CRISPRi-based circuits to control gene expression in plants.

The construction of synthetic gene circuits in plants has been limited by a lack of orthogonal and modular parts. Here, we implement a CRISPR (clustered regularly interspaced short palindromic repeats) interference (CRISPRi)-based reversible gene circuit platform in plants. We create a toolkit of engineered repressible promoters of different strengths and construct NOT and NOR gates in Arabidopsis thaliana protoplasts. We determine the optimal processing system to express single guide RNAs from RNA Pol II promoters to introduce NOR gate programmability for interfacing with host regulatory sequences. The performance of a NOR gate in stably transformed Arabidopsis plants demonstrates the system's programmability and reversibility in a complex multicellular organism. Furthermore, cross-species activity of CRISPRi-based logic gates is shown in Physcomitrium patens, Triticum aestivum and Brassica napus protoplasts. Layering multiple NOR gates together creates OR, NIMPLY and AND logic functions, highlighting the modularity of our system. Our CRISPRi circuits are orthogonal, compact, reversible, programmable and modular and provide a platform for sophisticated spatiotemporal control of gene expression in plants.

Rehabilitation Techniques for Adults Undergoing External Fixation Treatment for Lower Limb Reconstruction: A Systematic Review.

Introduction: External fixation devices are commonly used in orthopaedic surgery to manage a range of pathologies. In this patient population, there is currently no consensus on optimal rehabilitation techniques. There exists a large variation in practice, with a limited understanding of how these affect treatment outcomes. Methods: Following Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines, a systematic review was conducted of Allied and Complementary Medicine Database (AMED), Cumulative Index to Nursing and Allied Health Literature (CINAHL), MEDLINE, PEDro, and COCHRANE databases, grey literature sources and forward and backward searching of included articles. Studies were selected following rigorous screening with predefined inclusion criteria. Data quality was assessed using validated appraisal tools. Articles were synthesised by rehabilitation type and descriptive analysis was subsequently performed. Results: From 1,156 articles identified, 18 were eligible for inclusion. The overall quality was low, with clinical commentaries and case studies being the most common study type. Studies were synthesised by rehabilitation type, the most common themes being gait re-education, strengthening, therapy-assisted, active exercises and weight-bearing exercises. Conclusion: There is a lack of high-quality evidence to support meaningful recommendations and guide rehabilitation practices for this patient cohort. Further research for patients being treated in external fixation, especially related to the potential effects of physical rehabilitation on bone healing, return of strength, mobility and independent function is likely to have transferability within wider orthopaedic populations. Clinical significance: This systematic review is unable to provide clinical recommendations due to the poor quality of the available literature. However, it is hoped this paper will provide a foundation for further research to improve rehabilitation for patients being treated with external fixation. How to cite this article: Pawson JR, Church D, Fletcher J, et al. Rehabilitation Techniques for Adults Undergoing External Fixation Treatment for Lower Limb Reconstruction: A Systematic Review. Strategies Trauma Limb Reconstr 2024;19(1):45-55.

Acute and Complicated Inflammatory Pericarditis: A Guide to Contemporary Practice.

Inflammatory disease of the pericardium represents a relatively common presentation, especially among the young. For the most part, inflammatory pericardial disease can be expeditiously and effectively managed without significant sequelae. However, some individuals present with severe and recurrent illness, representing significant therapeutic challenges. During the past decade, there have been great strides made in developing an evidence-based approach to management of inflammatory pericardial disease, the result of which has been the development of (1) a systematic, protocoled approach to initial care; (2) targeted therapeutics; and (3) specialized, collaborative, and integrated care pathways. Herein we present a review of the current state of the art as it pertains to the diagnostic evaluation and therapeutic considerations in inflammatory pericardial disease with a focus on acute and complicated pericarditis.

Coupling Partial Nitritation, Anammox and n-DAMO in a membrane aerated biofilm reactor for simultaneous dissolved methane and nitrogen removal.

