KAT8 functions in redox homeostasis and mitochondrial dynamics during mouse oocyte meiosis progression.

As a histone acetyltransferase, lysine acetyltransferase 8 (KAT8) participates in diverse biological processes. However, the effect of KAT8 on oocyte maturation in mice remains unclear. In this study, we found that mouse oocytes overexpressing Kat8-OE induced maturation failure manifested reduced rates of GVBD and first polar body emission. In addition, immunostaining results revealed that Kat8 overexpressing oocytes showed inappropriate mitochondrial distribution patterns, overproduction of reactive oxygen species (ROS), accumulation of phosphorylated γH2AX, hyperacetylation of α-tubulin, and severely disrupted spindle/chromosome organization. Moreover, we revealed that Kat8 overexpression induced a decline in SOD1 proteins and KAT8's interaction with SOD1 in mouse ovaries via immunoprecipitation. Western blotting data confirmed that Kat8-OE induced downregulation of SOD1 expression, which is a key factor for the decline of oocyte quality in advanced maternal age. Also, the injection of Myc-Sod1 cRNA could partially rescue maternal age-induced meiotic defects in oocytes. In conclusion, our data demonstrated that high level of KAT8 inhibited SOD1 activity, which in turn induced defects of mitochondrial dynamics, imbalance of redox homeostasis, and spindle/chromosome disorganization during mouse oocyte maturation.

NET1 is a critical regulator of spindle assembly and actin dynamics in mouse oocytes.

BACKGROUND: Neuroepithelial transforming gene 1 (NET1) is a RhoA subfamily guanine nucleotide exchange factor that governs a wide array of biological processes. However, its roles in meiotic oocyte remain unclear. We herein demonstrated that the NET1-HACE1-RAC1 pathway mediates meiotic defects in the progression of oocyte maturation. METHODS: NET1 was reduced using a specific small interfering RNA in mouse oocytes. Spindle assembly, chromosomal alignment, the actin cap, and chromosomal spreads were visualized by immunostaining and analyzed under confocal microscopy. We also applied mass spectroscopy, and western blot analysis for this investigation. RESULTS: Our results revealed that NET1 was localized to the nucleus at the GV stage, and that after GVBD, NET1 was localized to the cytoplasm and predominantly distributed around the chromosomes, commensurate with meiotic progression. NET1 resided in the cytoplasm and significantly accumulated on the spindle at the MI and MII stages. Mouse oocytes depleted of Net1 exhibited aberrant first polar body extrusion and asymmetric division defects. We also determined that Net1 depletion resulted in reduced RAC1 protein expression in mouse oocytes, and that NET1 protected RAC1 from degradation by HACE1, and it was essential for actin dynamics and meiotic spindle formation. Importantly, exogenous RAC1 expression in Net1-depleted oocytes significantly rescued these defects. CONCLUSIONS: Our results suggest that NET1 exhibits multiple roles in spindle stability and actin dynamics during mouse oocyte meiosis.

Role of FBXL5 in redox homeostasis and spindle assembly during oocyte maturation in mice.

As an E3 ubiquitin ligase, F-box and leucine-rich repeat protein 5 (FBXL5) participates in diverse biologic processes. However, the role of Fbxl5 in mouse oocyte meiotic maturation has not yet been fully elucidated. The present study revealed that mouse oocytes depleted of Fbxl5 were unable to complete meiosis, as Fbxl5 silencing led to oocyte meiotic failure with reduced rates of GVBD and polar body extrusion. In addition, Fbxl5 depletion induced aberrant mitochondrial dynamics as we noted the overproduction of reactive oxygen species (ROS) and the accumulation of phosphorylated γH2AX with Fbxl5 knockdown. We also found that Fbxl5-KD led to the abnormal accumulation of CITED2 proteins in mouse oocytes. Our in vitro ubiquitination assay showed that FBXL5 interacted with CITED2 and that it mediated the degradation of CITED2 protein through the ubiquitination-proteasome pathway. Collectively, our data revealed critical functions of FBXL5 in redox hemostasis and spindle assembly during mouse oocyte maturation.

