Analysis of differences in intestinal flora associated with different BMI status in colorectal cancer patients.

BACKGROUND: Overweight is known to be an important risk factor for colorectal cancer (CRC), and the differences in intestinal flora among CRC patients with different BMI status have not been clearly defined. The purpose of this study was to elucidate the differences in the abundance, composition and biological function of intestinal flora in CRC patients with different BMI status. METHOD: A total of 170 CRC patients were included and grouped according to the BMI data of CRC patients. BMI ≥ 24 kg/m2 was defined as overweight group, and BMI within the range of 18.5-23.9 kg/m2 was defined as normal weight group. Preoperative stool collection of patients in both groups was used for 16S rRNA sequencing. Total RNA was extracted from 17 CRC tumor tissue samples for transcriptome sequencing, and then CIBERSORT algorithm was used to convert the transcriptome data into the relative content matrix of 22 kinds of immune cells, and the correlation between different intestinal flora and immune cells and immune-related genes under different BMI states was analyzed. Finally, we identified BMI-related differential functional pathways and analyzed the correlation between these pathways and differential intestinal flora. RESULT: There was no significant difference in α diversity and ß diversity analysis between overweight group and normal weight group. Partial least square discriminant analysis (PLS-DA) could divide the flora into two different clusters according to BMI stratification. A total of 33 BMI-related differential flora were identified by linear discriminant effect size analysis (LEfSe), among which Actinomyces, Desulfovibrio and Bacteroides were significantly enriched in overweight group. ko00514: Other types of O-glycan biosynthesis are significantly enriched in overweight group. There was a significant positive correlation between Clostridium IV and Macrophages M2 and T cells regulatory (Tregs). There was a significant negative correlation with Dendritic cells activated and T cells CD4 memory activated. CONCLUSIONS: The richness and diversity of intestinal flora of CRC patients may be related to different BMI status, and the enrichment of Actinomyces, Desulphurvibrio and Bacteroides may be related to overweight status of CRC patients. The tumor microenvironment in which BMI-related differential flora resides has different immune landscapes, suggesting that some intestinal flora may affect the biological process of CRC by regulating immune cell infiltration and immune gene expression, but further experiments are needed to confirm this.

Psychometric properties and measurement invariance of the Chinese version of the cognitive emotion regulation questionnaire-short in patients with cancer.

OBJECTIVE: The study aimed to evaluate the psychometric properties and measurement invariance (MI) of the Chinese version of the cognitive emotion regulation questionnaire-short (CERQ-short) in cancer patients. METHODS: This cross-sectional study included 505 cancer patients from mainland China. In addition to sociodemographic and clinical characteristics, the CERQ-short and the distress thermometer were included in the study measures. RESULTS: Item analysis indicated a promising result. And the results of CFA indicated that the CERQ-short demonstrated satisfactory factorial validity in cancer patients. Cronbach's alpha coefficients were between 0.663 and 0.910, while McDonald's omega coefficients were between 0.664 and 0.910. The CERQ-short had sufficient convergent, discriminant and concurrent validity among cancer patients. Lastly, MI supported that the CERQ-short demonstrated strong measurement equivalence across gender, residence and age. CONCLUSIONS: This study shows that the Chinese version of the CERQ-short has convincing psychometric properties and MI, which supports its use in cancer patients.

Molecular dynamics simulations revealed topological frustration in the binding-wrapping process of eIF4G with eIF4E.

Interaction between the cap-binding protein eIF4E and the scaffolding protein eIF4G is essential for the cap-dependent translation initiation in eukaryotes. In the Saccharomyces cerevisiae eIF4G/eIF4E complex, the intrinsically disordered eIF4E-binding domain of eIF4G folds into a bracelet-like structure upon binding to eIF4E. Aiming to unveil the molecular mechanism underlying the binding-wrapping process of eIF4G with eIF4E, we performed extensive coarse-grained molecular dynamics simulations and transition path analysis in this work. The major transition pathway revealed from our simulations showed that docking of the eIF4E-binding motif of eIF4G to the folded core of eIF4E initiates the binding process and then the disordered eIF4G wraps around the N-terminal tail of eIF4E. Additionally, we identified a minor transition pathway which indicates the involvement of topological frustration in the binding process. By manipulating the interaction strength of the wrapping contacts and the latching contacts, we further dissected factors affecting the formation of topological frustration and the binding transition kinetics. Our findings provide new clues for experimental studies on the binding mechanism of eIF4G to eIF4E in the future and exemplify the involvement of topological frustration in the binding process of intrinsically disordered proteins.

