Multi-omic analyses reveal key sectors of jasmonate-mediated defense responses in rice.

The phytohormone jasmonate (JA) plays a central role in plant defenses against biotic stressors. However, our knowledge of the JA signaling pathway in rice (Oryza sativa) remains incomplete. Here, we integrated multi-omic data from three tissues to characterize the functional modules involved in organizing JA-responsive genes. In the core regulatory sector, MYC2 transcription factor transcriptional cascades are conserved. in different species but with distinct regulators (e.g. bHLH6 in rice)., in which genes are early expressed across all tissues. In the feedback sector, MYC2 also regulates the expression of JA repressor and catabolic genes, providing negative feedback that truncates the duration of JA responses. For example, the MYC2-regulated NAC (NAM, ATAF1/2 and CUC2) transcription factor genes NAC1, NAC3, and NAC4 encode proteins that repress JA signaling and herbivore resistance. In the tissue-specific sector, many late-expressed genes are associated with the biosynthesis of specialized metabolites that mediate particular defensive functions. For example, the terpene synthase gene TPS35 is specifically induced in the leaf sheath and TPS35 functions in defense against oviposition by brown planthoppers and the attraction of this herbivore's natural enemies. Thus, by characterizing core, tissue-specific, and feedback sectors of JA-elicited defense responses, this work provides a valuable resource for future discoveries of key JA components in this important crop.

Sakuranetin protects rice from brown planthopper attack by depleting its beneficial endosymbionts.

Plants produce chemical defenses that poison insect herbivores or deter their feeding, but herbivores are also accompanied by microbial endosymbionts crucial for their nutrition, reproduction, and fitness. Hence, plant defenses could target a herbivore's beneficial endosymbionts, but this has not yet been demonstrated. Here, we studied flavonoids that are induced when rice is attacked by a phloem-feeding pest, the brown planthopper (BPH), which harbors beneficial yeast-like symbionts (YLS) essential for insect nutrition, such as by remedying deficiencies in sterols. BPH attack dramatically increased sakuranetin accumulations in leaf sheaths and phloem exudates. Sakuranetin is an antifungal phytoalexin derived from the antibacterial precursor, naringenin, via catalysis of naringenin-O-methyltransferase (NOMT). When added to artificial diets, sakuranetin decreased BPH survivorship, suggesting that it functions as an induced defense. Mutation of NOMT abolished sakuranetin accumulation and increased BPH oviposition and hatching rates. High-throughput amplicon sequencing revealed that BPH fed on sakuranetin-deficient nomt lines were enriched in YLS with only minor changes in the bacterial endosymbionts, compared to those feeding on sakuranetin-rich wild-type (WT) plants. In-vitro feeding of sakuranetin suggested that this flavonoid directly inhibited the growth of YLS. BPH feeding on nomt lines accumulated higher cholesterol levels, which might be attributed to increases in the supply of sterol precursors from the YLS, while nomt lines suffered more damage than WT plants did from BPH herbivory. BPH-elicited accumulation of sakuranetin requires intact jasmonate (JA) signaling. This study reveals that rice uses a JA-induced antifungal flavonoid phytoalexin in defense against BPH by inhibiting its beneficial endosymbionts.

Excitatory neurons in the lateral parabrachial nucleus mediate the interruptive effect of inflammatory pain on a sustained attention task.

BACKGROUND: Attentional deficits are among the most common pain-induced cognitive disorders. Pain disrupts attention and may excessively occupy attentional resources in pathological states, leading to daily function impairment and increased disability. However, the neural circuit mechanisms by which pain disrupts attention are incompletely understood. METHODS: We used a three-choice serial reaction time task (3CSRTT) to construct a sustained-attention task model in male C57BL/6J mice. Formalin or complete Freund's adjuvant was injected into a paw to establish an inflammatory pain model. We measured changes in 3CSRTT performance in the two inflammatory pain models, and investigated the neural circuit mechanisms of pain-induced attentional deficits. RESULTS: Acute inflammatory pain impaired 3CSRTT performance, while chronic inflammatory pain had no effect. Either inhibition of the ascending pain pathway by blockade of the conduction of nociceptive signals in the sciatic nerve using the local anesthetic lidocaine or chemogenetic inhibition of Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) neurons in the lateral parabrachial nucleus (LPBN) attenuated the acute inflammatory pain-induced impairment of 3CSRTT performance, while chemogenetic activation of CaMKIIα neurons in the LPBN disrupted the 3CSRTT. Furthermore, the activity of CaMKIIα neurons in the LPBN was significantly lower on Day 2 after complete Freund's adjuvant injection than on the day of injection, which correlated with the recovery of 3CSRTT performance during chronic inflammatory pain. CONCLUSIONS: Activation of excitatory neurons in the LPBN is a mechanism by which acute inflammatory pain disrupts sustained attention. This finding has implications for the treatment of pain and its cognitive comorbidities.

