Gut Microbial Metabolites Induce Changes in Circadian Oscillation of Clock Gene Expression in the Mouse Embryonic Fibroblasts

Circadian rhythm is an endogenous oscillation of about 24-h period in many physiological processes and behaviors. This daily oscillation is maintained by the molecular clock machinery with transcriptional-translational feedback loops mediated by clock genes including Period2 (Per2) and Bmal1. Recently, it was revealed that gut microbiome exerts a significant impact on the circadian physiology and behavior of its host; however, the mechanism through which it regulates the molecular clock has remained elusive. 3-(4-hydroxyphenyl)propionic acid (4-OH-PPA) and 3-phenylpropionic acid (PPA) are major metabolites exclusively produced by Clostridium sporogenes and may function as unique chemical messengers communicating with its host. In the present study, we examined if two C. sporogenes-derived metabolites can modulate the oscillation of mammalian molecular clock. Interestingly, 4-OH-PPA and PPA increased the amplitude of both PER2 and Bmal1 oscillation in a dosedependent manner following their administration immediately after the nadir or the peak of their rhythm. The phase of PER2 oscillation responded differently depending on the mode of administration of the metabolites. In addition, using an organotypic slice culture ex vivo, treatment with 4-OH-PPA increased the amplitude and lengthened the period of PER2 oscillation in the suprachiasmatic nucleus and other tissues. In summary, two C. sporogenes-derived metabolites are involved in the regulation of circadian oscillation of Per2 and Bmal1 clock genes in the host's peripheral and central clock machineries.1

Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

The mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) comprising the Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1) genes. The robustness of the TTFL is attributed to genetic redundancy among some essential clock genes, deterring genetic studies on molecular clocks using genome editing targeting single genes. To manipulate multiple clock genes in a streamlined and efficient manner, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for essential clock genes including Pers, Crys, or Bmal1. First, we tested several single guide RNAs (sgRNAs) targeting individual clock genes in silico and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and packaged into AAVs. CSAC efficiency was evident through protein downregulation in vitro and ablated molecular oscillation ex vivo. We also measured the efficiency of CSAC in vivo by assessing circadian rhythms after injecting CSAC into the suprachiasmatic nuclei of Cas9-expressing knock-in mice. Circadian locomotor activity and body temperature rhythms were severely disrupted in these mice, indicating that our CSAC is a simple yet powerful tool for investigating the molecular clock in vivo. © 2021, The Author(s).1

Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

Abstract Background MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level and are key modulators in neurodegenerative diseases. Overexpressed miRNAs play an important role in ALS; however, the pathogenic mechanisms of deregulated miRNAs are still unclear. Methods We aimed to assess the dysfunction of RNAs or miRNAs in fALS (SOD1 mutations). We compared the RNA-seq of subcellular fractions in NSC-34 WT (hSOD1) and MT (hSOD1 (G93A)) cells to find altered RNAs or miRNAs. We identified that Hif1α and Mef2c were upregulated, and Mctp1 and Rarb were downregulated in the cytoplasm of NSC-34 MT cells. Results SOD1 mutations decreased the level of miR-18b-5p. Induced Hif1α which is the target for miR-18b increased Mef2c expression as a transcription factor. Mef2c upregulated miR-206 as a transcription factor. Inhibition of Mctp1 and Rarb which are targets of miR-206 induces intracellular Ca2+ levels and reduces cell differentiation, respectively. We confirmed that miR-18b-5p pathway was also observed in G93A Tg, fALS (G86S) patient, and iPSC-derived motor neurons from fALS (G17S) patient. Conclusions Our data indicate that SOD1 mutation decreases miR-18b-5p, which sequentially regulates Hif1α, Mef2c, miR-206, Mctp1 and Rarb in fALS-linked SOD1 mutation. These results provide new insights into the downregulation of miR-18b-5p dependent pathogenic mechanisms of ALS

