Enhancer Control of Transcriptional Bursting.

Transcription is episodic, consisting of a series of discontinuous bursts. Using live-imaging methods and quantitative analysis, we examine transcriptional bursting in living Drosophila embryos. Different developmental enhancers positioned downstream of synthetic reporter genes produce transcriptional bursts with similar amplitudes and duration but generate very different bursting frequencies, with strong enhancers producing more bursts than weak enhancers. Insertion of an insulator reduces the number of bursts and the corresponding level of gene expression, suggesting that enhancer regulation of bursting frequency is a key parameter of gene control in development. We also show that linked reporter genes exhibit coordinated bursting profiles when regulated by a shared enhancer, challenging conventional models of enhancer-promoter looping.

Functional coordination between transcription factor clustering and gene activity.

The prevailing view of metazoan gene regulation is that transcription is facilitated through the formation of static activator complexes at distal regulatory regions. Here, we employed quantitative single-cell live-imaging and computational analysis to provide evidence that the dynamic assembly and disassembly process of transcription factor (TF) clusters at enhancers is a major source of transcriptional bursting in developing Drosophila embryos. We further show that the regulatory connectivity between TF clustering and burst induction is highly regulated through intrinsically disordered regions (IDRs). Addition of a poly-glutamine tract to the maternal morphogen Bicoid demonstrated that extended IDR length leads to ectopic TF clustering and burst induction from its endogenous target genes, resulting in defects in body segmentation during embryogenesis. Moreover, we successfully visualized the presence of "shared" TF clusters during the co-activation of two distant genes, which provides a concrete molecular explanation for the newly proposed "topological operon" hypothesis in metazoan gene regulation.

Visualizing the Role of Boundary Elements in Enhancer-Promoter Communication.

Formation of self-associating loop domains is a fundamental organizational feature of metazoan genomes. Here, we employed quantitative live-imaging methods to visualize impacts of higher-order chromosome topology on enhancer-promoter communication in developing Drosophila embryos. Evidence is provided that distal enhancers effectively produce transcriptional bursting from target promoters over distances when they are flanked with boundary elements. Importantly, neither inversion nor deletion of a boundary element abrogates this "enhancer-assisting activity," suggesting that they can facilitate intra-domain enhancer-promoter interaction and production of transcriptional bursting independently of topologically associating domain (TAD) formation. In contrast, domain-skipping activity of distal enhancers was lost after disruption of topological domains. This observation raises a possibility that intra-domain and inter-domain enhancer-promoter interactions are differentially regulated by chromosome topology.

Visualization of Transvection in Living Drosophila Embryos.

How remote enhancers interact with appropriate target genes persists as a central mystery in gene regulation. Here, we exploit the properties of transvection to explore enhancer-promoter communication between homologous chromosomes in living Drosophila embryos. We successfully visualized the activation of an MS2-tagged reporter gene by a defined developmental enhancer located in trans on the other homolog. This trans-homolog activation depends on insulator DNAs, which increase the stability-but not the frequency-of homolog pairing. A pair of heterotypic insulators failed to mediate transvection, raising the possibility that insulator specificity underlies the formation of chromosomal loop domains. Moreover, we found that a shared enhancer co-activates separate PP7 and MS2 reporter genes incis and intrans. Transvecting alleles weakly compete with one another, raising the possibility that they share a common pool of the transcription machinery. We propose that transvecting alleles form a trans-homolog "hub," which serves as a scaffold for the accumulation of transcription complexes.

Dynamic modulation of enhancer-promoter and promoter-promoter connectivity in gene regulation.

Enhancers are short segments of regulatory DNA that control when and in which cell-type genes should be turned on in response to a variety of extrinsic and intrinsic signals. At the molecular level, enhancers serve as a genomic scaffold that recruits sequence-specific transcription factors and co-activators to facilitate transcription from linked promoters. However, it remains largely unclear how enhancers communicate with appropriate target promoters in the context of higher-order genome topology. In this review, we discuss recent progress in our understanding of the functional interplay between enhancers, genome topology, and the molecular properties of transcription machineries in gene regulation. We suggest that the activities of transcription hubs are highly regulated through the dynamic rearrangement of enhancer-promoter and promoter-promoter connectivity during animal development.

Dynamic modulation of enhancer responsiveness by core promoter elements in living Drosophila embryos.

