Microbiota and metabolite alterations in pancreatic head and body/tail cancer patients.

Pancreatic head cancer (PHC) and pancreatic body/tail cancer (PBTC) have distinct clinical and biological behaviors. The microbial and metabolic differences in PHC and PBTC have not been studied. The pancreatic microbiota and metabolome of 15 PHC and 8 PBTC tissues and their matched nontumor tissues were characterized using 16S rRNA amplicon sequencing and untargeted metabolomics. At the genus level, Bradyrhizobium was increased while Corynebacterium and Ruminococcus were decreased in the PHC tissues (Head T) compared with the matched nontumor tissues (Head N) significantly. Shuttleworthia, Bacillus, and Bifidobacterium were significantly decreased in the PBTC tissues (Body/Tail T) compared with the matched nontumor tissues (Body/Tail N). Significantly, Ileibacterium was increased whereas Pseudoxanthomonas was decreased in Head T and Body/Tail T, and Lactobacillus was increased in Head T but decreased in Body/Tail T. A total of 102 discriminative metabolites were identified between Head T and Head N, which were scattered through linoleic acid metabolism and purine metabolism pathways. However, there were only four discriminative metabolites between Body/Tail T and Body/Tail N, which were related to glycerophospholipid metabolism and autophagy pathways. The differential metabolites in PHC and PBTC were commonly enriched in alpha-linolenic acid metabolism and choline metabolism in cancer pathways. Eubacterium decreased in Head T was positively correlated with decreased linoleic acid while negatively correlated with increased arachidyl carnitine and stearoylcarnitine. Bacillus decreased in Body/Tail T was negatively correlated with increased L-carnitine. These microbiota and metabolites deserve further investigations to reveal their roles in the pathogenesis of PHC and PBTC, providing clues for future treatments.

A convolutional neural network-based system for identifying neuroendocrine neoplasm and multiple types of lesions in the pancreas via endoscopic ultrasound (with videos).

BACKGROUND AND AIMS: Endoscopic ultrasound (EUS) is sensitive in detecting pancreatic neuroendocrine neoplasm (pNEN). However, the endoscopic diagnosis of pNEN is operator-dependent and time-consuming since pNEN mimics normal pancreas and other pancreatic lesions. We intended to develop a convolutional neural network (CNN)-based system named iEUS for identifying pNEN and multiple types of pancreatic lesions via EUS. METHODS: Retrospective data of 12,200 EUS images obtained from pNEN and non-pNEN pancreatic lesions, including pancreatic ductal adenocarcinoma (PDAC), autoimmune pancreatitis (AIP), and pancreatic cystic neoplasm (PCN), were used to develop iEUS. It was composed of a two-category (pNEN/ non-pNEN pancreatic lesion) classification model (CNN1) and a four-category (pNEN/ PDAC/ AIP/ PCN) classification model (CNN2). Videos from consecutive patients were prospectively collected for a human-iEUS contest to evaluate the performance of iEUS. RESULTS: A total of 573 patients were enrolled in this study. In the human-iEUS contest containing 203 videos, CNN1 and CNN2 showed an accuracy of 84.2% and 88.2% for diagnosing pNEN, respectively, which were significantly higher than that of novices (75.4%) and comparable with intermediate endosonographers (85.5%) and experts (85.5%). In addition, CNN2 showed an accuracy of 86.2%, 97.0%, and 97.0% for diagnosing PDAC, AIP, and PCN, respectively. With the assistance of iEUS, the sensitivity of endosonographers at all three levels in diagnosing pNEN has significantly improved (64.6% vs. 44.8%, 87.5% vs. 71.9%, 74.0% vs. 57.6%, respectively). CONCLUSIONS: The iEUS precisely diagnosed pNEN and other confusing pancreatic lesions, thus could assist endosonographers in achieving more accessible and accurate endoscopic diagnoses via EUS.

One-year results for myopia control with aspheric base curve orthokeratology lenses: A prospective randomised clinical trial.