Anaerobic technologies with downstream autotrophic nitrogen removal have been proposed to enhance bioenergy recovery and transform a wastewater treatment plant from an energy consumer to an energy exporter. However, approximately 20-50 % of the produced methane is dissolved in the anaerobically treated effluent and is easily stripped into the atmosphere in the downstream aerobic process, contributing to the release of greenhouse gas emissions. This study aims to develop a solution to beneficially utilize dissolved methane to support high-level nitrogen removal from anaerobically treated mainstream wastewater. A novel technology, integrating Partial Nitritation, Anammox and Methane-dependent nitrite/nitrate reduction (i.e. PNAM) was demonstrated in a membrane-aerated biofilm reactor (MABR). With the feeding of ∼50 mg NH4+-N/L and ∼20 mg/L dissolved methane at a hydraulic retention time of 15 h, around 90 % of nitrogen and ∼100 % of dissolved methane can be removed together in the MABR. Microbial community characterization revealed that ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), anammox bacteria, nitrite/nitrate-dependent anaerobic methane oxidation microorganisms (n-DAMO bacteria and archaea) and aerobic methanotrophs co-existed in the established biofilm. Batch tests confirmed the active microbial pathways and showed that AOB, anammox bacteria and n-DAMO microbes were jointly responsible for the nitrogen removal, and dissolved methane was mainly removed by the n-DAMO process, with aerobic methane oxidation making a minor contribution. In addition, the established system was robust against dynamic changes in influent composition. The study provides a promising technology for the simultaneous removal of dissolved methane and nitrogen from domestic wastewater, which can support the transformation of wastewater treatment from an energy- and carbon-intensive process, to one that is energy- and carbon-neutral.

Research priorities in regional anaesthesia: an international Delphi study.

BACKGROUND: Regional anaesthesia use is growing worldwide, and there is an increasing emphasis on research in regional anaesthesia to improve patient outcomes. However, priorities for future study remain unclear. We therefore conducted an international research prioritisation exercise, setting the agenda for future investigators and funding bodies. METHODS: We invited members of specialist regional anaesthesia societies from six continents to propose research questions that they felt were unanswered. These were consolidated into representative indicative questions, and a literature review was undertaken to determine if any indicative questions were already answered by published work. Unanswered indicative questions entered a three-round modified Delphi process, whereby 29 experts in regional anaesthesia (representing all participating specialist societies) rated each indicative question for inclusion on a final high priority shortlist. If ≥75% of participants rated an indicative question as 'definitely' include in any round, it was accepted. Indicative questions rated as 'definitely' or 'probably' by <50% of participants in any round were excluded. Retained indicative questions were further ranked based on the rating score in the final Delphi round. The final research priorities were ratified by the Delphi expert group. RESULTS: There were 1318 responses from 516 people in the initial survey, from which 71 indicative questions were formed, of which 68 entered the modified Delphi process. Eleven 'highest priority' research questions were short listed, covering themes of pain management; training and assessment; clinical practice and efficacy; technology and equipment. CONCLUSIONS: We prioritised unanswered research questions in regional anaesthesia. These will inform a coordinated global research strategy for regional anaesthesia and direct investigators to address high-priority areas.

Visualizing calcium-dependent signaling networks in plants.

Calcium-dependent protein kinases (CDPKs) are a multigene protein kinase family that have key regulatory roles in plants. However, imaging CDPK signals in plant cells remains challenging. The recently developed genetically encoded CDPK-Förster resonance energy transfer (FRET) reporter developed by Liese et al. allows visualization of calcium (Ca2+)-dependent conformational changes during activation or inactivation of CDPKs, providing a powerful tool for real-time monitoring of calcium decoding in plants.

Implementation of an office-based addiction treatment model for Medicaid enrollees: A mixed methods study.

