Kinome-wide siRNA screen identifies a DCLK2-TBK1 oncogenic signaling axis in clear cell renal cell carcinoma.

TANK-binding kinase 1 (TBK1) is a potential therapeutic target in multiple cancers, including clear cell renal cell carcinoma (ccRCC). However, targeting TBK1 in clinical practice is challenging. One approach to overcome this challenge would be to identify an upstream TBK1 regulator that could be targeted therapeutically in cancer specifically. In this study, we perform a kinome-wide small interfering RNA (siRNA) screen and identify doublecortin-like kinase 2 (DCLK2) as a TBK1 regulator in ccRCC. DCLK2 binds to and directly phosphorylates TBK1 on Ser172. Depletion of DCLK2 inhibits anchorage-independent colony growth and kidney tumorigenesis in orthotopic xenograft models. Conversely, overexpression of DCLK2203, a short isoform that predominates in ccRCC, promotes ccRCC cell growth and tumorigenesis in vivo. Mechanistically, DCLK2203 elicits its oncogenic signaling via TBK1 phosphorylation and activation. Taken together, these results suggest that DCLK2 is a TBK1 activator and potential therapeutic target for ccRCC.

Structural and Functional Insights into Host Death Domains Inactivation by the Bacterial Arginine GlcNAcyltransferase Effector.

Enteropathogenic E. coli NleB and related type III effectors catalyze arginine GlcNAcylation of death domain (DD) proteins to block host defense, but the underlying mechanism is unknown. Here we solve crystal structures of NleB alone and in complex with FADD-DD, UDP, and Mn2+ as well as NleB-GlcNAcylated DDs of TRADD and RIPK1. NleB adopts a GT-A fold with a unique helix-pair insertion to hold FADD-DD; the interface contacts explain the selectivity of NleB for certain DDs. The acceptor arginine is fixed into a cleft, in which Glu253 serves as a base to activate the guanidinium. Analyses of the enzyme-substrate complex and the product structures reveal an inverting sugar-transfer reaction and a detailed catalytic mechanism. These structural insights are validated by mutagenesis analyses of NleB-mediated GlcNAcylation in vitro and its function in mouse infection. Our study builds a structural framework for understanding of NleB-catalyzed arginine GlcNAcylation of host death domain.

USP13 promotes deubiquitination of ZHX2 and tumorigenesis in kidney cancer.

Clear cell renal cell carcinoma (ccRCC) is characterized by the loss of tumor suppressor Von Hippel Lindau (VHL) function. VHL is the component of an E3 ligase complex that promotes the ubiquitination and degradation of hypoxia inducible factor α (HIF-α) (including HIF1α and HIF2α) and Zinc Fingers And Homeoboxes 2 (ZHX2). Our recent research showed that ZHX2 contributed to ccRCC tumorigenesis in a HIF-independent manner. However, it is still unknown whether ZHX2 could be modified through deubiquitination even in the absence of pVHL. Here, we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and identified USP13 as a DUB that bound ZHX2 and promoted ZHX2 deubiquitination. As a result, USP13 promoted ZHX2 protein stability in an enzymatically dependent manner, and depletion of USP13 led to ZHX2 down-regulation in ccRCC. Functionally, USP13 depletion led to decreased cell proliferation measured by two-dimensional (2D) colony formation and three-dimensional (3D) anchorage-independent growth. Furthermore, USP13 was essential for ccRCC tumor growth in vivo, and the effect was partially mediated by its regulation on ZHX2. Our findings support that USP13 may be a key effector in ccRCC tumorigenesis.

Exploring causal correlations between circulating levels of cytokines and colorectal cancer risk: A Mendelian randomization analysis.