PURPOSE: To compare the effect of orthokeratology (ortho-k) using aspheric or spherical base curve (BCA vs. BCS) contact lenses on axial elongation and the relative peripheral refraction change (RPRC) in Chinese children. METHODS: Children aged 8-12 years with myopia between -0.75 and -4.00 D and astigmatism ≤1.00 D were randomly assigned to the BCA or BCS group. Peripheral refraction was assessed at 10°, 20° and 30° along the temporal and nasal retina at baseline and at the 12-month visit. Axial length (AL) was measured under cycloplegia at baseline and at the 6- and 12-month visits. Only right eye data were analysed. Repeated-measures analysis of covariance was performed to examine the differences in axial elongation and the RPRC between the BCA and BCS groups. RESULTS: The 1-year results from 31 BCA and 32 BCS subjects were analysed. No significant between-group differences were found at baseline (p ≥ 0.28). At the 12-month visit, the BCA lens produced a greater absolute RPRC along the horizontal meridian than the BCS lens (p < 0.001). Axial elongation was slower in the BCA group (0.19 ± 0.20 mm) than in the BCS group (0.29 ± 0.14 mm; p = 0.03). Axial elongation was correlated with the RPRC at 10° (r = 0.43, p = 0.02) and 20° (r = 0.39, p = 0.03) along the temporal retina in the BCA group; however, these correlations were not observed in the BCS group. CONCLUSION: The BCA ortho-k lens could improve the efficacy of slowing axial elongation in children. The improved myopia control observed in the BCA group may be the result of a larger myopic shift in relative peripheral refraction within 20° along the temporal retina.

Higher-Order Aberrations and Visual Performance in Myopic Children Treated With Aspheric Base Curve-Designed Orthokeratology.

OBJECTIVES: To investigate the impact of aspheric base curve (BC)-designed orthokeratology (ortho-k) (AOK) lenses on higher-order aberrations (HOA) at different pupil diameters and visual performance. METHODS: This prospective clinical study included subjects randomized to wear spherical BC-designed ortho-k (SOK) or AOK lenses. The Pediatric Refractive Error Profile (PREP) questionnaire was completed before and after 3 months of lens wear. The Strehl ratio (SR) and root mean square of ocular higher-order aberrations (HOAs), spherical aberration (SA), coma, and trefoil were measured under 4-mm, 5-mm, and 6-mm pupil diameters at baseline and 3-month visits. Corneal topography, uncorrected low-contrast (LC) visual acuity (VA), and high-contrast (HC) VA were measured at baseline and at 1 day, 1 week, 1 month, and 3 month follow-ups. RESULTS: Sixty-five participants completed the study. After 3 months with the ortho-k lens, there were no significant differences in ocular HOA, SA, coma, or trefoil between the SOK group and AOK group at 4-mm, 5-mm, and 6-mm pupil diameters (all P>0.05), except for a significant increase in SA in the AOK group (P=0.01). Stratified analyses showed that the AOK group exhibited greater HOA and SA at 5-mm and 6-mm pupil diameters in the lower myopia subgroup and greater SA at 6 mm in the higher myopia subgroup (all P<0.05). There were no significant differences between the groups in SR, HC VA, LC VA, or PREP scores (all P>0.05). CONCLUSION: Aspheric BC-designed ortho-k lenses produced a significantly greater SA than SOK lenses, with more significance at lower diopters, without sacrificing subjective visual performance.

Isolation of a novel bio-peptide from walnut residual protein inducing apoptosis and autophagy on cancer cells.

BACKGROUND: Walnut is unique because they have a perfect balance of n-6 and n-3 polyunsaturated fatty acids. The increasing market demand of walnut lipids results in the large amount of the oil extraction residue. The walnut residue is rich in nutritional proteins, and the uneconomic use of the by-product discouraged the development of walnut industry. Anticancer peptides have recently received attention as alternative chemotherapeutic agents that overcome the limits of current drugs. The aim of this study was to investigate whether anticancer bioactive peptide is contained in walnut. METHODS: Walnut residual protein was hydrolyzed separately by five different proteases. The sequential purification of the hydrolysates was carried out by ultra-filtration, gel filtration chromatography and RP-HPLC to obtain a cancer cell growth inhibitory peptide. Cell cycle distribution, Annexin V-FITC/PI double staining, TUNEL assay, western blot and immunofluorescence for LC3-II assay were used to detect apoptosis and autophagy on cells. Cytokine production was measured by ELISA kits, macrophage phagocytosis was measured by neutral red uptake assay, nitric oxide production was measured by Griess reagent. RESULTS: The hydrolysates of walnut residual protein produced by papain under the optimal conditions (5 % substrate concentration and an enzyme-substrate ratio of 10 % at temperature 60 C for 3 h), showed significant growth inhibitory activity on MCF-7. The amino acid sequence of the purified peptide was identified as CTLEW with a molecular weight of 651.2795 Da. It is a novel bio-peptide with an amphiphilic structure. CTLEW induced both apoptosis and autophagy on MCF-7 cells, inhibited the cancer cells growth of Caco-2 and HeLa significantly, but did not show any cytotoxic activity against non-cancerous IEC-6 cells. Moreover, the bio-peptide enhanced proliferation and IL-2 secretion of spleen lymphocytes, promoted phagocytosis and NO production of macrophages. CONCLUSION: These results suggested that a novel bio-peptide, CTLEW inducing apoptosis and autophagy on MCF-7 cells can be released from walnut residual protein through papain hydrolyzing under the certain condition. The bio-peptide shows selective inhibition towards cancer cells growth and immunomodulatory activity.

