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.

Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD.

Codon usage bias is a universal feature of all genomes, but its in vivo biological functions in animal systems are not clear. To investigate the in vivo role of codon usage in animals, we took advantage of the sensitivity and robustness of the Drosophila circadian system. By codon-optimizing parts of Drosophila period (dper), a core clock gene that encodes a critical component of the circadian oscillator, we showed that dper codon usage is important for circadian clock function. Codon optimization of dper resulted in conformational changes of the dPER protein, altered dPER phosphorylation profile and stability, and impaired dPER function in the circadian negative feedback loop, which manifests into changes in molecular rhythmicity and abnormal circadian behavioral output. This study provides an in vivo example that demonstrates the role of codon usage in determining protein structure and function in an animal system. These results suggest a universal mechanism in eukaryotes that uses a codon usage "code" within genetic codons to regulate cotranslational protein folding.

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.

hDNA2 nuclease/helicase promotes centromeric DNA replication and genome stability.

DNA2 is a nuclease/helicase that is involved in Okazaki fragment maturation, replication fork processing, and end resection of DNA double-strand breaks. Similar such helicase activity for resolving secondary structures and structure-specific nuclease activity are needed during DNA replication to process the chromosome-specific higher order repeat units present in the centromeres of human chromosomes. Here, we show that DNA2 binds preferentially to centromeric DNA The nuclease and helicase activities of DNA2 are both essential for resolution of DNA structural obstacles to facilitate DNA replication fork movement. Loss of DNA2-mediated clean-up mechanisms impairs centromeric DNA replication and CENP-A deposition, leading to activation of the ATR DNA damage checkpoints at centromeric DNA regions and late-S/G2 cell cycle arrest. Cells that escape arrest show impaired metaphase plate formation and abnormal chromosomal segregation. Furthermore, the DNA2 inhibitor C5 mimics DNA2 knockout and synergistically kills cancer cells when combined with an ATR inhibitor. These findings provide mechanistic insights into how DNA2 supports replication of centromeric DNA and give further insights into new therapeutic strategies.

Comprehensive mapping of abiotic stress inputs into the soybean circadian clock.

The plant circadian clock evolved to increase fitness by synchronizing physiological processes with environmental oscillations. Crop fitness was artificially selected through domestication and breeding, and the circadian clock was identified by both natural and artificial selections as a key to improved fitness. Despite progress in Arabidopsis, our understanding of the crop circadian clock is still limited, impeding its rational improvement for enhanced fitness. To unveil the interactions between the crop circadian clock and various environmental cues, we comprehensively mapped abiotic stress inputs to the soybean circadian clock using a 2-module discovery pipeline. Using the "molecular timetable" method, we computationally surveyed publicly available abiotic stress-related soybean transcriptomes to identify stresses that have strong impacts on the global rhythm. These findings were then experimentally confirmed using a multiplexed RNA sequencing technology. Specific clock components modulated by each stress were further identified. This comprehensive mapping uncovered inputs to the plant circadian clock such as alkaline stress. Moreover, short-term iron deficiency targeted different clock components in soybean and Arabidopsis and thus had opposite effects on the clocks of these 2 species. Comparing soybean varieties with different iron uptake efficiencies suggests that phase modulation might be a mechanism to alleviate iron deficiency symptoms in soybean. These unique responses in soybean demonstrate the need to directly study crop circadian clocks. Our discovery pipeline may serve as a broadly applicable tool to facilitate these explorations.

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.

Redox rhythm reinforces the circadian clock to gate immune response.

Recent studies have shown that in addition to the transcriptional circadian clock, many organisms, including Arabidopsis, have a circadian redox rhythm driven by the organism's metabolic activities. It has been hypothesized that the redox rhythm is linked to the circadian clock, but the mechanism and the biological significance of this link have only begun to be investigated. Here we report that the master immune regulator NPR1 (non-expressor of pathogenesis-related gene 1) of Arabidopsis is a sensor of the plant's redox state and regulates transcription of core circadian clock genes even in the absence of pathogen challenge. Surprisingly, acute perturbation in the redox status triggered by the immune signal salicylic acid does not compromise the circadian clock but rather leads to its reinforcement. Mathematical modelling and subsequent experiments show that NPR1 reinforces the circadian clock without changing the period by regulating both the morning and the evening clock genes. This balanced network architecture helps plants gate their immune responses towards the morning and minimize costs on growth at night. Our study demonstrates how a sensitive redox rhythm interacts with a robust circadian clock to ensure proper responsiveness to environmental stimuli without compromising fitness of the organism.

TRAF6 mediates human DNA2 polyubiquitination and nuclear localization to maintain nuclear genome integrity.