Leaf Senescence Regulation Mechanism Based on Comparative Transcriptome Analysis in Foxtail Millet.

Leaf senescence, a pivotal process in plants, directly influences both crop yield and nutritional quality. Foxtail millet (Setaria italica) is a C4 model crop renowned for its exceptional nutritional value and stress tolerance characteristics. However, there is a lack of research on the identification of senescence-associated genes (SAGs) and the underlying molecular regulatory mechanisms governing this process. In this study, a dark-induced senescence (DIS) experimental system was applied to investigate the extensive physiological and transcriptomic changes in two foxtail millet varieties with different degrees of leaf senescence. The physiological and biochemical indices revealed that the light senescence (LS) variety exhibited a delayed senescence phenotype, whereas the severe senescence (SS) variety exhibited an accelerated senescence phenotype. The most evident differences in gene expression profiles between these two varieties during DIS included photosynthesis, chlorophyll, and lipid metabolism. Comparative transcriptome analysis further revealed a significant up-regulation of genes related to polysaccharide and calcium ion binding, nitrogen utilization, defense response, and malate metabolism in LS. In contrast, the expression of genes associated with redox homeostasis, carbohydrate metabolism, lipid homeostasis, and hormone signaling was significantly altered in SS. Through WGCNA and RT-qPCR analyses, we identified three SAGs that exhibit potential negative regulation towards dark-induced leaf senescence in foxtail millet. This study establishes the foundation for a further comprehensive examination of the regulatory network governing leaf senescence and provides potential genetic resources for manipulating senescence in foxtail millet.

Djsnon, a downstream gene of Djfoxk1, is required for the regeneration of the planarian central nervous system.

Regulators of adult neurogenesis are crucial targets for neuronal repair. Freshwater planarians are ideal model systems for studying neuronal regeneration as they can regenerate their entire central nervous system (CNS) using pluripotent adult stem cells. Here, we identified Djfoxk1 in planarian Dugesia japonica to be required for planarian CNS regeneration. Knockdown of Djfoxk1 inhibits the regeneration of the cephalic ganglia, resulting in the failure of eye regeneration. By RNAi screening of Djfoxk1 downstream genes, we identified Djsnon as another regulator of planarian neuronal regeneration. Inhibition of Djsnon with RNA interference (RNAi) results in similar phenotypes caused by Djfoxk1 RNAi without affecting cell proliferation and wound healing. Our findings show that Djsnon as a downstream gene of Djfoxk1 regulates the regeneration of the planarian CNS.

Meis1 Controls the Differentiation of Eye Progenitor Cells and the Formation of Posterior Poles during Planarian Regeneration.

As a member of TALE family, Meis1 has been proven to regulate cell proliferation and differentiation during cell fate commitment; however, the mechanism is still not fully understood. The planarian, which has an abundance of stem cells (neoblasts) responsible for regenerating any organ after injury, is an ideal model for studying the mechanisms of tissue identity determination. Here, we characterized a planarian homolog of Meis1 from the planarian Dugesia japonica. Importantly, we found that knockdown of DjMeis1 inhibits the differentiation of neoblasts into eye progenitor cells and results in an eyeless phenotype with normal central nervous system. Furthermore, we observed that DjMeis1 is required for the activation of Wnt signaling pathway by promoting the Djwnt1 expression during posterior regeneration. The silencing of DjMeis1 suppresses the expression of Djwnt1 and results in the inability to reconstruct posterior poles. In general, our findings indicated that DjMeis1 acts as a trigger for the activation of eye and tail regeneration by regulating the differentiation of eye progenitor cells and the formation of posterior poles, respectively.

Maternal cigarette smoking before or during pregnancy increases the risk of birth congenital anomalies: a population-based retrospective cohort study of 12 million mother-infant pairs.