Humoral and cellular immunity against diverse SARS-CoV-2 variants.

Since the outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019, the virus has rapidly spread worldwide. This has led to an unprecedented global pandemic, marked by millions of COVID-19 cases and a significant number of fatalities. Over a relatively short period, several different vaccine platforms are developed and deployed for use globally to curb the pandemic. However, the genome of SARS-CoV-2 continuously undergoes mutation and/or recombination, resulting in the emergence of several variants of concern (VOC). These VOCs can elevate viral transmission and evade the neutralizing antibodies induced by vaccines, leading to reinfections. Understanding the impact of the SARS-CoV-2 genomic mutation on viral pathogenesis and immune escape is crucial for assessing the threat of new variants to public health. This review focuses on the emergence and pathogenesis of VOC, with particular emphasis on their evasion of neutralizing antibodies. Furthermore, the memory B cell, CD4+, and CD8+ T cell memory induced by different COVID-19 vaccines or infections are discussed, along with how these cells recognize VOC. This review summarizes the current knowledge on adaptive immunology regarding SARS-CoV-2 infection and vaccines. Such knowledge may also be applied to vaccine design for other pathogens.

The effects of base curve aspheric orthokeratology lenses on corneal topography and peripheral refraction: A randomized prospective trial.

BACKGROUND: To investigate the effects of orthokeratology (ortho-k) lenses with aspheric and spherical base curve designs on corneal refractive power (CRP) and peripheral refraction. METHODS: Children aged 8 to 12 years with myopia between -0.75 D to -4.00 D, astigmatism ≤1.00 D, and corneal astigmatism ≤1.50 D were randomly assigned to the base curve aspheric (BCA) and base curve spherical (BCS) ortho-k lens groups. CRP was assessed for the central 8 mm cornea along horizontal and vertical meridians, and peripheral refraction was measured at 10°, 20°, and 30° along the nasal and temporal retina. Primary measurements included relative corneal refractive power change (RCRPC) and relative peripheral refraction change (RPRC). RESULTS: The 3-month results of the 33 and 29 subjects (right eye only) in the BCA and BCS groups, respectively, were obtained. Nonsignificant differences were found in the baseline data between the two groups (p > 0.05). At the 3-month follow-up visit, the mean RCRPC in the BCA group (2.08 ± 0.65 D) was significantly greater than that in the BCS group (1.32 ± 0.81 D) (F1,51 = 25.25, p < 0.001). The BCA group (-1.82 ± 0.65 D) exhibited a larger absolute RPRC than the BCS group (-0.98 ± 0.54 D) (F1,57 = 33.73, p < 0.001). CONCLUSIONS: It was found that the BCA ortho-k lens resulted in a more aspheric treatment zone and a more myopic relative peripheral refraction (RPR) along the horizontal meridian. The more myopic RPR was contributed by a more hyperopic central refraction and a more myopic peripheral refraction in the BCA group.

The spliceosome component Usp39 controls B cell development by regulating immunoglobulin gene rearrangement.

