Circadian repression of CLOCK-BMAL1 by PERIOD and CRYPTOCHROME (CRY) in mammals lies at the core of the circadian timekeeping mechanism. CRY repression of CLOCK-BMAL1 and regulation of circadian ...period are proposed to rely primarily on competition for binding with coactivators on an α-helix located within the transactivation domain (TAD) of the BMAL1 C terminus. This model has, however, not been tested in vivo. Here, we applied CRISPR/Cas9-mediated mutagenesis in the monarch butterfly (Danaus plexippus), which possesses a vertebrate-like CRY (dpCRY2) and an ortholog of BMAL1, to show that insect CRY2 regulates circadian repression through TAD α-helix–dependent and –independent mechanisms. Monarch mutants lacking the BMAL1 C terminus including the TAD exhibited arrhythmic eclosion behavior. In contrast, mutants lacking the TAD α-helix but retaining the most distal C-terminal residues exhibited robust rhythms during the first day of constant darkness (DD1), albeit with a delayed peak of eclosion. Phase delay in this mutant on DD1 was exacerbated in the presence of a single functional allele of dpCry2, and rhythmicity was abolished in the absence of dpCRY2. Reporter assays in Drosophila S2 cells further revealed that dpCRY2 represses through two distinct mechanisms: a TAD-dependent mechanism that involves the dpBMAL1 TAD α-helix and dpCLK W328 and a TAD-independent mechanism involving dpCLK E333. Together, our results provide evidence for independent mechanisms of vertebrate-like CRY circadian regulation on the BMAL1 C terminus and the CLK PAS-B domain and demonstrate the importance of a BMAL1 TAD-independent mechanism for generating circadian rhythms in vivo.
The eastern North American monarch butterfly, Danaus plexippus, is an emerging model system to study the neural, molecular, and genetic basis of animal long-distance migration and animal clockwork ...mechanisms. While genomic studies have provided new insight into migration-associated and circadian clock genes, the general lack of simple and versatile reverse-genetic methods has limited in vivo functional analysis of candidate genes in this species. Here, we report the establishment of highly efficient and heritable gene mutagenesis methods in the monarch butterfly using transcriptional activator-like effector nucleases (TALENs) and CRISPR-associated RNA-guided nuclease Cas9 (CRISPR/Cas9). Using two clock gene loci, cryptochrome 2 and clock (clk), as candidates, we show that both TALENs and CRISPR/Cas9 generate high-frequency nonhomologous end-joining (NHEJ)-mediated mutations at targeted sites (up to 100%), and that injecting fewer than 100 eggs is sufficient to recover mutant progeny and generate monarch knockout lines in about 3 months. Our study also genetically defines monarch CLK as an essential component of the transcriptional activation complex of the circadian clock. The methods presented should not only greatly accelerate functional analyses of many aspects of monarch biology, but are also anticipated to facilitate the development of these tools in other nontraditional insect species as well as the development of homology-directed knock-ins.
Receptor activator of nuclear factor κB ligand (RANKL) is expressed by a number of cell types to participate in diverse physiological functions. We have previously identified 10 distal RANKL ...enhancers. Earlier studies have shown that RL-D5 is a multifunctional RANKL enhancer. Deletion of RL-D5 from the mouse genome leads to lower skeletal and lymphoid tissue RANKL, causing a high bone mass phenotype. Herein, we determine the physiological role and lineage specificity of 2 additional RANKL enhancers, RL-D6 and RL-T1, which are located 83 and 123 kb upstream of the gene's transcriptional start site, respectively. Lack of RL-D6 or RL-T1 did not alter skeletal RANKL or bone mineral density up to 48 weeks of age. Although both RL-D5 and RL-T1 contributed to activation induction of T-cell RANKL, RL-T1 knockout mice had drastically low lymphocyte and lymphoid tissue RANKL levels, indicating that RL-T1 is the major regulator of lymphocyte RANKL. Moreover, RL-T1 knockout mice had lower circulating soluble RANKL, suggesting that lymphocytes are important sources of circulating soluble RANKL. Under physiological conditions, lack of RL-D6 did not alter RANKL expression. However, lack of RL-D5 or RL-D6, but not of RL-T1, blunted the oncostatin M and lipopolysaccharide induction of RANKL ex vivo and in vivo, suggesting that RL-D5 and RL-D6 coregulate the inflammation-mediated induction of RANKL in osteocytes and osteoblasts while lack of RL-D6 did not alter secondary hyperparathyroidism or lactation induction of RANKL or bone loss. These results suggest that although RL-D5 mediates RANKL expression in multiple lineages, other cell type– or factor-specific enhancers are required for its appropriate control, demonstrating the cell type–specific and complex regulation of RANKL expression.