INTRODUCTION: Medications for opioid use disorder (MOUD) are the most effective treatment for opioid use disorder (OUD) but remain underutilized. To reduce barriers to MOUD prescribing and increase treatment access, New Jersey's Medicaid program implemented the Office-Based Addiction Treatment (OBAT) Program in 2019, which increased reimbursement for office-based buprenorphine prescribing and established newly reimbursable patient navigation services in OBAT clinics. Using a mixed-methods design, this study aimed to describe stakeholder experiences with the OBAT program and to assess implementation and uptake of the program. METHODS: This study used a concurrent, triangulated mixed-methods design, which integrated complementary qualitative (semi-structured interviews) and quantitative (Medicaid claims) data to gain an in-depth understanding of the implementation of the OBAT program. We elicited stakeholder perspectives through interviews with 22 NJ Medicaid MOUD providers and 8 policy key informants, and examined trends in OBAT program utilization using 2019-2020 NJ Medicaid claims for 5380 Medicaid enrollees who used OBAT services. We used cross-case analysis (provider interviews) and a case study approach (key informant interviews) in analyzing qualitative data, and calculated descriptive statistics and trends for quantitative data. RESULTS: Provider enrollment and utilization of OBAT services increased steadily during the first two years of program implementation. Interviewees reported that enhanced reimbursements for office-based MOUD incentivized greater MOUD prescribing, while coverage of patient navigation services improved patient care. Despite increasing enrollment in the OBAT program, the proportion of primary care physicians in the state who enrolled in the program remained limited. Key barriers to enrollment included: requirements for a patient navigator; concerns about administrative burdens and reimbursement delays from Medicaid; lack of awareness of the program; and beliefs that patients with OUD were better served in comprehensive care settings. Patient navigation was highlighted as a critical and valuable element of the program, but navigator enrollment and reimbursement challenges may have prevented greater uptake of this service. CONCLUSIONS: Implementation of an OBAT model that enhanced reimbursement and provided coverage for patient navigation likely expanded access to MOUD in NJ. Results support initiatives like the OBAT program in improving access to MOUD, but program adaptations, where feasible, could improve uptake and utilization.

Physiotherapist-led exercise versus usual care (waiting-list) control for patients awaiting rotator cuff repair surgery: A pilot randomised controlled trial (POWER).

BACKGROUND: Once a decision to undergo rotator cuff repair surgery is made, patients are placed on the waiting list. It can take weeks or months to receive surgery. There has been a call to move from waiting lists to 'preparation' lists to better prepare patients for surgery and to ensure it remains an appropriate treatment option for them. OBJECTIVE: To evaluate the feasibility, as measured by recruitment rates, treatment fidelity and follow-up rates, of a future multi-centre randomised controlled trial to compare the clinical and cost-effectiveness of undertaking a physiotherapist-led exercise programme while waiting for surgery versus usual care (waiting-list control). DESIGN: Two-arm, multi-centre pilot randomised controlled trial with feasibility objectives in six NHS hospitals in England. METHOD: Adults (n = 76) awaiting rotator cuff repair surgery were recruited and randomly allocated to a programme of physiotherapist-led exercise (n = 38) or usual care control (n = 38). RESULTS: Of 302 eligible patients, 76 (25%) were randomised. Of 38 participants randomised to physiotherapist-led exercise, 28 (74%) received the exercise programme as intended. 51/76 (67%) Shoulder Pain and Disability Index questionnaires were returned at 6-months. Of 76 participants, 32 had not received surgery after 6-months (42%). Of those 32, 20 were allocated to physiotherapist-led exercise; 12 to usual care control. CONCLUSIONS: A future multi-centre randomised controlled trial is feasible but would require planning for variable recruitment rates between sites, measures to improve treatment fidelity and opportunity for surgical exit, and optimisation of follow-up. A fully powered, randomised controlled trial is now needed to robustly inform clinical decision-making.

Racial And Ethnic Disparities In Buprenorphine Receipt Among Medicare Beneficiaries, 2015-19.