BACKGROUND: The associations of maternal cigarette smoking with congenital anomalies in offspring have been inconsistent. This study aimed to clarify the associations of the timing and intensity of maternal cigarette smoking with 12 subtypes of birth congenital anomalies based on a nationwide large birth cohort in the USA. METHODS: We used nationwide birth certificate data from the US National Vital Statistics System during 2016-2019. Women reported the average daily number of cigarettes they consumed 3 months before pregnancy and in each subsequent trimester during pregnancy. Twelve subtypes of congenital anomalies were identified in medical records. Poisson regression analysis was used to estimate the risk ratios (RRs) with 95% confidence intervals (CIs) for 12 subtypes of congenital anomalies associated with the timing (i.e., before pregnancy, and during three different trimesters of pregnancy) and intensity (i.e., number of cigarettes consumed per day) of maternal cigarette smoking. RESULTS: Among the 12,144,972 women included, 9.3% smoked before pregnancy and 7.0%, 6.0%, and 5.7% in the first, second, and third trimester, respectively. Maternal smoking before or during pregnancy significantly increased the risk of six subtypes of birth congenital anomalies (i.e., congenital diaphragmatic hernia, gastroschisis, limb reduction defect, cleft lip with or without cleft palate, cleft palate alone, and hypospadias), even as low as 1-5 cigarettes per day. The adjusted RRs (95% CIs) for overall birth congenital anomalies (defined as having any one of the congenital malformations above significantly associated with maternal cigarette smoking) among women who smoked 1-5, 6-10, and ≥ 11 cigarettes per day before pregnancy were 1.31 (1.22-1.41), 1.25 (1.17-1.33), and 1.35 (1.28-1.43), respectively. Corresponding values were 1.23 (1.14-1.33), 1.33 (1.24-1.42), 1.33 (1.23-1.43), respectively, for women who smoked cigarettes in the first trimester; 1.32 (1.21-1.44), 1.36 (1.26-1.47), and 1.38 (1.23-1.54), respectively, for women who smoked cigarettes in the second trimester; and 1.33 (1.22-1.44), 1.35 (1.24-1.47), and 1.35 (1.19-1.52), respectively, for women who smoked cigarettes in the third trimester. Compared with women who kept smoking before and throughout pregnancy, women who never smoked had significantly lower risk of congenital anomalies (RR 0.77, 95% CI 0.73-0.81), but women who smoked before pregnancy and quitted during each trimester of pregnancy had no reduced risk (all P > 0.05). CONCLUSIONS: Maternal smoking before or during pregnancy increased the risk of several birth congenital anomalies, even as low as 1-5 cigarettes per day. Maternal smokers who stopped smoking in the subsequent trimesters of pregnancy were still at an increased risk of birth congenital anomalies. Our findings highlighted that smoking cessation interventions should be implemented before pregnancy.

Epidemiology and genetic characterization of human metapneumovirus in pediatric patients from Hangzhou China.

Human metapneumovirus (HMPV), which is distributed worldwide, is a significant viral respiratory pathogen responsible for causing acute respiratory tract infections (ARTIs) in children. The aim of the present study was to investigate the epidemiological and genetic characteristics of HMPV in pediatric patients in Hangzhou China following the peak of onset of coronavirus disease 2019 (COVID-19). A total of 1442 throat swabs were collected from the pediatric patients with a diagnosis of ARTI from November 2020 to March 2021. The following viruses were detected by real-time polymerase chain reaction analysis: HMPV, RSV, adenovirus, hPIV1-3, influenza A, and influenza B. A two-step method was used to amplify the F genes of the HMPV-positive samples. Following sequencing, phylogenetic analyses were conducted using the MEGA version 7 software package. Among the 1442 samples, 103 (7.14%) were positive for HMPV. No significant differences were observed in the gender distribution. The highest incidence of HMPV occurred in children older than 6 years and the lowest was noted in children younger than 6 months. Lower respiratory tract infections were diagnosed at a higher rate than upper respiratory tract infections in HMPV-infected children. Only 10 HMPV-infected children (5.41%) were inpatients compared with 93 outpatients (7.39%). Co-infection was observed in 31 HMPV-positive samples including 24 samples of double infection and seven samples of triple infection. A total of 61F gene fragments of HMPV, which were approximately 727 bp in length were successfully sequenced. All the HMPVs belonged to the genotype B and were clustered into subgenotypes B1 (1.6%, 1/61) and B2 (98.4%, 60/61). A total of four specific amino acid substitutions were noted as follows: aa280, aa296, aa392, and aa396. These substitutions were present between sequences derived from the subgenotypes B1 and B2 in the fusion open reading frame from position 244 to 429. In conclusion, the present study provided significant information regarding the epidemiological and genetic characteristics of HMPV in children living in Hangzhou. Following the first peak of the COVID-19 pandemic, HMPV was considered an important viral respiratory pathogen present in children with ARTI.