Association of intimate partner violence with offspring growth in 32 low- and middle-income countries: a population-based cross-sectional study.

Intimate partner violence (IPV) against women presents a major public health challenge, especially in low-income and middle-income countries (LMICs), and its relationship with poor offspring growth is emerging but remains understudied. This study aimed to explore the impact of maternal exposure to IPV on offspring growth based on different approaches in LMICs. We conducted a population-based cross-sectional study using the most recent Demographic and Health Surveys from 32 LMICs; 81,652 mother-child dyads comprising women aged from 15 to 49 years with children aged 0 to 59 months were included. We applied logistic regression models to explore the independent and cumulative relationship between IPV, including emotional, physical, and sexual IPV, with poor child growth status, including stunting and wasting; 52.6% of mothers were under the age of 30 years with a 36% prevalence of any lifetime exposure to IPV. Maternal exposure to any IPV increased the odds of stunting, but only physical and sexual IPV were independently associated with an increased risk of stunting. Three different types of IPV exhibited a cumulative effect on stunting. Maternal exposure to physical IPV was significantly associated with an increased risk of wasting. Significant associations between maternal exposure to emotional IPV with offspring stunting and physical IPV with wasting were only observed in children aged 0 to 36 months. IPV against women remains high in LMICs and has adverse effects on offspring growth. Policy and program efforts are needed to prioritize the reduction of widespread physical and sexual IPV and to mitigate the impact of such violence.

Relationship Between Sleep Problems and Memory Impairment Among Nurses.

BACKGROUND: Sleep problems are common among nurses and can lead to various health problems. Further, the relationship between multiple sleep problems and memory impairment in clinical nurses remains unknown. Therefore, in this study, we aimed to explore the relationship between sleep problems and memory impairment among nurses. METHODS: Multistage cluster-stratified random sampling was performed from tertiary hospitals in Shandong, China. Overall, a total of 1833 nurses were included in the final analysis. The Chinese version of the Pittsburgh Sleep Quality Index and prospective and retrospective memory questionnaire were administered to the participants. RESULTS: The sleep quality of the nurses decreased during the normal epidemic prevention and control period compared with that before the epidemic in terms of sleep duration, sleep efficiency, and sleep disturbances. Nurses who reported cumulative or specific sleep problems (e.g., high sleep latency, sleep disturbances, and daytime dysfunction) and those in the "daily disturbances" and "poor sleep quality" groups had a higher risk of memory impairment than the others. CONCLUSION: Sleep problems might be important for memory impairment among nurses. These findings may help identify nurses at considerable risk of memory impairment in clinical practice.

Associations between parenting burnout and maternal adverse childhood experiences among postnatal women.

This study aims at exploring the relationships between adverse childhood experience (ACEs) and parental burnout. A total of 583 postnatal Chinese mothers were recruited in this cross-sectional study. Maternal ACEs were measured by Adverse Childhood Experience Questionnaire-Revised and parental burnout was measured by Parental Burnout Assessment. Multiple linear and binary logistic regression, and latent class analysis were used to explore the association between each type and cumulative ACEs and parental burnout. We found ACEs were associated with a higher risk of parental burnout. However, the association differed in the type of ACEs. The higher levels of physical abuse (B = 0.971 ~ 0.459, all p < 0.05) and emotional neglect (B = 1.010 ~ 1.407, all p < 0.05) in childhood were correlated with more serious parental burnout. The higher levels of self-threatened (B = 0.429 ~ 0.559, all p < 0.05) and self-deprived experience (B = 0.384 ~ 0.462, all p < 0.05), higher number of ACEs type (B = 2.909 ~ 3.918, all p < 0.05) were associated with more serious parental burnout. Results were consistent after combining four dimensions of parental burnout as a whole in LCA. This study indicated that maternal ACEs were associated with parental burnout. Women with self-deprived, self-threatened and more types of ACEs should be paid special attention.