Functional dissection of rice jasmonate receptors involved in development and defense.

The phytohormones, jasmonates (JAs), mediate many plant developmental processes and their responses to important environmental stresses, such as herbivore attack. Bioactive JAs are perceived by CORONATINE INSENSITIVE (COI)-receptors, and associated JAZ proteins, to activate downstream responses. To date, the JA receptors of the important monocot crop plant, rice, remain to be explored. Here, we studied all three rice COI proteins, OsCOI1a, OsCOI1b, and OsCOI2, by ligand binding, genome editing, and phenotyping and examining some of the responsible mechanisms for the different responses. OsCOI2 binds to most individual OsJAZs in the presence of endogenous JA ligands, as OsCOI1a /1b do, albeit with greater partner selectivity. Single mutants of each OsCOI and OsCOI1a/1b double mutants were constructed by CRIPSR-Cas9-based genome editing and used to phenotype developmental and defense responses. OsCOI1b is involved in root growth and grain-size control and plays overlapping roles with OsCOI1a in spikelet development, while OsCOI2 regulates leaf senescence, male sterility, root growth, and grain size. All OsCOIs mediated resistance to the devastating rice pest, the brown planthopper. However, the defense sectors regulated by OsCOI1a/1b and OsCOI2 clearly differed. Our results revealed that all three OsCOIs are functional JA receptors that play diverse roles in regulating downstream JA responses.

Sex-specific relationships between prenatal exposure to metal mixtures and birth weight in a Chinese birth cohort.

Birth weight is an indicator linking intrauterine environmental exposures to later-life diseases, and intrauterine metal exposure may affect birth weight in a sex-specific manner. We investigated sex-specific associations between prenatal exposure to metal mixtures and birth weight in a Chinese birth cohort. The birth weight of 1296 boys and 1098 girls were recorded, and 10 metals in maternal urine samples collected during pregnancy were measured using inductively coupled plasma mass spectrometry. Bayesian Kernel Machine Regression was used to estimate the association of individual metals or metal mixtures and birth weight for gestational age (BW for GA). The model showed a sex-specific relationship between prenatal exposure to metal mixtures and BW for GA with a significant negative association in girls and a non-significant positive association in boys. Cadmium (Cd) and nickel (Ni) were positively and negatively associated with BW for GA in girls, respectively. Moreover, increasing thallium (Tl) concentration lowered the positive association between Cd and BW for GA and enhanced the negative association between Ni and BW for GA in girls. When exposure to other metals increased, the positive association with Cd diminished, whereas the negative association with Ni or Tl increased. Our findings provide evidence supporting the complex effects of intrauterine exposure to metal mixtures on the birth weight of girls and further highlight the sex heterogeneity in fetal development influenced by intrauterine environmental factors.

The novel repressor Rce2 competes with Ace3 to regulate cellulase gene expression in the filamentous fungus Trichoderma reesei.

The filamentous fungus Trichoderma reesei is widely used for industrial cellulase production. In T. reesei, cellulase gene expression is tightly controlled by a regulatory network involving multiple transcription factors. Here, we isolated a novel protein, Rce2, using a pull-down assay and mass spectrometry analysis, from a partial carbon catabolite de-repression mutant, T. reesei Rut-C30, cultured under glucose-repressing conditions. Deletion and overexpression of Rce2 in T. reesei wild-type QM6a and mutant Rut-C30 revealed that Rce2 acts as a repressor of cellulase gene expression. DNase I footprinting assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Rce2 was located in the nucleus and bound to the consensus sequences 5'-(T/A)NNNNCCG-3' and 5'-CGGNNNN(T/A)-3' in the promoters of cellulase-related genes to repress their transcription. Additionally, Rce2 antagonized Ace3 binding to the cbh1 promoter to repress its transcription. However, Rce2 was not involved in Cre1-mediated carbon catabolite repression. These results demonstrate the mechanism through which Rce2 represses the expression of cellulase genes and provide novel insights into the regulatory system of cellulases and methods that can be used for the regulation of gene expression in T. reesei.

