Effect of dexmedetomidine added to retrobulbar blockade with lignocaine and bupivacaine in dogs undergoing enucleation surgery.

OBJECTIVE: To investigate the effect of the addition of dexmedetomidine (BLD) to retrobulbar blockade with combined lignocaine and bupivacaine on nociception. ANIMALS: A total of 17 eyes from 15 dogs. METHODS: Prospective, randomized, masked clinical comparison study. Dogs undergoing unilateral enucleation were randomly assigned into two groups; a retrobulbar administration of lignocaine and bupivacaine in a 1:2 volume ratio combined with either BLD or 0.9% saline (BLS). The total volume of the intraconal injection was calculated at 0.1 mL/cm cranial length. Intraoperative parameters were recorded: heart rate (HR), respiratory rate (RR), end-tidal CO2 (EtCO2 ) arterial blood pressure (BP), and inspired isoflurane concentration (ISOinsp). Pain scores, heart rate and RR were recorded postoperatively. RESULTS: Dogs receiving BLD (n = 8) had significantly lower intraoperative RR (p = 0.007), and significantly lower ISOinsp (p = 0.037) than dogs in the BLS group (n = 9). Postoperatively heart rate was significantly lower in the BLD group at 1 min (p = 0.025) and 1 h (p = 0.022). There were no other significant differences in intraoperative or postoperative parameters, or in postoperative pain scores (p = 0.354). Dogs receiving BLD had a higher rate of anesthetic events of bradycardia and hypertension (p = 0.027). Analgesic rescue was not needed in either group. CONCLUSIONS: The addition of BLD to retrobulbar anesthesia did not result in a detectable difference in pain scores relative to blockade with lignocaine and bupivacaine alone. Dogs receiving retrobulbar BLD had a significantly lower intraoperative RR and isoflurane requirement and an increased incidence of intraoperative bradycardia and hypertension.

Superficial keratectomy for the treatment of spontaneous chronic corneal epithelial defects in dogs.

OBJECTIVE: To report the outcome of superficial keratectomy with bandage contact lens placement for the treatment of spontaneous chronic corneal epithelial defects (SCCEDs) in dogs. METHODS: Patients that underwent a superficial keratectomy with bandage lens placement for the treatment of one or more SCCEDs were retrospectively included in the study. Signalment, eye(s) affected, prior medical therapy and any procedures performed, post-operative medical therapy, healing rate, and any post-operative complications were recorded. Superficial keratectomy was performed to approximately one-fifth of corneal depth under operating microscope guidance and a bandage lens was placed immediately post-operatively. Corneas were considered healed when the fluorescein stain was negative. RESULTS: One hundred and seven dogs met the inclusion criteria with 121 SCCEDs. The mean age of patients was 8.34 ± 2.89 years (1-15). Ninety-nine percent (120/121) of SCCEDS healed with no additional treatment within 21 days of surgery. One eye had a diamond burr debridement performed on Day 14 post-operatively and healed 2 weeks following the additional procedure. No post-operative complications were noted. CONCLUSIONS: This study found superficial keratectomy with bandage lens placement to be an effective treatment for SCCEDs.

Targeting the hepatitis B cccDNA with a sequence-specific ARCUS nuclease to eliminate hepatitis B virus in vivo.

Persistence of chronic hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human hepatocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL in vivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing ARCUS-POL mRNA. In both mouse and NHP, we observed a significant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure.

Age-specific survival rates, causes of death, and allowable take of golden eagles in the western United States.

