid (CSF) samples showing discrepant results between the FilmArray ME panel and routine PCR assays. A total of 327 CSF were analysed over 16 weeks in point-of-care (POC) A and B, over two 8-week periods, periods 1 and 2. The analysis yielded 30 (9·17%) detection of at least one pathogen including 21/30 (70%) viruses and 9/30 (30%) bacteria. During period 1, POC-A and POC-B manipulated CSF under a non-dedicated hood featuring laminar flow, whereas during period 2, CSFs were manipulated under a dedicated biosafety cabinet without any airflow in POC-A. During period 1, false positives were detected in 3/114 CSF (2·63%) in POC-A and 1/36 (2·77%) in POC-B (P = 0·97); during period 2, false positives were detected in 0/139 CSF (0%) in POC-A and 1/38 (2·63%) in POC-B (P = 0·23). All false positives were bacterial. The use of a dedicated cabinet without ventilation along with preventive measures during period 2 in POC-A significantly reduced the number of false-positive results (P = 0·05). Preventive measures described in this study can mitigate false positives when using PCR-based multiplex assays such as the BIOFIRE FilmArray ME Panel for the diagnosis of meningitis and other infectious diseases.

Independent and sensitive gait parameters for objective evaluation in knee and hip osteoarthritis using wearable sensors.

BACKGROUND: Although it is well-established that osteoarthritis (OA) impairs daily-life gait, objective gait assessments are not part of routine clinical evaluation. Wearable inertial sensors provide an easily accessible and fast way to routinely evaluate gait quality in clinical settings. However, during these assessments, more complex and meaningful aspects of daily-life gait, including turning, dual-task performance, and upper body motion, are often overlooked. The aim of this study was therefore to investigate turning, dual-task performance, and upper body motion in individuals with knee or hip OA in addition to more commonly assessed spatiotemporal gait parameters using wearable sensors. METHODS: Gait was compared between individuals with unilateral knee (n = 25) or hip OA (n = 26) scheduled for joint replacement, and healthy controls (n = 27). For 2 min, participants walked back and forth along a 6-m trajectory making 180° turns, with and without a secondary cognitive task. Gait parameters were collected using 4 inertial measurement units on the feet and trunk. To test if dual-task gait, turning, and upper body motion had added value above spatiotemporal parameters, a factor analysis was conducted. Effect sizes were computed as standardized mean difference between OA groups and healthy controls to identify parameters from these gait domains that were sensitive to knee or hip OA. RESULTS: Four independent domains of gait were obtained: speed-spatial, speed-temporal, dual-task cost, and upper body motion. Turning parameters constituted a gait domain together with cadence. From the domains that were obtained, stride length (speed-spatial) and cadence (speed-temporal) had the strongest effect sizes for both knee and hip OA. Upper body motion (lumbar sagittal range of motion), showed a strong effect size when comparing hip OA with healthy controls. Parameters reflecting dual-task cost were not sensitive to knee or hip OA. CONCLUSIONS: Besides more commonly reported spatiotemporal parameters, only upper body motion provided non-redundant and sensitive parameters representing gait adaptations in individuals with hip OA. Turning parameters were sensitive to knee and hip OA, but were not independent from speed-related gait parameters. Dual-task parameters had limited additional value for evaluating gait in knee and hip OA, although dual-task cost constituted a separate gait domain. Future steps should include testing responsiveness of these gait domains to interventions aiming to improve mobility.

Pulmonary embolism: clinical presentation and diagnosis in the oldest old.

The incidence of pulmonary embolism (PE) in the oldest old (persons aged ≥85) is increasing, but there are limited data on its clinical features and diagnosis. We performed a retrospective cohort study of 302 consecutive patients with confirmed PE and compared the oldest old to the young (aged <65) and the younger old (aged 65-84). The most common symptoms in the oldest old were dyspnoea (74.3%) and tachypnoea (71.4%), but the prevalence of chest pain decreased with advancing age. Delayed diagnosis was most common in the oldest old and was associated with increasing age, absence of dyspnoea, presence of cardiorespiratory disease and a higher Charlson Comorbidity index. Better age-specific diagnostic pathways are required in this population.

Preparing for an orthopedic consultation using an eHealth tool: a randomized controlled trial in patients with hip and knee osteoarthritis.

