Feasible and economic DIY procedures of a hand-held fluorescence detector set-FluorDetector to assist laboratory course development.

Fluorescence-related experimental techniques play an important role in biochemistry, molecular biology, and cell biology. However, fluorescence-related experiments are rarely included in the laboratory courses of most Chinese universities. This is mainly due to the conflict between large class size (50-60 students in one room) and funding/space limitations to purchase and accommodate enough fluorescence detection equipment. Here, we proposed feasible and economical Do It Yourself (DIY) procedures of a hand-held fluorescence detector set-FluorDetector to support the development of laboratory courses. Tested on several samples, clear fluorescence signals could be directly observed by FluorDetector and photographed with a smartphone. In addition, FluorDetector was able to turn a conventional stereomicroscope into a fluorescence stereomicroscope, detecting fluorescence signals with clean background. FluorDetector is easy to make with a 3D printer, with an extremely low cost ($200 each) when compared with a commercial fluorescence microscope or fluorescence stereomicroscope, and almost as sensitive as a microplate reader in measuring fluorescence. Therefore, FluorDetector is a possible strategy to solve the problem and help to integrate fluorescence-related experimental modules in laboratory courses.

Radiation-induced eCIRP impairs macrophage bacterial phagocytosis.

Macrophages are essential immune cells for host defense against bacterial pathogens after radiation injury. However, the role of macrophage phagocytosis in infection following radiation injury remains poorly examined. Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern that dysregulates host immune system responses such as phagocytosis. We hypothesized that radiation-induced eCIRP release impairs macrophage phagocytosis of bacteria. Adult healthy mice were exposed to 6.5-Gy total body irradiation (TBI). Primary peritoneal macrophages isolated from adult healthy mice were exposed to 6.5-Gy radiation. eCIRP-neutralizing monoclonal antibody (mAb) was added to the cell culture prior to irradiation. Bacterial phagocytosis by peritoneal macrophages was assessed using pHrodo Green-labeled E. coli 7 days after irradiation ex vivo and in vitro. Bacterial phagocytosis was also assessed after treatment with recombinant murine CIRP (rmCIRP). Rac1 and ARP2 protein expression in cell lysates and eCIRP levels in the peritoneal lavage were assessed by Western blotting. Bacterial phagocytosis by peritoneal macrophages was significantly decreased after irradiation compared to controls ex vivo and in vitro. Rac1 and ARP2 expression in the peritoneal macrophages were downregulated after TBI. TBI significantly increased eCIRP levels in the peritoneal cavity. rmCIRP significantly decreased bacterial phagocytosis in a dose-dependent manner. eCIRP mAb restored bacterial phagocytosis by peritoneal macrophages after irradiation. Ionizing radiation exposure impairs bacterial phagocytosis by macrophages after irradiation. Neutralization of eCIRP restores the phagocytic ability of macrophages after irradiation. Our findings elucidate a novel mechanism of immune dysfunction and provide a potential new therapeutic approach for limiting infection after radiation injury.

BMAL2 promotes eCIRP-induced macrophage endotoxin tolerance.