In the United States, the Bald and Golden Eagle Protection Act prohibits take of golden eagles (Aquila chrysaetos) unless authorized by permit, and stipulates that all permitted take must be sustainable. Golden eagles are unintentionally killed in conjunction with many lawful activities (e.g., electrocution on power poles, collision with wind turbines). Managers who issue permits for incidental take of golden eagles must determine allowable take levels and manage permitted take accordingly. To aid managers in making these decisions in the western United States, we used an integrated population model to obtain estimates of golden eagle vital rates and population size, and then used those estimates in a prescribed take level (PTL) model to estimate the allowable take level. Estimated mean annual survival rates for golden eagles ranged from 0.70 (95% credible interval = 0.66-0.74) for first-year birds to 0.90 (0.88-0.91) for adults. Models suggested a high proportion of adult female golden eagles attempted to breed and breeding pairs fledged a mean of 0.53 (0.39-0.72) young annually. Population size in the coterminous western United States has averaged ~31,800 individuals for several decades, with λ = 1.0 (0.96-1.05). The PTL model estimated a median allowable take limit of ~2227 (708-4182) individuals annually given a management objective of maintaining a stable population. We estimate that take averaged 2572 out of 4373 (59%) deaths annually, based on a representative sample of transmitter-tagged golden eagles. For the subset of golden eagles that were recovered and a cause of death determined, anthropogenic mortality accounted for an average of 74% of deaths after their first year; leading forms of take over all age classes were shooting (~670 per year), collisions (~611), electrocutions (~506), and poisoning (~427). Although observed take overlapped the credible interval of our allowable take estimate and the population overall has been stable, our findings indicate that additional take, unless mitigated for, may not be sustainable. Our analysis demonstrates the utility of the joint application of integrated population and prescribed take level models to management of incidental take of a protected species.

Social network analysis of publication collaboration of accelerating change in MedEd consortium.

INTRODUCTION: The American Medical Association formed the Accelerating Change in Medical Education Consortium through grants to effect change in medical education. The dissemination of educational innovations through scholarship was a priority. The objective of this study was to explore the patterns of collaboration of educational innovation through the consortium's publications. METHOD: Publications were identified from grantee schools' semi-annual reports. Each publication was coded for the number of citations, Altmetric score, domain of scholarship, and collaboration with other institutions. Social network analysis explored relationships at the midpoint and end of the grant. RESULTS: Over five years, the 32 Consortium institutions produced 168 publications, ranging from 38 papers from one institution to no manuscripts from another. The two most common domains focused on health system science (92 papers) and competency-based medical education (30 papers). Articles were published in 54 different journals. Forty percent of publications involved more than one institution. Social network analysis demonstrated rich publishing relationships within the Consortium members as well as beyond the Consortium schools. In addition, there was growth of the network connections and density over time. CONCLUSION: The Consortium fostered a scholarship network disseminating a broad range of educational innovations through publications of individual school projects and collaborations.

Evaluating kin and group selection as tools for quantitative analysis of microbial data.

Kin selection and multilevel selection theory are often used to interpret experiments about the evolution of cooperation and social behaviour among microbes. But while these experiments provide rich, detailed fitness data, theory is mostly used as a conceptual heuristic. Here, we evaluate how kin and multilevel selection theory perform as quantitative analysis tools. We reanalyse published microbial datasets and show that the canonical fitness models of both theories are almost always poor fits because they use statistical regressions misspecified for the strong selection and non-additive effects we show are widespread in microbial systems. We identify analytical practices in empirical research that suggest how theory might be improved, and show that analysing both individual and group fitness outcomes helps clarify the biology of selection. A data-driven approach to theory thus shows how kin and multilevel selection both have untapped potential as tools for quantitative understanding of social evolution in all branches of life.

Eptinezumab in episodic migraine: A randomized, double-blind, placebo-controlled study (PROMISE-1).

