In vitro and in vivo assays functional assays helped determine the oncogenic role of AKR1B1. Additionally, the knockdown of AKR1B1 expression level in GC cell lines could effectively suppress the AKT-mTOR pathway and inhibit the proliferation and migration of tumor cells. In conclusion, this study provides a theoretical basis to establish the potential association and regulatory mechanism of AKR1B1 while offering a new strategy for GC-targeted therapy.Gastric cancer (GC) is a common malignant tumor in the digestive tract and a major cause of global cancer death. Due to the limited access to early screening, many patients are diagnosed with advanced GC. Therefore, postoperative radiotherapy and chemotherapy possess limited efficacy in treating GC. AKR1B1 has been associated with tumorigenesis and metastasis across various tumors, becoming a potential therapeutic target for GC. However, its role and mechanism in GC remain unclear. In this study, AKR1B1 was elevated in GC tissue, depicting a poor prognosis. AKR1B1 is closely related to age, vascular and neural invasion, lymph node metastasis, and the TNM stage of GC. The developed survival prediction model suggested that AKR1B1 expression level is crucial in the prognosis of GC patients. Moreover, the expression level of AKR1B1 in GC tissues is closely associated with the AKT-mTOR pathway.  Gastric cancer (GC) is a common malignant tumor in the digestive tract and one of the major causes of global cancer death. , 2. The Aldo-keto reductases (AKRs) are oxidoreductases which rely on nicotinamide adenine dinucleotide phosphate (NADPH) and are the most glucose reductases. , 6. AldoThe aberrant AKR1B1 protein expression in human tissues has been related to the initiation and progression of various chronic diseases and cancers –9. StudiOur study recruited 115 patients with primary GC and paired contiguous normal stomach tissues after undergoing radical gastrectomy from the First Affiliated Hospital of Wannan Medical College during 2015–2016. Each subject participating in our study signed written informed consent forms. The agreement followed the ethical guidelines set out in the Declaration of Helsinki. The Affiliated Suzhou Hospital of Nanjing Medical University (ID: 2021240) and the institutional review board of the First Affiliated Hospital of Wannan Medical College (ID: 202248) approved this study.We fixed the GC specimens and paired contiguous normal stomach tissues with formalin. Then, they were buried in paraffin and cut into 5-μm slices for immunohistochemical staining as referred in a previous study . Section2 at 37°C.The included human GC cell strains were acquired from the Nanjing Medical University . They were sprouted in RPMI medium 1640  with 10% FBS , 100 units/ml penicillin G sodium, and 100 μg/mL streptomycin sulfate . The clones were cultivated in 5% COThese cells were cultured until 80% fusion and transfected through a lentivirus AKR1B1-shRNA technology  according to the company’s manuals . The celThe whole proteins were extracted with RIPA lysis buffer comprising proteinase and phosphatase inhibitors . The whole protein was separated with SDS-PAGE and shifted to a PVDF membrane which was sealed using 5% skimmed milk and probed using antibodies at 4°C all night long. We visualized the protein bands using chemiluminescence analysis and quantified them with ImageJ . Antibodies comprised in western-blot were anti-AKR1B1 , anti-p-AKT , anti-p-S6K1 , and anti-β-actin  antibodies.About a thousand cells were cultivated in every 6 pore plates for 10 days. The poietic cell lines were fixed by methanol and dyed with 0.1% crystal violet solution. Finally, they were calculated using an optical microscope  equipped with a digicam .Cells were inoculated into the upper cavities at a density of 10,000 cells/200 μl in a serum free medium. Each well in the lower cavities was full of complete medium of 800 μl. After incubation for twelve hours, the filters were fixed using 4% paraformaldehyde and stained by 0.1% crystal violet, and the mean value of 5 random fields in each sample was determined.The GC cell viability was measured with the Cell Counting Kit-8 (CCK-8) test  in 96-well plates with 2000 cells/well based on the manufacturer’s protocols.7 AKR1B1-KD or NC-shRNA AGS cells were injected to the right dorsal subcutaneous area of the mice on day 0. Then we measured the weight of mice fortnightly, and the dimensions of their subcutaneous neoplasms was determined. Four weeks later, the subcutaneous neoplasms were peeled and weighed. Ethical clearance by the Animal Ethics Committee of the Affiliated Suzhou Hospital of Nanjing Medical University  has been obtained for all the experiments in this study.SPF male BALB/c naked mice  were stochastically split into