BACKGROUND: To evaluate the effect of a stand-alone mobile and web-based educational intervention (eHealth tool) compared to usual preparation of a first orthopedic consultation of patients with hip or knee osteoarthritis (OA) on patients' satisfaction. METHODS: A two-armed randomized controlled trial involving 286 patients with (suspicion of) hip or knee OA, randomly allocated to either receiving an educational eHealth tool to prepare their upcoming consultation (n = 144) or usual care (n = 142). Satisfaction with the consultation on three subscales (range 1-4) of the Consumer Quality Index (CQI - primary outcome) and knowledge (assessed using 22 statements on OA, range 0-22), treatment beliefs (assessed by the Treatment beliefs in OsteoArthritis questionnaire, range 1-5), assessment of patient's involvement in consultation by the surgeon (assessed on a 5-point Likert scale) and patient satisfaction with the outcome of the consultation (numeric rating scale), were assessed. RESULTS: No differences between groups were observed on the 3 subscales of the CQI (group difference (95% CI): communication 0.009 (- 0.10, 0.12), conduct - 0.02 (- 0.12, 0.07) and information provision 0.02 (- 0.18, 0.21)). Between group differences (95% CI) were in favor of the intervention group for knowledge (1.4 (0.6, 2.2)), negative beliefs regarding physical activities (- 0.19 (- 0.37, - 0.002) and pain medication (- 0.30 (- 0.49, - 0.01)). We found no differences on other secondary outcomes. CONCLUSIONS: An educational eHealth tool to prepare a first orthopedic consultation for hip or knee OA does not result in higher patient satisfaction with the consultation, but it does influence cognitions about osteoarthritis. TRIAL REGISTRATION: Dutch Trial Register (trial number NTR6262). Registered 30 January 2017.

Defining the Dorsal STN Border Using 7.0-T MRI: A Comparison to Microelectrode Recordings and Lower Field Strength MRI.

BACKGROUND: 7.0-T T2-weighted MRI offers excellent visibility of the subthalamic nucleus (STN), which is used as a target for deep brain stimulation (DBS) in Parkinson's disease (PD). A comparison of 7.0-T MRI to microelectrode recordings (MER) for STN border identification has not been performed. OBJECTIVE: To compare representation of STN borders on 7.0-T T2 MRI with the borders identified during MER in patients undergoing DBS for PD and to evaluate whether STN identification on 7.0-T T2 MRI leads to alterations in stereotactic target planning. DESIGN/METHODS: STN border identification was done using volumetric 7.0-T T2 MRI acquisitions. This was compared to the STN borders identified by MER. STN target planning was independently performed by 3 DBS surgeons on T2 imaging using 1.5-, 3.0-, and 7.0-T MRI. RESULTS: A total of 102 microelectrode tracks were evaluated in 19 patients. Identification of the dorsal STN border was well feasible on 7-T T2, whereas the ventral STN was un-distinguishable from the substantia nigra. The dorsal STN border on MRI was located more dorsal than MER in 73% of trajectories. The average distance from MRI to MER border was 0.9 mm (range -4.4 to +3.5 mm). STN target planning showed high correspondence between the 3 field strengths. CONCLUSION: 7.0-T T2 MRI offers the possibility of easy identification of the dorsal border of the STN. However, higher field strength MRI does not change the planning of the target. Compared to MER, the dorsal border on MRI was located more dorsal in the majority of cases, situating MER activity within STN representation.

An Engineered Aro1 Protein Degradation Approach for Increased cis,cis-Muconic Acid Biosynthesis in Saccharomyces cerevisiae.