Regulatory interactions between enhancers and core promoters are fundamental for the temporal and spatial specificity of gene expression in development. The central role of core promoters is to initiate productive transcription in response to enhancer's activation cues. However, it has not been systematically assessed how individual core promoter elements affect the induction of transcriptional bursting by enhancers. Here, we provide evidence that each core promoter element differentially modulates functional parameters of transcriptional bursting in developing Drosophila embryos. Quantitative live imaging analysis revealed that the timing and the continuity of burst induction are common regulatory steps on which core promoter elements impact. We further show that the upstream TATA also affects the burst amplitude. On the other hand, Inr, MTE and DPE mainly contribute to the regulation of the burst frequency. Genome editing analysis of the pair-rule gene fushi tarazu revealed that the endogenous TATA and DPE are both essential for its correct expression and function during the establishment of body segments in early embryos. We suggest that core promoter elements serve as a key regulatory module in converting enhancer activity into transcription dynamics during animal development.

Mod(mdg4) variants repress telomeric retrotransposon HeT-A by blocking subtelomeric enhancers.

Telomeres in Drosophila are composed of sequential non-LTR retrotransposons HeT-A, TART and TAHRE. Although they are repressed by the PIWI-piRNA pathway or heterochromatin in the germline, the regulation of these retrotransposons in somatic cells is poorly understood. In this study, we demonstrated that specific splice variants of Mod(mdg4) repress HeT-A by blocking subtelomeric enhancers in ovarian somatic cells. Among the variants, we found that the Mod(mdg4)-N variant represses HeT-A expression the most efficiently. Subtelomeric sequences bound by Mod(mdg4)-N block enhancer activity within subtelomeric TAS-R repeats. This enhancer-blocking activity is increased by the tandem association of Mod(mdg4)-N to repetitive subtelomeric sequences. In addition, the association of Mod(mdg4)-N couples with the recruitment of RNA polymerase II to the subtelomeres, which reinforces its enhancer-blocking function. Our findings provide novel insights into how telomeric retrotransposons are regulated by the specific variants of insulator proteins associated with subtelomeric sequences.

Relationship between the Young's Modulus of the Achilles Tendon and Ankle Dorsiflexion Angle at Maximum Squat Depth in Healthy Young Males.

Background and Objective: Achilles tendon (AT) stiffness can reduce ankle dorsiflexion. However, whether AT stiffness affects the ankle dorsiflexion angle at a maximum squat depth remains unclear. Therefore, we aimed to investigate the relationship between the Young's modulus of the AT and ankle dorsiflexion angle at the maximum squat depth in healthy young males using shear-wave elastography (SWE). Materials and Methods: This cross-sectional study included 31 healthy young males. AT stiffness was measured using the Young's modulus through SWE. The ankle dorsiflexion angle at the maximum squat depth was measured as the angle between the vertical line to the floor and the line connecting the fibula head and the lateral malleolus using a goniometer. Results: Multiple regression analysis identified the Young's modulus of the AT at 10° of ankle dorsiflexion (standardized partial regression coefficient [ß] = -0.461; p = 0.007) and the ankle dorsiflexion angle in the flexed knee (ß = 0.340; p = 0.041) as independent variables for the ankle dorsiflexion angle at maximum squat depth. Conclusions: The Young's modulus of the AT may affect the ankle dorsiflexion angle at the maximum squat depth in healthy young males. Therefore, improving the Young's modulus of the AT may help increase the ankle dorsiflexion angle at maximum squat depth.

MicroRNAs block assembly of eIF4F translation initiation complex in Drosophila.

miRNAs silence their complementary target mRNAs by translational repression as well as by poly(A) shortening and mRNA decay. In Drosophila, miRNAs are typically incorporated into Argonaute1 (Ago1) to form the effector complex called RNA-induced silencing complex (RISC). Ago1-RISC associates with a scaffold protein GW182, which recruits additional silencing factors. We have previously shown that miRNAs repress translation initiation by blocking formation of the 48S and 80S ribosomal complexes. However, it remains unclear how ribosome recruitment is impeded. Here, we examined the assembly of translation initiation factors on the target mRNA under repression. We show that Ago1-RISC induces dissociation of eIF4A, a DEAD-box RNA helicase, from the target mRNA without affecting 5' cap recognition by eIF4E in a manner independent of GW182. In contrast, direct tethering of GW182 promotes dissociation of both eIF4E and eIF4A. We propose that miRNAs act to block the assembly of the eIF4F complex during translation initiation.

Large distances separate coregulated genes in living Drosophila embryos.