The multifunctional human DNA2 (hDNA2) nuclease/helicase is required to process DNA ends for homology-directed recombination repair (HDR) and to counteract replication stress. To participate in these processes, hDNA2 must localize to the nucleus and be recruited to the replication or repair sites. However, because hDNA2 lacks the nuclear localization signal that is found in its yeast homolog, it is unclear how its migration into the nucleus is regulated during replication or in response to DNA damage. Here, we report that the E3 ligase TRAF6 binds to and mediates the K63-linked polyubiquitination of hDNA2, increasing the stability of hDNA2 and promoting its nuclear localization. Inhibiting TRAF6-mediated polyubiquitination abolishes the nuclear localization of hDNA2, consequently impairing DNA end resection and HDR. Thus, the current study reveals a mechanism for the regulation of hDNA2 localization and establishes that TRAF6-mediated hDNA2 ubiquitination activates DNA repair pathways to maintain nuclear genome integrity.

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.

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.

Sequential posttranslational modifications program FEN1 degradation during cell-cycle progression.

We propose that cell-cycle-dependent timing of FEN1 nuclease activity is essential for cell-cycle progression and the maintenance of genome stability. After DNA replication is complete at the exit point of the S phase, removal of excess FEN1 may be crucial. Here, we report a mechanism that controls the programmed degradation of FEN1 via a sequential cascade of posttranslational modifications. We found that FEN1 phosphorylation stimulated its SUMOylation, which in turn stimulated its ubiquitination and ultimately led to its degradation via the proteasome pathway. Mutations or inhibitors that blocked the modification at any step in this pathway suppressed FEN1 degradation. Critically, the presence of SUMOylation- or ubiquitination-defective, nondegradable FEN1 mutant protein caused accumulation of Cyclin B, delays in the G1 and G2/M phases, and polyploidy. These findings may represent a newly identified regulatory mechanism used by cells to ensure precise cell-cycle progression and to prevent transformation.

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.

Heterologous expression and purification of a marine alginate lyase in Escherichia coli.

Alginate lyase digestion is an efficient way to degrade alginate into oligosaccharides, which are useful in various areas including chemistry, pharmacy and food industry. Here we determined the sequence of Vibrio sp. QY102 sourced alginate lyase, and set up its heterologous expression in E. coli. We improved its secretion efficiency by replacing the original secretive sequence by E. coli specific signal peptide ompA. We successfully purified the full-length protein in shake flask culture, however, degradation happened during fed batch cultivation. By domain and disorder examination, we found that the protein was completely functional by expressing the C terminal fragment alone. For the final strain we constructed (HMS-ompA-CF), the extracellular enzyme activity reached 375 U/ml in shake flask and 1789 U/ml in fed batch cultivation (5 L bioreactor). And the final protein yield reached 0.58 g/L in fed batch cultivation. We determined that the optimal pH and temperature for the shortened alginate lyase were 7.0 and 39 °C, respectively.

Non-optimal codon usage affects expression, structure and function of clock protein FRQ.

Codon-usage bias has been observed in almost all genomes and is thought to result from selection for efficient and accurate translation of highly expressed genes. Codon usage is also implicated in the control of transcription, splicing and RNA structure. Many genes exhibit little codon-usage bias, which is thought to reflect a lack of selection for messenger RNA translation. Alternatively, however, non-optimal codon usage may be of biological importance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. Here we show that, unlike most genes in Neurospora, frq exhibits non-optimal codon usage across its entire open reading frame. Optimization of frq codon usage abolishes both overt and molecular circadian rhythms. Codon optimization not only increases FRQ levels but, unexpectedly, also results in conformational changes in FRQ protein, altered FRQ phosphorylation profile and stability, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function. Our study provides an example of how non-optimal codon usage functions to regulate protein expression and to achieve optimal protein structure and function.

Codon usage is an important determinant of gene expression levels largely through its effects on transcription.

Codon usage biases are found in all eukaryotic and prokaryotic genomes, and preferred codons are more frequently used in highly expressed genes. The effects of codon usage on gene expression were previously thought to be mainly mediated by its impacts on translation. Here, we show that codon usage strongly correlates with both protein and mRNA levels genome-wide in the filamentous fungus Neurospora Gene codon optimization also results in strong up-regulation of protein and RNA levels, suggesting that codon usage is an important determinant of gene expression. Surprisingly, we found that the impact of codon usage on gene expression results mainly from effects on transcription and is largely independent of mRNA translation and mRNA stability. Furthermore, we show that histone H3 lysine 9 trimethylation is one of the mechanisms responsible for the codon usage-mediated transcriptional silencing of some genes with nonoptimal codons. Together, these results uncovered an unexpected important role of codon usage in ORF sequences in determining transcription levels and suggest that codon biases are an adaptation of protein coding sequences to both transcription and translation machineries. Therefore, synonymous codons not only specify protein sequences and translation dynamics, but also help determine gene expression levels.

B-1a cells protect mice from sepsis-induced acute lung injury.

