Preclinical evaluation and phase 1 study of the PI3Kα/δ inhibitor TQ-B3525 in Chinese patients with advanced cancers.

BACKGROUND: Phosphatidylinositol 3-kinase (PI3K) inhibitors transformed management of various malignancies. This study preclinically characterized TQ-B3525 (dual PI3Kα/δ inhibitor) and assessed the recommended phase 2 dose (RP2D), safety, efficacy, and pharmacokinetics in relapsed or refractory (R/R) lymphoma or advanced solid tumors (STs). METHODS: Oral TQ-B3525 was given at eight dose levels on a 28-day cycle. Primary end points were dose-limiting toxicity (DLT), maximum tolerated dose (MTD), and safety. RESULTS: TQ-B3525 showed high selectivity and suppressed tumor growth. Between June 12, 2018, and November 18, 2020, 80 patients were enrolled (63 in dose-escalation cohort; 17 in dose-expansion cohort). Two DLTs occurred in two (two of 63, 3.2%) DLT-evaluable patients; MTD was not identified. TQ-B3525 at 20 mg once daily was selected as RP2D. Grade 3 or worse treatment-related adverse events mainly included hyperglycemia (16.3%), neutrophil count decreased (15.0%), and diarrhea (10.0%). Two (2.5%) treatment-related deaths were reported. Sixty patients with R/R lymphoma and 11 advanced STs demonstrated objective response rates of 68.3% and 9.1%, disease control rates of 91.7% and 54.6%, median progression-free survivals of 12.1 and 1.1 months; median overall survivals were not reached. CONCLUSION: TQ-B3525 exhibited rapid absorption and a nearly proportional increase in exposure. Acceptable safety and promising efficacy support further investigation of TQ-B3525 (20 mg once daily) for R/R lymphoma.

Selinexor With Anti-PD-1 Antibody as a Potentially Effective Regimen for Patients With Natural Killer/T-Cell Lymphoma Failing Prior L-Asparaginase and PD-1 Blockade.

BACKGROUND: Natural killer/T-cell lymphoma (NKTCL) is a rare and heterogeneous tumor type of non-Hodgkin's lymphoma (NHL) with a poor clinical outcome. There is no standardized salvage treatment failing l-asparaginase-based regimens. Here we report our retrospective results of the combined use of selinexor and PD-1 blockade (tislelizumab) in 5 patients with NKTCL who had exhausted almost all available treatments. PATIENTS AND METHODS: A total of 5 patients with relapsed/refractory(R/R) NK/T-cell lymphomas failing prior l-asparaginase and anti-PD-1 antibody were retrospectively collected. They were treated with at least one cycle of XPO1 inhibitor plus the same anti-PD-1 antibody. Anti-PD-1 antibody (Tislelizumab) was administrated at 200 mg on day 1 every 3 weeks and selinexor doses and schedules ranged from 40 mg weekly for 2 weeks per 21-day cycle to 60 mg weekly per cycle. RESULTS: Five patients with relapsed NKTCL with extensive organ involvement including 4 central nervous system (CNS) infiltration patients were included. Four patients achieved objective responses including 3 complete responses (CR) and 1 partial response (PR). After a median follow-up time of 14.5 (range, 5-22) months, 1 patient was still in remission with CR, and the other 4 patients discontinued due to disease progression with a median progression-free survival (PFS) of 6 months and median overall survival (OS) of 12 months. Four patients with CNS involvement achieved a median OS of 8 months. Our data suggest that selinexor in combination with an anti-PD-1 antibody is a promising small molecule and immunotherapy combination regimen for patients with relapsed or refractory NKTCL.

POU2F1/DNMT3a Pathway Participates in Neuropathic Pain by Hypermethylation-Mediated LRFN4 Downregulation Following Oxaliplatin Treatment.