Colorectal cancer has the highest mortality rate of all digestive system diseases. Considering the debate about cytokines and biases that exist in traditional observational study designs, we performed a two-sample Mendelian randomization (MR) analysis to explore the association of circulating cytokines with CRC risk. In this study, we used cytokine genetic variants from a recently published genome-wide association study (GWAS) including 14,824 European-ancestry participants. Summary-level data for colorectal cancer were obtained from genome-wide association analyses of the FinnGen consortium. In addition, we conducted independent supplementary analyses using genetic variation data of colorectal cancer and cytokines from a large public GWAS in 2021. Among 91 circulating factors, we only found IL-12B to be significantly associated with CRC risk (odds ratio [OR]: 1.19; 95% confidence interval [CI]: 1.00-1.42; p = .046). We used 2021 data for analysis and found that higher Interleukin-12p70 levels (IL-12p70) were revealed to have a significant positive association with CRC risk (OR: 1.27; 95% CI: 1.13-1.43; p < 1.22 × 10-3). Moreover, CRC was suggestively correlated with an elevated level of vascular endothelial growth factor (VEGF) (OR: 1.17; 95% CI: 1.02-1.35; p = .026), macrophage colony-stimulating factor (M-CSF) (OR: 0.85; 95% CI: 0.76-0.96; p = .005), IL-13 (OR: 1.15; 95% CI: 1.02-1.30; p = .028), IL-10 (OR: 1.23; 95% CI: 1.01-1.49; p = .037), and IL-7 (OR: 1.19; 95% CI: 1.02-1.39; p = .024). Our MR studies support that one cytokine IL-12 is significantly associated with CRC risk and that five cytokines VEGF, M-CSF, IL-13, IL-10, and IL-7 are associated with CRC risk.

Development and Validation of a Post-Radiotherapy Prediction Model for Bowel Dysfunction After Rectal Cancer Resection.

BACKGROUND & AIMS: The benefit of radiotherapy for rectal cancer is based largely on a balance between a decrease in local recurrence and an increase in bowel dysfunction. Predicting postoperative disability is helpful for recovery plans and early intervention. We aimed to develop and validate a risk model to improve the prediction of major bowel dysfunction after restorative rectal cancer resection with neoadjuvant radiotherapy using perioperative features. METHODS: Eligible patients more than 1 year after restorative resection following radiotherapy were invited to complete the low anterior resection syndrome (LARS) score at 3 national hospitals in China. Clinical characteristics and imaging parameters were assessed with machine learning algorithms. The post-radiotherapy LARS prediction model (PORTLARS) was constructed by means of logistic regression on the basis of key factors with proportional weighs. The accuracy of the model for major LARS prediction was internally and externally validated. RESULTS: A total of 868 patients reported a mean LARS score of 28.4 after an average time of 4.7 years since surgery. Key predictors for major LARS included the length of distal rectum, anastomotic leakage, proximal colon of neorectum, and pathologic nodal stage. PORTLARS had a favorable area under the curve for predicting major LARS in the internal dataset (0.835; 95% CI, 0.800-0.870, n = 521) and external dataset (0.884; 95% CI, 0.848-0.921, n = 347). The model achieved both sensitivity and specificity >0.83 in the external validation. In addition, PORTLARS outperformed the preoperative LARS score for prediction of major events. CONCLUSIONS: PORTLARS could predict major bowel dysfunction after rectal cancer resection following radiotherapy with high accuracy and robustness. It may serve as a useful tool to identify patients who need additional support for long-term dysfunction in the early stage. CLINICALTRIALS: gov, number NCT05129215.

Intracorporeal versus Extracorporeal Anastomosis in Laparoscopic Right Hemicolectomy: An Updated Systematic Review and Meta-Analysis of Randomized Control Trials.

INTRODUCTION: Laparoscopic right hemicolectomy has become the standard surgical procedure for the treatment of right colon disease; however, the choice of anastomosis remains controversial. This study aimed to compare the safety and efficacy of intracorporeal anastomosis and extracorporeal anastomosis in laparoscopic right hemicolectomy. METHODS: A systematic literature search was performed in PubMed, Embase, Web of Science, and Cochrane Library. Randomized controlled trials that compared intracorporeal anastomosis with extracorporeal anastomosis in patients with laparoscopic right hemicolectomy until June 4, 2023, are selected. The primary outcomes measured were incidence of anastomotic leakage within 30 days post-operation. Statistical analyses were performed using Review Manager (version 5.4.1). RESULTS: Seven RCTs, including 720 patients, were eligible for the meta-analysis. The incidence of anastomotic leakage showed no significant difference between the intracorporeal anastomosis group and the extracorporeal anastomosis group (RR 0.93, 95% CI: 0.49, 1.76, p = 0.83, and I2 = 0%). However, the intracorporeal anastomosis group had significantly lower rates of postoperative ileus (RR 0.67, 95% CI: 0.45-0.99, p = 0.04, I2 = 46%) and surgical site infections (RR 0.34, 95% CI: 0.16-0.74, p = 0.007, I2 = 0%) compared to the extracorporeal anastomosis group. Additionally, patients in the intracorporeal anastomosis group experienced earlier postoperative passage of gas and stool (WMD -0.39, 95% CI: -0.60, -0.19, p = 0.0002, and I2 = 67%; WMD -0.53, 95% CI: -0.85, -0.21, p = 0.001, and I2 = 75%), as well as shorter hospital stays (WMD -0.46, 95% CI: -0.74, -0.18, p = 0.001, and I2 = 34%). CONCLUSION: In laparoscopic right hemicolectomy, intracorporeal anastomosis does not increase the incidence of anastomotic leakage within 30 days post-operation compared to extracorporeal anastomosis. In addition, intracorporeal anastomosis resulted in faster recovery of bowel function. This suggests that intracorporeal anastomosis is safe and effective.