OBJECTIVE: To evaluate the efficacy and safety of eptinezumab, a humanized anti-calcitonin gene-related peptide monoclonal antibody, in the preventive treatment of episodic migraine. METHODS: The PRevention Of Migraine via Intravenous ALD403 Safety and Efficacy-1 (PROMISE-1) study was a phase 3, multicenter, randomized, double-blind, placebo-controlled, parallel-group study. Adults with episodic migraine were randomized to eptinezumab 30 mg, 100 mg, 300 mg, or placebo for up to four intravenous (IV) doses administered every 12 weeks. The primary endpoint was change from baseline in monthly migraine days (MMDs) over weeks 1-12. RESULTS: A total of 888 patients received treatment across 84 study sites. Mean MMDs at baseline was ∼8.6 across treatment groups. Eptinezumab 100 mg and 300 mg met the primary endpoint, significantly reducing MMDs across weeks 1-12 compared with placebo (30 mg, -4.0; 100 mg, -3.9, p = 0.0182; 300 mg, -4.3; placebo, -3.2, p = 0.0001). Treatment-emergent adverse events were reported by 58.4% (30 mg), 63.2% (100 mg), 57.6% (300 mg), and 59.5% (placebo) of patients. Treatment-emergent adverse events reported by ≥2% of eptinezumab-treated patients at an incidence greater than placebo included: upper respiratory tract infection (30 mg, 11.4%; 100 mg, 9.9%; 300 mg, 10.3%; placebo, 7.2%), and fatigue (30 mg, 2.3%; 100 mg, 3.6%; 300 mg, 3.6%; placebo, <1%). CONCLUSION: Eptinezumab (100 mg or 300 mg) significantly reduced migraine frequency, was well tolerated, and had an acceptable safety profile when used for the preventive treatment of migraine in adults with episodic migraine. ClinicalTrials.gov identifier: NCT02559895.

IAEA contribution to the development of 64Cu radiopharmaceuticals for theranostic applications.

Copper-64 is a very attractive radioisotope with unique nuclear properties that allow using it as both a diagnostic and therapeutic agent, thus providing an almost ideal example of a theranostic radionuclide. A characteristic of Cu-64 stems from the intrinsic biological nature of copper ions that play a fundamental role in a large number of cellular processes. Cu-64 is a radionuclide that reflects the natural biochemical pathways of Cu-64 ions, therefore, can be exploited for the detection and therapy of certain malignancies and metabolic diseases. Beside these applications of Cu-64 ions, this radionuclide can be also used for radiolabelling bifunctional chelators carrying a variety of pharmacophores for targeting different biological substrates. These include peptide-based substrates and immunoconjugates as well as small-molecule bioactive moieties. Fueled by the growing interest of Member States (MS) belonging to the International Atomic Energy Agency (IAEA) community, a dedicated Coordinated Research Project (CRP) was initiated in 2016, which recruited thirteen participating MS from four continents. Research activities and collaborations between the participating countries allowed for collection of an impressive series of results, particularly on the production, preclinical evaluation and, in a few cases, clinical evaluation of various 64Cu-radiopharmaceuticals that may have potential impact on future development of the field. Since this CRP was finalized at the beginning of 2020, this short review summarizes outcomes, outputs and results of this project with the purpose to propagate to other MS and to the whole scientific community, some of the most recent achievements on this novel class of theranostic 64Cu-pharmaceuticals.

Eptinezumab Demonstrated Efficacy in Sustained Prevention of Episodic and Chronic Migraine Beginning on Day 1 After Dosing.

OBJECTIVE: To determine the onset of preventive efficacy with eptinezumab in patients with migraine. BACKGROUND: Eptinezumab is a monoclonal antibody inhibiting calcitonin gene-related peptide approved as an intravenously administered treatment for the prevention of migraine. METHODS: Patients who received eptinezumab 100 mg, eptinezumab 300 mg, or placebo in PROMISE7-1 (episodic migraine; 100 mg, n = 221; 300 mg, n = 222; placebo, n = 222) or PROMISE7-2 (chronic migraine; 100 mg, n = 356; 300 mg, n = 350; placebo, n = 366) were included. Testing of the percentage of patients with a migraine on day 1 after dosing was prespecified and alpha-controlled. In further exploration of this prespecified endpoint, a post hoc closed testing procedure, which controlled the false-positive (type 1) error rate, provided a statistically rigorous evaluation of migraine prevention onset. The procedure involved up to 84 tests of significance, all of which were performed in sequence until the first nonsignificant result. RESULTS: For both studies, all tests for significance for eptinezumab 100 and 300 mg, from days 1-84 through day 1 alone, achieved nominal significance (P < .05), indicating that eptinezumab was fully effective beginning on day 1. Over each interval, the treatment effect was comparable to the effect over weeks 1-12. Mean changes from baseline in monthly migraine days for the primary endpoint period ranged from -3.9 to -4.9, -4.1 to -4.9, and -2.2 to -3.2 for eptinezumab 100, 300 mg, and placebo, respectively, in PROMISE7-1 and from -7.2 to -8.0, -7.9 to -8.2, and -4.3 to -5.6, respectively, in PROMISE7-2. The difference from placebo (95% confidence interval) in day 1 treatment effect was -2.2 (-4.1, -0.3) and -2.5 (-4.4, -0.6) days/month for eptinezumab 100 and 300 mg, respectively, in PROMISE7-1, and was -3.8 (-5.6, -2.0) and -4.0 (-5.8, -2.1) days/month for 100 and 300 mg, respectively, in PROMISE7-2. CONCLUSIONS: The migraine preventive effect of eptinezumab is rapid and sustained in patients with episodic or chronic migraine, with onset of optimal preventive efficacy observed on the day following the initial dose.