Evidence demonstrates that DNA methylation is associated with the occurrence and development of various neurological diseases. However, the potential target genes undergoing DNA methylation, as well as their involvement in the chemotherapy drug oxaliplatin-induced neuropathic pain, are still unclear. Here, Lrfn4, which showed hypermethylation in the promoter regions, was screened from the SRA methylation database (PRJNA587622) following oxaliplatin treatment. MeDIP and qPCR assays identified that oxaliplatin treatment increased the methylation in Lrfn4 promoter region and decreased the expression of LRFN4 in the spinal dorsal horn. The assays with gain and loss of LRFN4 function demonstrated that LRFN4 downregulation in spinal dorsal horn contributed to the oxaliplatin-induced mechanical allodynia and cold hyperalgesia. Moreover, oxaliplatin treatment increased the DNA methyltransferases DNMT3a expression and the interaction between DNMT3a and Lrfn4 promoter, while inhibition of DNMT3a prevented the downregulation of LRFN4a induced by oxaliplatin. We also observed that the transcriptional factor POU2F1 can bind to the predicted sites in DNMT3a promoter region, oxaliplatin treatment upregulated the expression of transcriptional factor POU2F1 in dorsal horn neurons. Intrathecal injection of POU2F1 siRNA prevented the DNMT3a upregulation and the LRFN4 downregulation induced by oxaliplatin. Additionally, intrathecal injection of DNMT3a siRNA or POU2F1 siRNA alleviated the mechanical allodynia induced by oxaliplatin. These findings suggested that transcription factor POU2F1 upregulated the expression of DNMT3a, which subsequently decreased LRFN4 expression through hypermethylation modification in spinal dorsal horn, thereby mediating neuropathic pain following oxaliplatin treatment.

Induction with MEDA regimen and consolidation with Auto-HSCT for stage IV NKTCL patients: A prospective multicenter study.

Optimal treatment strategies for natural killer/T-cell lymphoma (NKTCL) patients with stage IV disease have not been well defined. In this prospective phase 2 study, we evaluated the treatment using MEDA (methotrexate, etoposide, dexamethasone and pegaspargase) as induction chemotherapy and autologous hematopoietic stem cell transplantation (Auto-HSCT) for consolidation. Patients with stage IV disease without prior L-asparaginase-based chemotherapy were eligible. Four cycles of MEDA were administered as induction treatment. Patients with complete response (CR, necessary to have complete metabolic remission of PET/CT, negative plasma EBV-DNA and negative EBER staining of bone marrow biopsy tissue) were consolidated by Auto-HSCT. A total of 53 patients were enrolled. The overall response (OR) rate and CR rate after four cycles of MEDA chemotherapy were 75.5% and 56.6%, respectively. Among them, 25 patients underwent Auto-HSCT. The 4-year overall survival (OS) rate and progression-free survival (PFS) rate were 58.0% (95% CI, 43.4%-70.0%) and 43.4% (95% CI, 29.9%-56.1%), respectively. Patients who underwent Auto-HSCT had a 4-year OS rate of 92.0% (95% CI, 71.6%-97.9%) and a 4-year PFS rate of 80.0% (95% CI, 58.4%-91.1%). Grade 3/4 neutropenia and thrombocytopenia occurred in 28.3% and 17.0% of the patients, respectively. MEDA chemotherapy is an effective induction regimen with reduced grade 3/4 hematological toxicities for stage IV NKTCL. Consolidation with Auto-HSCT can be considered as a potential approach to improve the long-term survival of CR patients after induction treatment.

GELAD chemotherapy with sandwiched radiotherapy for patients with newly diagnosed stage IE/IIE natural killer/T-cell lymphoma: a prospective multicentre study.

