SNAC1-OsERF103-OsSDG705 module mediates drought response in rice.

Drought stress profoundly hampers both plant growth and crop yield. To combat this, plants have evolved intricate transcriptional regulation mechanisms as a pivotal strategy. Through a genetic screening with rice genome-scale mutagenesis pool under drought stress, we identified an APETALA2/Ethylene Responsive Factor, namely OsERF103, positively responds to drought tolerance in rice. Combining chromatin immunoprecipitation sequencing and RNA sequencing analyses, we pinpointed c. 1000 genes directly influenced by OsERF103. Further results revealed that OsERF103 interacts with Stress-responsive NAC1 (SNAC1), a positive regulator of drought tolerance in rice, to synergistically regulate the expression of key drought-related genes, such as OsbZIP23. Moreover, we found that OsERF103 recruits a Su(var)3-9,enhancer of zeste and trithorax-domain group protein 705, which encodes a histone 3 lysine 4 (H3K4)-specific methyltransferase to specifically affect the deposition of H3K4me3 at loci like OsbZIP23 and other genes linked to dehydration responses. Additionally, the natural alleles of OsERF103 are selected during the domestication of both indica and japonica rice varieties and exhibit significant geographic distribution. Collectively, our findings have unfurled a comprehensive mechanistic framework underlying the OsERF103-mediated cascade regulation of drought response. This discovery not only enhances our understanding of drought signaling but also presents a promising avenue for the genetic improvement of drought-tolerant rice cultivars.

Propofol attenuates renal ischemia/reperfusion injury by regulating the MALAT1/miR-126-5p axis.

BACKGROUND: Propofol (PPF) plays a protective role in ischemia-reperfusion (I/R) in multiple organs, including renal ischemia-reperfusion injury (RIRI). The present study aimed to investigate the underlying mechanisms by which PPF exerts its protective functions in RIRI. METHODS: BALB/c mice were employed for the construction of RIRI animal model. PPF pre-treatment was carried out before I/R. An in vitro I/R model was established with HK-2 cells after hypoxia/reoxygenation (H/R) culture, and PPF was utilized to treat the cells before H/R. A quantitative-polymerase chain reaction (qPCR) was conducted to detect long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and miR-126-5p expression levels. Flow cytometry was adopted to detect the apoptosis of HK-2 cells. Bioinformatics analysis, qPCR, a luciferase reporter gene experiment and a RNA immunoprecipitation experiment were used to determine the regulatory relationship between MALAT1 and miR-126-5p. The expression level of vascular endothelial growth factor A (VEGFA) was examined by western blotting. RESULTS: MALAT1 expression was augmented and miR-126-5p was decreased in RIRI models. PPF pre-treatment remarkably reduced creatinine and urea nitrogen levels in the serum of BALB/c mice with RIRI, and diminished the apoptosis of HK-2 cells treated with H/R. In addition, PPF pre-treatment markedly restrained the expression of MALAT1 in both in vivo and in vitro models and up-regulated miR-126-5p expression. MALAT1 could adsorb miR-126-5p to repress it and up-regulate VEGFA. MALAT1 overexpression reversed the protective effects of PPF on RIRI. CONCLUSIONS: PPF protects the kidney against RIRI by inhibiting MALAT1 and up-regulating miR-126-5p expression, as well as indirectly inhibiting the expression of VEGFA.

Single-Point Mutant Inverts the Stereoselectivity of a Carbonyl Reductase toward ß-Ketoesters with Enhanced Activity.