Liquid - liquid phase separation of tau: Driving forces, regulation, and biological implications.

The past 15 years have witnessed an explosion in the studies of biomolecular condensates that are implicated in numerous biological processes and play vital roles in human health and diseases. Recent findings demonstrate that the microtubule-associated protein tau forms liquid condensates through liquid-liquid phase separation (LLPS) in in vitro experiments using purified recombinant proteins and cell-based experiments. Although in vivo studies are lacking, liquid condensates have emerged as an important assembly state of physiological and pathological tau and LLPS can regulate the function of microtubules, mediate stress granule formation, and accelerate tau amyloid aggregation. In this review, we summarize recent advances in tau LLPS, aiming to unveiling the delicate interactions driving tau LLPS. We further discuss the association of tau LLPS with physiology and disease in the context of the sophisticated regulation of tau LLPS. Deciphering the mechanisms underlying tau LLPS and the liquid-to-solid transition enables rational design of molecules that inhibit or delay the formation of tau solid species, thus providing novel targeted therapeutic strategies for tauopathies.

Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway.

4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2Δhsd4A1) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2Δhsd4A1 mutant, HGMS2Δhsd4A1/Δkstd1, enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2Δhsd4A1/kstd2 and HGMS2Δkstd1/Δhsd4A1/kshA1B1, efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

Exploring the relationship between postnatal depressive symptoms and parental burnout from the perspective of the population and individual level.

BACKGROUND: Parental burnout has become increasingly common, which can lead to a range of adverse outcomes. Postnatal mothers are vulnerable and mothers with high postpartum depression scores may be more prone to parental burnout. This study aims to investigate the association between postnatal depressive symptoms and parental burnout at both the population and individual levels. METHODS: This study comprised a cross-sectional study design and participants were recruited using convenience sampling. A total of 560 postnatal mothers answered a questionnaire on their general information, postnatal depressive symptoms and parental burnout. Multiple linear regression and binary logistic regression analysis were used to examine the association between postnatal depressive symptoms and parental burnout. Furthermore, latent class analysis was used to identify subtypes of parental burnout. Finally, binary logistic regression was used to examine the differences in postnatal depressive symptoms between latent classes comprising parental burnout. RESULTS: The prevalence of burnout was approximately 10%. At the population level, postnatal depressive symptoms were positively associated with parental burnout (all P < 0.05). At the individual level, two latent classes were identified (i.e., "low parental burnout class" and "high parental burnout class"). Moreover, mothers with postnatal depressive symptoms were more likely to be associated with high parental burnout (PB) class than the low parental burnout class (OR = 1.12, 95% CI:1.03 to 1.23). CONCLUSION: This study found a positive relationship between postnatal depressive symptoms and parental burnout. It provided evidence for developing depression-targeted programs for parental burnout, which could bring great benefits for both mothers and infants.

Effectiveness of Digital Guided Self-help Mindfulness Training During Pregnancy on Maternal Psychological Distress and Infant Neuropsychological Development: Randomized Controlled Trial.