Early-stage natural killer/T-cell lymphoma (NK/TCL) patients usually receive a combination of chemotherapy and radiotherapy, but the optimal treatment approach has not yet been established. This study aimed to investigate the efficacy and safety profile of a novel chemotherapy regimen and sandwiched radiotherapy in early-stage NK/TCL. Patients with newly diagnosed stage IE/IIE disease were eligible. Patients were initially treated with two courses of the GELAD regimen (gemcitabine 1·0 g/m2 day 1, etoposide 60 mg/m2 days 1-3, pegaspargase 2000 units/m2 day 4, and dexamethasone 40 mg days 1-4), followed by intensity-modulated radiotherapy (IMRT; 50-56 Gy in 25-28 fractions) and two additional courses of GELAD chemotherapy. A total of 52 patients were enrolled. The overall response rate and complete response rate per Lugano 2014 criteria were 94·2% and 92·3% respectively. With a median follow-up of 32 months, the estimated four-year overall survival rate and progression-free survival rate were 94·2% [95% confidence interval (CI), 83·2% to 93·1%] and 90·4% (95% CI, 78·4% to 95·9%) respectively. The most common adverse events were related to pegaspargase. Haematological toxicities were mild, with grade 3/4 neutropenia in 15·4% of patients. Our study provides a new approach with high activity and improved safety for the treatment of early-stage NK/TCL patients. This study was registered at www.clinicaltrials.gov as NCT02733458.

Isolating an active and inactive CACTA transposon from lettuce color mutants and characterizing their family.

Dietary flavonoids play an important role in human nutrition and health. Flavonoid biosynthesis genes have recently been identified in lettuce (Lactuca sativa); however, few mutants have been characterized. We now report the causative mutations in Green Super Lettuce (GSL), a natural light green mutant derived from red cultivar NAR; and GSL-Dark Green (GSL-DG), an olive-green natural derivative of GSL. GSL harbors CACTA 1 (LsC1), a 3.9-kb active nonautonomous CACTA superfamily transposon inserted in the 5' untranslated region of anthocyanidin synthase (ANS), a gene coding for a key enzyme in anthocyanin biosynthesis. Both terminal inverted repeats (TIRs) of this transposon were intact, enabling somatic excision of the mobile element, which led to the restoration of ANS expression and the accumulation of red anthocyanins in sectors on otherwise green leaves. GSL-DG harbors CACTA 2 (LsC2), a 1.1-kb truncated copy of LsC1 that lacks one of the TIRs, rendering the transposon inactive. RNA-sequencing and reverse transcription quantitative PCR of NAR, GSL, and GSL-DG indicated the relative expression level of ANS was strongly influenced by the transposon insertions. Analysis of flavonoid content indicated leaf cyanidin levels correlated positively with ANS expression. Bioinformatic analysis of the cv Salinas lettuce reference genome led to the discovery and characterization of an LsC1 transposon family with a putative transposon copy number greater than 1,700. Homologs of tnpA and tnpD, the genes encoding two proteins necessary for activation of transposition of CACTA elements, were also identified in the lettuce genome.

Non-vernalization requirement in Chinese kale caused by loss of BoFLC and low expressions of its paralogs.

KEY MESSAGE: We identified the loss of BoFLC gene as the cause of non-vernalization requirement in B. oleracea. Our developed codominant marker of BoFLC gene can be used for breeding program of B. oleracea crops. Many species of the Brassicaceae family, including some Brassica crops, require vernalization to avoid pre-winter flowering. Vernalization is an unfavorable trait for Chinese kale (Brassica oleracea var. chinensis Lei), a stem vegetable, and therefore it has been lost during its domestication/breeding process. To reveal the genetics of vernalization variation, we constructed an F2 population through crossing a Chinese kale (a non-vernalization crop) with a kale (a vernalization crop). Using bulked segregant analysis (BSA) and RNA-seq, we identified one major quantitative trait locus (QTL) controlling vernalization and fine-mapped it to a region spanning 80 kb. Synteny analysis and PCR-based sequencing results revealed that compared to that of the kale parent, the candidate region of the Chinese kale parent lost a 9,325-bp fragment containing FLC homolog (BoFLC). In addition to the BoFLC gene, there are four other FLC homologs in the genome of B. oleracea, including Bo3g005470, Bo3g024250, Bo9g173370, and Bo9g173400. The qPCR analysis showed that the BoFLC had the highest expression among the five members of the FLC family. Considering the low expression levels of the four paralogs of BoFLC, we speculate that its paralogs cannot compensate the function of the lost BoFLC, therefore the presence/absence (PA) polymorphism of BoFLC determines the vernalization variation. Based on the PA polymorphism of BoFLC, we designed a codominant marker for the vernalization trait, which can be used for breeding programs of B. oleracea crops.