groups, each group of 5. After one week of adaptation, 10t-test or Mann–Whitney U test. The IHC results were analysed using Chi-squared or Fisher's exact tests. P < 0.05 was determined statistically significant.The data were shown as mean ± S.D. of a minimum of three stand-alone experiments. Data were analyzed using SPSS 22.0, GraphPad Prism 8, and R software (version 3.6.1). Two groups were compared using the unpaired and two-tailed Data will be made available on request.P = 0.003, P < 0.001, P < 0.001, Firstly, the AKR1B1 expression was detected in 115 GC tissues and contiguous normal stomach tissues using IHC. The IHC points were decided based on the intensity and extent of the staining . The posP = 0.044), vascular invasion (P = 0.030), neural infiltration (P = 0.017), lymphaden metastasis (P < 0.001), and the TNM classification  and AKR1B1neg (AKR1B1 negative) expressions. The survival rate of AKR1B1pos sufferers was obviously lower than AKR1B1neg sufferers  or patients with TNM stage III (P = 0.618). Conversely, increased AKR1B1 expression was related to worse prognosis in GC patients with the tumor invasion depth T3-4 (P = 0.021) or TNM stage I-II (P = 0.018). Meanwhile, high AKR1B1 expression could not predict a significant effect on survival, irrespective of whether they belonged to lymph node (P = 0.097) or no lymphaden (P = 0.232) metastases. Consequently, the AKR1B1 expression level can be detected in GC tissues to evaluate patient survival depending on the clinicopathological indicators.A subgroup survival analysis was performed according to additional clinical pathological indexes to clarify the predictive effect of AKR1B1 expression on survival across different clinicopathological subgroups. The survival rate of AKR1B1P < 0.05, The single factor analysis of Cox’s proportional risk model proved that differentiation degree, vascular invasion, neural invasion, tumor invasion depth, lymphaden metastasis, and AKR1B1 expression  , 4B. WheAKR1B1 in GC tissues had a tight connection with the expression levels of AKT (P = 0.006) and mTOR  as it inhibits the phosphorylation of mTOR, Raptor, eIF4E, S6K, and 4E-BP1, thus inhibiting tumor growth. These regulatory pathways can synthesize the regulatory effects of AKR1B1 on mTOR-related signaling networks [The AKT-mTOR signaling pathway modulates the migration, proliferation, metabolism, and drug resistance of cancer cells , 36. Thenetworks , 40.in vitro and in vivo experiments showed that AKR1B1 could accelerate GC cell migration and proliferation ability while affecting the nutritional level by controlling the AKT-mTOR pathway. These findings may provide the theoretical foundation for confirming the prospective connection between AKR1B1 and AKT-mTOR pathway and its regulatory mechanism, providing novel strategies for targeted GC therapy.Abnormal elevated expression of AKR1B1 in GC could lead to poor prognosis among GC sufferers. AKR1B1 could be an independent prognostic factor for postoperative GC patients. The prognostic model established according to AKR1B1 expression depicted its considerable impact on patient prognosis. Additionally,


H11N9 viruses in wild birds might have provided the NA gene of human H7N9 virus in early 2013 in China, which evolved with highly pathogenic strains in 2017 and caused severe fatalities. To investigate the prevalence and evolution of the H11N9 influenza viruses, 16,781 samples were collected and analyzed during 2016–2020. As a result, a novel strain of influenza A (H11N9) virus with several characteristics that increase virulence was isolated. This strain had reduced pathogenicity in chicken and mice and was able to replicate in mice without prior adaptation. Phylogenetic analyses showed that it was a sextuple‐reassortant virus of H11N9, H3N8, H3N6, H7N9, H9N2, and H6N8 viruses present in China, similar to the H11N9 strains in Japan and Korea during the same period. This was the H11N9 strain isolated from China most recently, which add a record to viruses in wild birds. This study identified a new H11N9 reassortant in a wild bird with key mutation contributing to virulence. Therefore, comprehensive surveillance and enhanced biosecurity precautions are particularly important for the prediction and prevention of potential pandemics resulting from reassortant viruses with continuous evolution and expanding geographic distributions.The online version contains supplementary material available at 10.1007/s11262-023-02009-8. Avian influenza viruses are ribonucleic acid viruses of the family Orthomyxoviridae and possess 8 negative-sense RNA segments encoding 11 known proteins , 3. CurrInfluenza A viruses can be divided into two distinct groups of high or low pathogenicity on the basis of their pathogenicity in chickens. All