Muconic acid (MA) is a chemical building block and precursor to adipic and terephthalic acids used in the production of nylon and polyethylene terephthalate polymer families. Global demand for these important materials, coupled to their dependence on petrochemical resources, provides substantial motivation for the microbial synthesis of MA and its derivatives. In this context, the Saccharomyces cerevisiae yeast shikimate pathway can be sourced as a precursor for the formation of MA. Here we report a novel strategy to balance MA pathway performance with aromatic amino acid prototrophy by destabilizing Aro1 through C-terminal degron tagging. Coupling of a composite MA production pathway to degron-tagged Aro1 in an aro3Δ aro4Δ mutant background led to the accumulation of 5.6 g/liter protocatechuic acid (PCA). However, metabolites downstream of PCA were not detected, despite the inclusion of genes mediating their biosynthesis. Because CEN.PK family strains of S. cerevisiae lack the activity of Pad1, a key enzyme supporting PCA decarboxylase activity, chromosomal expression of intact PAD1 alleviated this bottleneck, resulting in nearly stoichiometric conversion (95%) of PCA to downstream products. In a fed-batch bioreactor, the resulting strain produced 1.2 g/liter MA under prototrophic conditions and 5.1 g/liter MA when supplemented with amino acids, corresponding to a yield of 58 mg/g sugar.IMPORTANCE Previous efforts to engineer a heterologous MA pathway in Saccharomyces cerevisiae have been hindered by a bottleneck at the PCA decarboxylation step and the creation of aromatic amino acid auxotrophy through deleterious manipulation of the pentafunctional Aro1 protein. In light of these studies, this work was undertaken with the central objective of preserving amino acid prototrophy, which we achieved by employing an Aro1 degradation strategy. Moreover, resolution of the key PCA decarboxylase bottleneck, as detailed herein, advances our understanding of yeast MA biosynthesis and will guide future strain engineering efforts. These strategies resulted in the highest titer reported to date for muconic acid produced in yeast. Overall, our study showcases the effectiveness of careful tuning of yeast Aro1 activity and the importance of host-pathway dynamics.

Deep brain stimulation for Parkinson's disease: defining the optimal location within the subthalamic nucleus.

BACKGROUND: Individual motor improvement after deep brain stimulation (DBS) of the subthalamic nucleus (STN) for Parkinson's disease (PD) varies considerably. Stereotactic targeting of the dorsolateral sensorimotor part of the STN is considered paramount for maximising effectiveness, but studies employing the midcommissural point (MCP) as anatomical reference failed to show correlation between DBS location and motor improvement. The medial border of the STN as reference may provide better insight in the relationship between DBS location and clinical outcome. METHODS: Motor improvement after 12 months of 65 STN DBS electrodes was categorised into non-responding, responding and optimally responding body-sides. Stereotactic coordinates of optimal electrode contacts relative to both medial STN border and MCP served to define theoretic DBS 'hotspots'. RESULTS: Using the medial STN border as reference, significant negative correlation (Pearson's correlation -0.52, P<0.01) was found between the Euclidean distance from the centre of stimulation to this DBS hotspot and motor improvement. This hotspot was located at 2.8 mm lateral, 1.7 mm anterior and 2.5 mm superior relative to the medial STN border. Using MCP as reference, no correlation was found. CONCLUSION: The medial STN border proved superior compared with MCP as anatomical reference for correlation of DBS location and motor improvement, and enabled defining an optimal DBS location within the nucleus. We therefore propose the medial STN border as a better individual reference point than the currently used MCP on preoperative stereotactic imaging, in order to obtain optimal and thus less variable motor improvement for individual patients with PD following STN DBS.

Does deep brain stimulation improve lower urinary tract symptoms in Parkinson's disease?

AIMS: To investigate whether deep brain stimulation (DBS) of the globus pallidus pars interna (GPi) or the subthalamic nucleus (STN) improve lower urinary tract symptoms (LUTS) in advanced Parkinson's disease (PD). METHODS: An exploratory post-hoc analysis was performed of specific LUTS items of questionnaires used in a randomized clinical trial with 128 patients (NSTAPS study). First, we compared scores on LUTS items at baseline and 12 months for the GPi DBS and STN DBS group separately. Second, we divided the group by sex, instead of DBS location; to assess a possible gender associated influence of anatomical and pathophysiological differences, again comparing scores at baseline and 12 months. Third, we reported on Foley-catheter use at baseline and after 12 months. RESULTS: Urinary incontinence and frequency improved after both GPi DBS and STN DBS at 12 months, postoperatively, but this was only statistically significant for the STN DBS group (P = 0.004). The improvements after DBS were present in both men (P = 0.01) and women (P = 0.05). Nocturia and urinary incontinence did not improve significantly after any type of DBS, irrespective of sex. At 12 months, none of the patients had a Foley-catheter. CONCLUSIONS: Urinary incontinence and frequency significantly improved after STN DBS treatment in male and female patients with PD. Nocturia and nighttime incontinence due to parkinsonism did not improve after DBS, irrespective of gender.

Preliminary effects of a regional approached multidisciplinary educational program on healthcare utilization in patients with hip or knee osteoarthritis: an observational study.