Background: The disruption of the circadian clock is associated with inflammatory and immunological disorders. BMAL2, a critical circadian protein, forms a dimer with CLOCK, activating transcription. Extracellular cold-inducible RNA-binding protein (eCIRP), released during sepsis, can induce macrophage endotoxin tolerance. We hypothesized that eCIRP induces BMAL2 expression and promotes macrophage endotoxin tolerance through triggering receptor expressed on myeloid cells-1 (TREM-1). Methods: C57BL/6 wild-type (WT) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Serum levels of eCIRP 20 h post-CLP were assessed by ELISA. Peritoneal macrophages (PerM) were treated with recombinant mouse (rm) CIRP (eCIRP) at various doses for 24 h. The cells were then stimulated with LPS for 5 h. The levels of TNF-α and IL-6 in the culture supernatants were assessed by ELISA. PerM were treated with eCIRP for 24 h, and the expression of PD-L1, IL-10, STAT3, TREM-1 and circadian genes such as BMAL2, CRY1, and PER2 was assessed by qPCR. Effect of TREM-1 on eCIRP-induced PerM endotoxin tolerance and PD-L1, IL-10, and STAT3 expression was determined by qPCR using PerM from TREM-1-/- mice. Circadian gene expression profiles in eCIRP-treated macrophages were determined by PCR array and confirmed by qPCR. Induction of BMAL2 activation in bone marrow-derived macrophages was performed by transfection of BMAL2 CRISPR activation plasmid. The interaction of BMAL2 in the PD-L1 promoter was determined by computational modeling and confirmed by the BIAcore assay. Results: Serum levels of eCIRP were increased in septic mice compared to sham mice. Macrophages pre-treated with eCIRP exhibited reduced TNFα and IL-6 release upon LPS challenge, indicating macrophage endotoxin tolerance. Additionally, eCIRP increased the expression of PD-L1, IL-10, and STAT3, markers of immune tolerance. Interestingly, TREM-1 deficiency reversed eCIRP-induced macrophage endotoxin tolerance and significantly decreased PD-L1, IL-10, and STAT3 expression. PCR array screening of circadian clock genes in peritoneal macrophages treated with eCIRP revealed the elevated expression of BMAL2, CRY1, and PER2. In eCIRP-treated macrophages, TREM-1 deficiency prevented the upregulation of these circadian genes. In macrophages, inducible BMAL2 expression correlated with increased PD-L1 expression. In septic human patients, blood monocytes exhibited increased expression of BMAL2 and PD-L1 in comparison to healthy subjects. Computational modeling and BIAcore assay identified a putative binding region of BMAL2 in the PD-L1 promoter, suggesting BMAL2 positively regulates PD-L1 expression in macrophages. Conclusion: eCIRP upregulates BMAL2 expression via TREM-1, leading to macrophage endotoxin tolerance in sepsis. Targeting eCIRP to maintain circadian rhythm may correct endotoxin tolerance and enhance host resistance to bacterial infection.

The effect of different flushing and locking techniques on catheter occlusion rates in central venous catheters: protocol for a multicentre, randomized controlled, parallel-group, open-label, superiority clinical trial.

BACKGROUND: Maintaining venous access is of great clinical importance. Running a slow continuous infusion to keep the vein open (KVO) is often used in peripheral intravenous catheters (PIVCs). Previous studies have compared the effects of intermittent flushing and continuous infusion via peripherally inserted central catheters (PICCs). In this study, we applied KVO to central venous catheters (CVCs) and compared the occlusion rate of this technique with that of the intermittent flushing technique. METHOD: This is a randomized controlled trial of 14 hospitals in China. A total of 250 patients will be recruited in this study, and they will be randomized at a 1:1 ratio. After study inclusion, patients who will undergo CVC insertion will receive intermittent flushing with prefilled saline syringes (control group) or KVO infusion with elastic pumps (test group). All the catheters will be checked for patency by scoping Catheter Injection and Aspiration (CINAS) Classification on Days 3 and 7. The primary outcome is the rate of catheter occlusion in 7 days. Patients will be followed up until 9 days after CVC insertion, catheter occlusion, or catheter removal. The secondary outcomes are the rate of catheter occlusion in 3 days, nurse satisfaction, cost-effectiveness, adverse event rate, catheter-related bloodstream infection rate, catheter-related thrombosis rate, extravasation rate, phlebitis rate, and catheter migration. DISCUSSION: We expect that the trial will generate findings that can provide an evidence-based basis for the improvement and optimization of clinical catheter flushing techniques. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR2200064007. Registered on 23 September 2022. https://www.chictr.org.cn/showproj.html?proj=177311 .

Structure-specific nucleases in genome dynamics and strategies for targeting cancers.

Nucleases are a super family of enzymes that hydrolyze phosphodiester bonds present in genomes. They widely vary in substrates, causing differentiation in cleavage patterns and having a diversified role in maintaining genetic material. Through cellular evolution of prokaryotic to eukaryotic, nucleases become structure-specific in recognizing its own or foreign genomic DNA/RNA configurations as its substrates, including flaps, bubbles, and Holliday junctions. These special structural configurations are commonly found as intermediates in processes like DNA replication, repair, and recombination. The structure-specific nature and diversified functions make them essential to maintaining genome integrity and evolution in normal and cancer cells. In this article, we review their roles in various pathways, including Okazaki fragment maturation during DNA replication, end resection in homology-directed recombination repair of DNA double-strand breaks, DNA excision repair and apoptosis DNA fragmentation in response to exogeneous DNA damage, and HIV life cycle. As the nucleases serve as key points for the DNA dynamics, cellular apoptosis, and cancer cell survival pathways, we discuss the efforts in the field in developing the therapeutic regimens, taking advantage of recently available knowledge of their diversified structures and functions.