BACKGROUND: Maternal psychological distress during pregnancy is associated with unfavorable outcomes in infants. Mindfulness-based interventions (MBIs) can effectively alleviate psychological distress, but there are often barriers to the access of face-to-face interventions. OBJECTIVE: This study aimed to investigate the effectiveness of a digital guided self-help (GSH) MBI (GSH-MBI) in reducing maternal psychological distress and improving infant neuropsychological performance. METHODS: This was a randomized controlled trial. We recruited 160 women who were 12 to 20 weeks pregnant and exhibited psychological distress. We randomized them into a digital GSH-MBI group and a control group (usual perinatal care). The digital GSH-MBI consisted of a 6-week intervention through a WeChat mini program, with a daily reminder sent to the participants by a research assistant via WeChat. The primary outcomes consisted of maternal psychological distress, including depression, anxiety, and pregnancy-related anxiety symptoms, which were assessed at 6 time points from baseline to 6 months post partum (only pregnancy-related anxiety symptoms were assessed 3 times during pregnancy). The secondary outcomes were infant neuropsychological outcomes, including temperament and developmental behaviors, which were assessed at 6 weeks and 6 months post partum. RESULTS: Compared with the control group, the digital GSH-MBI group showed a significant reduction in depression, anxiety, and pregnancy-related anxiety symptoms. In addition, the scores of the digital GSH-MBI group were lower than those of the control group for the 3 types of infant temperament at 6 weeks post partum, including quality of mood, distractibility, and adaptability. CONCLUSIONS: Digital GSH-MBIs are effective in alleviating psychological distress among pregnant women and protecting infant outcomes. TRIAL REGISTRATION: Chinese Clinical Trial Register ChiCTR2000040717; https://www.chictr.org.cn/showproj.aspx?proj=65376.

Effects and Mechanisms of a Web- and Mobile-Based Acceptance and Commitment Therapy Intervention for Anxiety and Depression Symptoms in Nurses: Fully Decentralized Randomized Controlled Trial.

BACKGROUND: Acceptance and commitment therapy (ACT) is a promising intervention for improving mental health. However, there is limited evidence on its effectiveness for nurses, particularly in web- and mobile-based intervention forms, in mitigating anxiety and depression symptoms. OBJECTIVE: In this study, we aimed to examine the effect and underlying psychological mechanisms of a web- and mobile-based ACT intervention on nurses' anxiety and depression symptoms. METHODS: In this fully decentralized randomized controlled trial, nurses were recruited nationwide across China through advertisements and posters. They were randomly assigned to either the 5-week fully automated intervention or the waiting group. Primary outcomes (anxiety and depression symptoms); secondary outcomes (sleep quality, burnout, and work performance); and mediators (psychological flexibility, cognitive defusion, mindfulness, and values) were assessed using the Wenjuanxing platform. Data collectors were blinded to the group assignments throughout the study period. RESULTS: A total of 145 nurses with anxiety or depression symptoms were randomly assigned to either the intervention group (n=72, 49.7%) or the control group (n=73, 50.3%); 97.2% (n=141) were female. During the study, 36 (24.8%) nurses were lost to follow-up, and 53 (73.6%) completed the entire intervention. Nurses in the intervention group showed significant improvement in anxiety (d=0.67, 95% CI 0.33-1.00) and depression symptoms (d=0.58, 95% CI 0.25-0.91), and the effects were sustained for 3 months after the intervention (anxiety: d=0.55, 95% CI 0.22-0.89; depression: d=0.66, 95% CI 0.33-1.00). Changes in psychological flexibility, cognitive defusion, and values mediated the effect of the intervention on anxiety and depression symptoms, while mindfulness did not have a mediating effect. CONCLUSIONS: The web- and mobile-based ACT intervention used in this study significantly improved nurses' anxiety and depression symptoms by improving psychological flexibility, cognitive defusion, and values. The results provide new ideas for hospital administrators to prevent and intervene in nurses' psychological issues. TRIAL REGISTRATION: Chinese Clinical Trial Register ChiCTR2200059218; https://tinyurl.com/4mb4t5y9.

Association of Workplace Bullying with Suicide Ideation and Attempt Among Chinese Nurses During the COVID-19 Pandemic.

Nurses experience a high incidence of workplace bullying and are at a higher risk of suicide than the general population. However, there is no empirical evidence on how exposure to workplace bullying is associated with suicide ideation and attempts among nurses. Nurses were recruited from tertiary hospitals in Shandong Province, China, using stratified cluster sampling. Suicide ideation and attempts were assessed using two items, and the Workplace Psychologically Violent Behaviors Instrument was used to measure subtypes of workplace bullying. The prevalence of workplace bullying, suicide ideation, and suicide attempts was 30.6%, 16.8%, and 10.8%, respectively. After adjusting for covariates, victims of workplace bullying were at a high risk of suicide ideation and attempts. Among workplace bullying subtypes, individuals' isolation from work and direct negative behaviors were predictors of both suicide ideation and attempts; attack on personality only predicted suicide attempts. The more bullying subtypes experienced by nurses, the greater their likelihood of suicide ideation and attempts. These findings suggested that workplace bullying was associated with an increased risk of suicide ideation and attempts in nurses, with both independent and cumulative risks. Interventions should focus on prevention and managing the effects of workplace bullying among nurses.