Hyperoxia impairs intraflagellar transport and causes dysregulated metabolism with resultant decreased cilia length.

Supplemental oxygen is a lifesaving measure in infants born premature to facilitate oxygenation. Unfortunately, it may lead to alveolar simplification and loss of proximal airway epithelial cilia. Little is known about the mechanism by which hyperoxia causes ciliary dysfunction in the proximal respiratory tract. We hypothesized that hyperoxia causes intraflagellar transport (IFT) dysfunction with resultant decreased cilia length. Differentiated basal human airway epithelial cells (HAEC) were exposed to hyperoxia or air for up to 48 h. Neonatal mice (<12 h old) were exposed to hyperoxia for 72 h and recovered in room air until postnatal day (PND) 60. Cilia length was measured from scanning electron microscopy images using a MATLAB-derived program. Proteomics and metabolomics were carried out in cells after hyperoxia. After hyperoxia, there was a significant time-dependent reduction in cilia length after hyperoxia in HAEC. Proteomic analysis showed decreased abundance of multiple proteins related to IFT including dynein motor proteins. In neonatal mice exposed to hyperoxia, there was a significant decrease in acetylated α tubulin at PND10 followed by recovery to normal levels at PND60. In HAEC, hyperoxia decreased the abundance of multiple proteins associated with complex I of the electron transport chain. In HAEC, hyperoxia increased levels of malate, fumarate, and citrate, and reduced the ATP/ADP ratio at 24 h with a subsequent increase at 36 h. Exposure to hyperoxia reduced cilia length, and this was associated with aberrant IFT protein expression and dysregulated metabolism. This suggests that hyperoxic exposure leads to aberrant IFT protein expression in the respiratory epithelium resulting in shortened cilia.

Dynamic regulation of eEF1A1 acetylation affects colorectal carcinogenesis.

The dysregulation of the translation elongation factor families which are responsible for reprogramming of mRNA translation has been shown to contribute to tumor progression. Here, we report that the acetylation of eukaryotic Elongation Factor 1 Alpha 1 (eEF1A1/EF1A1) is required for genotoxic stress response and maintaining the malignancy of colorectal cancer (CRC) cells. The evolutionarily conserved site K439 is identified as the key acetylation site. Tissue expression analysis demonstrates that the acetylation level of eEF1A1 K439 is higher than paired normal tissues. Most importantly, hyperacetylation of eEF1A1 at K439 negatively correlates with CRC patient survival. Mechanistically, CBP and SIRT1 are the major acetyltransferase and deacetylase of eEF1A1. Hyperacetylation of eEF1A1 at K439 shows a significant tumor-promoting effect by increasing the capacity of proliferation, migration, and invasion of CRC cells. Our findings identify the altered post-translational modification at the translation machines as a critical factor in stress response and susceptibility to colorectal carcinogenesis.

Decreased plasma exposure of clopidogrel active metabolite in rats after long-term treatment with clopidogrel.