Bridging the biochemistry lecture and laboratory courses: Construction and application of the "Innovative Experimental Design" module.

Both lecture and laboratory courses of biochemistry are important professional courses for undergraduates with biology related majors. Course optimization and update is crucial but challenging, especially for the laboratory course. Although taught separately, here we showed a strategy to bridge the two courses and promote the improvement of both. In addition to knowledge teaching, we implanted the "Innovative Experimental Design" module in the lecture course in which students were required to design and present their own experimental ideas. After evaluation by the faculty group, the best idea was supported for further experimental test. Here we described the preliminary experiments and optimization procedures about the idea of microbial fuel cells. This experiment is ready to be included into the laboratory course program in spring 2023.

Alter codon bias of the P. pastoris genome to overcome a bottleneck in codon optimization strategy development and improve protein expression.

Apart from its role in translation, codon bias is also an important mechanism to regulate mRNA levels. The traditional frequency-based codon optimization strategy is rather efficient in organisms such as N. crassa, but much less in yeast P. pastoris which is a popular host for heterologous protein expression. This is because that unlike N. crassa, the preferred codons of P. pastoris are actually AU-rich and hence codon optimization for extremely low GC content comes with issues of pre-mature transcriptional termination or low RNA stability in spite of translational advantages. To overcome this bottleneck, we focused on three reporter genes in P. pastoris first and confirmed the great advantage of GC-prone codon optimization on mRNA levels. Then we altered the codon bias profile of P. pastoris by introducing additional rare tRNA gene copies. Prior to that we constructed IPTG-regulated tRNA species to enable chassis cells to switch between different codon bias status. As demonstrated again with reporter genes, protein yield of luc and 0788 was successfully increased by 4-5 folds in chassis cells. In summary, here we provide an alternative codon optimization strategy for genes with unsatisfactory performance under traditional codon frequency-based optimization.

Brain activation during standing balance control in dual-task paradigm and its correlation among older adults with mild cognitive impairment: a fNIRS study.

BACKGROUND: This study aimed to compare the balance ability and functional brain oxygenation in the prefrontal cortex (PFC) among older adults with mild cognitive impairment (MCI) under single and dual tasks, and also investigate their relationship. Neural regulatory mechanisms of the brain in the MCI were shed light on in balance control conditions. METHODS: 21 older adults with MCI (female = 12, age: 71.19 ± 3.36 years) were recruited as the experimental group and 19 healthy older adults (female = 9, age: 70.16 ± 4.54 years) as the control group. Participants completed balance control of single task and dual task respectively. Functional near-infrared spectroscopy (fNIRS) and force measuring platform are used to collect hemodynamic signals of the PFC and center of pressure (COP) data during the balance task, respectively. RESULTS: The significant Group*Task interaction effect was found in maximal displacement of the COP in the medial-lateral (ML) direction (D-ml), 95% confidence ellipse area (95%AREA), root mean square (RMS), the RMS in the ML direction (RMS-ml), the RMS in the anterior-posterior (AP) direction (RMS-ap), sway path (SP), the sway path in the ML direction (SP-ml), and the sway path in the AP direction (SP-ap). The significant group effect was detected for five regions of interest (ROI), namely the left Brodmann area (BA) 45 (L45), the right BA45 (R45), the right BA10 (R10), the left BA46 (L46), and the right BA11 (R11). Under single task, maximal displacement of the COP in the AP direction (D-ap), RMS, and RMS-ap were significantly negatively correlated with R45, L45, and R11 respectively. Under dual task, both RMS and 95%AREA were correlated positively with L45, and both L10 and R10 were positively correlated with RMS-ap. CONCLUSION: The MCI demonstrated worse balance control ability as compared to healthy older adults. The greater activation of PFC under dual tasks in MCI may be considered a compensatory strategy for maintaining the standing balance. The brain activation was negatively correlated with balance ability under single task, and positively under dual task. TRIAL REGISTRATION: ChiCTR2100044221 , 12/03/2021.

Effects of the low-speed continuous infusion catheter technique on double-lumen central venous catheters: A randomized controlled trial.