Transcriptional enhancers are short segments of DNA that switch genes on and off in response to a variety of cellular signals. Many enhancers map quite far from their target genes, on the order of tens or even hundreds of kilobases. There is extensive evidence that remote enhancers are brought into proximity with their target promoters via long-range looping interactions. However, the exact physical distances of these enhancer-promoter interactions remain uncertain. Here, we employ high-resolution imaging of living Drosophila embryos to visualize the distances separating linked genes that are coregulated by a shared enhancer. Cotransvection assays (linked genes on separate homologs) suggest a surprisingly large distance during transcriptional activity: at least 100-200 nm. Similar distances were observed when a shared enhancer was placed into close proximity with linked reporter genes in cis. These observations are consistent with the occurrence of "transcription hubs," whereby clusters (or condensates) of multiple RNA polymerase II complexes and associated cofactors are periodically recruited to active promoters. The dynamics of this process might be responsible for rapid fluctuations in the distances separating the transcription of coregulated reporter genes during transvection. We propose that enhancer-promoter communication depends on a combination of classical looping and linking models.

Continuous Renal Replacement Therapy for a Patient with Severe COVID-19.

The outbreak of coronavirus disease 2019 (COVID-19) is a global health threat. It is a respiratory disease, and acute kidney injury (AKI) is rare; however, if a patient develops severe AKI, renal replacement therapy (RRT) should be considered. Recently, we had a critically ill COVID-19 patient who developed severe AKI and needed continuous RRT (CRRT). To avoid the potential risk of infection from CRRT effluents, we measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic material in the effluents by qRT-PCR, and low copy numbers of the viral genome were detected. Due to unstable hemodynamic status in critically ill patients, CRRT should be the first choice for severe AKI in COVID-19 patients. We suggest prevention of clinical infection and control during administration of RRT in the acute phase of COVID-19 patients with AKI or multiple organ failure.

Direct hemoperfusion using a polymyxin B-immobilized polystyrene column for COVID-19.

OBJECTIVE: To evaluate the efficacy and safety of direct hemoperfusion using a polymyxin B-immobilized polystyrene column (PMX-DHP) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive pneumonia patients. METHODS: This study was a case series conducted at a designated infectious diseases hospital. Twelve SARS-CoV-2-positive patients with partial pressure of arterial oxygen/percentage of inspired oxygen (P/F) ratio < 300 were treated with PMX-DHP on two consecutive days each during hospitalization. We defined day 1 as the first day when PMX-DHP was performed. PMX-DHP efficacy was assessed on days 7 and 14 after the first treatment based on eight categories. Subsequently, improvement in P/F ratio and urinary biomarkers on days 4 and 8, malfunctions, and ventilator and extracorporeal membrane oxygenation avoidance rates were also evaluated. RESULTS: On day 14 after the first treatment, disease severity decreased in 58.3% of the patients. P/F ratio increased while urine ß2-microglobulin decreased on days 4 and 8. Cytokine measurement pre- and post-PMX-DHP revealed decreased levels of interleukin-6 and the factors involved in vascular endothelial injury, including vascular endothelial growth factor. Twenty-two PMX-DHPs were performed, of which seven and five PMX-DHPs led to increased inlet pressure and membrane coagulation, respectively. When the membranes coagulated, the circuitry needed to be reconfigured. Circuit problems were usually observed when D-dimer and fibrin degradation product levels were high before PMX-DHP. CONCLUSIONS: Future studies are expected to determine the therapeutic effect of PMX-DHP on COVID-19. Because of the relatively high risk of circuit coagulation, coagulation capacity should be assessed beforehand.

Temporal dynamics of pair-rule stripes in living Drosophila embryos.

Traditional studies of gene regulation in the Drosophila embryo centered primarily on the analysis of fixed tissues. These methods provided considerable insight into the spatial control of gene activity, such as the borders of eve stripe 2, but yielded only limited information about temporal dynamics. The advent of quantitative live-imaging and genome-editing methods permits the detailed examination of the temporal control of endogenous gene activity. Here, we present evidence that the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) undergo dynamic shifts in gene expression. We observe sequential anterior shifting of the stripes along the anterior to posterior axis, with stripe 1 exhibiting movement before stripe 2 and the more posterior stripes. Conversely, posterior stripes shift over greater distances (two or three nuclei) than anterior stripes (one or two nuclei). Shifting of the ftz and eve stripes are slightly offset, with ftz moving faster than eve This observation is consistent with previous genetic studies, suggesting that eve is epistatic to ftz The precision of pair-rule temporal dynamics might depend on enhancer-enhancer interactions within the eve locus, since removal of the endogenous eve stripe 1 enhancer via CRISPR/Cas9 genome editing led to precocious and expanded expression of eve stripe 2. These observations raise the possibility of an added layer of complexity in the positional information encoded by the segmentation gene regulatory network.

Influence of the Amount of Change in Quadriceps Tendon Young's Modulus on Amount of Change in Walking Speed before and after Total Knee Arthroplasty.