Rituximab plus cladribine versus R-CHOP in frontline management of marginal zone lymphoma in China: a propensity-score matched multicenter study.

Marginal zone lymphoma (MZL) is an uncommon subtype of non-Hodgkin lymphoma (NHL). Combination of rituximab and cladribine (R-2CdA) is a potential option for indolent NHL (iNHL) and mantle cell lymphoma (MCL) patients. The goal of this multicenter retrospective study was to assess the efficacy and safety of R-2CdA in MZL to support consensus-reaching in first-line therapy in advanced-stage patients. We searched electronic medical records databases of eight centers in China. Between November 2014 and December 2019, 183 symptomatic advanced MZL patients (42 treated with R-2CdA and 141 with rituximab plus cyclophosphamide, adriamycin, vincristine, and prednisone [R-CHOP]) were identified. After propensity score matching (PSM) (1:1) to adjust for clinical characteristics, 39 patients from each treatment arm were selected. The overall response rate (ORR) (84.6% vs. 94.9%, P = 0.263) and complete response rate (59.0% vs. 66.7%, P = 0.487) were comparable between two protocols. Neither progression-free survival (PFS), including the 5-year PFS (67.7% vs. 56.1%, P = 0.352), nor overall survival was improved by R-2CdA versus R-CHOP. However, R-2CdA was more tolerable than R-CHOP in MZL patients regarding grade 3/4 hematological adverse events (odds ratio [OR] 0.565, 95% confidence interval [CI] neutropenic fever (OR 0.795, 95% CI 0.678-0.932), and infections (OR 0.800, 95% CI 0.640-1.000). Overall, our study demonstrated that R-2CdA is potentially as effective as but safer than R-CHOP in advanced MZL.

Ketoconazole 2% cream alters the skin fungal microbiome in seborrhoeic dermatitis: a cohort study.

BACKGROUND: Seborrhoeic dermatitis (SD) is a common chronic inflammatory dermatosis. Current theories on the pathogenesis of SD highlight the role of microbes on the skin surface. Ketoconazole is commonly used for the treatment of SD; however, there are limited data focusing on the effects of ketoconazole in shaping the skin microbiome in patients with SD. AIM: In this prospective cohort study, we used a high-throughput DNA sequencing method to characterize the cutaneous microbial communities of patients with SD before and after topical ketoconazole treatment. METHODS: In total, 30 patients with facial SD and 15 age- and sex-matched healthy controls (HCs) were enrolled in this study. Skin swabs were collected from SD lesional sites of the cheek at baseline, after ketoconazole treatment and 2 weeks post-treatment. DNA was extracted from skin samples. The bacterial 16S V3V4 rRNA and fungal internal transcribed spacer 1-5F regions were sequenced, and the microbial community compositions were analysed. RESULTS: Significantly lower bacterial and fungal diversities were detected at the lesional sites of facial SD compared with HCs. A decreased relative abundance of Cutibacterium and increased abundances of Malassezia and Staphylococcus were found in facial SD. Disease diversity was positively correlated with the relative abundances of Malassezia, Staphylococcus and Corynebacterium, while transepidermal water loss was negatively associated with the relative abundance of Cutibacterium. After ketoconazole treatment, fungal Shannon diversity and the relative abundances of Candida and Aspergillus were significantly increased at the lesional sites, and the relative abundance of Malassezia showed a decreasing trend. These changing trends were maintained until 2 weeks post-treatment. CONCLUSION: Facial SD showed lower fungal diversity accompanied by increased relative abundances of Malassezia and Staphylococcus and decreased relative abundance of Cutibacterium. Ketoconazole treatment reduced Malassezia and increased fungal diversity to restore skin microbial communities.

Dysbiosis of skin mycobiome in atopic dermatitis.