Enzyme stereoselectivity control is still a major challenge. To gain insight into the molecular basis of enzyme stereo-recognition and expand the source of antiPrelog carbonyl reductase toward ß-ketoesters, rational enzyme design aiming at stereoselectivity inversion was performed. The designed variant Q139G switched the enzyme stereoselectivity toward ß-ketoesters from Prelog to antiPrelog, providing corresponding alcohols in high enantiomeric purity (89.1-99.1 % ee). More importantly, the well-known trade-off between stereoselectivity and activity was not found. Q139G exhibited higher catalytic activity than the wildtype enzyme, the enhancement of the catalytic efficiency (kcat /Km ) varied from 1.1- to 27.1-fold. Interestingly, the mutant Q139G did not lead to reversed stereoselectivity toward aromatic ketones. Analysis of enzyme-substrate complexes showed that the structural flexibility of ß-ketoesters and a newly formed cave together facilitated the formation of the antiPrelog-preferred conformation. In contrast, the relatively large and rigid structure of the aromatic ketones prevents them from forming the antiPrelog-preferred conformation.

miR-485-5p suppresses Schwann cell proliferation and myelination by targeting cdc42 and Rac1.

Schwann cells, a crucial element in peripheral nervous system, play important roles after peripheral nerve injury. In recent years, the role of miR-485-5p has been discovered in neurological diseases. However, the involvement of miR-485-5p and peripheral nerve injury remains unknown. Mice were subjected to sciatic nerve crush to mimic peripheral nerve injury and the expression of miR-485-5p was detected in sciatic nerve stumps by real-time PCR. BrdU assay was used to analyze the proliferation of Schwann cells after transfection with miR-485-5p mimic and miR-485-5p inhibitor. The effect of miR-485-5p on Schwann cell myelination was determined by evaluating levels of cyclic adenosine monophosphate (cAMP)-induced myelin-associated proteins, including Krox20 and MBP, as well as the coculture of Schwann cells and dorsal root ganglion (DRG) neurons via immunostaining with anti-MBP antibodies. The regulation mechanism of miR-485-5p was measured by bioinformatics analysis, luciferase reporter assay, and real-time PCR and Western blot. We found miR-485-5p expression was downregulated post nerve injury. miR-485-5p mimic significantly suppressed the proliferation and cAMP-induced expression levels of Krox20 and MBP in Schwann cells. Conversely, miR-485-5p inhibitor promoted these changes in Schwann cells. Also, miR-485-5p inhibitor elevated MBP-positive myelinated fibers. Cdc42 and Rac1 are targets of miR-485-5p in Schwann cells. Downregulation of cdc42 reversed the effect of miR-485-5p inhibitor on the proliferation of Schwann cells. And reducing Rac1 expression attenuated the effect of miR-485-5p silencing on Schwann cell myelination. In conclusion, this study indicated that miR-485-5p suppressed the proliferation and myelination of Schwann cells via targeting cdc42 and Rac1. Which may provide a novel method for the treatment of peripheral nerve injury.

Long noncoding RNA NEAT1 promotes the growth of human retinoblastoma cells via regulation of miR-204/CXCR4 axis.

Retinoblastoma (RB) is an aggressive eye cancer of infancy and childhood with high mortality. Studies have shown that long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) is closely related to the progression of multiple cancers. However, its role in RB remains unknown. This study aimed to investigate the role and underlying mechanism of NEAT1 in RB. We first detected the expression of NEAT1 in human RB tissues and cell lines. The effects of NEAT1 on the proliferation, migration, and apoptosis of RB cells were analyzed by loss-of-function. The underlying mechanism of NEAT1 in RB was mainly focused on the microRNA 204/C-X-C chemokine receptor type 4 (miR-204/CXCR4) axis. In addition, the role and mechanism of NEAT1 in RB were further evaluated in a mouse xenograft tumor model. We found NEAT1 and CXCR4 expression levels were elevated, whereas miR-204 expression was decreased in RB tissues and cells. Downregulation of NEAT1 significantly decreased the proliferation and migration but promoted the apoptosis of RB cells. NEAT1 functioned as a competing endogenous RNA for miR-204 to regulate CXCR4 expression. Knockdown of NEAT1 suppressed the tumor volume, tumor weight, and CXCR4 expression, whereas increased miR-204 expression in mice. In conclusion, NEAT1 promotes the development of RB via miR-204/CXCR4 axis, which provides a new target for the treatment of RB disease.

Association between serum zinc levels and lung cancer: a meta-analysis of observational studies.