We examined Medicare Part D claims from the period 2015-19 to identify state and national racial and ethnic disparities in buprenorphine receipt among Medicare disability beneficiaries with diagnosed opioid use disorder or opioid overdose. Racial and ethnic disparities in buprenorphine use remained persistently high during the study period, especially for Black beneficiaries, suggesting the need for targeted interventions and policies.

Evolutionary analysis and quality assessment of ζ-carbonic anhydrase sequences from environmental microbiome.

Carbonic anhydrase (CA) is one of the most vital enzymes in living cells. This study has been performed due to the significance of this metalloenzyme for life and the novelty of some CA families like ζ-CA to evaluate evolutionary processes and quality check their sequences. In this study, bioinformatics methods revealed the presence of ζ-CA in some eukaryotic and prokaryotic microorganisms. Notably, it has not been previously reported in prokaryotes. The coexistence of ß- and ζ-CAs in some microorganisms is also a novel finding as well. Also, our analysis identified several CA proteins with 6-14 amino acid intervals between histidine and cysteine in the second highly conserved motif, which can be classified as the novel ζ-CA subfamily members that emerged under the Zn deficiency of aquatic ecosystems and selection pressure in these environments. There is also a possibility that the achieved results are rooted in the contamination of samples from the environmental microbiome genome with genomes of diatom species and the occurrence of errors was observed in the DNA sequencing outcomes. Combining of all results from evolutionary analysis to quality control of ζ-CA DNA sequences is the incentive motivation to explore more the hidden aspects of ζ-CAs.

Antigravitropic PIN polarization maintains non-vertical growth in lateral roots.

Lateral roots are typically maintained at non-vertical angles with respect to gravity. These gravitropic setpoint angles are intriguing because their maintenance requires that roots are able to effect growth response both with and against the gravity vector, a phenomenon previously attributed to gravitropism acting against an antigravitropic offset mechanism. Here we show how the components mediating gravitropism in the vertical primary root-PINs and phosphatases acting upon them-are reconfigured in their regulation such that lateral root growth at a range of angles can be maintained. We show that the ability of Arabidopsis lateral roots to bend both downward and upward requires the generation of auxin asymmetries and is driven by angle-dependent variation in downward gravitropic auxin flux acting against angle-independent upward, antigravitropic flux. Further, we demonstrate a symmetry in auxin distribution in lateral roots at gravitropic setpoint angle that can be traced back to a net, balanced polarization of PIN3 and PIN7 auxin transporters in the columella. These auxin fluxes are shifted by altering PIN protein phosphoregulation in the columella, either by introducing PIN3 phosphovariant versions or via manipulation of levels of the phosphatase subunit PP2A/RCN1. Finally, we show that auxin, in addition to driving lateral root directional growth, acts within the lateral root columella to induce more vertical growth by increasing RCN1 levels, causing a downward shift in PIN3 localization, thereby diminishing the magnitude of the upward, antigravitropic auxin flux.

Multimodality Imaging in Differentiating Constrictive Pericarditis From Restrictive Cardiomyopathy: A Comprehensive Overview for Clinicians and Imagers.

In the evaluation of heart failure, 2 differential diagnostic considerations include constrictive pericarditis and restrictive cardiomyopathy. The often outwardly similar clinical presentation of these 2 pathologic entities routinely renders their clinical distinction difficult. Consequently, initial assessment requires a keen understanding of their separate pathophysiology, epidemiology, and hemodynamic effects. Following a detailed clinical evaluation, further assessment initially rests on comprehensive echocardiographic investigation, including detailed Doppler evaluation. With the combination of mitral inflow characterization, tissue Doppler assessment, and hepatic vein interrogation, initial differentiation of constrictive pericarditis and restrictive cardiomyopathy is often possible with high sensitivity and specificity. In conjunction with a compatible clinical presentation, successful differentiation enables both an accurate diagnosis and subsequent targeted management. In certain cases, however, the diagnosis remains unclear despite echocardiographic assessment, and additional evaluation is required. With advances in noninvasive tools, such evaluation can often continue in a stepwise, algorithmic fashion noninvasively, including both cross-sectional and nuclear imaging. Should this additional evaluation itself prove insufficient, invasive assessment with appropriate expertise may ultimately be necessary.