Topological frustration leading to backtracking in a coupled folding-binding process.

Intrinsically disordered proteins (IDPs) are abundant in all species. Their discovery challenges the traditional "sequence-structure-function" paradigm of protein science because IDPs play important roles in various biological processes without preformed folded structures. Bioinformatic analysis reveals that the intrinsically conformational disorder of IDPs as well as their conformational transition upon binding to their targets is encoded by their amino acid sequences. The rRNase domain of colicin E3 and the immunity protein Im3 are a pair of proteins involved in bacterial survival. While the N-terminal segment and the central segment of E3 make comparable intermolecular contacts with Im3 in the bound state, binding of E3 with Im3 is dominantly triggered by the central segment of E3. In this work, to further investigate the binding mechanism of disordered E3 with Im3, we performed systematic free energy and transition path analysis through coarse-grained molecular dynamics simulations. We observed backtracking of the N-terminal segment of E3 in the binding process, whose occurrence depends on salt concentration. Conformational analysis revealed that initial binding of the N-terminal segment of E3 to Im3 usually leads to misorientation of a central hairpin of E3 on Im3, which generates topological frustration and results in backtracking of the N-terminal segment. Our results not only provide deeper mechanistic insights into the coupled folding-binding process of the E3/Im3 complex, but also suggest that topological frustration could be present in the coupled folding-binding process of IDPs and play an important role in regulating the binding transition pathways.

The structure and phase of tau: from monomer to amyloid filament.

Tau is a microtubule-associated protein involved in regulation of assembly and spatial organization of microtubule in neurons. However, in pathological conditions, tau monomers assemble into amyloid filaments characterized by the cross-ß structures in a number of neurodegenerative diseases known as tauopathies. In this review, we summarize recent progression on the characterization of structures of tau monomer and filament, as well as the dynamic liquid droplet assembly. Our aim is to reveal how post-translational modifications, amino acid mutations, and interacting molecules modulate the conformational ensemble of tau monomer, and how they accelerate or inhibit tau assembly into aggregates. Structure-based aggregation inhibitor design is also discussed in the context of dynamics and heterogeneity of tau structures.

Work-related potential traumatic events and job burnout among operating room nurses: Independent effect, cumulative risk, and latent class approaches.

AIMS: The objective of this study is to explore and compare the strength of associations between work-related potential traumatic events and burnout among operating room nurses based on three different approaches. DESIGN: The study followed a multisite cross-sectional design. METHODS: A stratified sampling method was conducted. Cities in the Shandong Province were divided into four groups, and two tertiary hospitals were randomly selected from all tertiary hospitals in cities of each group. A total of 361 eligible operating room nurses provided valid questionnaires between June and November 2019. Work-related potential traumatic events questionnaire and the Chinese version of the Maslach Burnout Inventory were conducted. Associations between individual, cumulative potential traumatic events, as well as latent class analysis-derived patterns of potential traumatic experiences with burnout were examined using logistic regression analysis. RESULTS: Specific traumatic events (e.g., patients' sudden death, bullying and ostracism from colleagues) were independently associated with an increased risk of burnout. Work-related potential traumatic events had a cumulative effect on burnout, whereby operating room nurses exposed to cumulative potential traumatic events carried a higher risk for burnout than others. The "multiple work-related potential traumatic events" pattern derived by latent class analysis was related to an increased risk of high depersonalization. CONCLUSION: Operating room nurses who reported specific or multiple work-related potential traumatic events were at high risk for burnout. It is of great significance to provide qualified and timely counselling or support. IMPACT: This study is the first to focus on work-related potential traumatic events among operating room nurses and examine their relationship with burnout based on three perspectives. The findings could help identify those operating room nurses who are at high risk of burnout in clinical practice. Hospital managers should develop targeted interventions to prevent or mitigate the harmful impact of potential traumatic events on occupational health.