The spliceosome is a large ribonucleoprotein complex responsible for pre-mRNA splicing and genome stability maintenance. Disruption of the spliceosome activity may lead to developmental disorders and tumorigenesis. However, the physiological role that the spliceosome plays in B cell development and function is still poorly defined. Here, we demonstrate that ubiquitin-specific peptidase 39 (Usp39), a spliceosome component of the U4/U6.U5 tri-snRNP complex, is essential for B cell development. Ablation of Usp39 in B cell lineage blocks pre-pro-B to pro-B cell transition in the bone marrow, leading to a profound reduction of mature B cells in the periphery. We show that Usp39 specifically regulates immunoglobulin gene rearrangement in a spliceosome-dependent manner, which involves modulating chromatin interactions at the Igh locus. Moreover, our results indicate that Usp39 deletion reduces the pre-malignant B cells in Eµ-Myc transgenic mice and significantly improves their survival.

Cross-Activation of Hemichannels/Gap Junctions and Immunoglobulin-Like Domains in Innate-Adaptive Immune Responses.

Hemichannels (HCs)/gap junctions (GJs) and immunoglobulin (Ig)-like domain-containing proteins (IGLDCPs) are involved in the innate-adaptive immune response independently. Despite of available evidence demonstrating the importance of HCs/GJs and IGLDCPs in initiating, implementing, and terminating the entire immune response, our understanding of their mutual interactions in immunological function remains rudimentary. IGLDCPs include immune checkpoint molecules of the immunoglobulin family expressed in T and B lymphocytes, most of which are cluster of differentiation (CD) antigens. They also constitute the principal components of the immunological synapse (IS), which is formed on the cell surface, including the phagocytic synapse, T cell synapse, B cell synapse, and astrocytes-neuronal synapse. During the three stages of the immune response, namely innate immunity, innate-adaptive immunity, and adaptive immunity, HCs/GJs and IGLDCPs are cross-activated during the entire process. The present review summarizes the current understanding of HC-released immune signaling factors that influence IGLDCPs in regulating innate-adaptive immunity. ATP-induced "eat me" signals released by HCs, as well as CD31, CD47, and CD46 "don't eat me" signaling molecules, trigger initiation of innate immunity, which serves to regulate phagocytosis. Additionally, HC-mediated trogocytosis promotes antigen presentation and amplification. Importantly, HC-mediated CD4+ T lymphocyte activation is critical in the transition of the innate immune response to adaptive immunity. HCs also mediate non-specific transcytosis of antibodies produced by mature B lymphocytes, for instance, IgA transcytosis in ovarian cancer cells, which triggers innate immunity. Further understanding of the interplay between HCs/GJs and IGLDCPs would aid in identifying therapeutic targets that regulate the HC-Ig-like domain immune response, thereby providing a viable treatment strategy for immunological diseases. The present review delineates the clinical immunology-related applications of HC-Ig-like domain cross-activation, which would greatly benefit medical professionals and immunological researchers alike. HCs/GJs and IGLDCPs mediate phagocytosis via ATP; "eat me and don't eat me" signals trigger innate immunity; HC-mediated trogocytosis promotes antigen presentation and amplification in innate-adaptive immunity; HCs also mediate non-specific transcytosis of antibodies produced by mature B lymphocytes in adaptive immunity.

Bracovirus Sneaks Into Apoptotic Bodies Transmitting Immunosuppressive Signaling Driven by Integration-Mediated eIF5A Hypusination.

A typical characteristics of polydnavirus (PDV) infection is a persistent immunosuppression, governed by the viral integration and expression of virulence genes. Recently, activation of caspase-3 by Microplitis bicoloratus bracovirus (MbBV) to cleave Innexins, gap junction proteins, has been highlighted, further promoting apoptotic cell disassembly and apoptotic body (AB) formation. However, whether ABs play a role in immune suppression remains to be determined. Herein, we show that ABs transmitted immunosuppressive signaling, causing recipient cells to undergo apoptosis and dismigration. Furthermore, the insertion of viral-host integrated motif sites damaged the host genome, stimulating eIF5A nucleocytoplasmic transport and activating the eIF5A-hypusination translation pathway. This pathway specifically translates apoptosis-related host proteins, such as P53, CypA, CypD, and CypJ, to drive cellular apoptosis owing to broken dsDNA. Furthermore, translated viral proteins, such Vank86, 92, and 101, known to complex with transcription factor Dip3, positively regulated DHYS and DOHH transcription maintaining the activation of the eIF5A-hypusination. Mechanistically, MbBV-mediated extracellular vesicles contained inserted viral fragments that re-integrated into recipients, potentially via the homologous recombinant repair system. Meanwhile, this stimulation regulated activated caspase-3 levels via PI3K/AKT 308 and 473 dephosphorylation to promote apoptosis of granulocyte-like recipients Sf9 cell; maintaining PI3K/AKT 473 phosphorylation and 308 dephosphorylation inhibited caspase-3 activation leading to dismigration of plasmatocyte-like recipient High Five cells. Together, our results suggest that integration-mediated eIF5A hypusination drives extracellular vesicles for continuous immunosuppression.