Mapping the Binding Interactions between Human Gasdermin D and Human Caspase-1 Using Carbene Footprinting.

Carbene footprinting is a recently developed mass spectrometry-based chemical labeling technique that probes protein interactions and conformation. Here, we use the methodology to investigate binding interactions between the protease human Caspase-1 (C285A) and full-length human Gasdermin D (hGSDMD), which are important in inflammatory cell death. GSDMD is cleaved by Caspase-1, releasing its N-terminal domain which oligomerizes in the membrane to form large pores, resulting in lytic cell death. Regions of reduced carbene labeling (masking), caused by protein binding, were observed for each partner in the presence of the other and were consistent with hCaspase-1 exosite and active-site interactions. Most notably, the results showed direct occupancy of hCaspase-1 (C285A) active-site by hGSDMD for the first time. Differential carbene labeling of full-length hGSDMD and the pore-forming N-terminal domain assembled in liposomes showed masking of the latter, consistent with oligomeric assembly and insertion into the lipid bilayer. Interactions between Caspase-1 and the specific inhibitor VRT-043198 were also studied by this approach. In wild-type hCaspase-1, VRT-043198 modifies the active-site Cys285 through the formation of a S,O-hemiacetal. Here, we showed by carbene labeling that this inhibitor can noncovalently occupy the active site of a C285A mutant. These findings add considerably to our knowledge of the hCaspase-1-hGSDMD system.

Opioid use disorder treatment and the role of New Jersey Medicaid policy changes: perspectives of office-based buprenorphine providers.

Background: In the US, seventy percent of drug-related deaths are attributed to opioids. In response to the ongoing opioid crisis, New Jersey's (NJ) Medicaid program implemented the MATrx model to increase treatment access for Medicaid participants with opioid use disorder (OUD). The model's goals include increasing the number of office-based treatment providers, enhancing Medicaid reimbursement for certain treatment services, and elimination of prior authorizations for OUD medications.Objectives: To explore office-based addiction treatment providers' experiences delivering care in the context of statewide policy changes and their perspectives on treatment access changes and remaining barriers.Methods: This qualitative study used purposive sampling to recruit office-based New Jersey medications for opioid use disorder (MOUD) providers . Twenty-two providers (11 females, 11 males) discussed treatment experiences since the policy changes in 2019, including evaluations of the current state of OUD care in New Jersey and perceived outcomes of the MATrx model policy changes.Results: Providers reported the MOUD climate in NJ improved as Medicaid implemented policies intended to reduce barriers to care and increase treatment access. Elimination of prior authorizations was noted as important, as it reduced provider burden and allowed greater focus on care delivery. However, barriers remained, including stigma, pharmacy supply issues, and difficulty obtaining injectable or non-generic medication formulations.Conclusion: NJ policies may have improved access to care for Medicaid beneficiaries by reducing barriers to care and supporting providers in prescribing MOUD. Yet, stigma and lack of psychosocial supports still need to be addressed to further improve access and care quality.

Antipsychotic Medication Use In Medicaid-Insured Children Decreased Substantially Between 2008 And 2016.

After the rapid growth of pediatric antipsychotic prescribing in the early 2000s, especially in the Medicaid population, concerns regarding the safety and appropriateness of such prescribing increased. Many states implemented policy and educational initiatives aimed at safer and more judicious antipsychotic use. Antipsychotic use leveled off in the late 2000s, but there have been no recent national estimates of trends in antipsychotic use in children enrolled in Medicaid, and it is unclear how use varied by race and ethnicity. This study observed a sizable decline in antipsychotic use among children ages 2-17 between 2008 and 2016. Although the magnitude of change varied, declines were observed across foster care status, age, sex, and racial and ethnic groups studied. The proportion of children with an antipsychotic prescription who received any diagnosis associated with a pediatric indication that was approved by the Food and Drug Administration increased from 38 percent in 2008 to 45 percent in 2016, which may indicate a trend toward more judicious prescribing.