Introducing intrinsic disorder reduces electrostatic steering in protein-protein interactions.

Protein-protein interactions underlie many critical biology functions, such as cellular signaling and gene expression, in which electrostatic interactions can play a critical role in mediating the specificity and stability of protein complexes. A substantial portion of proteins are intrinsically disordered, and the influences of structural disorder on binding kinetics and thermodynamics have been widely investigated. However, whether the effect of electrostatic steering depends on structural disorder remains unexplored. In this work, we addressed the consequence of introducing intrinsic disorder in the electrostatic steering of the E3/Im3 complex using molecular dynamics simulation. Our results recapitulated the experimental observations that the responses of stability and kinetics to salt concentration for the ordered E3/Im3 complex were larger than those for the disordered E3/Im3 complex. Mechanistic analysis revealed that the native contact interactions involved in the encounter state and the transition state were essentially identical for both ordered and disordered E3. Therefore, the observed difference in electrostatic steering between ordered E3 and disordered E3 may result from their difference in conformation rather than their difference in binding mechanism. Because charged residues are frequently involved in protein-protein interactions, our results suggest that increasing structural disorder is expected to generally modulate the effect of electrostatic steering.

Withdrawn: Invisible State of MdmX and Design of its Inhibitors.

Withdrawal: Xiyao Cheng, Yongqi Huang, and Zhengding Su. Invisible State of MdmX and Design of its Inhibitors. The FASEB Journal. 34:s1. doi: 10.1096/fasebj.2020.34.s1.09193. The above abstract, published online on April 20, 2020, in Wiley Online Library (wileyonlinelibrary.com), has been withdrawn by agreement between the authors, FASEB, and Wiley Periodicals, Inc. The abstract has been withdrawn at the request of the authors.

Withdrawn: A Controllable Protein Translation Strategy for Exploring Cellular Signal Transduction Based on Reading through Premature Termination Codons.

Withdrawal: Xiyao Cheng, Yongqi Huang, and Zhengding Su. A Controllable Protein Translation Strategy for Exploring Cellular Signal Transduction Based on Reading through Premature Termination Codons. The FASEB Journal. 34:s1. doi: 10.1096/fasebj.2020.34.s1.09195. The above abstract, published online on April 19, 2020, in Wiley Online Library (wileyonlinelibrary.com), has been withdrawn by agreement between the authors, FASEB, and Wiley Periodicals, Inc. The abstract has been withdrawn at the request of the authors.

Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria.

4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2Δkstd211 + ΔkshB122, showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2kstd2 + Δkstd211+ΔkshB122 and HGMS2kshA51 + Δkstd211+ΔkshA226, by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

The recovery of KaiA's activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock.

Circadian rhythms are the endogenous oscillation of biological reactions and behaviors in most organisms on Earth. Circadian clocks are the pacemakers regulating circadian rhythms, and the transcription-translation dependent feedback loop (TTFL) model was supposed to be the sole model of circadian clocks. However, recent years have witnessed rapid progresses in the study of non-TTFL circadian clocks. The cyanobacterial circadian clock consists of three proteins (KaiA, KaiB, and KaiC), and is extensively studied as a non-TTFL circadian clock model. Although containing only three proteins, the molecular mechanism of the KaiABC circadian clock remains elusive. We recently noticed that KaiA has an auto-inhibition conformation during the oscillation, but how this auto-inhibition is regulated is unclear. Here, we started from the design of light modulated KaiAs to investigate this mechanism. We designed different KaiA constructs fused with the light modulable LOV2 protein, and used light-modulated KaiAs to regulate the phosphorylation and dephosphorylation of KaiC. Our data indicated that the N-terminal domain of KaiA is important for KaiA's reversible off/on switching during the unidirectional oscillation of the KaiABC system. This work provides an updated model to explain the molecular mechanism of the KaiABC circadian clock.