Background and Objectives: Walking speed after total knee arthroplasty (TKA) is an important outcome. However, the effect of quadriceps tendon stiffness on walking speed remains unclear. This study aimed to clarify the influence of the amount of change in quadriceps tendon stiffness on the degree of change in walking speed before and after TKA. Materials and Methods: Sixteen patients who underwent TKA for knee osteoarthritis participated in this study (median age: 74.0 years (interquartile range: 64.5-75.8)). Shear-wave elastography was deployed to measure quadriceps tendon stiffness using Young's modulus. A motion analysis system was used to assess kinematic parameters and walking speed. Participants' knee circumference, range of motion, extension strength, one-leg standing time, walking pain level, and activity level were measured preoperatively and one year after TKA, and changes in values were calculated. We used path analysis to clarify the influence of the amount of change in the quadriceps tendon Young's modulus on the change in walking speed. Results: The quadriceps tendon Young's modulus negatively affected the knee flexion angle during swing (standardized partial regression coefficients (ß) = -0.513, p = 0.042). The knee flexion angle during swing positively affected step length (ß = 0.586, p = 0.017). Step length positively affected cadence (ß = 0.733, p = 0.001). Step length and cadence positively affected walking speed (ß = 0.563, p < 0.001, ß = 0.502, p < 0.001, respectively). Conclusions: The amount of change in the quadriceps tendon Young's modulus may affect the degree of change in walking speed after TKA through the amount of change in the knee flexion angle during swing, step length, and cadence. Clinically, reducing quadriceps tendon stiffness can be addressed in rehabilitation programs to increase walking speed after TKA.

A Simulation Case Study of Knee Joint Compressive Stress during the Stance Phase in Severe Knee Osteoarthritis Using Finite Element Method.

Background and Objectives: Medial knee osteoarthritis is known to increase the mechanical load on the medial compartment of the knee joint during walking; however, it is not visually understood how much the mechanical load increases nor where in the medial compartment of the knee joint that load is focused. Therefore, we conducted a simulation study to determine the location and amount of the mechanical load in the medial compartment of the knee joint during the stance phase. Materials and Methods: Subject was a patient with right medial knee osteoarthritis. Computed tomography imaging and gait analysis were performed on subject. The CT image of the right knee was calculated using finite element analysis software. Since this software can set the flexion angle arbitrarily while maintaining the nonuniform material properties of the bone region, the model is constructed by matching the knee joint extension image obtained by CT to the loading response phase of gait analysis. The data of muscle exertion tension and vertical ground reaction force were inserted into the knee joint model created from the computed tomography-based finite element method, and the knee joint compressive stress was calculated. Results: With regard to compressive stress, the tibia showed high stress at 4.10 to 5.36 N/mm2. The femur showed high stress at 4.00 to 6.48 N/mm2. The joint compressive stress on the medial compartment of the knee joint was found to concentrate on the edge of the medial tibial condyle in the medial knee osteoarthritis subject. Conclusions: The measurement method of knee joint compressive stress by computed tomography-based finite element method can visually be a reliable method of measuring joint compressive stress in the medial knee osteoarthritis. This reflects the clinical findings because concentration of stress on the medial knee joint was observed at the medial osteophyte.

Detection of SARS-CoV-2 in Hemodialysis Effluent of Patient with COVID-19 Pneumonia, Japan.

We report detection of severe acute respiratory syndrome coronavirus 2 RNA in hemodialysis effluent from a patient in Japan with coronavirus disease and prolonged inflammation. Healthcare workers should observe strict standard and contact precautions and use appropriate personal protective equipment when handling hemodialysis circuitry from patients with diagnosed coronavirus disease.

MicroRNAs mediate gene silencing via multiple different pathways in drosophila.

MicroRNAs (miRNAs) guide RNA-induced silencing complex (RISC) that contains an Argonaute family protein to complementary target messenger RNAs (mRNAs). Via RISC, miRNAs silence the expression of target mRNAs by shortening the poly(A) tail-which leads to mRNA decay-and by repressing translation. It has been suggested that GW182, an Argonaute-associating protein, plays the central role in such microRNA actions. Here we show that, although GW182 is obligatory for poly(A) shortening, translational repression by microRNAs occurs even in the absence of GW182. Yet, GW182 is also capable of inducing translational repression independently. Both of these translational repression mechanisms block formation of 48S and 80S ribosomal complexes. Thus microRNAs utilize at least three distinct silencing pathways: GW182-mediated deadenylation and GW182-dependent and -independent repression of early translation initiation. Differential contribution from these multiple pathways may explain previous, apparently contradictory observations of how microRNAs inhibit protein synthesis.