BACKGROUND: Providing relevant information on disease and self-management helps patients to seek timely contact with care providers and become actively involved in their own care process. Therefore, health professionals from primary care, multiple hospitals and health organisations jointly decided to develop an educational program on osteoarthritis (OA). The objective of the present study was to determine preliminary effects of this OA educational program on healthcare utilization and clinical outcomes. METHODS: We developed an educational group-based program consisting of 2 meetings of 1.5 h, provided by a physiotherapist, a general practitioner (GP) and orthopaedic surgeon or specialized nurse. The program included education on OA, (expectations regarding) treatment options and self-management. Patients were recruited through searching the GPs' electronic patients records and advertisements in local newspapers. At baseline and at 3 months follow-up participating OA patients completed questionnaires. Paired-sample t-tests, McNemar's test and Wilcoxon Signed-Rank test were used to estimate the preliminary effects of the program. RESULTS: A total of 146 participants in 3 districts attended the sessions, of whom 143 agreed to participate in this study; mean age 69.1 years (SD10.2).107 (75%) participants completed both baseline and follow up assessments. The proportion of participants who had visited their GP in the 3 months after the program was lower than 3 months previous to the program (40% versus 25%, p-value 0.01). Also, we observed a decrease in proportion of patients who visited the physio- and exercise therapist, (36.1% versus 25.0%, p-value 0.02). Both illness perceptions and knowledge on OA and treatment options changed positively (Δ-1.8, 95%CI:0.4-3.4, and Δ2.4, 95%CI:-3.0 - -1.6 respectively). No changes in BMI, pain, functioning and self-efficacy were found. However, a trend towards an increase in physical activity was observed. CONCLUSIONS: Our results show that a multidisciplinary educational program may result in a decrease in healthcare utilization and has a positive effect on illness perceptions and knowledge on OA due to clear and consistent information on OA and it treatment options. TRIAL REGISTRATION: Netherlands Trial Register ( NTR5472 ). Registered 22 September 2015.

Substituting the Target After Unsatisfactory Outcome of Deep Brain Stimulation in Advanced Parkinson's Disease: Cases From the NSTAPS Trial and Systematic Review of the Literature.

BACKGROUND: Deep brain stimulation (DBS) of the globus pallidus pars interna (GPi) and the subthalamic nucleus (STN) are established treatment option in Parkinson's disease (PD). If DBS does not provide the desired effect, re-operation to the alternative target is a treatment option, but data on the effect of re-operation are scarce. OBJECTIVES: The objective of this study is to evaluate the clinical effect of re-operation the alternative target after failure of initial STN or GPi DBS for Parkinson's disease. MATERIALS AND METHODS: We descriptively analyzed the baseline characteristics, the effect of initial surgery and re-operation of eight NSTAPS (Netherlands SubThalamic and Pallidal Stimulation) patients and six previously published cases that underwent re-operation to a different target. RESULTS: In the NSTAPS cohort, two of the eight patients showed more than 30% improvement of off-drug motor symptoms after re-operation. The initial DBS leads of these patients were off target. In the cases from the literature, 30% off-drug motor improvement was seen in all three patients re-operated from GPi to STN and none of the three patients re-operated from STN to GPi. Only one of the three cases from the literature where any improvement was seen with the operation had a confirmed on target lead location after the first surgery, while the other two patients did not undergo post-operative imaging after the first surgery. CONCLUSIONS: Re-operation to a different target due to lack of effect appears to have a limited chance of leading to objective improvement if the leads were correctly placed during initial surgery.

An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose.

Benzylisoquinoline alkaloids (BIAs) are a diverse family of plant-specialized metabolites that include the pharmaceuticals codeine and morphine and their derivatives. Microbial synthesis of BIAs holds promise as an alternative to traditional crop-based manufacturing. Here we demonstrate the production of the key BIA intermediate (S)-reticuline from glucose in Saccharomyces cerevisiae. To aid in this effort, we developed an enzyme-coupled biosensor for the upstream intermediate L-3,4-dihydroxyphenylalanine (L-DOPA). Using this sensor, we identified an active tyrosine hydroxylase and improved its L-DOPA yields by 2.8-fold via PCR mutagenesis. Coexpression of DOPA decarboxylase enabled what is to our knowledge the first demonstration of dopamine production from glucose in yeast, with a 7.4-fold improvement in titer obtained for our best mutant enzyme. We extended this pathway to fully reconstitute the seven-enzyme pathway from L-tyrosine to (S)-reticuline. Future work to improve titers and connect these steps with downstream pathway branches, already demonstrated in S. cerevisiae, will enable low-cost production of many high-value BIAs.