Background Approximately 25% of distal clavicle fractures are unstable. Unstable patterns have longer times to union and higher nonunion rates. Stable restoration of the distal clavicle is important ...in decreasing the nonunion rate in distal clavicle fractures. The purpose of this study was to biomechanically compare operative constructs for the treatment of unstable, comminuted distal-third clavicle fractures in a cadaveric model using a locking plate and coracoclavicular reconstruction. We hypothesized that the combination of coracoclavicular reconstruction and a distal clavicle locking plate is biomechanically superior to either construct used individually. Materials and methods An unstable distal clavicle fracture was created in 21 thawed fresh-frozen cadaveric specimens. The 21 specimens were divided into 3 treatment groups of 7: distal-third locking plate, acromioclavicular (AC) TightRope (Arthrex, Naples, FL, USA), and distal-third locking plate and AC TightRope together. After fixation, each specimen was cyclically tested with recording of displacement to determine the stiffness and stability of each construct, followed by load-to-failure testing in tension and compression to determine the maximum load. Results The combined construct of the locking distal clavicle plate and coracoclavicular reconstruction resulted in increased stiffness, maximum resistance to compression, and decreased displacement compared with either construct alone. Conclusion Greater fracture stability was achieved with the combination of the AC TightRope and locking clavicle plate construct than with either alone, suggesting a possibility for increased fracture-healing rates.
The hydroformylation of 1-dodecene on a rhodium-biphephos catalyst complex exploiting a thermomorphic multicomponent solvent system was studied experimentally in a batch reactor in order to describe ...the kinetics of the main and the most relevant side reactions. The formation of the active catalyst was studied in preliminary experiments. Based on a postulated catalytic cycle mechanistic kinetic models were developed considering isomerization, hydrogenation and hydroformylation reactions as well as the formation of not catalytically active Rh-species. The complex overall network was decomposed to support parameter estimation. The isomerization of 1-dodecene, the hydrogenations of iso- and 1-dodecene and the hydroformylations of iso-dodecene and 1-dodecene were investigated as a function of temperature, total pressure and partial pressures of carbon monoxide and hydrogen, respectively. These four sub-networks of increasing size and the total network were analyzed sequentially in order to identify kinetic models and to estimate the corresponding parameters applying model reduction techniques based on singular value decomposition combined with rank revealing QR factorization.
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•Hydroformylation of 1-dodecene in a TMS system on rhodium-biphephos complex studied.•Sub-networks of increasing size analyzed sequentially by experiments and modeling.•Catalytic cycle for isomerization, hydrogenation and hydroformylation developed.•Mechanistic kinetic models derived, reduced and parameters estimated.•Model reduction by singular value decomposition and rank revealing QR factorization.
Limited expression and distribution of nectin-1, the major herpes simplex virus (HSV) type-1 entry-receptor, within tumors has been proposed as an impediment to oncolytic HSV (oHSV) therapy. To ...determine whether resistance to oHSVs in malignant peripheral nerve sheath tumors (MPNSTs) was explained by this hypothesis, nectin-1 expression and oHSV viral yields were assessed in a panel of MPNST cell lines using γ134.5-attenuated (Δγ134.5) oHSVs and a γ134.5 wild-type (wt) virus for comparison. Although there was a correlation between nectin-1 levels and viral yields with the wt virus (R=0.75, P =0.03), there was no correlation for Δγ134.5 viruses (G207, R7020 or C101) and a modest trend for the second-generation oHSV C134 (R=0.62, P=0.10). Nectin-1 overexpression in resistant MPNST cell lines did not improve Δγ134.5 oHSV output. While multistep replication assays showed that nectin-1 overexpression improved Δγ134.5 oHSV cell-to-cell spread, it did not confer a sensitive phenotype to resistant cells. Finally, oHSV yields were not improved with increased nectin-1 in vivo. We conclude that nectin-1 expression is not the primary obstacle of productive infection for Δγ134.5 oHSVs in MPNST cell lines. In contrast, viruses that are competent in their ability to counter the antiviral response may derive benefit with higher nectin-1 expression.