highly pathogenic avian influenza viruses (HPAIVs) known to date that mutate from Low pathogenic avian influenza viruses (LPAIVs) have been restricted to subtypes H5 and H7 . HPAIVs The NA gene of human influenza A(H7N9) virus might have originated from influenza A  viruses that circulated in eastern China –14. MostConsidering that H11N9 viruses might contributed to H7N9 reassortment and have the threat to public health, active surveillance on influenza was required urgently. Recently, H11N9 viruses were detected in China Feb 2016)  and Sout16  and Shttps://www.ncbi.nlm.nih.gov/genomes/FLU/annotation), then deposited in GenBank. The sequence feature was annotated on Influenza Research Database .To respond to the H7N9 outbreak, a total of 16,781 swab and fecal samples were collected from waterfowl in natural reserves in Shanghai and Jiangxi during 2016–2020. For virus isolation, 9 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs were inoculated with the sample supernatants. Viral RNA was extracted from 200 ml of allantoic fluid and subjected to reverse transcriptase polymerase chain reaction (RT-PCR). First, reverse transcription was performed with the primer Uni12 and GoScript™ Reverse Transcriptase System (Promega). This was followed by PCR in which the reverse transcription product(s) was amplified by the universal primer set MBTuni-12 and -13 to amplify the short segments for hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M), and nonstructural gene (NS). Segment-specific primers were used to amplify the long segments for polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acidic protein (PA) (Table S1) , 22. Fulhttps://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database) and the EpiFlu database from the Global Initiative on Sharing All Influenza Data .For phylogenetic analysis, the sequences of full genomes of the top 100 basic local alignment search tool (BLAST) hits for the new isolate were downloaded from NCBI Multiple sequence alignments were produced using MUSCLE in MEGA7. Maximum clade credibility phylogenetic trees were generated for the full-genome sequences of the top 100 BLAST hits. ModelTest-NG was used to select the best-fit model for nucleotide substitutions. We used relaxed molecular clock models  to estimate divergence times. Markov chain Monte Carlo (MCMC) chains were run for 100–1100 million iterations according to the number of sequences. The best-fit substitution and tree models are listed in Table S2. TRACER 1.6 was used to confirm appropriate burn-in and adequate effective sample sizes (ESS > 200) for the MCMC analyses . All phyTo determine pathogenicity of the influenza virus in chickens, Intravenous Pathogenicity Index (IVPI) was essentially conducted as described in OIE manual . Specifi3 EID50 of designated virus in a volume of 50 µl. The control group was injected with an equal volume of phosphate-buffered saline. On 3 days post inoculation (dpi), three inoculated mice were sacrificed, and we collected organs including lungs, nasal turbinate, spleen, trachea, brain, and colon for viral titrations by Real-Time Polymerase Chain Reaction (qPCR). The results were extrapolated from corresponding standard curves and expressed as EID50/g equivalents [To determine pathogenicity of the influenza virus in mice, groups of 6 6-week-old female SPF BALB/c mice (weighing ≥ 18 g)  were inoculated intranasally with 10ivalents . The remA novel strain of the H11N9 subtype AIV was isolated from a spot-billed duck and named A/Anas poecilorhyncha/shanghai/SH2/2016 . SH2 harbors a single basic residue at the cleavage site and was classified as an LPAI virus   were most closely related to isolates from Korea and China (Table S3). Phylogenetic analysis showed similar results , and caused no clinical signs in chickens with an IVPI of 0.0, justifying classification of the isolate tested to be of a low pathogenic phenotype . A few c of mice , 20, 38.In addition, full-genome sequenced progeny viruses from duck-derived SH2 H11N9 isolate included a series of known pathogenicity markers that play a role in determining high pathogenicity . Though A/wild bird/Anhui Shengjin Lake/S119/2014 (H11N9), A/wild bird/Anhui Caizi Lake/L306/2014 (H11N9), and A/waterfowl/Korea/S353/2016 [In summary, we isolated a novel H11N9 strain in late 2016 that possesses new characteristics of increasing virulence. Although our phylogenetic analysis was based on strains with limited genetic diversity, SH2 is most likely a reassortant virus that originated from domestic birds in China. So, continued influenza surveillance in wild birds is extremely essential to prevent a potent pandemic caused by virus reassortment.Supplementary file1 (PDF 1821 kb)Below is the link to the electronic supplementary material.