Clopidogrel (Clop) is oxidized by cytochrome P450s (CYPs) to an active thiol metabolite, Clop-AM, to inhibit platelet activation and aggregation. As an irreversible inhibitor of CYP2B6 and CYP2C19, clopidogrel may inhibit its own metabolism after long-term administration. The study compared the pharmacokinetic profiles of clopidogrel and its metabolites in rats receiving a single or a 2 week administration of Clop. The mRNA and protein levels of hepatic clopidogrel-metabolizing enzymes and their enzymatic activities were analyzed to explore their contribution to any altered plasma exposure of Clop and its metabolites. The results showed that long-term treatment with clopidogrel significantly decreased the AUC(0-t) and Cmax values of Clop-AM in rats, accompanied with markedly impaired catalytic activities of Clop-metabolizing CYPs including CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4. It suggests that consecutive administration of Clop to rats decreases hepatic CYPs activities, which may, in turn, inhibit clopidogrel metabolism and then reduce Clop-AM plasma exposure. Therefore, long-term treatment with clopidogrel has the potential to reduce its anti-platelet activity and to increase the risk of drug-drug interaction.

Genome of the parasitoid wasp Cotesia chilonis sheds light on amino acid resource exploitation.

BACKGROUND: A fundamental feature of parasitism is the nutritional exploitation of host organisms by their parasites. Parasitoid wasps lay eggs on arthropod hosts, exploiting them for nutrition to support larval development by using diverse effectors aimed at regulating host metabolism. However, the genetic components and molecular mechanisms at the basis of such exploitation, especially the utilization of host amino acid resources, remain largely unknown. To address this question, here, we present a chromosome-level genome assembly of the parasitoid wasp Cotesia chilonis and reconstruct its amino acid biosynthetic pathway. RESULTS: Analyses of the amino acid synthetic pathway indicate that C. chilonis lost the ability to synthesize ten amino acids, which was confirmed by feeding experiments with amino acid-depleted media. Of the ten pathways, nine are known to have been lost in the common ancestor of animals. We find that the ability to synthesize arginine was also lost in C. chilonis because of the absence of two key genes in the arginine synthesis pathway. Further analyses of the genomes of 72 arthropods species show that the loss of arginine synthesis is common in arthropods. Metabolomic analyses by UPLC-MS/MS reveal that the temporal concentrations of arginine, serine, tyrosine, and alanine are significantly higher in host (Chilo suppressalis) hemolymph at 3 days after parasitism, whereas the temporal levels of 5-hydroxylysine, glutamic acid, methionine, and lysine are significantly lower. We sequence the transcriptomes of a parasitized host and non-parasitized control. Differential gene expression analyses using these transcriptomes indicate that parasitoid wasps inhibit amino acid utilization and activate protein degradation in the host, likely resulting in the increase of amino acid content in host hemolymph. CONCLUSIONS: We sequenced the genome of a parasitoid wasp, C. chilonis, and revealed the features of trait loss in amino acid biosynthesis. Our work provides new insights into amino acid exploitation by parasitoid wasps, and this knowledge can specifically be used to design parasitoid artificial diets that potentially benefit mass rearing of parasitoids for pest control.

Structural basis for the DNA-binding activity of human ARID4B Tudor domain.

Human ARID4A and ARID4B are homologous proteins that are important in controlling gene expression and epigenetic regulation but have distinct functions. Previous studies have shown that the N-terminal domain of ARID4A is an unusual interdigitated double Tudor domain with DNA-binding activity. However, how the Tudor domain of ARID4B differs from that of ARID4A remains unknown. Here, we found that the ARID4B Tudor domain has significantly weaker DNA affinity than the ARID4A Tudor domain despite sharing more than 80% sequence identity. Structure determination and DNA titration analysis indicated that the ARID4B Tudor domain is also an interdigitated double Tudor domain with a DNA-binding surface similar to ARID4A. We identified a residue close to the DNA-binding site of the Tudor domain that differs between ARID4A and ARID4B. The Leu50 in ARID4A is Glu50 in ARID4B, and the latter forms salt bridges with two lysine residues at the DNA-binding surface. This causes a decrease in the strength of positive charge, thus reducing DNA-binding affinity while significantly increasing protein stability. We also found that a C-terminal extension region enhances the DNA-binding affinity of the ARID4B Tudor domain. This C-terminal extension is disordered and contains a positively charged RGR motif, providing an additional DNA-binding site. Finally, sequence and phylogenetic analyses indicated that the residue differences and the presence of the RGR extension region are conserved. These results provide new insight into the functional differences between ARID4A and ARID4B proteins, as well as elucidating the function of the disordered regions in these proteins.

Bone morphogenetic protein 4 inhibits pulmonary fibrosis by modulating cellular senescence and mitophagy in lung fibroblasts.

