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.

[Exploration of an integrated competency development model for undergraduates training by participating the international Genetic Engineering Machine Competition].

Starting from participating the high-level professional competition, our school has built a talent training system with the spirit of "biomaker" and an innovative practical ability training system. Such system takes the interest of student as the starting point, and relies on the strong scientific research and teaching infrastructure. The programme gives full play to students' initiatives and enhances the scientific research literacy and comprehensive ability of undergraduates majoring in biotechnology. It is an effective exploration of the traditional university education model and meets the urgent demand for innovative talents training in the era of rapid development of life sciences.

Codon usage bias regulates gene expression and protein conformation in yeast expression system P. pastoris.

BACKGROUND: Protein synthesis is one of the extremely important anabolic pathways in the yeast expression system Pichia pastoris. Codon optimization is a commonly adopted strategy for improved protein expression, although unexpected failures did appear sometimes waiting for further exploration. Recently codon bias has been studied to regulate protein folding and activity in many other organisms. RESULTS: Here the codon bias profile of P. pastoris genome was examined first and a direct correlation between codon translation efficiency and usage frequency was identified. By manipulating the codon choices of both endogenous and heterologous signal peptides, secretion abilities of N-terminal signal peptides were shown to be tolerant towards codon changes. Then two gene candidates with different levels of structural disorder were studied, and full-length codon optimization was found to affect their expression profiles differentially. Finally, more evidences were provided to support possible protein conformation change brought by codon optimization in structurally disordered proteins. CONCLUSION: Our results suggest that codon bias regulates gene expression by modulating several factors including transcription and translation efficiency, protein folding and activity. Because of sequences difference, the extent of affection may be gene specific. For some genes, special codon optimization strategy should be adopted to ensure appropriate expression and conformation.

Damage-Associated Molecular Patterns As Double-Edged Swords in Sepsis.

Significance: Sepsis is defined as a life-threatening organ dysfunction caused by dysregulated host response to infection. This leads to an uncontrolled inflammatory response at the onset of infection, followed by immunosuppression. The development of a specific treatment modality for sepsis is still challenging, reflecting our inadequate understanding of its pathophysiology. Understanding the mechanism and transition of the early hyperinflammation to late stage of immunosuppression in sepsis is critical for developing sepsis therapeutics. Recent Advances: Damage-associated molecular patterns (DAMPs) are intracellular molecules and released upon tissue injury and cell death in sepsis. DAMPs are recognized by pattern recognition receptors to initiate inflammatory cascades. DAMPs not only elicit an inflammatory response but also they subsequently induce immunosuppression, both are equally important for exacerbating sepsis. Recent advances on a new DAMP, extracellular cold-inducible RNA-binding protein for fueling inflammation and immunosuppression in sepsis, have added a new avenue into the dual functions of DAMPs in sepsis. Critical Issues: The molecular modification of DAMPs and their binding to pattern recognition receptors transit dynamically by the cellular environment in pathophysiologic conditions. Correlation between the dynamic changes of the impacts of DAMPs and the clinical outcomes in sepsis still lacks adequate understanding. Here, we focus on the impacts of DAMPs that cause inflammation as well as induce immunosuppression in sepsis. We further discuss the therapeutic potential by targeting DAMPs to attenuate inflammation and immunosuppression for mitigating sepsis. Future Directions: Uncovering pathways of the transition from inflammation to immunosuppression of DAMPs is a potential therapeutic avenue for mitigating sepsis.

Correction of immunosuppression in aged septic rats by human ghrelin and growth hormone through the vagus nerve-dependent inhibition of TGF-ß production.