BACKGROUND: Central venous catheters are widely used in clinical practice, and the incidence of central venous catheter occlusion is between 25 % and 38 %. The turbulence caused by the pulsatile flushing technique is harmful to the vascular endothelium and may lead to phlebitis. The low-speed continuous infusion catheter technique is a new type of continuous infusion that ensures that the catheter is always in a keep-vein-open state by continuous low-speed flushing; hence, avoiding the problem of catheter occlusion. OBJECTIVE: To investigate the effectiveness of the low-speed continuous infusion catheter technique and the routine care of double-lumen central venous catheters. DESIGN: This was a prospective, randomized, controlled, open-label trial. SETTING: Patients were recruited from 14 medical institutions in China between February and June 2023. PARTICIPANTS: In total, 251 patients were recruited, with 125 in the intervention group and 126 in the control group. METHODS: Patients who used double-lumen central venous catheters for infusion treatment were selected, and those who met the sampling criteria were randomly divided into intervention and control groups using the random envelope method. The intervention group used the low-speed continuous infusion catheter technique to maintain catheter patency, whereas the control group used routine care with a trial period of 7 days. The primary outcome was the occlusion rate. The secondary outcomes included nursing satisfaction and complication rates of the two groups. RESULTS: After 7 days, the rate of catheter occlusion was 28.0 % (35/125, 95 % confidence interval (CI):0.203, 0.367) in the intervention group and 53.97 % (68/126, 95 % CI: 0.449-0.629) in the control group, with a statistically significant difference (χ2 = 17.488, p < 0.001); at 3 days of intervention, the rate of catheter blockage was 8.0 % (10/125, 95 % CI: 0.039-0.142) in the intervention group and 23.8 % (30/126, 0.167-0.322) in the control group, with a statistically significant difference (χ2 = 11.707, p < 0.001). Nurse satisfaction was significantly higher in the intervention group (115/125, 92.0 %, 95 % CI: 0.858-0.961) than in the control group (104/126, 82.54 %, 95 % CI: 0.748-0.887) (χ2 = 5.049, p = 0.025). There were no statistically significant complication rates in either group (p = 0.622). CONCLUSION: The low-speed continuous infusion catheter technique helps maintain catheter patency, improves nurse satisfaction, and provides a high level of safety. REGISTRATION: Chinese Clinical Trial Registry (ChiCTR2200064007, www.chictr.org.cn). The first recruitment was conducted in February. https://www.chictr.org.cn/showproj.html?proj=177311.

The static balance ability on soft and hard support surfaces in older adults with mild cognitive impairment.

OBJECTIVE: This study aimed to assess the static balance ability of the older adults with mild cognitive impairment (MCI) while standing on soft and hard support surfaces. METHODS: Forty older adults participated in this study (21 in the MCI group and 19 in the control group). Participants were required to perform balance tests under four conditions of standing: standing on a hard support surface with eyes open, standing on a soft support surface with eyes open, standing on a hard support surface with eyes closed, and standing on a soft support surface with eyes closed. Each test was measured in three trials and each trial lasted 30 seconds. Participants were asked to take off their shoes and place their feet in a parallel position with a 20-centimeter distance for bipedal support. The trajectories of the center of pressure (COP) were measured using a Kistler force platform with a frequency of 1000 Hz to assess balance while standing in both groups, with larger COP trajectories indicating poorer static balance in older adults. RESULTS: With eyes open, the displacement of COP in the anterior-posterior direction(D-ap) (hard support surface: P = 0.003) and the 95% confidence ellipse area(95%AREA-CE) (soft support surface: P = 0.001, hard support surface: P < 0.001) of the COP in the MCI group standing on hard and soft support surfaces were significantly larger than the control group. The 95%AREA-CE (P < 0.001) of the COP in the MCI group on the soft support surface was significantly larger than on the hard support surface. With eyes closed, the root mean square distance(RDIST), root mean square distance-ML(RDISTml), and 95%AREA-CE of the COP were no significant between-group differences when standing on hard support surfaces. However, the RDIST (P = 0.014), RDISTml (P = 0.014), and 95%AREA-CE (P = 0.001) of the COP in the MCI group on the soft support surfaces were significantly larger than the control group. The 95%AREA-CE (P < 0.001), RDIST (P < 0.001), and RDISTml (P < 0.001) of the COP in the MCI group on the soft support surface were significantly larger than the hard support surface. CONCLUSION: With eyes open, the older adults with MCI showed poorer static balance ability compared to the older adults with normal cognition on soft and hard support surfaces. With eyes closed, the older adults with MCI showed poorer static balance on soft support surfaces, but no differences on hard support surfaces compared with the older adults with normal cognition. With eyes open and closed, the older adults with MCI showed poorer static balance on soft support surfaces as compared to hard support surfaces.