Effect of early-life exposure to PM2.5 on childhood asthma/wheezing: a birth cohort study.

BACKGROUND: Although studies have investigated the association between early-life exposure to fine particulate matter (PM2.5 ) and childhood asthma/wheezing, results are inconsistent and the susceptible exposure window remains largely unknown. METHODS: A prospective birth cohort study was conducted to recruit pregnant women during their early pregnancy, and to follow up them and their children up to 3-4 years old. Diagnosis of asthma/wheezing was extracted from children's medical records. A spatiotemporal land-use regression (ST-LUR) model was used to assess maternal exposure to PM2.5 during pregnancy and their children's exposure after birth. The Cox proportional hazards model and accelerated failure time model (for violation of proportional hazards assumption) were applied to estimate the effects of prenatal and postnatal exposures to PM2.5 on the risk of childhood asthma/wheezing. RESULTS: A total of 3725 children were included, and 392 children (10.52%) were diagnosed with asthma/wheezing. Both prenatal and postnatal exposures to PM2.5 were positively associated with the risk of asthma/wheezing. Each interquartile range (IQR) increment in PM2.5 exposure during the entire pregnancy (4.8 µg/m3 ) and the period from birth to the end of follow-up (1.5 µg/m3 ) was associated with adjusted hazard ratios (HRs) of 1.44 [95% confidence interval (CI): 1.13, 1.85] and 2.74 (95% CI: 2.59, 2.91), respectively. Subgroup analyses showed greater HRs for PM2.5 exposures during the pseudoglandular stage (6-16 gestational weeks [GWs]: IQR = 4.8 µg/m3 , HR = 1.10, 95% CI: 1.02, 1.18) and canalicular stage (16-24 GWs: IQR = 4.8 µg/m3 , HR = 1.13, 95% CI:1.03, 1.23) than other stages, and also showed significant effects in the first three-year period after birth (IQR = 1.5 µg/m3 , HR = 2.37, 95% CI: =2.24, 2.51). CONCLUSION: Higher prenatal and postnatal PM2.5 exposures may increase the risk of childhood asthma/wheezing. The pseudoglandular stage, canalicular stage, and the first three years after birth may be key susceptible to exposure windows.

Tetradentate Pt(II) Complexes with Peripheral Hindrances for Highly Efficient Solution-Processed Blue Phosphorescent OLEDs.

Two tetradentate Pt(II) complexes with peripheral bulky-group hindrances [Pt(pzpyOczpy-B1) and Pt(pzpyOczpy-B2)] were synthesized and fully investigated for their structural and blue phosphorescent properties. Both X-ray crystallography and computational simulation revealed that bulky substituents incorporated into the C-pyrazolyl and C-pyridinyl positions lie out of the cyclometallated plane, thus alleviating the intramolecular distortions as well as reducing the intermolecular interaction in the solid state. In dichloromethane, their emission peaks at 460 nm with a narrow full width at half-maximum (FWHM) of less than 50 nm, and the photoluminescent quantum yields are over 95% with short decay lifetimes (<5 µs). Solution-processed blue devices are fabricated based on the two complexes. Device A based on Pt(pzpyOczpy-B1) shows excellent electroluminescent performances with the maximum current efficiency, power efficiency, and external quantum efficiency of 47.0 cd/A, 24.6 lm/W, and 22.9%, respectively. The understanding on inert peripheral hindrances provides an effective approach to designing Pt(II) complexes for high-quality blue phosphorescent emitters.

Effect of maternal ozone exposure before and during pregnancy on wheezing risk in offspring: A birth cohort study in Guangzhou, China.