Multiplex detection of eight different viral enteropathogens in clinical samples, combining RT-PCR technology with melting curve analysis.

BACKGROUND: Early and accurate identification of infection viruses among children can benefit the personalized medical treatment and management, and reduce the future occurrence of serious symptoms. Thus, it is critical to develop a high-throughput multiplex real-time RT-PCR method to improve the accuracy and efficiency in routine clinical lab tests. METHODS: We developed a real time RT-PCR combined with melting curve analysis (RRCMC) method for simultaneous detection of rotavirus A, B, C, norovirus GI and GII, adenovirus, astrovirus and sapovirus. RESULTS: Stool samples were collected from 160 children with acute diarrhea and tested by RRCMC assay. A total of 71 patients were tested positive with norovirus, adenovirus or rotavirus. The RRCMC assay has high specificity. There is no internal cross-reaction among the 8 diarrhea viruses and no cross-reaction of other commonly intestinal pathogens and human genome. The limit detection was ranged from 1 × 102 to 1 × 105 nucleic acid copies/ml for each diarrhea virus. CONCLUSION: The RRCMC method is a suitable rapid clinical test for infectious viruses, with the advantages of high-throughput, low cost, high sensitivity and specificity.

Life-Course Cumulative Burden of Body Mass Index and Blood Pressure on Progression of Left Ventricular Mass and Geometry in Midlife: The Bogalusa Heart Study.

RATIONALE: Data are limited regarding the influence of life-course cumulative burden of increased body mass index (BMI) and elevated blood pressure (BP) on the progression of left ventricular (LV) geometric remodeling in midlife. OBJECTIVE: To investigate the dynamic changes in LV mass and LV geometry over 6.4 years during midlife and to examine whether the adverse progression of LV geometric remodeling is influenced by the cumulative burden of BMI and BP from childhood to adulthood. METHODS AND RESULTS: The study consisted of 877 adults (604 whites and 273 blacks; 355 males; mean age=41.4 years at follow-up) who had 5 to 15 examinations of BMI and BP from childhood and 2 examinations of LV dimensions at baseline and follow-up 6.4 years apart during adulthood. The area under the curve (AUC) was calculated as a measure of long-term burden (total AUC) and trends (incremental AUC) of BMI and systolic BP (SBP). After adjusting for age, race, sex, smoking, alcohol drinking, and baseline LV mass index, the annual increase rate of LV mass index was associated with all BMI measures (ß=0.16-0.36, P<0.05 for all), adult SBP (ß=0.07, P=0.04), and total AUC of SBP (ß=0.09, P=0.01) but not with childhood and incremental AUC values of SBP. All BMI and SBP measures (except childhood SBP) were significantly associated with increased risk of incident LV hypertrophy, with odds ratios of BMI (odds ratio=1.85-2.74, P<0.05 for all) being significantly greater than those of SBP (odds ratio=1.09-1.34, P<0.05 for all except childhood SBP). In addition, all BMI measures were significantly and positively associated with incident eccentric and concentric LV hypertrophy. CONCLUSIONS: Life-course cumulative burden of BMI and BP is associated with the development of LV hypertrophy in midlife, with BMI showing stronger associations than BP. Visual Overview: An online visual overview is available for this article.