Phenolic acid-induced phase separation and translation inhibition mediate plant interspecific competition.

Phenolic acids (PAs) secreted by donor plants suppress the growth of their susceptible plant neighbours. However, how structurally diverse ensembles of PAs are perceived by plants to mediate interspecific competition remains a mystery. Here we show that a plant stress granule (SG) marker, RNA-BINDING PROTEIN 47B (RBP47B), is a sensor of PAs in Arabidopsis. PAs, including salicylic acid, 4-hydroxybenzoic acid, protocatechuic acid and so on, directly bind RBP47B, promote its phase separation and trigger SG formation accompanied by global translation inhibition. Salicylic acid-induced global translation inhibition depends on RBP47 family members. RBP47s regulate the proteome rather than the absolute quantity of SG. The rbp47 quadruple mutant shows a reduced sensitivity to the inhibitory effect of the PA mixture as well as to that of PA-rich rice when tested in a co-culturing ecosystem. In this Article, we identified the long sought-after PA sensor as RBP47B and illustrated that PA-induced SG-mediated translational inhibition was one of the PA perception mechanisms.

De novo biosynthesis of 2'-fucosyllactose in engineered Pichia pastoris.

PURPOSE: Pichia pastoris is well known for its ability to produce short and low-immunogenic humanized glycosyl chains onto recombinant glycoproteins, it was thus speculated to be applicable to synthesize oligosaccharides. In this study, generally recognized as safe (GRAS) microorganism Pichia pastoris GS115 was tested for its potential to be used as a new synthetic chassis to produce the most abundant human milk oligosaccharide 2'-fucosyllactose (2'-FL). METHODS: To enable the de novo synthesis of 2'-FL, lactose transporter lac12, two enzymes of gmd, gmer, and fucosyltransferases futC were integrated into the genome of P. pastoris, under the control of constitutive PGAP promoter. RESULTS: The resulting recombinant yeasts yielded up to 0.276 g/L through culture optimization in a 5 L bioreactor. CONCLUSION: To our knowledge, this is the first report of 2'-FL production in engineered Pichia pastoris. This work is a good starting point to produce 2'-FL using Pichia pastoris as a viable chassis.

Extracellular CIRP dysregulates macrophage bacterial phagocytosis in sepsis.

In sepsis, macrophage bacterial phagocytosis is impaired, but the mechanism is not well elucidated. Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern that causes inflammation. However, whether eCIRP regulates macrophage bacterial phagocytosis is unknown. Here, we reported that the bacterial loads in the blood and peritoneal fluid were decreased in CIRP-/- mice and anti-eCIRP Ab-treated mice after sepsis. Increased eCIRP levels were correlated with decreased bacterial clearance in septic mice. CIRP-/- mice showed a marked increase in survival after sepsis. Recombinant murine CIRP (rmCIRP) significantly decreased the phagocytosis of bacteria by macrophages in vivo and in vitro. rmCIRP decreased the protein expression of actin-binding proteins, ARP2, and p-cofilin in macrophages. rmCIRP significantly downregulated the protein expression of ßPIX, a Rac1 activator. We further demonstrated that STAT3 and ßPIX formed a complex following rmCIRP treatment, preventing ßPIX from activating Rac1. We also found that eCIRP-induced STAT3 phosphorylation was required for eCIRP's action in actin remodeling. Inhibition of STAT3 phosphorylation prevented the formation of the STAT3-ßPIX complex, restoring ARP2 and p-cofilin expression and membrane protrusion in rmCIRP-treated macrophages. The STAT3 inhibitor stattic rescued the macrophage phagocytic dysfunction induced by rmCIRP. Thus, we identified a novel mechanism of macrophage phagocytic dysfunction caused by eCIRP, which provides a new therapeutic target to ameliorate sepsis.

Strong negative correlation between codon usage bias and protein structural disorder impedes protein expression after codon optimization.