Ozone (O3) exposure may lead to the development and exacerbation of asthma or wheezing in postnatal children; however, it has rarely been studied before and during pregnancy. Wheezing is one of the most common symptoms when diagnosing of asthma; thus, we investigated the associations of O3 exposure before and during pregnancy with wheezing in preschool children and the potential susceptible exposure windows from a heavily polluted city in China. This population-based birth cohort study, which included 3725 mother-child pairs from Guangzhou, began in 2016, and the follow-up period ended on July 31, 2020. We used a spatiotemporal land-use-regression model combined with activity patterns to estimate the daily O3 exposure levels during the pre-pregnancy period and each trimester, and wheezing was recorded by reviewing medical records. We used the Cox proportional hazard model to quantify the effects of O3 exposure on childhood wheezing adjusted for potential confounders. No significant association was detected between pre-pregnancy exposure to O3 and childhood wheezing. However, increased ambient O3 exposures throughout pregnancy and in the second trimester were positively associated with the risk of childhood wheezing, with hazard ratios (HRs) and 95% confident intervals (CIs) per interquartile range (IQR) increment of 1.22 (95% CI: 1.04-1.44) and 1.31 (95% CI: 1.09-1.58), respectively. The effects of maternal O3 exposure on childhood wheezing risk was stronger when the exposure occurred in the warm conception season (P < 0.05). Significant childhood wheezing risk could be attributable to maternal O3 exposure, especially during the second trimester and with warm-season conception in Guangzhou. Further cohorts of children, particularly school age children who have more robust asthma diagnoses, should be investigated in the future.

Roles of PKAc1 and CRE1 in cellulose degradation, conidiation, and yellow pigment synthesis in Trichoderma reesei QM6a.

PURPOSE: This study aimed to reveal the roles of the protein kinase A catalytic subunit 1 (pkac1) and carbon catabolite repressor cre1 genes in cellulase production by Trichoderma reesei wild-type strain QM6a. Our strategy might be useful to construct a high-yielding cellulase strain for its wide application. METHODS: This paper describes cellulase activity, plate conidiation, and yellow pigment synthesis assays of QM6a with the disruption of pkac1 and cre1. RESULTS: Deletion of pkac1 (Δpkac1) had no effect on cellulase production or transcript levels of major cellulase genes in the presence of cellulose. Disruption of cre1 (Δcre1) resulted in a remarkable increase in cellulase production and expression of the four major cellulase genes. Double disruption of pkac1 and cre1 significantly improved enzyme activity and protein production. The double disruption also resulted in a significant reduction in yellow pigment production and abrogated conidial production. CONCLUSION: Double deletion of pkac1 and cre1 led to increased hydrolytic enzyme production in T. reesei using cellulose as a carbon source.

Designing Hollow Carbon Sphere with Hierarchal Porous for Na-S Systems with Ultra-Long Cycling Stabilities.

Captured by the low-cost and high theoretical specific capacity, Na-S systems have garnered much attention. However, their intermediate products (dissolved polysulfide) are always out of control. Considering the excellent space confinements and conductivity, they have been regarded as promising candidates. Herein, the hollow spheres with suitable thickness shell (~20 nm) are designed as hosting materials, accompanied by in-depth complexing. Benefitting from the abundant micro-pores (mainly about conical-type and slits-type pores < 1.0 nm), the active S4 molecules are successfully filled in the pores through vacuum tube sealing technology, effectively avoiding the process from solid S8 to liquid Na2S6. As cathode for Na-S systems, their capacity could remain at 920 mAh g−1 at 0.1 C after 100 cycles. Even at 10.0 C, the capacity still remained at about 310 mAh g−1 after 7000 cycles. Supported by the detailed kinetic behaviors, the improvement of ions diffusion behaviors is noted, bringing about the effective thorough redox reactions. Moreover, the enhanced surface-controlling behaviors further induces the evolution of rate properties. Therefore, their stable phase changing is further confirmed through in situ resistances. Thus, the work is anticipated to offer significant design for hosting carbon materials and complexing manners.

Folic acid: a potential inhibitor against SARS-CoV-2 nucleocapsid protein.