Bracovirus-mediated innexin hemichannel closure in cell disassembly.

Cell-cell communication is necessary for cellular immune response. Hemichannel closure disrupts communication between intracellular and extracellular environments during polydnavirus-induced immunosuppression in invertebrates. However, the effects of hemichannel closure on cellular immune response are unclear. Here, we examined apoptotic body formation triggered by hemichannel closure in hemocytes of Spodoptera litura infected with bracovirus from the parasitic wasp, Microplitis bicoloratus. We showed that Microplitis bicoloratus bracovirus (MbBV) induced apoptotic cell disassembly, accompanied by hemichannel closure. Hemocyte apoptotic body formation was caused by the dysregulation of the innexins (Inxs), Inx1, Inx2, Inx3, and Inx4, during the MbBV-mediated inhibition of pI3K/AKT signaling and activation of caspase-3, which cleaved gap junction Inx proteins. Our results showed that hemichannel opening or closure in response to various stimuli, which induces the modulation of Inx levels, could inhibit or activate apoptotic body formation, respectively. Therefore, the "hemichannel open and close" model may regulate the cellular immune response.

Interactions of Vank proteins from Microplitis bicoloratus bracovirus with host Dip3 suppress eIF4E expression.

Microplitis bicoloratus bracovirus (MbBV) inhibits the immune response of the host Spodoptera litura by disrupting nuclear factor (NF)-κB signaling and downstream gene expression. However, the underlying molecular mechanisms are not well understood. Herein, we report that viral ankyrin (Vank) proteins interacted with host dorsal-interacting protein 3 (Dip3) to selectively inhibit the transcription of eukaryotic translation initiation factor 4 E (eIF4E). Dip3 and Vank proteins were co-expressed and colocalized in the nucleus. Furthermore, ectopic expression of Dip3 rescued the transcription of some NF-κB-dependent genes suppressed by Vank proteins, including eIF4E. Co-immunoprecipitation and pull-down assays confirmed that Vank proteins interacted with and bound to full-length Dip3, which including MADF, DNA-binding protein, BESS, and protein-protein interaction motifs as well as non-motif sequences. In vivo, RNAi-mediated dip3 silencing decreased eIF4E levels and was accompanied by an immunosuppressive phenotype in S. litura. Our results provided novel insights into the regulation of host transcription during immune suppression by viral proteins that modulate nuclear NF-κB signaling.

Connexins and cAMP Cross-Talk in Cancer Progression and Metastasis.

Connexin-containing gap junctions mediate the direct exchange of small molecules between cells, thus promoting cell-cell communication. Connexins (Cxs) have been widely studied as key tumor-suppressors. However, certain Cx subtypes, such as Cx43 and Cx26, are overexpressed in metastatic tumor lesions. Cyclic adenosine monophosphate (cAMP) signaling regulates Cx expression and function via transcriptional control and phosphorylation. cAMP also passes through gap junction channels between adjacent cells, regulating cell cycle progression, particularly in cancer cell populations. Low levels of cAMP are sufficient to activate key effectors. The present review evaluates the mechanisms underlying Cx regulation by cAMP signaling and the role of gap junctions in cancer progression and metastasis. A deeper understanding of these processes might facilitate the development of novel anticancer drugs.

Connexins in Lung Cancer and Brain Metastasis.

Connexins (Cxs) are involved in the brain metastasis of lung cancer cells. Thus, it is necessary to determine whether gap junction-forming Cxs are involved in the communication between lung cancer cells and the host cells, such as endothelial cells, forming the brain-blood-barrier, and cells in the central nervous system. Data from multiple studies support that Cxs function as tumor suppressors during lung cancer occurrence. However, recent evidence suggests that during metastasis to the brain, cancer cells establish communication with the host. This review discusses junctional or non-junctional hemichannel studies in lung cancer development and brain metastasis, highlighting important unanswered questions and controversies.