BACKGROUND: Accumulation of myofibroblasts is critical to fibrogenesis in idiopathic pulmonary fibrosis (IPF). Senescence and insufficient mitophagy in fibroblasts contribute to their differentiation into myofibroblasts, thereby promoting the development of lung fibrosis. Bone morphogenetic protein 4 (BMP4), a multifunctional growth factor, is essential for the early stage of lung development; however, the role of BMP4 in modulating lung fibrosis remains unknown. METHODS: The aim of this study was to evaluate the role of BMP4 in lung fibrosis using BMP4-haplodeleted mice, BMP4-overexpressed mice, primary lung fibroblasts and lung samples from patients with IPF. RESULTS: BMP4 expression was downregulated in IPF lungs and fibroblasts compared to control individuals, negatively correlated with fibrotic genes, and BMP4 decreased with transforming growth factor (TGF)-ß1 stimulation in lung fibroblasts in a time- and dose-dependent manner. In mice challenged with bleomycin, BMP4 haploinsufficiency perpetuated activation of lung myofibroblasts and caused accelerated lung function decline, severe fibrosis and mortality. BMP4 overexpression using adeno-associated virus 9 vectors showed preventative and therapeutic efficacy against lung fibrosis. In vitro, BMP4 attenuated TGF-ß1-induced fibroblast-to-myofibroblast differentiation and extracellular matrix (ECM) production by reducing impaired mitophagy and cellular senescence in lung fibroblasts. Pink1 silencing by short-hairpin RNA transfection abolished the ability of BMP4 to reverse the TGF-ß1-induced myofibroblast differentiation and ECM production, indicating dependence on Pink1-mediated mitophagy. Moreover, the inhibitory effect of BMP4 on fibroblast activation and differentiation was accompanied with an activation of Smad1/5/9 signalling and suppression of TGF-ß1-mediated Smad2/3 signalling in vivo and in vitro. CONCLUSION: Strategies for enhancing BMP4 signalling may represent an effective treatment for pulmonary fibrosis.

Antibiotics-induced disruption of gut microbiota increases systemic exposure of clopidogrel active metabolite in type 2 diabetic rats.

Gut microbiota play an important role in the pathophysiology of type 2 diabetic mellitus (T2DM) and biodisposition of drugs. Our previous study demonstrated that T2DM rats had the decreased plasma exposure of clopidogrel active metabolite (Clop-AM) due to upregulation of P-glycoprotein (P-gp). However, whether the change to clopidogrel (Clop) disposition under T2DM condition is associated with gut microbiota needs to be elucidated. In the study, we used an antibiotic cocktail consisting of ampicillin, vancomycin, metronidazole, and neomycin to disrupt gut microbiota and observed their influence on pharmacokinetic profiles of Clop-AM. Antibiotic administration markedly alleviated T2DM rats' phenotype including hyperglycemia, insulin resistance, oxidative stress, inflammation, hyperlipidemia, and liver dysfunction. Meanwhile, treatment with antibiotics significantly reversed the reduced systemic exposure of Clop-AM in T2DM rats relative to control rats, which was associated with the decreased intestinal P-gp level that might promote Clop absorption, resulting in more Clop transformation to Clop-AM. Fecal microbiome analysis exhibited a serious disruption of gut microbiota after antibiotic treatment with the sharply reduced microbial load and the altered microbial composition. Interestingly, an in vitro study showed that antibiotics had no influence on P-gp mRNA leve in SW480 cells, suggesting the microbiome disruption, not the direct role of antibiotics on P-gp expression, contributes to the altered P-gp level and Clop disposition in T2DM rats. The findings add new insights into the potential impact of gut microbiota on Clop biodisposition. Significance Statement 1.Antibiotics increase systemic exposure of Clop-AM in T2DM rats, which is associated with the downregulation of P-gp level.2.Antibiotics-induced disruption of gut microbiota, not direct effect of antibiotics on P-gp and CYPs expression, contributes to the altered Clop disposition.3.Antibiotics also alleviate T2DM phenotype including hyperglycemia, hyperlipidemia, insulin resistance, liver dysfunction and inflammation.

Involvement of miRNA-34a regulated Krüppel-like factor 4 expression in hyperoxia-induced senescence in lung epithelial cells.