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease with an increasing prevalence worldwide. The aetiology and pathogenesis of AD have not been fully elucidated. Previous studies have suggested the role of fungi as a triggering factor in the development AD. Here we conducted a systematic review to investigate the skin mycobiome profiles in AD and to address whether there is an association between fungal dysbiosis and AD. We searched Medline/PubMed, Embase and Web of Science for research studies published in English between January 1st, 2010 and April 21st, 2021. A total of 11 human studies and 3 animal studies were included in this analysis. Fungal dysbiosis was observed in AD lesions with a depleted amount of Malassezia and a higher abundance of filamentous fungi. A positive correlation between Candida and Staphylococcus was also demonstrated in AD. We supposed that specific species of Malassezia spp. and Candida spp. may play a role in the pathogenesis of AD by interacting with the pathogenic bacteria. Topical application of emollients could improve the skin barrier function and restore the skin fungal flora by increasing the amount of Malassezia. Further studies focusing on the complex interplay between specific skin fungi and the host can provide better insight into the role of microorganisms in the pathogenesis of AD.

Altered skin fungal and bacterial community compositions in tinea capitis.

BACKGROUND: Tinea capitis is an infection of the scalp and hair shaft caused by dermatophytes that predominantly occurs in children. Skin fungal infections have been found to be associated with alterations in the overall bacterial and fungal communities. However, the scalp microbiome in tinea capitis have not been fully investigated. OBJECTIVES: To investigate and compare the scalp bacterial and fungal microbiomes between children with tinea capitis and healthy children and between children and adults. METHODS: Skin samples were collected from the scalp. Bacterial and fungal community compositions were analysed by amplification sequencing of the V3-V4 of 16S rDNA and ITS1-5F, respectively. RESULTS: The predominant fungi detected using amplicon sequencing were consistent with the culture- or real-time PCR-positive pathogens in most samples. Children with tinea capitis had lower fungal and higher bacterial Shannon diversity than healthy children. A higher relative abundance of pathogenic fungi and significant alterations in the bacterial community in the lesional sites of tinea capitis than healthy scalps. Compared with adults, healthy children were characterised by higher Shannon diversities with significantly lower relative abundances of Malassezia and Cutibacterium and higher relative abundances of Candida and Streptococcus. CONCLUSIONS: We demonstrated that tinea capitis was characterised by significant alterations in both fungal and bacterial communities and amplicon sequencing could be a complementary method for pathogen identification.

Skin microbiome alterations in seborrheic dermatitis and dandruff: A systematic review.

Seborrheic dermatitis (SD) and dandruff (DF) are common chronic inflammatory skin diseases characterized by recurrent greasy scales, sometimes with erythema and itchiness. Although the exact pathophysiology of the disease is still unclear, current theories highlight the role of microbes on the skin surface in the pathogenesis of SD. Here, we conducted a systematic review to investigate the skin microbiome alterations in patients with SD/DF. We searched Medline/PubMed, Embase and Web of Science for research studies published in English between 1 January 2000 and 31 December 2020. A total of 12 studies with 706 SD/DF samples and 379 healthy samples were included in this study. The scalp and face were predominated by the fungi of Ascomycota and Basidiomycota and the bacteria of Actinobacteria and Firmicutes. In general, the included studies demonstrated an increased Malassezia restricta/Malassezia globosa ratio and a reduction in the Cutibaterium/Staphylococcus ratio in the setting of SD/DF. Staphylococcus was associated with epidermal barrier damage, including elevated levels of trans-epidermal water loss and pH, while Cutibacterium had a positive correlation with water content. Malassezia was also found to be related to an increased itching score and disease severity. Further studies focusing on the interactions between various microbes and the host and microbes can help us to better understand the pathogenesis of SD/DF.

Downregulation of T7 RNA polymerase transcription enhances pET-based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis.