BACKGROUND: Inconsistent results according to numerous studies that had investigated the association between serum zinc levels and lung cancer risk were reported. The aim of this study was to explore whether serum zinc levels were lower in lung cancer patients than that in controls. METHODS: We systematically retrieved the databases of PubMed, Wanfang, Cochrane, ScienceDirect website, CNKI, and SinoMed databases for comprehensive relevant studies published before December 2018 and conducted a meta-analysis. Standard mean differences (SMD) were pooled using a random effects model. RESULTS: Thirty-two articles were eligible to investigate the correlation between serum zinc levels and lung cancer risk, involving 2894 cases and 9419 controls. The pooled results showed sufficient evidence approving the association between serum zinc levels and lung cancer risk. And the serum zinc levels in lung cancer were significantly lower than that in controls (summary SMD = - 0.88, 95% confidence interval (CI) = - 0.94, - 0.82). Meanwhile, consistent results were obtained both in European populations and Asian populations. No publication bias was detected in our analysis. CONCLUSIONS: The present meta-analysis suggested that serum zinc levels were significantly lower in lung cancer patients than that in controls.

Directed evolution of mandelate racemase by a novel high-throughput screening method.

Optically pure methyl (R)-o-chloromandelate and (R)-acetyl-o-mandelic acid are key intermediates for the synthesis of (S)-clopidogrel, which could be prepared with 100 % theoretical yield by sequential hydrolysis and racemization. At the moment, efficient sequential hydrolysis and racemization are hindered by the low catalytic activity of mandelate racemase (MR) toward (S)-o-chloromandelic acid ((S)-2-CMA). In the present work, we proposed to improve the catalytic performance of MR toward (S)-2-CMA by directed evolution and developed an enantioselective oxidation system for high-throughput screening (HTS) of MR libraries. Based on this HTS method, a triple mutant V22I/V29I/Y54F (MRDE1) with 3.5-fold greater relative activity as compared to the native MR was obtained. Kinetic analysis indicated that the enhanced catalytic efficiency mainly arose from the elevated k cat. Further insight into the source of improved catalytic activity was gained by molecular simulations, finding that substrate binding and product release were possibly made easier by decreased steric bulk and increased hydrophobicity of substrate binding sites. In addition, the substrate (S)-2-CMA in the enzyme-substrate complex of MRDE1 seemed to have a lower binding free energy comparing with the complex of wild-type MR. The HTS method developed in this work and the successful directed evolution of MR based on this method provide an example for racemase engineering and may inspire directed evolution of other racemases toward enhanced catalytic performance on non-natural substrates.

Identification and elimination of metabolic bottlenecks in the quinone modification pathway for enhanced coenzyme Q10 production in Rhodobacter sphaeroides.

In this report, UbiE and UbiH in the quinone modification pathway (QMP) were identified in addition to UbiG as bottleneck enzymes in the CoQ10 biosynthesis by Rhodobacter sphaeroides. The CoQ10 content was enhanced after co-overexpression of UbiE and UbiG, however, accompanied by the accumulation of the intermediate 10P-MMBQ. UbiH was then co-overexpressed to pull the metabolic flux towards downstream, resulting in an elevated CoQ10 productivity and decreased biomass. On the other hand, the expression levels of UbiE and UbiG were tuned to eliminate the intermediate accumulation, however at the sacrifice of productivity. To alleviate the detrimental effect on either productivity or cell growth, we tried to fuse UbiG with UbiE and localize them onto the membrane to elevate intermediate conversion. By fusing UbiE and UbiG to pufX, CoQ10 was accumulated to 108.51±2.76mg/L with a biomass of 12.2±0.9g/L. At last, we combined the optimized QMP and the previously engineered 2-methyl-d-erythritol-4-phosphate pathway (MEP) to further boost CoQ10 biosynthesis, resulting in a strain with 138±2.64mg/L CoQ10 production.

Biocatalysis of CO2 and CH4: Key enzymes and challenges.