BACKGROUND: Premature infants, subjected to supplemental oxygen and mechanical ventilation, may develop bronchopulmonary dysplasia, a chronic lung disease characterized by alveolar dysplasia and impaired vascularization. We and others have shown that hyperoxia causes senescence in cultured lung epithelial cells and fibroblasts. Although miR-34a modulates senescence, it is unclear whether it contributes to hyperoxia-induced senescence. We hypothesized that hyperoxia increases miR-34a levels, leading to cellular senescence. METHODS: We exposed mouse lung epithelial (MLE-12) cells and primary human small airway epithelial cells to hyperoxia (95% O2/5% CO2) or air (21% O2/5% CO2) for 24 h. Newborn mice (< 12 h old) were exposed to hyperoxia (> 95% O2) for 3 days and allowed to recover in room air until postnatal day 7. Lung samples from premature human infants requiring mechanical ventilation and control subjects who were not mechanically ventilated were employed. RESULTS: Hyperoxia caused senescence as indicated by loss of nuclear lamin B1, increased p21 gene expression, and senescence-associated secretory phenotype factors. Expression of miR-34a-5p was increased in epithelial cells and newborn mice exposed to hyperoxia, and in premature infants requiring mechanical ventilation. Transfection with a miR-34a-5p inhibitor reduced hyperoxia-induced senescence in MLE-12 cells. Additionally, hyperoxia increased protein levels of the oncogene and tumor-suppressor Krüppel-like factor 4 (KLF4), which were inhibited by a miR-34a-5p inhibitor. Furthermore, KLF4 knockdown by siRNA transfection reduced hyperoxia-induced senescence. CONCLUSION: Hyperoxia increases miR-34a-5p, leading to senescence in lung epithelial cells. This is dictated in part by upregulation of KLF4 signaling. Therefore, inhibiting hyperoxia-induced senescence via miR-34a-5p or KLF4 suppression may provide a novel therapeutic strategy to mitigate the detrimental consequences of hyperoxia in the neonatal lung.

Upregulating carnitine palmitoyltransferase 1 attenuates hyperoxia-induced endothelial cell dysfunction and persistent lung injury.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature infants that may cause long-term lung dysfunction. Accumulating evidence supports the vascular hypothesis of BPD, in which lung endothelial cell dysfunction drives this disease. We recently reported that endothelial carnitine palmitoyltransferase 1a (Cpt1a) is reduced by hyperoxia, and that endothelial cell-specific Cpt1a knockout mice are more susceptible to developing hyperoxia-induced injury than wild type mice. Whether Cpt1a upregulation attenuates hyperoxia-induced endothelial cell dysfunction and lung injury remains unknown. We hypothesized that upregulation of Cpt1a by baicalin or L-carnitine ameliorates hyperoxia-induced endothelial cell dysfunction and persistent lung injury. METHODS: Lung endothelial cells or newborn mice (< 12 h old) were treated with baicalin or L-carnitine after hyperoxia (50% and 95% O2) followed by air recovery. RESULTS: We found that incubation with L-carnitine (40 and 80 mg/L) and baicalin (22.5 and 45 mg/L) reduced hyperoxia-induced apoptosis, impaired cell migration and angiogenesis in cultured lung endothelial cells. This was associated with increased Cpt1a gene expression. In mice, neonatal hyperoxia caused persistent alveolar and vascular simplification in a concentration-dependent manner. Treatment with L-carnitine (150 and 300 mg/kg) and baicalin (50 and 100 mg/kg) attenuated neonatal hyperoxia-induced alveolar and vascular simplification in adult mice. These effects were diminished in endothelial cell-specific Cpt1a knockout mice. CONCLUSIONS: Upregulating Cpt1a by baicalin or L-carnitine ameliorates hyperoxia-induced lung endothelial cell dysfunction, and persistent alveolar and vascular simplification. These findings provide potential therapeutic avenues for using L-carnitine and baicalin as Cpt1a upregulators to prevent persistent lung injury in premature infants with BPD.

Rationale and design of a prospective, multicenter, phase II clinical trial of safety and efficacy evaluation of long course neoadjuvant chemoradiotherapy plus tislelizumab followed by total mesorectal excision for locally advanced rectal cancer (NCRT-PD1-LARC trial).