Dinuclear zinc-catalyzed enantioselective formal [3 + 2] cycloaddition of N-2,2,2-trifluoroethylisatin ketimines with low reactivity aurone derivatives.

Highly enantioselective formal [3 + 2] cycloaddition of N-2,2,2-trifluoroethylisatin ketimines with aurone derivatives of low reactivity using chiral dinuclear zinc catalysts has been developed via a Brønsted base and Lewis acid cooperative activation model. These transformations involving a domino Michael/Mannich reaction sequence led to efficient construction of a range of chiral spiro[benzofuran-pyrrolidine] scaffolds bearing three biologically relevant heterocyclic moieties and two adjacent spiro quaternary stereocenters in high yields (up to 95%) and with good enantioselectivities (up to 99% ee).

Comparison of the Efficacy and Safety of the American Thyroid Association Guidelines and American College of Radiology TI-RADS.

OBJECTIVE: To evaluate the 2015 American Thyroid Association (ATA) guidelines and 2017 American College of Radiology (ACR) Thyroid Imaging, Reporting and Data System (TI-RADS) for their efficacy in predicting malignant thyroid nodules and safety in recommending fine needle aspiration (FNA). METHODS: We reviewed data of 970 thyroid nodules from 908 patients with core needle biopsy pathology. We calculated the accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for each guideline to predict malignancies. We compared the areas under the curve and FNA recommendations between the 2 guidelines. RESULTS: According to the core needle biopsy pathology, 59.9% (581/970) of the thyroid nodules were malignant. Accuracy, sensitivity, specificity, positive predictive value, and negative predictive value was 68%, 91%, 33%, 67%, and 70%, respectively, for the ATA guidelines and 70%, 84%, 49%, 71%, and 68%, respectively, for the ACR TI-RADS. Areas under the curve (ATA: 0.71 vs ACR TI-RADS: 0.74; P = .054) were similar when predicting malignancies. For the 545 nodules with maximum diameter ≥1.0 cm, the ACR TI-RADS recommended FNA less often than the ATA guidelines (83.3% [454/545] vs 87.7% [478/545]; P = .01). For the 321 malignant nodules with maximum diameter ≥1.0 cm, the proportions of FNA recommendations were not significantly different (ACR TI-RADS: 90.7% [291/321] vs ATA: 92.5% [297/321]; P = .06). CONCLUSION: The 2015 ATA guidelines and 2017 ACR TI-RADS showed a similar ability in predicting malignancies. Reducing FNA recommendations by the ACR TI-RADS would not lead to a significant decrease in the FNA recommendations given for malignancies with maximum diameter ≥1.0 cm.

Manipulation of femtosecond laser filamentation by a gaseous lattice.

Manipulation of femtosecond laser filamentation is essential for many potential applications. We report the simulations of the manipulation of femtosecond laser filamentation by introducing a novel gaseous lattice medium with the alternating positive and negative refractive index distribution at different stages of filamentation. The results show that the filament length has greatly been extended and a multi-filament array can be formed by the gas lattice medium. It has been found that additional focusing and discrete diffraction provided by the gas lattice medium contribute to a new dynamic equilibrium in the filamentation. As a result, a varied cross-section pattern, higher field intensity, and electron density along the filamentation are obtained. Our approach provides a new way to manipulate filamentation for many practical photonic applications.

Catalytic Asymmetric Synthesis of Tetrahydrofuran Spirooxindoles via a Dinuclear Zinc Catalyst.

An asymmetric Michael/hemiketalization and Fridel-Crafts reaction has been reported through a one-pot reaction. A number of structurally novel tetrahydrofuran spirooxindoles are synthesized in the presence of a 10 mol % dinuclear zinc catalyst with diastereomer ratios (dr) of 3:1-13:1 and an enantiomeric excess (ee) of 75-99%. The reaction can be performed on a gram scale without impacting its efficiency. The absolute configuration of products is confirmed by X-ray single crystal structure analysis, and a possible mechanism is proposed.

Long-Term Excessive Body Weight and Adult Left Ventricular Hypertrophy Are Linked Through Later-Life Body Size and Blood Pressure: The Bogalusa Heart Study.