Eptinezumab for the prevention of chronic migraine: efficacy and safety through 24 weeks of treatment in the phase 3 PROMISE-2 (Prevention of migraine via intravenous ALD403 safety and efficacy-2) study.

BACKGROUND: PROMISE-2 was a phase 3, randomized, double-blind, placebo-controlled study that evaluated the efficacy and safety of repeat intravenous (IV) doses of the calcitonin gene-related peptide-targeted monoclonal antibody eptinezumab (ALD403) for migraine prevention in adults with chronic migraine. This report describes the results of PROMISE-2 through 24 weeks of treatment. METHODS: Patients received up to two 30-min IV administrations of eptinezumab 100 mg, 300 mg, or placebo separated by 12 weeks. Patients recorded migraine and headache endpoints in a daily eDiary. Additional assessments, including patient-reported outcomes, were performed at regularly scheduled clinic visits throughout the 32-week study period (screening, day 0, and weeks 2, 4, 8, 12, 16, 20, 24, and 32). RESULTS: A total of 1072 adults received treatment: eptinezumab 100 mg, n = 356; eptinezumab 300 mg, n = 350; placebo, n = 366. The reduction in mean monthly migraine days observed during the first dosing interval (100 mg, - 7.7 days; 300 mg, - 8.2 days; placebo, - 5.6 days) was further decreased after an additional dose (100 mg, - 8.2 days; 300 mg, - 8.8 days; placebo, - 6.2 days), with both doses of eptinezumab demonstrating consistently greater reductions from baseline compared to placebo. The ≥50% and ≥ 75% migraine responder rates (MRRs) increased after a second dose, with more eptinezumab-treated patients experiencing migraine response than placebo patients (≥50% MRRs weeks 13-24: 100 mg, 61.0%; 300 mg, 64.0%; placebo, 44.0%; and ≥ 75% MRRs weeks 13-24: 100 mg, 39.3%; 300 mg, 43.1%; placebo, 23.8%). The percentages of patients who improved on patient-reported outcomes, including the Headache Impact Test and Patient Global Impression of Change, increased following the second dose administration at week 12, and were greater with eptinezumab than with placebo at all time points. No new safety concerns were identified with the second dose regarding the incidence, nature, and severity of treatment-emergent adverse events. CONCLUSION: Eptinezumab 100 mg or 300 mg administered IV at day 0 and repeated at week 12 provided sustained migraine preventive benefit over a full 24 weeks and demonstrated an acceptable safety profile in patients with chronic migraine. TRIAL REGISTRATION: ClinicalTrials.gov (Identifier: NCT02974153 ). Registered November 23, 2016.

Eptinezumab for prevention of chronic migraine: A randomized phase 2b clinical trial.