BACKGROUND: Long course radiotherapy plus neoadjuvant chemotherapy followed by resection (total mesorectal excision, TME) has accepted widespread recognized in the treatment of locally advanced rectal cancer (LARC). Tislelizumab, an anti-PD1 humanized IgG4 monoclonal antibody, has been demonstrated with clinical activity and is approved for treating recurrent/refractory classical Hodgkin lymphoma and locally advanced/metastatic urothelial carcinoma in China. However, the safety and efficacy of long course (neoadjuvant chemoradiotherapy, NCRT) plus tislelizumab followed by TME for LARC is still uncertain. METHODS: This NCRT-PD1-LARC trial will be a prospective, multicenter and phase II clinical trial designed to evaluate the safety and efficacy of LARC patients treated with long course NCRT plus tislelizumab followed by TME. This trial will consecutively enroll 50 stage II/III LARC patients (cT3N0M0 and cT1-3N1-2M0) with the tumor distal location ≤ 7 cm from anal verge at 7 centers in China. The enrolled patients will receive long course radiotherapy (50 Gy/25 f, 2 Gy/f, 5 days/week) and three 21-day cycles capecitabine (1000 mg/m2, bid, po, day1-14) plus three 21-day cycles tislelizumab (200 mg, iv.gtt, day8), followed by TME 6-8 weeks after the end of radiotherapy. The primary efficacy endpoint will be the pathological complete response (pCR) rate, which is defined as absence of viable tumor cells in the primary tumor and lymph nodes. DISCUSSION: To our knowledge, this trial is the first multicenter clinical trial in China to assess the safety and efficacy of NCRT plus anti-PD1 therapy followed by TME to treat patients with LARC. NCRT followed by TME was recognized as the most recommended treatment against LARC while could not be completely satisfied in clinic. This study expects to provide a solid basis and encouraging outcomes for this promising combination of radiotherapy, chemotherapy and immunotherapy in LARC. TRIAL REGISTRATION: Name of the registry: ClinicalTrials.gov. TRIAL REGISTRATION NUMBER: NCT04911517. Date of registration: 23 May 2021. URL of trial registry record: https://www. CLINICALTRIALS: gov/ct2/show/NCT04911517?id=BFH-NCRTPD&draw=2&rank=1 .

Experimental Insight into the Enigmatic Persistence of Marine Refractory Dissolved Organic Matter.

More than 90% of marine dissolved organic matter (DOM) is biologically recalcitrant. This recalcitrance has been attributed to intrinsically refractory molecules or to low concentrations of molecules, but their relative contributions are a long-standing debate. Characterizing the molecular composition of marine DOM and its bioavailability is critical for understanding this uncertainty. Here, using different sorbents, DOM was solid-phase extracted from coastal, epipelagic, and deep-sea water samples for molecular characterization and was subjected to a 180-day incubation. 1H nuclear magnetic resonance spectroscopy and ultra-high-resolution mass spectrometry (UHRMS) analyses revealed that all of the DOM extracts contained refractory carboxyl-rich alicyclic molecules, accompanied with minor bio-labile components, for example, carbohydrates. Furthermore, dissolved organic carbon concentration analysis showed that a considerable fraction of the extracted DOM (86-95%) amended in the three seawater samples resisted microbial decomposition throughout the 180-day heterotrophic incubation, even when concentrated threefold. UHRMS analysis revealed that DOM composition remained mostly invariant in the 180-day deep-sea incubations. These results underlined that the dilution and intrinsic recalcitrance hypotheses are not mutually exclusive in explaining the recalcitrance of oceanic DOM, and that the intrinsically refractory DOM likely has a relatively high contribution to the solid-phase extractable DOM in the ocean.

Crystal structure of the phosphorylated Arabidopsis MKK5 reveals activation mechanism of MAPK kinases.

The mitogen-activated protein kinase (MAPK) signaling pathways are highly conserved in eukaryotes, regulating various cellular processes. The MAPK kinases (MKKs) are dual specificity kinases, serving as convergence and divergence points of the tripartite MAPK cascades. Here, we investigate the biochemical characteristics and three-dimensional structure of MKK5 in Arabidopsis (AtMKK5). The recombinant full-length AtMKK5 is phosphorylated and can activate its physiological substrate AtMPK6. There is a conserved kinase interacting motif (KIM) at the N-terminus of AtMKK5, indispensable for specific recognition of AtMPK6. The kinase domain of AtMKK5 adopts active conformation, of which the extended activation segment is stabilized by the phosphorylated Ser221 and Thr215 residues. In line with sequence divergence from other MKKs, the αD and αK helices are missing in AtMKK5, suggesting that the AtMKK5 may adopt distinct modes of upstream kinase/substrate binding. Our data shed lights on the molecular mechanisms of MKK activation and substrate recognition, which may help design specific inhibitors targeting human and plant MKKs.