RATIONALE: Childhood adiposity is associated with cardiac structure in later life, but little is known regarding to what extent childhood body weight affects adult left ventricular geometric patterns through adult body size and blood pressure (BP). OBJECTIVE: Determine quantitatively the mediation effect of adult body weight and BP on the association of childhood body mass index (BMI) with adult left ventricular (LV) hypertrophy. METHODS AND RESULTS: This longitudinal study consisted of 710 adults, aged 26 to 48 years, who had been examined for BMI and BP measured ≥4× during childhood and ≥2× during adulthood, with a mean follow-up period of 28.0 years. After adjusting for age, race, and sex, adult BMI had a significant mediation effect (76.4%; P<0.01) on the childhood BMI-adult LV mass index association. The mediation effects of adult systolic BP (15.2%), long-term burden (12.1%), and increasing trends of systolic BP (7.9%) were all significant (P<0.01). Furthermore, these mediators also had significant mediation effects on the association of childhood BMI with adult LV hypertrophy, eccentric hypertrophy, and concentric hypertrophy. Importantly, the mediation effects of adult BMI were all significantly stronger than those of adult systolic BP on LV mass index, LV hypertrophy, and LV remodeling patterns (P<0.01). Additionally, the mediation effect of systolic BP on concentric hypertrophy was significantly stronger than that on eccentric hypertrophy (P<0.01). CONCLUSIONS: These findings suggest that increased childhood BMI has long-term adverse impact on subclinical changes in adult cardiac structure, and early life excessive body weight and adult LV hypertrophy are linked through later life excessive body weight and elevated BP.

Enantiomeric separation of vilanterol trifenatate by chiral liquid chromatography.

Vilanterol trifenatate is a novel chiral long-acting ß2-agonist developed. Vilanterol combined with inhaled corticosteroids can treat COPD and asthma. A simple liquid chromatographic method is developed for the quantitative determination of R-vilanterol and S-vilanterol (impurity S). HPLC separation was achieved on Chiralpak ID (250 × 4.6 mm; particle size 5 µm) column using hexane-ethanol-ethanolamine (75:25:0.1, v/v/v) as mobile phase at a flow rate of 1.0 mL/min. The resolution is greater than 3.3. Ethanolamine in the mobile phase is vital to enhance chromatographic efficiency and resolution between the isomers. The method was validated with respect to accuracy, specificity, precision, LOD, LOQ, linearity, and robustness as ICH guidelines.

Recombinant expression, purification and antimicrobial activity of a novel antimicrobial peptide PaDef in Pichia pastoris.

The antimicrobial peptide PaDef was isolated from Mexican avocado fruit and was reported to inhibit the growth of Escherichia coli and Staphylococcus aureus in 2013. In this study, an N-terminal 6 × His tagged recombinant PaDef (rPaDef) with a molecular weight of 7.5 KDa, for the first time, was expressed as a secreted peptide in Pichia pastoris. The optimal culture condition for rPaDef expression was determined to be incubation with 1.5% methanol for 72 h at 28 °C under pH 6.0. Under this condition, the amount of the rPaDef accumulation reached as high as 79.6 µg per 1 ml of culture medium. Once the rPaDef peptide was purified to reach a 95.7% purity using one-step nickel affinity chromatography, its strong and concentration-dependent antimicrobial activity was detected to be against a broad-spectrum of bacteria of both Gram-negative and Gram-positive. The growth of these bacterial pathogens was almost completely inhibited when the rPaDef peptide was at a concentration of as low as 90 µg/ml. In summary, our data showed that rPaDef derived from Mexican avocado fruit can be expressed and secreted efficiently when P. pastoris was used as a cell factory. This is the first report on heterologous expression of PaDef in P. pastoris and the approach described holds great promise for antibacterial drug development.

Migration mechanism of mesenchymal stem cells studied by QD/NSOM.