BACKGROUND: Calcitonin gene-related peptide plays an important role in migraine pathophysiology. We evaluated eptinezumab, an intravenous (IV) anti-calcitonin gene-related peptide monoclonal antibody, for the prevention of chronic migraine. OBJECTIVE: To determine the safety, tolerability, and effectiveness of four dose levels of eptinezumab and to inform the phase 3 development program. METHODS: This was a phase 2b, parallel-group, double-blind, randomized, placebo-controlled, dose-ranging clinical trial. Men and women (N = 616) aged 18-55 years were included if they had a diagnosis of chronic migraine, with onset at age ≤35 years and history of chronic migraine ≥1 year. During the 28-day screening period, patients must have had ≥15 headache days, including ≥8 migraine days, with ≥5 migraine attacks as recorded in the electronic diary. Patients were assigned in a 1:1:1:1:1 ratio to eptinezumab 300, 100, 30, 10 mg or placebo, administered as a single IV infusion. The primary endpoint was the percentage of patients with a ≥75% decrease in monthly migraine days over weeks 1-12 compared with the 28-day screening period. RESULTS: The ≥75% migraine responder rates over weeks 1-12 for eptinezumab 300, 100, 30, and 10 mg were 33.3%, 31.4%, 28.2%, and 26.8%, respectively, versus 20.7% for placebo (p = 0.033, 0.072, 0.201, 0.294 vs. placebo). Secondary efficacy endpoints (e.g. ≥50% responder rate, change from baseline in frequency of migraine/headache days, and percentage of severe migraines) had results favoring the three higher eptinezumab doses versus placebo. Eptinezumab was well tolerated and adverse event rates were similar to placebo. CONCLUSIONS: The results of this trial demonstrate that eptinezumab appears effective and well-tolerated for the preventive treatment of chronic migraine and justifies the conduct of pivotal phase 3 trials for migraine prevention. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02275117.

Hexokinase 2 as a novel selective metabolic target for rheumatoid arthritis.

OBJECTIVES: Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Hexokinases (HKs) catalyse the first step in glucose metabolism, and HK2 constitutes the principal HK inducible isoform. We hypothesise that HK2 contributes to the synovial lining hypertrophy and plays a critical role in bone and cartilage damage. METHODS: HK1 and HK2 expression were determined in RA and osteoarthritis (OA) synovial tissue by immunohistochemistry. RA FLS were transfected with either HK1 or HK2 siRNA, or infected with either adenovirus (ad)-GFP, ad-HK1 or ad-HK2. FLS migration and invasion were assessed. To study the role of HK2 in vivo, 108 particles of ad-HK2 or ad-GFP were injected into the knee of wild-type mice. K/BxN serum transfer arthritis was induced in HK2F/F mice harbouring Col1a1-Cre (HK2Col1), to delete HK2 in non-haematopoietic cells. RESULTS: HK2 is particular of RA histopathology (9/9 RA; 1/8 OA) and colocalises with FLS markers. Silencing HK2 in RA FLS resulted in a less invasive and migratory phenotype. Consistently, overexpression of HK2 resulted in an increased ability to migrate and invade. It also increased extracellular lactate production. Intra-articular injection of ad-HK2 in normal knees dramatically increased synovial lining thickness, FLS activation and proliferation. HK2 was highly expressed in the synovial lining after K/BxN serum transfer arthritis. HK2Col1 mice significantly showed decreased arthritis severity, bone and cartilage damage. CONCLUSION: HK2 is specifically expressed in RA synovial lining and regulates FLS aggressive functions. HK2 might be an attractive selective metabolic target safer than global glycolysis for RA treatment.

Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells.

Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19+ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+ tumors in an in vivo mouse model.

Five years of health promoting work with bottle shops on the Central Coast of NSW Australia. How can we best ensure outlets check ID?

ISSUE ADDRESSED: Australian surveys indicate that a large proportion of packaged liquor outlets do not check identification for young people before selling alcohol to them. There are a substantial number of presentations to Emergency Departments from young people aged 15 to 17 years. This subgroup is second only to those aged 18 to 24 years. In the 15- to 17-year-old age group, supply from direct purchase or underage friends, who have purchased alcohol, represents substantial sources of alcohol that is more likely to be consumed without parental supervision. METHOD: Teenagers 18-19 years of age approached a randomly selected sample of bottle shops, on the NSW Central Coast Region, to attempt to purchase alcohol without producing identification (ID). Legally we are unable to test with teens under the age of 18. If outlets do not check ID for customers 18 or 19 years of age, we propose they might not check identification for 15- to 17-year-olds. A raft of local interventions was employed over four-survey periods to attempt to reduce selling rates. RESULTS: The lowest alcohol sales without ID occurred in 2015 when NSW Liquor and Gaming successfully prosecuted a Central Coast outlet for an underage sale. The rate of alcohol sales without checking ID each year was as follows: 2012-43.8%, 2014-37.55%, 2015-21.5% and 2016-45%. CONCLUSION: Alcohol sales to young customers without checking ID are common, widespread and seemingly resistant to nonpunitive interventions. The NSW Liquor Act could be modified to allow compliance testing and much more practical enforcement. While Central Coast bottle shops have a better record than other Australian areas showing some improvements with our nonpunitive industry education interventions, the results need to improve substantially to stifle primary supply.