CONTEXT: Coronavirus disease 2019 is a global pandemic. Studies suggest that folic acid has antiviral effects. Molecular docking shown that folic acid can act on SARS-CoV-2 Nucleocapsid Phosphoprotein (SARS-CoV-2 N). OBJECTIVE: To identify novel molecular therapeutic targets for SARS-CoV-2. MATERIALS AND METHODS: Traditional Chinese medicine targets and virus-related genes were identified with network pharmacology and big data analysis. Folic acid was singled out by molecular docking, and its potential target SARS-CoV-2 N was identified. Inhibition of SARS-CoV-2 N of folic acid was verified at the cellular level. RESULTS: In total, 8355 drug targets were potentially involved in the inhibition of SARS-CoV-2. 113 hub genes were screened by further association analysis between targets and virus-related genes. The hub genes related compounds were analysed and folic acid was screened as a potential new drug. Moreover, molecular docking showed folic acid could target on SARS-CoV-2 N which inhibits host RNA interference (RNAi). Therefore, this study was based on RNAi to verify whether folic acid antagonises SARS-CoV-2 N. Cell-based experiments shown that RNAi decreased mCherry expression by 81.7% (p < 0.001). This effect was decreased by 8.0% in the presence of SARS-CoV-2 N, indicating that SARS-CoV-2 N inhibits RNAi. With increasing of folic acid concentration, mCherry expression decreased, indicating that folic acid antagonises the regulatory effect of SARS-CoV-2 N on host RNAi. DISCUSSION AND CONCLUSIONS: Folic acid may be an antagonist of SARS-CoV-2 N, but its effect on viruses unclear. In future, the mechanisms of action of folic acid against SARS-CoV-2 N should be studied.

Trichoderma reesei ACE4, a Novel Transcriptional Activator Involved in the Regulation of Cellulase Genes during Growth on Cellulose.

The filamentous fungus Trichoderma reesei is a model strain for cellulase production. Cellulase gene expression in T. reesei is controlled by multiple transcription factors. Here, we identified by comparative genomic screening a novel transcriptional activator, ACE4 (activator of cellulase expression 4), that positively regulates cellulase gene expression on cellulose in T. reesei. Disruption of the ace4 gene significantly decreased expression of four main cellulase genes and the essential cellulase transcription factor-encoding gene ace3. Overexpression of ace4 increased cellulase production by approximately 22% compared to that in the parental strain. Further investigations using electrophoretic mobility shift assays, DNase I footprinting assays, and chromatin immunoprecipitation assays indicated that ACE4 directly binds to the promoter of cellulase genes by recognizing the two adjacent 5'-GGCC-3' sequences. Additionally, ACE4 directly binds to the promoter of ace3 and, in turn, regulates the expression of ACE3 to facilitate cellulase production. Collectively, these results demonstrate an important role for ACE4 in regulating cellulase gene expression, which will contribute to understanding the mechanism underlying cellulase expression in T. reesei. IMPORTANCET. reesei is commonly utilized in industry to produce cellulases, enzymes that degrade lignocellulosic biomass for the production of bioethanol and bio-based products. T. reesei is capable of rapidly initiating the biosynthesis of cellulases in the presence of cellulose, which has made it useful as a model fungus for studying gene expression in eukaryotes. Cellulase gene expression is controlled through multiple transcription factors at the transcriptional level. However, the molecular mechanisms by which transcription is controlled remain unclear. In the present study, we identified a novel transcription factor, ACE4, which regulates cellulase expression on cellulose by binding to the promoters of cellulase genes and the cellulase activator gene ace3. Our study not only expands the general functional understanding of the novel transcription factor ACE4 but also provides evidence for the regulatory mechanism mediating gene expression in T. reesei.

Association of frailty with quality of life in older hypertensive adults: a cross-sectional study.

BACKGROUND: Hypertension is one of the most common chronic cardiovascular diseases in older adults. Frailty and hypertension often coexist in older people, but few studies have explored frailty in older hypertensive adults. We aimed to explore the association of frailty with quality of life in older hypertensive adults. METHOD: We collected the data of 291 patients with hypertension aged ≥ 60 years. Blood pressure was measured with a standard aneroid sphygmomanometer and an ambulatory blood pressure monitor. The characteristics of the Fried phenotype were used to assess physical frailty. The Medical Outcomes Study's 36-Item Short Form Health Survey (SF-36) was used to assess the quality of life. RESULTS: Forty-eight (16.5%) patients were frail. Compared with pre-frail or robust older hypertensive patients, those who were frail were older, had higher incidences of living alone, a longer duration of hypertension, lower grip strength, and slower walking speed. Moreover, frail patients had a lower diastolic blood pressure (DBP) and mean arterial pressure (MAP), more chronic diseases, a lower proportion of beta-blocker usage, and worse quality of life. Frailty was positively correlated with pulse pressure and negatively correlated with DBP and MAP, but these associations disappeared after adjustment by age. The SF-36 score was negatively correlated with frailty and positively correlated with grip strength and walking speed. After adjusting for age, the SF-36 total score remained negatively correlated with frailty and positively correlated with walking speed. Frailty states remained significantly associated with the SF-36 score. CONCLUSION: Frailty was associated with a worse quality of life in older adults with hypertension. Frailty prevention and intervention may help improve the quality of life of older adults with hypertension.