Clinical benefits of PD-1/PD-L1 inhibitors in patients with metastatic colorectal cancer: a systematic review and meta-analysis.

BACKGROUND: Immunotherapy for colorectal cancer has developed rapidly in the past decade. Many high-quality clinical trials examining the application of PD-1/PD-L1 inhibitors in patients with metastatic colorectal cancer (mCRC) have been conducted in recent years. However, the clinical benefits, including the efficacy and safety of these treatments against mCRC, remain controversial. Hence, we conducted this meta-analysis on the clinical benefits of PD-1/PD-L1 inhibitors in patients with mCRC. METHODS: We searched online databases including MEDLINE, Embase, Cochrane Library, and Web of Science, from inception to January 4, 2021. The outcomes related to efficacy and safety were extracted and analyzed. Subgroup analyses were conducted according to the categories of dMMR-MSI-H (tumors with mismatch repair deficiency and high levels of microsatellite instability) ≥ 5% vs. dMMR-MSI-H < 5%, monotherapy vs. combination therapy, PD-1 inhibitors vs. PD-L1 inhibitors, and nivolumab vs. pembrolizumab. RESULTS: Fourteen studies including 1129 subjects were included in our systematic review. The overall complete response (CR), partial response (PR), stable disease (SD), and progression of disease (PD) rates were 0.01 (95% CI 0.00-0.04), 0.04 (95% CI 0.05-0.26), 0.27 (95% CI 0.22-0.32), and 0.44 (95% CI 0.30-0.58), respectively. The overall objective response rate (ORR) and disease control rate (DCR) were 0.16 (95%CI 0.06-0.31) and 0.50 (95%CI 0.35-0.65), respectively. The overall rate of adverse events (AEs) and severe adverse responses (SAEs) were 0.84 (95% CI 0.72-0.92) and 0.30 (95% CI 0.20-0.41), respectively. The ORRs of the dMMR-MSI-H ≥ 5% and dMMR-MSI-H < 5% subgroups were 0.40 (95% CI 0.30-0.51) and 0.04 (95% CI 0.00-0.09), respectively. CONCLUSIONS: PD-1/PD-L1 inhibitors produced encouraging clinical benefits including the response rate in the treatment of dMMR-MSI-H mCRC. They actually have been influenced by the present state of mCRC therapy including pMMR-MSI-L mCRC. Nevertheless, additional multi-center prospective studies are still expected. TRIAL REGISTRATION: We have registered this study in the International Prospective Register of Systematic Reviews (PROSPERO), and the registration number is CRD42021249601 .

The taTME learning curve for mid-low rectal cancer: a single-center experience in China.

PURPOSE: As transanal total mesorectal excision (taTME) is performed worldwide, the optimization of existing training and guidance programs to enhance new taTME learners' competence in performing this procedure is warranted. This study aimed to evaluate the taTME learning curve in patients with mid-low rectal cancer. METHODS: Patients who underwent taTME for mid-low rectal cancer between October 2015 and August 2021 at a single center were included. A cumulative sum (CUSUM) learning curve analysis was performed with the total operation time as the study outcome. The learning curve was analyzed using risk-adjusted CUSUM analysis, with postoperative complications and anastomotic leakage (AL) as outcomes. RESULTS: In total, 104 consecutive patients were included in this study. The CUSUM learning curve for total operative time started declining after 42 cases (309.1 ± 84.4 vs. 220.2 ± 46.4, P < 0.001). The risk-adjusted CUSUM (RA-CUSUM) learning curve for postoperative complications fluctuated in cases 44-75 and declined significantly after case 75. The RA-CUSUM learning curve for AL declined after 68 cases. CONCLUSIONS: taTME had learning curves of 42, 75, and 68 cases for total operative time, postoperative complications, and AL, respectively. A surgeon may require 42 and 75 cases to achieve "proficiency" and "mastery" in taTME procedures, respectively.