Mitigating greenhouse gas emissions is a critical challenge for promoting global sustainability. The utilization of CO2 and CH4 as substrates for the production of valuable products offers a promising avenue for establishing an eco-friendly economy. Biocatalysis, a sustainable process utilizing enzymes to facilitate biochemical reactions, plays a significant role in upcycling greenhouse gases. This review provides a comprehensive overview of the enzymes and associated reactions involved in the biocatalytic conversion of CO2 and CH4. Furthermore, the challenges facing the field are discussed, paving the way for future research directions focused on developing robust enzymes and systems for the efficient fixation of CO2 and CH4.

An AP2/ERF transcription factor confers chilling tolerance in rice.

Cold stress, a prominent adverse environmental factor, severely hinders rice growth and productivity. Unraveling the complex mechanisms governing chilling tolerance in rice is crucial for molecular breeding of cold-tolerant varieties. Here, we identify an APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factor, OsERF52, as a positive modulator in response to low temperatures. OsERF52 directly regulates the expression of C-Repeat Binding Factor (CBF) genes in rice. In addition, Osmotic Stress/ABA-Activated Protein Kinase 9-mediated phosphorylation of OsERF52 at S261 enhances its stability and interaction with Ideal Plant Architecture 1 and OsbHLH002/OsICE1. This collaborative activation leads to the expression of OsCBFs, thereby initiating the chilling response in rice. Notably, plants with base-edited OsERF52S261D-3HA exhibit enhanced chilling resistance without yield penalty. Our findings unveil the mechanism orchestrated by a regulatory framework involving a protein kinase and transcription factors from diverse families, offering potential genetic resources for developing chilling-tolerant rice varieties.

Immunoactivation by Cutaneous Blue Light Irradiation Inhibits Remote Tumor Growth and Metastasis.

An improved innate immunity will respond quickly to pathogens and initiate efficient adaptive immune responses. However, up to now, there have been limited clinical ways for effective and rapid consolidation of innate immunity. Here, we report that cutaneous irradiation with blue light of 450 nm rapidly stimulates the innate immunity through cell endogenous reactive oxygen species (ROS) regulation in a noninvasive way. The iron porphyrin-containing proteins, mitochondrial cytochrome c (Cyt-c), and cytochrome p450 (CYP450) can be mobilized by blue light, which boosts electron transport and ROS production in epidermal and dermal tissues. As a messenger of innate immune activation, the increased level of ROS activates the NF-κB signaling pathway and promotes the secretion of immunomodulatory cytokines in skin. Initiated from skin, a regulatory network composed of cytokines and immune cells is established through the circulation system for innate immune activation. The innate immunity activated by whole-body blue light irradiation inhibits tumor growth and metastasis by increasing the infiltration of antitumor neutrophils and tumor-associated macrophages. Our results elucidate the remote immune modulation mechanism of blue light and provide a clinically applicable way for innate immunity activation.

Biocatalytic synthesis of chiral intermediate of pregabalin with high substrate loading by a newly isolated Morgarella morganii ZJB-09203.

The chemoenzymatic process involving biocatalytic resolution of rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE, 1) has been the most competitive and attractive route for pregabalin. A new esterase-producing strain ZJB-09203, which exhibited high hydrolytic activity, excellent enantioselectivity, and diastereoselectivity towards CNDE, has been successfully isolated from soil samples with a pH indicator agar plate method. The isolate was identified as Morgarella morganii by the ATB system (ID 32 GN) and the 16S rDNA sequence. In order to suppress product inhibition during enzymatic hydrolysis of CNDE, an adsorptive biocatalytic process was developed by utilizing anion-exchange resin D201 as adsorbent for selective removal of (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid (2) from the reaction medium. This approach allowed the substrate loading to be increased up to 1.5 M and the chiral intermediate 2 was produced in 682 mM, 45.3 % conversion, and 95 % ee. These results imply that M. morganii ZJB-09203 esterase is a promising biocatalyst in the development of chemenzymatic manufacturing process for pregabalin.

Combining Protein and Organelle Engineering for Linalool Overproduction in Saccharomyces cerevisiae.