BACKGROUND: Sepsis morbidity and mortality are aggravated by acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Mouse B-1a cells are a phenotypically and functionally unique sub-population of B cells, providing immediate protection against infection by releasing natural antibodies and immunomodulatory molecules. We hypothesize that B-1a cells ameliorate sepsis-induced ALI. METHODS: Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). PBS or B-1a cells were adoptively transferred into the septic mice intraperitoneally. After 20 h of CLP, lungs were harvested and assessed by PCR and ELISA for pro-inflammatory cytokines (IL-6, IL-1ß) and chemokine (MIP-2) expression, by histology for injury, by TUNEL and cleaved caspase-3 for apoptosis, and by myeloperoxidase (MPO) assay for neutrophil infiltration. RESULTS: We found that septic mice adoptively transferred with B-1a cells significantly decreased the mRNA and protein levels of IL-6, IL-1ß and MIP-2 in the lungs compared to PBS-treated mice. Mice treated with B-1a cells showed dramatic improvement in lung injury compared to PBS-treated mice after sepsis. We found apoptosis in the lungs was significantly inhibited in B-1a cell injected mice compared to PBS-treated mice after sepsis. B-1a cell treatment significantly down-regulated MPO levels in the lungs compared to PBS-treated mice in sepsis. The protective outcomes of B-1a cells in ALI was further confirmed by using B-1a cell deficient CD19-/- mice, which showed significant increase in the lung injury scores following sepsis as compared to WT mice. CONCLUSIONS: Our results demonstrate a novel therapeutic potential of B-1a cells to treat sepsis-induced ALI.

Serine/threonine kinase PpkA coordinates the interplay between T6SS2 activation and quorum sensing in the marine pathogen Vibrio alginolyticus.

Type VI secretion systems (T6SS) are multiprotein secretion machines that can mediate killing of bacterial cells and thereby modify the composition of bacterial communities. The mechanisms that control the production of and secretion of these killing machines are incompletely understood, although quorum sensing (QS) and the PpkA kinase modulate T6SS activity in some organisms. Here we investigated control the T6S in the marine organism Vibrio alginolyticus EPGS, which encodes two T6SS systems (T6SS1 and T6SS2). We found that the organism principally relies on T6SS2 for interbacterial competition. We further carried out a phosphoproteomic screen to identify substrates of the T6SS2-linked PpkA2 kinase. Substrates of PpkA2 encoded within the T6SS2 cluster as well proteins that are apparently not linked to T6SS-related processes were identified. Similar to other organisms, PpkA2 autophosphorylation was critical for T6SS2 function. Notably, phosphorylation of a polypeptide encoded outside of the T6SS2 cluster, VtsR, was critical for T6SS2 expression and function because it augments the expression of luxR, a key regulator of QS that also promotes T6SS2 gene expression. Thus, PpkA2 controls a phosphorylation cascade that mediates a positive regulatory loop entwining T6SS and QS, thereby coordinating these pathways to enhance the competitive fitness of V. alginolyticus.

Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris.

Pichia pastoris expression system has been widely used in recombinant protein production. So far the majority of heterologous proteins are expressed by methanol inducible promoter PAOX1 and constitutive promoter PGAP . The use of other promoters is rather limited. Here we selected 16 potentially efficient and regulatory promoter candidates based on the RNA-seq and RNA folding free energy ΔG data. GFP and recombinant amylase were inserted after these promoters to reveal their strength and efficiency under different carbon sources and culture scales. Two novel promoters were successfully identified and could possibly be applied in recombinant protein expression: the methanol-inducible promoter P0547 and the constitutive promoter P0472 .

Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development.

Flap endonuclease 1 (FEN1) phosphorylation is proposed to regulate the action of FEN1 in DNA repair as well as Okazaki fragment maturation. However, the biologic significance of FEN1 phosphorylation in response to DNA damage remains unknown. Here, we report an in vivo role for FEN1 phosphorylation, using a mouse line carrying S187A FEN1, which abolishes FEN1 phosphorylation. Although S187A mouse embryonic fibroblast cells showed normal proliferation under low oxygen levels (2%), the mutant cells accumulated oxidative DNA damage, activated DNA damage checkpoints, and showed G1-phase arrest at atmospheric oxygen levels (21%). This suggests an essential role for FEN1 phosphorylation in repairing oxygen-induced DNA damage and maintaining proper cell cycle progression. Consistently, the mutant cardiomyocytes showed G1-phase arrest due to activation of the p53-mediated DNA damage response at the neonatal stage, which reduces the proliferation potential of the cardiomyocytes and impairs heart development. Nearly 50% of newborns with the S187A mutant died in the first week due to failure to undergo the peroxisome proliferator-activated receptor signaling-dependent switch from glycolysis to fatty acid oxidation. The adult mutant mice developed dilated hearts and showed significantly shorter life spans. Altogether, our results reveal an important role of FEN1 phosphorylation to counteract oxygen-induced stress in the heart during the fetal-to-neonatal transition.-Zhou, L., Dai, H., Wu, J., Zhou, M., Yuan, H., Du, J., Yang, L., Wu, X., Xu, H., Hua, Y., Xu, J., Zheng, L., Shen, B. Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development.