Escherichia coli BL21 (DE3) is an excellent and widely used host for recombinant protein production. Many variant hosts were developed from BL21 (DE3), but improving the expression of specific proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), a significant industrial enzyme, severe cell autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other recombinant proteins. This precludes a further increase of the produced enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial enzyme production costs. Analysis of membrane structure and messenger RNA expression analysis showed that cells could underwent a form of programmed cell death (PCD) during the autolysis period. However, blocking three known PCD pathways in BL21 (DE3) did not completely alleviate autolysis completely. Consequently, we attempted to develop a strong expression host resistant to autolysis by controlling the speed of recombinant protein expression. To find a more suitable protein expression rate, the high- and low-strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5-1A and lac-1G. The results showed that only one base in lac promoter needs to be changed, and the A at the +1 position was changed to a G, resulting in the improved host BL21 (DE3-lac1G), which resistant to autolysis. As a consequence, the GDH activity at 43 h was greatly increased from 37.5 to 452.0 U/ml. In scale-up fermentation, the new host was able to produce the model enzyme with a high rate of 89.55 U/ml/h at 43 h, compared to only 3 U/ml/h achieved using BL21 (DE3). Importantly, BL21 (DE3-lac1G) also successfully improved the production of 10 other enzymes. The engineered E. coli strain constructed in this study conveniently optimizes recombinant protein overexpression by suppressing cell autolysis, and shows great potential for industrial applications.

Mirabegron in medical expulsive therapy for distal ureteral stones: a prospective, randomized, controlled study.

OBJECTIVE: To observe the efficacy and safety of Mirabegron in patients with distal, ureteral stones ≤ 10 mm. PATIENTS AND METHODS: A total of 90 patients with distal ureteral stones ≤ 10 mm were prospectively randomized into two groups. Forty-five cases in the study group and 45 cases as control. The stone-free rates (SFRs) and renal colic episodes between two groups were compared at the 1st, 2nd and 4th week end by imaging examinations. RESULT: All of 90 patients were randomly assigned to two groups. In patients with ≤ 5 mm stones, the SFRs in the 1st week (63.6% vs. 33.3%, P = 0.040), the 2nd week (86.4% vs. 54.2%, P = 0.018), and the 4th week (90.9% vs. 66.7%, P = 0.046) after treatment were all significantly higher than that in the control group by the stratification analysis of stone size. Even though SFRs were all higher for patients with > 5 mm stones in study group, there was no statistically significant difference (All P > 0.05). In terms of renal colic episodes, the frequency of occurrence of the study group was significantly lower than that of the control group and need less antalgic. CONCLUSIONS: The MET with Mirabegron has a significant role in improve SFR for the patients with distal ureteral stones ≤ 5 mm and no effect in > 5 mm stones. Furthermore, Mirabegron reduces the need for antalgic in ≤ 10 mm stones with low incidence of adverse effects.

Congruence about working alliance in Chinese context: The moderating effect of therapists' self-efficacy and the relationship between congruence and psychotherapy outcome.

The present study was conducted to (a) identify the clients and therapists' perceptual directional discrepancy and temporal congruence in different aspects of working alliance (i.e., goals/tasks and bond), (b) examine the moderating effect of therapists' client-specific self-efficacy on the directional discrepancy and congruence, (c) clarify the relationship between specific working alliance aspects congruence and the next-session symptom, and (d) test the relationship between the congruence of goals/tasks or bond and the psychotherapy outcome under different levels of therapists' self-efficacy. Clients (n = 87, 80.9% female, average age = 21.78 [1.90]) and therapists (n = 43, 65% female) in a Chinese university mental health center evaluated the working alliance session by session. Clients self-reported session-level symptom severity were assessed at the beginning of every session and therapists' self-efficacy for a specific client were assessed at the end of the first session. The truth-and-bias approach was used to analyze the perceptual directional discrepancy and temporal congruence in goals/tasks and bond, and examine the moderating effect of therapists' self-efficacy. Multilevel polynomial regression and response surface analysis were used to clear the relationship between congruence/incongruence and client symptom level in the next session. (a) Clients and therapists temporally agreed on both the goals/tasks and bond dimensions of the working alliance. Averagely, therapists tended to rate goals/tasks agreement lower than clients but did not rate more or less intense bond than clients. (b) Therapists with low or medium self-efficacy for specific client underrated goals/tasks and bond more than therapists with high client-specific efficacy, and among the therapist-client dyads, the higher therapists' client-specific self-efficacy, the higher temporal congruence in bond rather than goals/tasks. (c) For both goals/tasks and bond, when clients and therapists were in agreement, client symptoms decreased as the congruent combinations of therapists' and clients' goals/tasks increased. Clients' symptom level increased more sharply as the degree of discrepancy increases for goals/tasks, not affected by the direction. (d) However, for therapists with high client-specific self-efficacy, their underestimations were more associated with the less severe next-session symptoms of their clients than their overestimations. This association was not found among clients whose therapists' self-efficacies for them were low. The findings provide a deeper insight into the congruence of the working alliance. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Dectin-1 Contributes to Myocardial Ischemia/Reperfusion Injury by Regulating Macrophage Polarization and Neutrophil Infiltration.