The migration of mesenchymal stem cells (MSCs) plays a key role in tumor-targeted delivery vehicles and tumor-related stroma formation. However, there so far has been no report on the distribution of cell surface molecules during the VEGF-induced migration of MSCs. Here, we have utilized near-field scanning optical microscopy (NSOM) combined with fluorescent quantum dot (QD)-based nano-technology to capture the functional relationship between CD44 and CD29 adhesion molecules on MSCs and the effect of their spatial rearrangements. Before VEGF-induced migration of MSCs, both CD44 and CD29 formed 200-220 nm nano-domains respectively, with little co-localization between the two types of domains. Surprisingly, the size of the CD44 nano-domain rapidly increased in size to 295 nm and apparently larger aggregates were formed following MSC treatment with VEGF for 10 min, while the area of co-localization increased to 0.327 µm2. Compared with CD44, CD29 was activated obviously later, for the fact that CD29 aggregation didn't appear until 30 min after VEGF treatment. Consistently, its co-localization area increased to 0.917 µm2. The CD44 and CD29 nano-domains further aggregated into larger nano-domains or even formed micro-domains on the membrane of activated MSCs. The aggregation and co-localization of these molecules promoted FAK formation and cytoskeleton rearrangement. All of the above changes induced by VEGF contributed to MSC migration. Taken together, our data of NSOM-based dual color fluorescent imaging demonstrated for the first time that CD44, together with CD29, involved in VEGF-induced migration of MSCs through the interaction between CD44 and its co-receptor of VEGFR-2.

Therapeutic efficacy of an anti-PD-L1 antibody based immunocytokine in a metastatic mouse model of colorectal cancer.

Immunocytokines (antibody-cytokine fusions) have been proved to be a promising class of therapeutic agents for tumors. Anti-PD-L1 antibodies or IL-2 have been used to treat a variety of cancers. Here, in order to remove T cell inhibition and increasing the IL-2 concentration in the tumor microenvironment, we engineered a novel anti-PD-L1 antibody based immunocytokine by fusing hIL-2 to the C-Term of atezolizumab, denoted as BIPI. Our results revealed that BIPI was effective in stimulating T cell activation in vitro and could selectively localize to the tumor. Furthermore, tumor regression and prolonged survival were also observed in the metastatic colorectal cancer mouse model. The obviously longer survival mice in BIPI treatment group turned out depending on the function of CD8+ T cells. The IFN- secreted from CD8+ T cells in the spleen also contributed to the better tumor inhibition profile in BIPI treatment group than in anti-PD-L1 or IL-2 treatment alone. Taken together, our data evidenced the enhanced antitumor potency of BIPI, suggesting its potential use for cancers with a low response to the anti-PD-L1 or IL-2 treatment.

Feedback mechanisms between M2 macrophages and Th17 cells in colorectal cancer patients.

IL-17 and IL-22 are linked to the development of intestinal inflammation and colorectal cancer (CRC). However, the maintenance of IL-17 and IL-22 production, as well as the cell type (Th17) that mediates these cytokines in CRC patients, remains unknown. To examine this, untreated CRC patients and healthy controls were recruited in this study. We first observed that CRC patients contained significantly elevated levels of IL-17- and IL-22-producing CD4+ T cells. The vast majority of IL-22-expressing CD4+ T cells also expressed IL-17. We then found that the production of both IL-17 and IL-22 required support from autologous monocytes, since the depletion of monocytes significantly downregulated IL-17 and IL-22 secretion. Naive T cells from CRC patients did not secrete IL-17 or IL-22 initially, but long-term coculture with autologous monocytes significantly upregulated IL-17 and IL-22 production in an IL-6-dependent manner. Blockade of IL-6 significantly reduced the levels of both IL-17 and IL-22. We then observed that CD163+ M2 macrophages were the main contributor of IL-6. Interestingly, incubation of monocytes with CCR4+CCR6+ Th17 cells resulted in significantly higher levels of CD163+ macrophages as well as higher IL-6 secretion, than incubation with non-Th17 CD4+ T cells. Together, our study discovered a positive feedback mechanism between Th17 and M2 macrophages in CRC patients.