Associations of COVID-19 lockdown with gestational length and preterm birth in China.

BACKGROUND: The effects of COVID-19 lockdown measures on maternal and fetal health remain unclear. We examined the associations of COVID-19 lockdown with gestational length and preterm birth (PTB) in a Chinese population. METHODS: We obtained medical records of 595,396 singleton live infants born between 2015 and 2020 in 5 cities in Guangdong Province, South China. The exposed group (N = 101,900) included women who experienced the COVID-19 Level I lockdown (1/23-2/24/2020) during pregnancy, while the unexposed group (N = 493,496) included women who were pregnant during the same calendar months in 2015-2019. Cumulative exposure was calculated based on days exposed to different levels of emergency responses with different weighting. Generalized linear regression models were applied to estimate the associations of lockdown exposure with gestational length and risk of PTB (< 37 weeks). RESULTS: The exposed group had a shorter mean gestational length than the unexposed group (38.66 vs 38.74 weeks: adjusted ß = - 0.06 week [95%CI, - 0.07, - 0.05 week]). The exposed group also had a higher risk of PTB (5.7% vs 5.3%; adjusted OR = 1.08 [95%CI, 1.05, 1.11]). These associations seemed to be stronger when exposure occurred before or during the 23rd gestational week (GW) than during or after the 24th GW. Similarly, higher cumulative lockdown exposure was associated with a shorter gestational length and a higher risk of PTB. CONCLUSIONS: The COVID-19 lockdown measures were associated with a slightly shorter gestational length and a moderately higher risk of PTB. Early and middle pregnancy periods may be a more susceptible exposure window.

The transcription factor ACE3 controls cellulase activities and lactose metabolism via two additional regulators in the fungus Trichoderma reesei.

Fungi of the genus Trichoderma are a rich source of enzymes, such as cellulases and hemicellulases, that can degrade lignocellulosic biomass and are therefore of interest for biotechnological approaches seeking to optimize biofuel production. The essential transcription factor ACE3 is involved in cellulase production in Trichoderma reesei; however, the mechanism by which ACE3 regulates cellulase activities is unknown. Here, we discovered that the nominal ace3 sequence in the T. reesei genome available through the Joint Genome Institute is erroneously annotated. Moreover, we identified the complete ace3 sequence, the ACE3 Zn(II)2Cys6 domain, and the ACE3 DNA-binding sites containing a 5'-CGGAN(T/A)3-3' consensus. We found that in addition to its essential role in cellulase production, ace3 is required for lactose assimilation and metabolism in T. reesei Transcriptional profiling with RNA-Seq revealed that ace3 deletion down-regulates not only the bulk of the major cellulase, hemicellulase, and related transcription factor genes, but also reduces the expression of lactose metabolism-related genes. Additionally, we demonstrate that ACE3 binds the promoters of many cellulase genes, the cellulose response transporter gene crt1, and transcription factor-encoding genes, including xyr1 We also observed that XYR1 dimerizes to facilitate cellulase production and that ACE3 interacts with XYR1. Together, these findings uncover how two essential transcriptional activators mediate cellulase gene expression in T. reesei On the basis of these observations, we propose a model of how the interactions between ACE3, Crt1, and XYR1 control cellulase expression and lactose metabolism in T. reesei.

A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription.

OBJECTIVE: To simplify CRISPR/Cas9 genome editing in the industrial filamentous fungus Trichoderma reesei based on in vivo guide RNA (gRNA) transcription. RESULTS: Two putative RNA polymerase III U6 snRNA genes were identified in the genome of T. reesei QM6a by BLASTN using Myceliophthora. thermophila U6 snRNA gene as the template. The regions approximately 500 bp upstream of two U6 genes were efficient promoters for the in vivo expression of gRNA. The CRISPR system consisting of Cas9 and in vivo synthesized gRNA under control of the T. reesei U6 snRNA promoters was sufficient to cause a frameshift mutation in the ura5 gene via non-homologous end-joining-mediated events. CONCLUSIONS: We report a simple gene editing method using a CRISPR/Cas9-coupled in vivo gRNA transcription system in T. reesei.