Loss of col8a1a function during zebrafish embryogenesis results in congenital vertebral malformations.

Congenital vertebral malformations (CVM) occur in 1 in 1000 live births and in many cases can cause spinal deformities, such as scoliosis, and result in disability and distress of affected individuals. Many severe forms of the disease, such as spondylocostal dystostosis, are recessive monogenic traits affecting somitogenesis, however the etiologies of the majority of CVM cases remain undetermined. Here we demonstrate that morphological defects of the notochord in zebrafish can generate congenital-type spine defects. We characterize three recessive zebrafish leviathan/col8a1a mutant alleles ((m531, vu41, vu105)) that disrupt collagen type VIII alpha1a (col8a1a), and cause folding of the embryonic notochord and consequently adult vertebral column malformations. Furthermore, we provide evidence that a transient loss of col8a1a function or inhibition of Lysyl oxidases with drugs during embryogenesis was sufficient to generate vertebral fusions and scoliosis in the adult spine. Using periodic imaging of individual zebrafish, we correlate focal notochord defects of the embryo with vertebral malformations (VM) in the adult. Finally, we show that bends and kinks in the notochord can lead to aberrant apposition of osteoblasts normally confined to well-segmented areas of the developing vertebral bodies. Our results afford a novel mechanism for the formation of VM, independent of defects of somitogenesis, resulting from aberrant bone deposition at regions of misshapen notochord tissue.

Male-sterile maize plants produced by targeted mutagenesis of the cytochrome P450-like gene (MS26) using a re-designed I-CreI homing endonuclease.

The I-CreI homing endonuclease from Chlamydomonas reinhardti has been used as a molecular tool for creating DNA double-strand breaks and enhancing DNA recombination reactions in maize cells. The DNA-binding properties of this protein were re-designed to recognize a 22 bp target sequence in the 5th exon of MS26, a maize fertility gene. Three versions of a single-chain endonuclease, called Ems26, Ems26+ and Ems26++, cleaved their intended DNA site within the context of a reporter assay in a mammalian cell line. When the Ems26++ version was delivered to maize Black Mexican Sweet cells by Agrobacterium-mediated transformation, the cleavage resulted in mutations at a co-delivered extra-chromosomal ms26-site in up to 8.9% of the recovered clones. Delivery of the same version of Ems26 to immature embryos resulted in mutations at the predicted genomic ms26-site in 5.8% of transgenic T(0) plants. This targeted mutagenesis procedure yielded small deletions and insertions at the Ems26 target site consistent with products of double-strand break repair generated by non-homologous end joining. One of 21 mutagenized T(0) plants carried two mutated alleles of the MS26 gene. As expected, the bi-allelic mutant T(0) plant and the T(1) progeny homozygous for the ms26 mutant alleles were male-sterile. This paper described the second maize chromosomal locus (liguless-1 being the first one) mutagenized by a re-designed I-CreI-based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.

Fruiting bodies of the social amoeba Dictyostelium discoideum increase spore transport by Drosophila.