BACKGROUND: Macrophage-associated immune response plays an important role in myocardial ischemia/reperfusion (IR) injury. Dectin-1, expressed mainly on activated myeloid cells, is crucial for the regulation of immune homeostasis as a pattern recognition receptor. However, its effects and roles during the myocardial IR injury remain unknown. METHODS: Genetic ablation, antibody blockade, or Dectin-1 activation, along with the adoptive bone marrow transfer chimeric model, was used to determine the functional significance of Dectin-1 in myocardial IR injury. Immune cell filtration and inflammation were examined by flow cytometry, quantitative real-time polymerase chain reaction, and immunohistochemistry. Moreover, Dectin-1+ cells were analyzed by flow cytometry in the blood of patients with ST-segment-elevation myocardial infarction and stable patients with normal coronary artery (control). RESULTS: We demonstrated that Dectin-1 expression observed on the bone marrow-derived macrophages is increased in the heart during the early phase after IR injury. Dectin-1 deficiency and antibody-mediated Dectin-1 inhibition led to a considerable improvement in cardiac function, accompanied by a reduction in cardiomyocyte apoptosis, which was associated with a decrease in M1 macrophage polarization and Ly-6C+ monocyte and neutrophil infiltration. Activation of Dectin-1 with its agonist had the opposite effects. Furthermore, Dectin-1 contributed to neutrophil recruitment through the regulation of Cxcl1 and granulocyte colony-stimulating factor expression. In addition, Dectin-1-dependent interleukin-23/interleukin-1ß production was shown to be essential for interleukin-17A expression by γδT cells, leading to neutrophil recruitment and myocardial IR injury. Furthermore, we demonstrated that circulating Dectin-1+CD14++CD16- and Dectin-1+CD14++CD16+ monocyte levels were significantly higher in patients with ST-segment-elevation myocardial infarction than in controls and positively correlated with the severity of cardiac dysfunction. CONCLUSIONS: Our results reveal a crucial role of Dectin-1 in the process of mouse myocardial IR injury and provide a new, clinically significant therapeutic target.

Characterization of four polymorphic genes controlling red leaf colour in lettuce that have undergone disruptive selection since domestication.

Anthocyanins protect plants from biotic and abiotic stressors and provide great health benefits to consumers. In this study, we cloned four genes (Red Lettuce Leaves 1 to 4: RLL1 to RLL4) that contribute to colour variations in lettuce. The RLL1 gene encodes a bHLH transcription factor, and a 5-bp deletion in some cultivars abolishes its function to activate the anthocyanin biosynthesis pathway. The RLL2 gene encodes an R2R3-MYB transcription factor, which was derived from a duplication followed by mutations in its promoter region. The RLL3 gene encodes an R2-MYB transcription factor, which down-regulates anthocyanin biosynthesis through competing with RLL2 for interaction with RLL1; a mis-sense mutation compromises the capacity of RLL3 to bind RLL1. The RLL4 gene encodes a WD-40 transcription factor, homologous to the RUP genes suppressing the UV-B signal transduction pathway in Arabidopsis; a mis-sense mutation in rll4 attenuates its suppressing function, leading to a high concentration of anthocyanins. Sequence analysis of the RLL1-RLL4 genes from wild and cultivated lettuce showed that their function-changing mutations occurred after domestication. The mutations in rll1 disrupt anthocyanin biosynthesis, while the mutations in RLL2, rll3 and rll4 activate anthocyanin biosynthesis, showing disruptive selection for leaf colour during domestication of lettuce. The characterization of multiple polymorphic genes in this study provides the necessary molecular resources for the rational breeding of lettuce cultivars with distinct levels of red pigments and green cultivars with high levels of health-promoting flavonoids.