Hypoxia regulates angeogenic-osteogenic coupling process via up-regulating IL-6 and IL-8 in human osteoblastic cells through hypoxia-inducible factor-1α pathway.

Inappropriate angiogenesis and osteogenesis are considered as the crucial factors of osteoporotic fracture. Hypoxia is a primary driving force for regulating the angiogenic-osteogenic coupling process. Our recent results indicated that hypoxia could improve angiogenesis as well as differentiation and activity of osteoblastic cells via up-regulating VEGF through HIF-1α pathway. Here we demonstrated that in human osteoblastic MG-63, U2-OS and Saos-2 cells, besides VEGF, the other two pro-angiogenic factors IL-6 and IL-8 were also up-regulated by hypoxia and CoCl2 (a mimic of hypoxia). Mechanism studies indicated overexpression of HIF-1α (generated from transfection with a plasmid encoding sense HIF-1α) markedly increased the levels of IL-6 and IL-8 in osteoblastic cells. Furthermore, a luciferase reporter assay was performed using the reporter vector containing the IL-6 or IL-8 promoter sequence to illustrate observably increased activity of hypoxia-induced IL-6 and IL-8 promoter caused by overexpression of HIF-1α. Additionally, chromatin immune-precipitation analysis showed hypoxia increased the DNA binding ability of HIF-1α to IL-6 or IL-8 promoter. Analysis in vitro by MTT test and Boyden chamber assay showed exogenous IL-6 and IL-8 (a relatively short period of treatment with recombinant IL-6 or IL-8 equivalent to the autocrine levels) could significantly promote the proliferation of human osteoblastic, endothelial and monocytic cells, as well as the migration of human endothelial cells. Taken together, these results indicate that IL-6 and IL-8 in osteoblastic cells may also contribute to the angiogenic-osteogenic coupling process via HIF-1α pathway. Besides VEGF, IL-6- or IL-8-targeted adjunctive therapy maybe a new strategy to improve the treatment of osteoporosis.

Enhancement of cellulase production in Trichoderma reesei RUT-C30 by comparative genomic screening.

BACKGROUND: Cellulolytic enzymes produced by the filamentous fungus Trichoderma reesei are commonly used in biomass conversion. The high cost of cellulase is still a significant challenge to commercial biofuel production. Improving cellulase production in T. reesei for application in the cellulosic biorefinery setting is an urgent priority. RESULTS: Trichoderma reesei hyper-cellulolytic mutant SS-II derived from the T. reesei NG14 strain exhibited faster growth rate and more efficient lignocellulosic biomass degradation than those of RUT-C30, another hyper-cellulolytic strain derived from NG14. To identify any genetic changes that occurred in SS-II, we sequenced its genome using Illumina MiSeq. In total, 184 single nucleotide polymorphisms and 40 insertions and deletions were identified. SS-II sequencing revealed 107 novel mutations and a full-length wild-type carbon catabolite repressor 1 gene (cre1). To combine the mutations of RUT-C30 and SS-II, the sequence of one confirmed beneficial mutation in RUT-C30, cre196, was introduced in SS-II to replace full-length cre1, forming the mutant SS-II-cre196. The total cellulase production of SS-II-cre196 was decreased owing to the limited growth of SS-II-cre196. In contrast, 57 genes mutated only in SS-II were selected and knocked out in RUT-C30. Of these, 31 were involved in T. reesei growth or cellulase production. Cellulase activity was significantly increased in five deletion strains compared with that in two starter strains, RUT-C30 and SS-II. Cellulase production of T. reesei Δ108642 and Δ56839 was significantly increased by 83.7% and 70.1%, respectively, compared with that of RUT-C30. The amount of glucose released from pretreated corn stover hydrolyzed by the crude enzyme from Δ108642 increased by 11.9%. CONCLUSIONS: The positive attribute confirmed in one cellulase hyper-producing strain does not always work efficiently in another cellulase hyper-producing strain, owing to the differences in genetic background. Genome re-sequencing revealed novel mutations that might affect cellulase production and other pathways indirectly related to cellulase formation. Our strategy of combining the mutations of two strains successfully identified a number of interesting phenotypes associated with cellulase production. These findings will contribute to the creation of a gene library that can be used to investigate the involvement of various genes in the regulation of cellulase production.