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

REMI: Defect prediction for efficient API testing

Quality assurance for common APIs is important since the the reliability of APIs affects the quality of other systems using the APIs. Testing is a common practice to ensure the quality of APIs, but it is a challenging and laborious task especially for industrial projects. Due to a large number of APIs with tight time constraints and limited resources, it is hard to write enough test cases for all APIs. To address these challenges, we present a novel technique, Remi that predicts high risk APIs in terms of producing potential bugs. Remi allows developers to write more test cases for the high risk APIs. We evaluate Remi on a real-world industrial project, Tizen-wearable, and apply Remi to the API development process at Samsung Electronics. Our evaluation results show that Remi predicts the bug-prone APIs with reasonable accuracy (0.681 f-measure on average). The results also show that applying Remi to the Tizen-wearable development process increases the number of bugs detected, and reduces the resources required for executing test cases

SNMP-based enterprise IP network topology discovery

Precise network topology information is required to perform management activities such as fault detection, root cause analysis, performance monitoring, and load balancing in enterprise networks. To accomplish these management tasks, both network discovery and connectivity information are essential. This paper examines various problems with the existing topology discovery mechanisms and proposes an SNMP-based approach to discover physical as well as logical topology. We present algorithms for identifying network device types and discovering connectivity among them. The connectivity of end host and management information base (MIB)-enabled devices with switches and routers is discussed and evaluated. We also present an algorithm for discovering logical topology, such as VLAN and subnet connectivity. Finally, we present a combination of graph and tree layouts, to visualize connectivity information. Copyright (C) 2010 John Wiley & Sons, Ltd.X1165Nsciescopu

Use of fluorine-doped tin oxide instead of indium tin oxide in highly efficient air-fabricated inverted polymer solar cells

The stability and efficiency of organic solar cells (OSCs) were improved using thermally stable fluorine-doped tin oxide (FTO) as the bottom electrode and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) and TiO(2) as the buffer layers. The TiO(2) layer between FTO and the P3HT:PCBM active layer improved the interface characteristics for a better charge transfer. The PEDOT:PSS layer retarded the oxygen diffusion to the active layer. A maximum power conversion efficiency of 4.3% was obtained for the inverted structure of FTO/TiO(2)/P3HT:PCBM/PEDOT:PSS/Ag with a stable performance, and the cell retained over 65% of its initial efficiency after 500 h. Additionally, the OSCs were fabricated using all-solution based vacuum-free processes with screen printing for the Ag electrode and the results were comparable to the device that used an evaporated Ag electrode

Seed-mediated synthesis of ultra-long copper nanowires and their application as transparent conducting electrodes

Owing to a recent push toward one-dimensional nanomaterials, in this study, we report a seed-mediated synthetic strategy for copper nanowires (Cu NWs) production involving thermal decomposition of metal-surfactant complexes in an organic medium. Ultra-long Cu NWs with a high aspect ratio and uniform diameter were obtained by separating nucleation and growth steps. The underlying mechanism for nanowire formation was investigated, in addition, properties of the obtained Cu NWs were also characterized using diverse analysis techniques. The performance of resulting Cu NWs as transparent electrodes was demonstrated for potential application. This article can provide information on both new synthetic pathway and potential use of Cu NWs

microRNA-25 as a novel modulator of circadian Period2 gene oscillation

Circadian rhythm: microRNAs keep the clock in order A newly identified microRNA plays a key role in fine-tuning the genetic interactions governing the circadian rhythms in mammals, according to researchers in South Korea. Numerous studies have suggested that the Period genes, which negatively regulate the CLOCK and BMAL1 genes to produce a 24-hour feedback loop, may be further modified by microRNAs after they are transcribed. Kyungjin Kim at Daegu Gyeongbuk Institute of Science and Technology, South Korea, and co-workers confirmed that a novel microRNA, miR-25-3p, reduces the expression of a Period gene, Per2, in mice. When miR-25-3p is over-expressed, it dampens and shortens the oscillations of Per2 levels. Interestingly, the researchers showed that natural miR-25-3p expression levels varied across different parts of the brain, supporting the theory that different tissues of the body maintain their own unique circadian cycles