Exosome-transferred hsa_circ_0014235 promotes DDP chemoresistance and deteriorates the development of non-small cell lung cancer by mediating the miR-520a-5p/CDK4 pathway.

BACKGROUND: Circular RNAs (circRNAs) play crucial roles in the development and progression of human cancers, including non-small cell lung cancer (NSCLC). However, most of these circRNAs, such as hsa_circ_0014235, are not fully identified in functions and mechanisms. METHODS: The isolated exosomes from serum specimens were identified using transmission electron microscopy (TEM). The expression of hsa_circ_0014235, miR-520a-5p and cyclin-dependent kinase 4 (CDK4) was detected by real-time quantitative polymerase chain reaction (qPCR). For functional assays, cell proliferation, colony formation ability, migration, invasion, cell apoptosis and cell cycle progression were determined using cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay, transwell assay and flow cytometry assay, respectively. The expression of CDK4 and other indicated marker proteins was detected by western blot. The predicted target relationship between miR-520a-5p and hsa_circ_0014235 or cyclin-dependent kinase 4 (CDK4) was verified by dual-luciferase reporter assay or RNA immunoprecipitation (RIP) assay. RESULTS: The expression of hsa_circ_0014235 was notably elevated in NSCLC serum-derived exosomes, tumor tissues and cells. NSCLC serum-derived exosomes promoted NSCLC cell resistance to cisplatin (DDP), cell proliferation, migration and invasion in vitro, as well as tumor growth and DDP resistance in vivo. Hsa_circ_0014235 overexpression enhanced DDP resistance and facilitated cell malignant behaviors. MiR-520a-5p was a target of hsa_circ_0014235, and rescue experiments showed that miR-520a-5p restoration reversed the effects of hsa_circ_0014235 overexpression. Moreover, CDK4 was a target of miR-520a-5p, and rescue experiments showed that CDK4 knockdown reversed the aggressive effects of miR-520a-5p inhibition on NSCLC progression. CONCLUSIONS: Exosome-transmitted hsa_circ_0014235 promoted NSCLC malignant development by mediating the miR-520a-5p/CDK4 regulatory axis.

Optimized strategy for in vivo Cas9-activation in Drosophila.

While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.

Dectin-2 Deficiency Modulates Th1 Differentiation and Improves Wound Healing After Myocardial Infarction.

RATIONALE: Macrophages are involved in wound healing after myocardial infarction (MI). The role of Dectin-2, a pattern recognition receptor mainly expressed on myeloid cells, in the infarct healing remains unknown. OBJECTIVE: The aim of this study is to determine whether Dectin-2 signaling is involved in the healing process and cardiac remodeling after MI and to elucidate the underlying molecular mechanisms. METHODS AND RESULTS: In a mouse model of permanent coronary ligation, Dectin-2, mainly expressed in macrophages, was shown to be increased in the early phase after MI. Dectin-2 knockout mice showed an improvement in the infarct healing and cardiac remodeling, compared with wild-type mice, which was demonstrated by significantly lower mortality because of cardiac rupture, increased wall thickness, and better cardiac function. Increased expression of α-smooth muscle actin and collagen I/III was observed, whereas the levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 were decreased in the hearts of Dectin-2 knockout mice after MI. Dectin-2 deficiency inhibited the rate of apoptotic and necrotic cell death. However, Dectin-2 did not affect immune cell infiltration and macrophage polarization, but it led to a stronger activation of the Th1/interferon-γ immune reaction, through the enhancement of interleukin-12 production in the heart. Interferon-γ was shown to downregulate transforming growth factor-ß-induced expression of α-smooth muscle actin and collagen I/III in isolated cardiac fibroblasts, leading to a decrease in migration and myofibroblast differentiation. Finally, Dectin-2 knockout improved myocardial ischemia-reperfusion injury and infarct healing. CONCLUSIONS: Dectin-2 leads to an increase in cardiac rupture, impairs wound healing, and aggravates cardiac remodeling after MI through the modulation of Th1 differentiation.