As a common phenomenon existing in almost all genomes, codon usage bias has been studied for a long time. Codon optimization is a frequently used strategy to accelerate protein synthesis rate. Besides regulating protein translation speed, codon usage bias has also be reported to affect co-translation folding and transcription. P. pastoris is a well-developed expression system, whose efficiency is tightly correlated with commercial value. However, few studies focus on the role of codon usage bias in affecting protein expression in P. pastoris. Besides, many genes in P. pastoris genome show significant negative correlation between codon usage bias and protein structural disorder tendency. It's not known whether this feature is important for their expression. In order to answer these questions, we picked 4P. pastoris gene candidates with strong negative correlation between codon usage bias and protein structural disorder. We then performed full-length codon optimization which completely eliminated the correlation. Protein and RNA assays were then used to compare protein and mRNA levels before and after codon optimization. As a result, codon optimization failed to elevate their protein expression levels, and even resulted in a decrease. As represented by the trypsin sensitivity assays, codon optimization also altered the protein structure of 0616 and 0788. Besides protein, codon optimization also affected mRNA levels. Shown by in vitro and in vivo RNA degradation assays, the mRNA stability of 0616, 0788 and 0135 were also altered by codon optimization. For each gene, the detailed effect may be related with its specific sequence and protein structure. Our results suggest that codon usage bias is an important factor to regulate gene expression level, as well as mRNA and protein stabilities in P. pastoris. "Extreme" codon optimization in genes with strong negative correlation between codon usage bias and protein structural disorder tendency may not be favored. Compromised strategies should be tried if expression is not successful. Besides, codon optimization may affect protein structural conformation more severely in structural disordered proteins.

Error-prone, stress-induced 3' flap-based Okazaki fragment maturation supports cell survival.

How cells with DNA replication defects acquire mutations that allow them to escape apoptosis under environmental stress is a long-standing question. Here, we report that an error-prone Okazaki fragment maturation (OFM) pathway is activated at restrictive temperatures in rad27Δ yeast cells. Restrictive temperature stress activated Dun1, facilitating transformation of unprocessed 5' flaps into 3' flaps, which were removed by 3' nucleases, including DNA polymerase δ (Polδ). However, at certain regions, 3' flaps formed secondary structures that facilitated 3' end extension rather than degradation, producing alternative duplications with short spacer sequences, such as pol3 internal tandem duplications. Consequently, little 5' flap was formed, suppressing rad27Δ-induced lethality at restrictive temperatures. We define a stress-induced, error-prone OFM pathway that generates mutations that counteract replication defects and drive cellular evolution and survival.

Online bioinformatics teaching practice: Comparison of popular docking programs using SARS-CoV-2 spike RBD-ACE2 complex as a benchmark.

In this information era, there is an urgent need for tighter integration of bioinformatics and experimental biology. The enormous amount of data generated by biological experiments calls for extensive computational analysis. Many bioinformatics textbooks at present mainly focus on theories, which hinders the vigorous development of scientific research. As a result, most students are simply familiar with the bioinformatics theories but lack the opportunity to put them into practice. Here, we present our bioinformatics docking project conducted during the self-isolation period of the COVID-19 pandemic. Five students used the RBD-ACE2 complex as a benchmark to conduct a systematic comparison of several open-source online molecular docking programs. The virus surface spike protein mediates the entry of the SARS-CoV-2 virus into human cells by binding to its receptor, angiotensin-converting enzyme 2 (ACE2), through its receptor-binding domain (RBD). Through docking and comparing predicted structures to the crystal structure, students gained the opportunity to practice different bioinformatics tools independently and conduct research collaboratively. It opens a window for students to reach out to the state-of-the-art bioinformatics techniques and to keep up with the research trends. The online workshop has also proven to be an innovative method for bioinformatics teaching. We hope our work can inspire other educators to develop strategies to expose undergraduate students to modern bioinformatics and turn every temporary difficulty into a possible learning opportunity.

Assessing Global Circadian Rhythm Through Single-Time-Point Transcriptomic Analysis.

Plant circadian clock has emerged as a central hub integrating various endogenous signals and exogenous stimuli to coordinate diverse plant physiological processes. The intimate relationship between crop circadian clock and key agronomic traits has been increasingly appreciated. However, due to the lack of fundamental genetic resources, more complex genome structures and the high cost of large-scale time-course circadian expression profiling, our understanding of crop circadian clock is still very limited. To study plant circadian clock, conventional methods rely on time-course experiments, which can be expensive and time-consuming. Different from these conventional approaches, the molecular timetable method can estimate the global rhythm using single-time-point transcriptome datasets, which has shown great promises in accelerating studies of crop circadian clock. Here we describe the application of the molecular timetable method in soybean and provide key technical caveats as well as related R Markdown scripts.