Long-term onsite monitoring of a sewage sludge drying pan finds methane emissions consistent with IPCC default emission factor.

As the wastewater sector moves towards achieving net zero greenhouse gas (GHG) emissions, quantifying and understanding fugitive emissions from various sewage treatment steps is crucial for developing effective GHG abatement strategies. Methane (CH4) emissions from a sludge drying pan (SDP) were measured at a wastewater treatment plant in Australia for more than a year, using a micrometeorological technique paired with open-path lasers. The emission rate was tightly associated with sludge additions, climatology, and operational processes. The mean emission rate during the 90 weeks after initial sludge addition was 2.3 (± 0.8) g m-2 d-1, with cumulative emissions of approximately 32 t of CH4. A dynamic temporal pattern of emissions was observed, highlighting the importance of continuous (or near-continuous) measurements for quantifying SDP emissions. A Methane Correction Factor (MCF) expressed as a fraction of the measured chemical oxygen demand of the sludge, was determined to be 0.17 after 63 weeks (the median operational cycle duration at the facility). This is broadly consistent with, albeit slightly less than, the IPCC default value of 0.2 for shallow anaerobic lagoons. These emission measurements will support wastewater utilities that employ open air sludge drying processes to develop effective GHG abatement strategies.

The TOPLESS corepressor regulates developmental switches in the bryophyte Physcomitrium patens that were critical for plant terrestrialisation.

The plant-specific TOPLESS (TPL) family of transcriptional corepressors is integral to multiple angiosperm developmental processes. Despite this, we know little about TPL function in other plants. To address this gap, we investigated the roles TPL plays in the bryophyte Physcomitrium patens, which diverged from angiosperms approximately 0.5 billion years ago. Although complete loss of PpTPL function is lethal, transgenic lines with reduced PpTPL activity revealed that PpTPLs are essential for two fundamental developmental switches in this plant: the transitions from basal photosynthetic filaments (chloronemata) to specialised foraging filaments (caulonemata) and from two-dimensional (2D) to three-dimensional (3D) growth. Using a transcriptomics approach, we integrated PpTPL into the regulatory network governing 3D growth and we propose that PpTPLs represent another important class of regulators that are essential for the 2D-to-3D developmental switch. Transcriptomics also revealed a previously unknown role for PpTPL in the regulation of flavonoids. Intriguingly, 3D growth and the formation of caulonemata were crucial innovations that facilitated the colonisation of land by plants, a major transformative event in the history of life on Earth. We conclude that TPL, which existed before the land plants, was co-opted into new developmental pathways, enabling phytoterrestrialisation and the evolution of land plants.

One-year stable pilot-scale operation demonstrates high flexibility of mainstream anammox application.

Mainstream nitrogen removal via anammox is widely recognized as a promising wastewater treatment process. However, its application is challenging at large scale due to unstable suppression of nitrite-oxidizing bacteria (NOB). In this study, a pilot-scale mainstream anammox process was implemented in an Integrated Fixed-film Activated Sludge (IFAS) configuration. Stable operation with robust NOB suppression was maintained for over one year. This was achieved through integration of three key control strategies: i) low dissolved oxygen (DO = 0.4 ± 0.2 mg O2/L), ii) regular free nitrous acid (FNA)-based sludge treatment, and iii) residual ammonium concentration control (NH4 + with a setpoint of ∼8 mg N/L). Activity tests and FISH demonstrated that NOB barely survived in sludge flocs and were inhibited in biofilms. Despite receiving organic-deficient wastewater from a pilot-scale High-Rate Activated Sludge (HRAS) system as the feed, the system maintained a stable effluent total nitrogen concentration mostly below 10 mg N/L, which was attributed to the successful retention of anammox bacteria. This study successfully demonstrated large-scale long-term mainstream anammox application and generated new practical knowledge for NOB control and anammox retention.