Abstract Background Administration of tranexamic acid topically and intravenously has demonstrated effectiveness in decreasing blood loss and transfusion rates. Methods We randomized 131 patients ...undergoing primary total knee arthroplasty to receive either intracapsular (69) or intravenous tranexamic acid (62). Postoperative blood loss was calculated using the formula derived by Nadler et al. The number of units transfused was recorded, as well as length of hospital stay. Results We found no statistically significant difference on calculated blood loss (postoperative day POD 1: 624 ± 326 vs 644 ± 292; P = .71, POD 2: 806 ± 368 vs 835 ± 319; P = .64, and POD 3: 1076 ± 419 vs 978 ± 343; P = .55). There was no difference in number of blood transfusions, length of stay, or complications. Conclusion Intracapsular tranexamic acid is not inferior to intravenous tranexamic acid in decreasing blood loss and blood transfusion rate in primary total knee arthroplasty.
BACKGROUND
Point-of-care ultrasound (POCUS) has extensive clinical utility in internal medicine, but formal and uniform curricula in internal medicine are lacking.
OBJECTIVE
To determine the ...effectiveness of a longitudinal, flipped-classroom, academic half-day curriculum on internal medicine resident confidence, utilization, and changes in clinical management.
METHODS
We implemented an asynchronous, flipped-classroom, academic half-day curriculum from November 2020 to November 2021 and conducted an evaluation with a prospective, before-after cohort study. Curriculum included 4 rotating sessions comprised of 20 to 30 min of image interpretation followed by 1.5 to 2 h of image acquisition. Confidence was rated via Likert scale. Utilization was reported via indicating never, 1 to 2, 3 to 4, 5 to 6, or >6 times per month (recorded as 1-5, respectively). Image interpretation was assessed via a 6-question, multiple-choice video assessment.
RESULTS
Nineteen of 99 potential residents (19%) completed a pre- and post-curriculum evaluation. Residents attended a median of 4 sessions. Confidence improved from 2.47 to 3.53 (P = .002). Utilization did not improve overall (2.11-2.42, P = .22), but utilization of left ventricular function assessment (1.53-2.00, P = .046) and pulmonary assessment (1.53-2.00, P = .039) increased. The percentage of residents that had ever changed their clinical management by POCUS increased from 47% to 84% after implementation of the curriculum. Cardiac, pulmonary/pleural, volume assessment, and abdominal free fluid exams were reported as the most clinically useful.
CONCLUSION
Implementation of a longitudinal, academic half-day curriculum for POCUS resulted in improved confidence, increased POCUS utilization for the cardiac and pulmonary examination, and changes in clinical management based on POCUS.
•The hydroformylation of 1-dodecene was studied in a TMS system.•A suitable solvent and catalyst systems were determined.•Semi-batch experiments including perturbations were performed.•The main ...branches of the reaction network could be identified.•For this reaction network a corresponding catalytic cycle is suggested.
The hydroformylation of 1-dodecene was studied using Rh(acac)(CO)2 and a ligand as a catalyst in a thermomorphic multicomponent solvent (TMS) system consisting of N,N-dimethylformamide, decane and the olefin. High n-aldehyde/iso-aldehydes ratios were obtained with the bidentate phosphite ligand biphephos. In systematic preliminary investigations suitable catalyst/ligand-ratios and catalyst concentrations were determined. In order to derive a simplified reaction network, semi-batch experiments were performed measuring responses to perturbations of pressure and feed composition. From the results obtained the main branches of the reaction network could be identified comprising also isomerizations and hydrogenations of n- and iso-dodecenes. For this simplified reaction network a catalytic cycle is suggested providing the basis for the formulation of a more detailed mechanistic kinetic model.
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