Linalool, a plant-derived high-value monoterpene, is widely used in the perfume, cosmetic, and pharmaceutical industries. Recently, engineering microbes to produce linalool has become an attractive alternative to plant extraction or chemical synthesis approaches. However, the low catalytic activity of linalool synthase and the shortage of precursor pools have been considered as two key factors for low yields of linalool. In this study, we rationally engineered the entrance of the substrate-binding pocket of linalool synthase (t67OMcLISM) and successfully increased the catalytic efficiency of this enzyme toward geranyl pyrophosphate. Specifically, F447E and F447A, with decreased entrance hydrophobicity and steric hindrance, increased linalool production by 2.2 and 1.9 folds, respectively. Subsequently, cytoplasm and peroxisomes were harnessed to boost linalool synthesis in Saccharomyces cerevisiae, achieving a high titer of linalool (219.1 mg/L) in shake-flask cultivation. Finally, the engineered diploid strain produced 2.6 g/L of linalool by 5 L fed-batch fermentation, which was the highest production in yeast to date. The protein engineering and biosynthetic pathway compartmentalization in the peroxisome provide references for the microbial production of other monoterpenes.

Comparison of immunogenicity and porcine-to-rhesus lamellar corneal xenografts survival between fresh preserved and dehydrated porcine corneas.

BACKGROUND: To compare the immunogenicity of fresh preserved and dehydrated lamellar porcine corneas in porcine-to-mouse heterotopic transplantation and to investigate the survival of preserved porcine corneas as xenografts in porcine-to-rhesus lamellar corneal transplantation. METHODS: Dehydrated and fresh preserved, endothelium deprived, porcine corneas were cut into fragments and grafted beneath the kidney capsule of BALB/c mice. Porcine-specific delayed type hypersensitivity (DTH) and antibody (IgM, IgG) immune responses of the recipient mice were assessed. In addition, fresh preserved and dehydrated porcine corneas were used in porcine-to-rhesus lamellar corneal xenotransplantation. The rhesus recipients were divided into three groups. Dehydrated corneas were applied to Group 1 and 3, and fresh preserved corneas were applied to Group 2. Only Group 3 received subconjunctival injections with triamcinolone acetonide for 1 month. All xenografts were evaluated by slit-lamp microscopy for 6 months. Two recipients in each group were examined by in vivo confocal microscopy and then killed for corneal histopathological staining 3 months after surgery. RESULTS: Neither fresh preserved nor dehydrated corneal fragments evoked any measurable change in mice recipient humoral immune status, but both could induce porcine-specific DTH at 1, 2, and 4 week after being grafted. However, the intensity of the DTH responses evoked by dehydrated corneas was lower than that evoked by fresh preserved corneas. In porcine-to-rhesus lamellar keratoplasty, with the exception one graft in Group 2 that developed characteristics of rejection, all the xenografts remained transparent or translucent up to 6 month after surgery. Histopathological examination of Group 1 showed that the infratemporal xenografts were much thicker and displayed some inflammatory cell infiltration in the peripheral portion of the graft and bed interface. However, in Group 3, which was treated with triamcinolone acetonide, there was an easily identified scar at the lamellar interface with no inflammatory cells present. In the rejected graft in Group 2, infiltrating cells included a few eosinophils and massive lymphocytes. Confocal microscopy examination showed that activated keratocytes localized in the anterior stroma and highly reflective tissue at the interface of the graft and bed. CONCLUSIONS: Porcine corneas might be an ideal source for clinical lamellar corneal xenotransplantation. In cases of tectonic lamellar transplantation, the possibility to use dehydrated pig material may become an option in the future.

Long Noncoding RNA TRPM2-AS Promotes the Growth, Migration, and Invasion of Retinoblastoma via miR-497/WEE1 Axis.