BACKGROUND: Many microbial phenotypes are the product of cooperative interactions among cells, but their putative fitness benefits are often not well understood. In the cellular slime mold Dictyostelium discoideum, unicellular amoebae aggregate when starved and form multicellular fruiting bodies in which stress-resistant spores are held aloft by dead stalk cells. Fruiting bodies are thought to be adaptations for dispersing spores to new feeding sites, but this has not been directly tested. Here we experimentally test whether fruiting bodies increase the rate at which spores are acquired by passing invertebrates. RESULTS: Drosophila melanogaster accumulate spores on their surfaces more quickly when exposed to intact fruiting bodies than when exposed to fruiting bodies physically disrupted to dislodge spore masses from stalks. Flies also ingest and excrete spores that still express a red fluorescent protein marker. CONCLUSIONS: Multicellular fruiting bodies created by D. discoideum increase the likelihood that invertebrates acquire spores that can then be transported to new feeding sites. These results thus support the long-hypothesized dispersal benefits of altruism in a model system for microbial cooperation.

In the social amoeba Dictyostelium discoideum, density, not farming status, determines predatory success on unpalatable Escherichia coli.

BACKGROUND: The social amoeba Dictyostelium discoideum interacts with bacteria in a variety of ways. It is a predator of bacteria, can be infected or harmed by bacteria, and can form symbiotic associations with bacteria. Some clones of D. discoideum function as primitive farmers because they carry bacteria through the normally sterile D. discoideum social stage, then release them after dispersal so the bacteria can proliferate and be harvested. Some farmer-associated bacteria produce small molecules that promote host farmer growth but inhibit the growth of non-farmer competitors. To test whether the farmers' tolerance is specific or extends to other growth inhibitory bacteria, we tested whether farmer and non-farmer amoebae are differentially affected by E. coli strains of varying pathogenicity. Because the numbers of each organism may influence the outcome of amoeba-bacteria interactions, we also examined the influence of amoeba and bacteria density on the ability of D. discoideum to grow and develop on distinct bacterial strains. RESULTS: A subset of E. coli strains did not support amoeba proliferation on rich medium, independent of whether the amoebae were farmers or non-farmers. However, amoebae could proliferate on these strains if amoebae numbers are high relative to bacteria numbers, but again there was no difference in this ability between farmer and non-farmer clones of D. discoideum. CONCLUSIONS: Our results show that farmer and non-farmers did not differ in their abilities to consume novel strains of E. coli, suggesting that farmer resistance to their own carried bacteria does not extend to foreign bacteria. We see that increasing the numbers of bacteria or amoebae increases their respective likelihood of competitive victory over the other, thus showing Allee effects. We hypothesize that higher bacteria numbers may result in higher concentrations of a toxic product or in a reduction of resources critical for amoeba survival, producing an environment inhospitable to amoeba predators. Greater amoeba numbers may counter this growth inhibition, possibly through reducing bacterial numbers via increased predation rates, or by producing something that neutralizes a potentially toxic bacterial product.

Public attitudes towards COVID-19 vaccine mandates and vaccine certificates in Canada: a time series study.

INTRODUCTION: Since the beginning of the pandemic, numerous public health measures such as COVID-19 vaccines, vaccine mandates and vaccination certificates have been introduced to mitigate the spread of COVID-19. Public opinion and attitudes towards these measures have fluctuated in response to the dynamic political, social, and cultural landscape of the pandemic. METHODS: We conducted a time-series study consisting of national cross-sectional surveys between November 2021 to March 2022 to evaluate the Canadian public's attitudes towards COVID-19 vaccine mandates and vaccine certificates. RESULTS: When examining public sentiment towards COVID-19 vaccine certificates and proof of vaccination measures, there was a shift in responses over time. The proportion of participants "strongly supporting" these measures decreased from 66.0 to 43.1% between W25(Capacity Limits), -W32 (Mask Mandate Removed), whereas "strongly oppose" was the second most common response and rose from 15.9 to 20.6% during this same time period. Concurrently, when examining participants views surrounding mandates, many participants believed that their province was reopening at "about the right pace", which remained relatively stable over time (33.0-35.4%) between W28 (Emergency Act)-W32 (Mask Mandate Removed). CONCLUSION: Our study's findings on the public's attitudes towards COVID-19 vaccine mandates and vaccine certificates in Canada may aid to guide and streamline the implementation of future similar public health interventions. Future research should include extended follow-up and a more comprehensive examination of trust in government institutions and polarized perspectives on vaccine mandates.