Relationship between Quadriceps Tendon Young's Modulus and Maximum Knee Flexion Angle in the Swing Phase of Gait in Patients with Severe Knee Osteoarthritis.

Background and objectives: Decreased knee flexion in the swing phase of gait can be one of the causes of falls in severe knee osteoarthritis (OA). The quadriceps tendon is one of the causes of knee flexion limitation; however, it is unclear whether the stiffness of the quadriceps tendon affects the maximum knee flexion angle in the swing phase. The purpose of this study was to clarify the relationship between quadriceps tendon stiffness and maximum knee flexion angle in the swing phase of gait in patients with severe knee OA. Materials and Methods: This study was conducted from August 2018 to January 2020. Thirty patients with severe knee OA (median age 75.0 (interquartile range 67.5-76.0) years, Kellgren-Lawrence grade: 3 or 4) were evaluated. Quadriceps tendon stiffness was measured using Young's modulus by ShearWave Elastography. The measurements were taken with the patient in the supine position with the knee bent at 60° in a relaxed state. A three-dimensional motion analysis system measured the maximum knee flexion angle in the swing phase. The measurements were taken at a self-selected gait speed. The motion analysis system also measured gait speed, step length, and cadence. Multiple regression analysis by the stepwise method was performed with maximum knee flexion angle in the swing phase as the dependent variable. Results: Multiple regression analysis identified quadriceps tendon Young's modulus (standardized partial regression coefficients [ß] = -0.410; p = 0.013) and gait speed (ß = 0.433; p = 0.009) as independent variables for maximum knee flexion angle in the swing phase (adjusted coefficient of determination = 0.509; p < 0.001). Conclusions: Quadriceps tendon Young's modulus is a predictor of the maximum knee flexion angle. Clinically, decreasing Young's modulus may help to increase the maximum knee flexion angle in the swing phase in those with severe knee OA.

Influence of a Foot Insole for a Down Syndrome Patient with a Flat Foot: A Case Study.

Background and Objectives: Patients with Down syndrome have many orthopedic problems including flat foot. Insertion of an insole for a flat foot provides support to the medial longitudinal arch; thus, insole therapy is often used to treat a flat foot. However, the influence of an insole insertion on the knee joint kinematics for a patient with Down syndrome is unknown. This study aimed to elucidate the influence of an insole for a flat foot on the knee kinematics during gait for a patient with Down syndrome. Materials and Methods: The subject was a 22-year-old male with Down syndrome who had a flat foot. The knee joint angle during the gait was measured using a 3D motion capture system that consisted of eight infrared cameras. Results: The gait analysis demonstrated a reduction in the knee flexion angle during double knee action. The knee valgus and tibial internal rotation angles also decreased during the loading response phase while wearing shoes that contained the insole. Conclusions: As the angle of the knee joint decreased during the gait, it was considered that the stability of the knee joint improved by inserting the insole. In particular, there was a large difference in the tibial internal rotation angle when the insole was inserted. It is thus hypothesized that the insole contributes to the rotational stability of the knee joint. This study suggests that knee stability may improve and that gait becomes more stable when a Down syndrome patient with a flat foot wears an insole.

Development of a Knee Joint CT-FEM Model in Load Response of the Stance Phase During Walking Using Muscle Exertion, Motion Analysis, and Ground Reaction Force Data.

Background and objectives: There are no reports on articular stress distribution during walking based on any computed tomography (CT)-finite element model (CT-FEM). This study aimed to develop a calculation model of the load response (LR) phase, the most burdensome phase on the knee, during walking using the finite element method of quantitative CT images. Materials and Methods: The right knee of a 43-year-old man who had no history of osteoarthritis or surgeries of the knee was examined. An image of the knee was obtained using CT and the extension position image was converted to the flexion angle image in the LR phase. The bone was composed of heterogeneous materials. The ligaments were made of truss elements; therefore, they do not generate strain during expansion or contraction and do not affect the reaction force or pressure. The construction of the knee joint included material properties of the ligament, cartilage, and meniscus. The extensor and flexor muscles were calculated and set as the muscle exercise tension around the knee joint. Ground reaction force was vertically applied to suppress the rotation of the knee, and the thigh was restrained. Results: An FEM was constructed using a motion analyzer, floor reaction force meter, and muscle tractive force calculation. In a normal knee, the equivalent stress and joint contact reaction force in the LR phase were distributed over a wide area on the inner upper surface of the femur and tibia. Conclusions: We developed a calculation model in the LR phase of the knee joint during walking using a CT-FEM. Methods to evaluate the heteromorphic risk, mechanisms of transformation, prevention of knee osteoarthritis, and treatment may be developed using this model.