BACKGROUND: Co-administration of human ghrelin and growth hormone (GH) reverse immunosuppression in septic aged animals, but the mechanism remains elusive. Here, we hypothesize that ghrelin and GH co-treatment restores the immune response in aged septic rats by inhibiting the production of transforming growth factor-ß (TGF-ß), an immunoregulatory cytokine, through the vagus nerve. METHODS: Male aged Fischer rats (22-23-month-old) were made septic by cecal ligation and puncture (CLP) with or without dissecting the vagus nerve (vagotomy). Human ghrelin and GH or vehicle (PBS) were administrated subcutaneously at 5 h post CLP. After 20 h of CLP, serum and spleens were harvested. RESULTS: Serum TGF-ß levels were increased in septic aged rats, while ghrelin and GH treatment significantly reduced its levels. Expression of TGF-ß in the spleen was upregulated after sepsis, while ghrelin and GH treatment significantly inhibited its expression. TNF-α and IL-6 levels were significantly reduced after ex vivo LPS stimulation of splenocytes from rats that underwent CLP compared to sham rats; while these levels were significantly higher in splenocytes from ghrelin and GH-treated CLP rats compared to vehicle-treated CLP rats. Ghrelin and GH treatment reduced program death receptor-1 (PD-1) expression, increased human leukocyte antigen-DR (HLA-DR) expression, attenuated lymphopenia, and cleaved caspase-3 levels in the spleen of septic aged rats. Vagotomy diminished the beneficial effects of ghrelin and GH treatment in septic rats. In vitro, the addition of ghrelin, GH, or ghrelin and GH together had no effect on restoring immune response in splenocytes from CLP rats following LPS stimulation, indicating the requirement of the vagus nerve for ghrelin and GH's effect. CONCLUSIONS: Ghrelin and GH attenuate immunosuppression in aged septic rats through the vagus nerve-dependent inhibition of TGF-ß production.

Codon usage bias affects α-amylase mRNA level by altering RNA stability and cytosine methylation patterns in Escherichia coli.

Codon usage bias exists in almost every organism and is reported to regulate protein translation efficiency and folding. Besides translation, the preliminary role of codon usage bias on gene transcription has also been revealed in some eukaryotes such as Neurospora crassa. In this study, we took as an example the α-amylase-coding gene (amyA) and examined the role of codon usage bias in regulating gene expression in the typical prokaryote Escherichia coli. We confirmed the higher translation efficiency on codon-optimized amyA RNAs and found that the RNA level itself was also affected by codon optimization. The decreased RNA level was caused at least in part by altered mRNA stability at the post-transcriptional level. Codon optimization also altered the number of cytosine methylation sites. Examination on dcm knockouts suggested that cytosine methylation may be a minor mechanism adopted by codon bias to regulate gene RNA levels. More studies are required to verify the global effect of codon usage and to reveal its detailed mechanism on transcription.

Identification and study of InV as an inverse autotransporter family representative in Edwardsiella piscicida.

Invasins and intimins, members of virulence-related adhesin family which is involved in attachment and adherence to epithelial cells during infection, are found in various pathogens. These pathogens can attach to enterocytes and lead to the formation of a pedestal-like structure. Invasins and intimins belong to type Ve secretion systems, and the N-terminal ß-barrel domain acts as a translocation pore to secrete the C-terminal passenger domain. However, the relationship between invasins/intimins and type III secretion system (T3SS) has been poorly studied. Based on the transposon insertion mutant library of Edwardsiella piscicida, we got a transposon insertion mutant with significant T3SS defect and identified the mutated gene ETAE_0323 (named inV later). This gene encoded a protein with 2359 amino acid residues and was predicted to be an invasin. To study the relationship between InV and T3SS, strains with N-terminus or C-terminus deleted InV fragments were made. However, none of them was able to copy the phenotype of the transposon insertion mutant previously identified. The localization of InV in ΔT3SS strain was not significantly different from WT, suggesting that the T3SS defect in the transposon insertion mutant was likely to be caused by polar effect. Nevertheless, depletion of inV still showed dramatic internalization and virulence defect in HeLa cell and zebrafish model, respectively, suggesting InV as a virulence related protein.