Responses of microbial community to different concentration of perchlorate in the Qingyi River.

Perchlorate is a refractory and mobile contaminant that is wildly distributed in surface water, and due to its tremendous inhibitory effect on mammalian thyroid function, it has gained much attention in recent years. Numerous studies have focused on environmental detection of perchlorate, especially in water. However, less attention has been paid to the effects of perchlorate on the composition of the microbial community in rivers. Upstream of the Qingyi River, an important source of drinking water for local residents, there are two perchlorate manufacturers. In this study, we selected eight study sites from upstream to downstream of the Qingyi River, including sites located upstream and downstream of the perchlorate manufacturers. Our results indicated that perchlorate was detected in all sites except for QYR2, QYR3, and QYR10. The concentration of perchlorate in the five study sites was much higher than the reference dose proposed by the National Academy of Science, and ranged from 187 to 9647.00 µg/L. We utilized 16S rDNA high throughput sequencing to analyze changes in the composition of the microbial community, based on the Illumina 2 × 250 MiSeq platform. The results showed that, when microbial communities were exposed to high concentration of perchlorate, there was an increase in the ratio of Betaproteobacteria, Bacteroidetes, Actinobacteria, and Saccharibacteria in the microbial community along with a decrease in the ratio of Chloroflexi, Verrucomicrobia, and Gammaproteobacteria. Our study has provided a theoretical basis for the alteration of the microbial community caused by the perchlorate pollution, which maybe have a truly important impact on the quality of groundwater.

Microplitis bicoloratus bracovirus modulates innate immune suppression through the eIF4E-eIF4A axis in the insect Spodoptera litura.

Eukaryotic initiation factor 4E (eIF4E) is regulated during the innate immune response. However, its translational regulation under innate immune suppression remains largely unexplored. Microplitis bicoloratus bracovirus (MbBV), a symbiotic virus harbored by the parasitoid wasp, Microplitis bicoloratus, suppresses innate immunity in parasitized Spodoptera litura. Here, we generated eIF4E dsRNA and used it to silence the eIF4E gene of S. litura, resulting in a hallmark immunosuppressive phenotype characterized by increased apoptosis of hemocytes and retardation of head capsule width development. In response to natural parasitism, loss of eIF4E function was associated with similar immunosuppression, and we detected no significant differences between the response to parasitism and treatment with eIF4E RNAi. Under MbBV infection, eIF4E overexpression significantly suppressed MbBV-induced increase in apoptosis and suppressed apoptosis to the same extent as co-expression of both eIF4E and eIF4A. There were no significant differences between MbBV-infected and uninfected larvae in which eIF4E was overexpressed. More importantly, in the eIF4E RNAi strain, eIF4A RNAi did not increase apoptosis. Collectively, our results indicate that eIF4E plays a nodal role in the MbBV-suppressed innate immune response via the eIF4E-eIF4A axis.

[Absorption and distribution of K, Na and Mg in Avicennia marina seedlings under cadmium stress].

In this paper, mangrove seedlings Avicennia marina were treated with various contents of cadmium (0, 0.5, 5, 25, 50, 100, 150 mg · L(-1)). These seedlings were cultivated by man-made seawater with a salinity of 15 in sand for 90 days in a greenhouse. The absorption and distribution of elements contents (K, Na and Mg) under cadmium stress were investigated at 45th and 90th day, respectively. The results showed that the enrichment of cadmium in the different components of seedlings increased with the increasing cadmium stress level and exposure time. The cadmium contents in roots and cotyledons were relatively higher than in the other components, accounting for 66.9% and 16.3% of cadmium in the seedlings under the 150 mg · L(-1) cadmium stress, respectively. The fall of cotyledons could reduce the damage of cadmium stress to the whole seedlings. The Na contents increased in roots and stems and decreased in leaves and cotyledons after cadmium stress for 90 days. The K content decreased in roots and cotyledons, while had no significant change in stems and leaves. The Mg content in roots, stems, leaves and cotyledons of seedlings treated with cadmium for 90 days were lower than those of the control, and were negatively related to the cadmium content.