Long noncoding RNAs (lncRNAs) exhibit vital roles in many types of cancer, including retinoblastoma (RB), the most common primary intraocular malignancy tumor of infancy. A novel lncRNA TRPM2-AS has been demonstrated to be related to multiple cancers; however, its role in RB remains unclear. Here, we aimed to investigate the function of TRPM2-AS in RB. In this study, TRPM2-AS expression in 35 human RB tissues and RB cell lines was detected by real-time PCR. And, the relationship between its expression and clinicopathological characteristics of RB patients was analyzed. RB cells' proliferation, migration, invasion, apoptosis, and cell cycle were explored after silencing TRPM2-AS. The mechanism of TRPM2-AS in RB was focused on miR-497/WEE1 axis. Additionally, the role and mechanism of TRPM2-AS were confirmed in a xenograft mouse model. We found TRPM2-AS expression was enhanced in RB tissues and cells. And, higher TRPM2-AS expression was related to advanced clinical stage and optic nerve invasion in patients. Downregulation of TRPM2-AS significantly inhibited proliferation, migration, and invasion, elevated apoptosis, attenuated G2/M phase arrest in RB cells, and suppressed tumor growth in vivo. TRPM2-AS acted as a ceRNA for miR-497 to positively regulate WEE1 expression. miR-497 inhibitor or WEE1 overexpression dramatically reversed the effects of TRPM2-AS downregulating on the malignant phenotypes of RB cells. Therefore, TRPM2-AS is an oncogenic lncRNA in RB, and it functions largely through the miR-497/WEE1 pathway. Despite the limited sample size, this study indicates that TRPM2-AS may be a candidate target in RB therapies.

Blue light-triggered Fe2+-release from monodispersed ferrihydrite nanoparticles for cancer iron therapy.

Site-specific Fe2+ generation is promising for tumor therapy. Up to now, reported materials or systems for Fe2+ delivery do not naturally exist in the body, and their biological safety and toxicity are concerned. Herein, inspired by the natural biomineral ferrihydrite in ferritin, we synthesized monodispersed ferrihydrite nanoparticles and demonstrated a light triggered Fe2+ generation on tumor sites. Ferrihydrite nanoparticles of 20-30 nm in diameter possessed high cellular uptake efficiency and good biocompatibility. Under common blue light illumination, a large amount of Fe2+ could be released from ferrihydrite and promote the iron/reactive oxygen species (ROS)-related irreversible DNA fragmentation and glutathione peroxidase 4 (GPX4) inhibition, which led to the apoptosis- and ferroptosis-depended cancer cell proliferation inhibition. On mice, this method induced tumor associated macrophage (TAM) polarization from the tumor-promoting M2 type to the tumor-killing M1 type. With the intravenous pre-injection of ferrihydrite, the combinational effects of the light/Fe2+-approach attenuated pulmonary metastasis on mice. These results demonstrated a novel external light controlled Fe2+-generation approach based on biomineral, which will fully tap the anti-cancer potential of Fe2+ in chemo-dynamic, photo-dynamic and immune-activating therapies.

Ferrihydrite nanoparticles as the photosensitizer augment microbial infected wound healing with blue light.

Visible blue light exerts microbicidal effects with reduced deleterious effects compared with UV light. However, the lack of specific photosensitizers restricts the use of blue light on wound tissues. Here, we report the use of biomimetic ferrihydrite nanoparticles (Fhn) as the sensitizer to augment not only the antimicrobial but also the healing effects of blue light on S. aureus-infected wound tissue. Based on the excellent photo-Fenton active Fhn under blue light illumination (450 nm, 35 630 lux), the Fhn-sensitized blue-light therapy completely cured acute wound within 7 days in sessions of one hour per day and diminished bacterial and fungal colony-forming units more than 5 log (99.999%) and 2 log (99%) in vitro. Mechanistic studies revealed that hydroxyl radicals (˙OH) generated by the combined therapy could effectively damage the microbe genome and membranes without significant damage to wound tissues. Interestingly, these two naturally occurring nonantibiotic modalities (Fhn with blue light) significantly stimulate the angiogenesis and decrease the inflammatory response on the wound site, which accelerates the wound healing synergically. The results demonstrated the use of biomimetic Fhn as the general photosensitizer for enhanced antimicrobial, anti-inflammatory and wound healing effects of blue light-based therapy.

[Systematic review of penetrating keratoplasty rejection treated by immunosuppressants].

OBJECTIVE: To investigate the validity and side-effect of immunosuppressants for preventing and treating of immune rejection after penetrating keratoplasty (PKP). METHODS: Randomized and non-randomized controlled trials of immunosuppressants after PKP were searched from Pubmed, EMbase.com, Cochrane library, CNKI and Wanfang database; methodological quality and meta-analysis were carried out according to Evidence-Based Medicine(EBM). RESULTS: Thirty-one studies in all were evaluated, of which twenty-three were about the prevention, and nine were about the treatment after PKP. The rate of immune rejection after normal PKP is 4.9%-28.9% when using corticosteroids to prevent immune rejection, especially for long-time use. According to meta-analysis: the effectiveness of local cyclosporine A and local FK-506 in preventing immune rejection after PKP is significant, and FK-506 is more effective than CsA topically; systemic CsA and MMF could effectively prevent immune rejection after high-risk PKP; as far as treating immune rejection, corticosteroid, whether topical or systemic, was effective; however additional topical CsA could not improve the treatment effect. CONCLUSION: The use of immunosuppressants such as corticosteroids and CsA whether topical or systemic can effectively prevent the occurrence of immune rejection after high-risk PKP.

Long non-coding RNA CASC9 promotes the progression of retinoblastoma via interacting with miR-145-5p.

Abnormal expression of long non-coding RNA cancer susceptibility candidate 9 (CASC9) has been found to play vital roles in many human tumors. However, the role and the regulatory mechanism of CASC9 have not yet been demonstrated in retinoblastoma (RB). Hence, we performed this study to explore the function and mechanism of CASC9 in RB. CASC9 expression was firstly detected in human RB tissues and cells. The influence of CASC9 on the malignant phenotypes of RB cells, including cell proliferation, invasion, epithelial-mesenchymal transition (EMT) and apoptosis, was analyzed by overexpressing or silencing CASC9. The association between CASC9, miR-145-5p and E2F transcription factor 3 (E2F3) was determined by dual-luciferase reporter assay and RNA immunoprecipitation. We found that CASC9 expression was elevated in RB tissues and cells. Overexpression of CASC9 significantly facilitated the proliferation, invasion and EMT of RB cells. On the contrary, knockdown of CASC9 inhibited the proliferation, invasion and EMT, while enhanced the apoptosis of RB cells. CASC9 acted as a competing endogenous RNA (ceRNA) for miR-145-5p to regulate E2F3. Additionally, miR-145-5p inhibitor and E2F3 overexpression both partly reversed the malignant phenotypes of RB cells affected by CASC9 knockdown. However, miR-145-5p overexpression further strengthened these features induced by CASC9 downregulation. These findings suggested that CASC9 contributed to RB development by regulating E2F3 via sponging miR-145-5p. CASC9 might be a possible therapeutic target for RB.

Heterologous overexpression of a novel halohydrin dehalogenase from Pseudomonas pohangensis and modification of its enantioselectivity by semi-rational protein engineering.

A gene encoding a halohydrin dehalogenase from Pseudomonas pohangensis (PpHHDH) was identified, synthesized and expressed in Escherichia coli. Subsequently, we used protein engineering to enhance the enzyme's enantioselectivity. We created two enantiocomplementary HHDH mutants, N160L and Q159L, which exhibited higher S- and R-selectivity toward PGE, respectively. The exchange of Leu at 159 for Gln led to a 2.3-fold increase in enantioselectivity (E-value of 22.2) compared to the wild-type. In addition, the N160L mutant displayed an inverted enantioselectivity (from ER = 9.8 to ES = 21.6) toward PGE. The wild-type PpHHDH and its variants were purified and characterized. They all displayed maximum activity at pH 7.5. The optimum temperature of mutant Q159L and N160L was increased from 35 °C to 40 °C. The wild-type PpHHDH and N160L mutant had good pH stability at pH 5.0-7.5, and Q159L showed an even wider range of pH tolerance, from pH 4.5 to pH 8.0. The mutants N160L and Q159L showed slightly better thermostability than wild-type PpHHDH. For most tested substrates, the two variants showed higher enantioselectivity. These findings further confirmed the importance of amino acid residues at positions 159 and 160 for the enantioselectivity of PpHHDH.