Although acting primarily on intestine, kidney and bone, their use in pig nutrition has shown a wide range of effects also in peripheral tissues. However, there is an ambiguity in the existing literature about whether the effects of Vit D3 and 25(OH)D3 differ in attributing the molecular and phenotypic outcomes in pigs. We searched Web of Science and PubMed databases concerning the efficacy of Vit D3 in comparison with 25(OH)D3 on pig physiology, i.e. reproductive capacities, growth performance, immunity and bone development. Dietary intake of Vit D3 or 25(OH)D3 did not influence the reproductive capacity of sows. Unlike Vit D3, the maternal intake of 25(OH)D3 significantly improved the growth performance of piglets, which might be attributed to maternally induced micronutrient efficiency. Consequently, even in the absence of maternal vitamin D supplementation, 25(OH)D3-fed offspring also demonstrated better growth than the offspring received Vit D3. Moreover, a similar superior impact of 25(OH)D3 was seen with respect to serum markers of innate and humoral immunity. Last but not least, supplements containing 25(OH)D3 were found to be more effective than Vit D3 to improve bone mineralisation and formation, especially in pigs receiving basal diets low in Ca and phosphorus. The insights are of particular value in determining the principal dietary source of vitamin D to achieve its optimum utilisation efficiency, nutritional benefits and therapeutic potency and to further improve animal welfare across different management types.Vitamin D In recent years, 25(OH)D3 is being studied as an alternative to Vit D3, as it is more bioavailable, efficiently absorbed, bypasses hepatic metabolism and is three to five times more potent than Vit D3. Nevertheless, both dietary forms of Vit D are biologically inactive and require two (Vit D3) and one sequential hydroxylation reaction (25(OH)D3) for activation (3 is transported via Vit D-binding protein (DBP) and hydroxylated via Vit D 25-hydroxylase (encoded by CYP2R1) in liver to form 25(OH)D3 and via 25-hydroxyvitamin D 1-alpha-hydroxylase (encoded by CYP27B1) in kidney to 1,25(OH)2D3 (the active form of Vit D). The excess amount of 1,25(OH)2D3 is subject to renal elimination following CYP24A1-mediated hydroxylation. Nevertheless, both synthesis and elimination also occur in non-renal tissues (i.e. intestine). After activation, 1,25(OH)2D3 migrates via DBP to various target tissues such as intestine, bone, muscle, immune system, kidney, parathyroid glands and reproductive system to attribute its functions.Vitamin Dtivation . After i2D3 complex. This complex interaction initiates the formation of two autonomous protein interaction surfaces on the VDR; one of them modulates interplay with the retinoid X receptor (RXR) for DNA binding and the other recruits co-regulators to control gene expression. After dimerisation of VDR-1,25(OH)2D3 with RXR, the heterodimer translocates to the nucleus, binds to the Vit D responsive element (VDRE), which in turn regulates the expression of numerous genes (> 900), and implements the specific function of Vit D in particular tissues,5.The mediation of the biological functions of Vit D depends on its successful binding with the intracellular Vit D receptor  forming the VDR-1,25(OH),7. The tissue-wide expression of VDR and other genes involving Vit D metabolism underscores the fact that the function of Vit D is not limited to osteogenesis or serum mineral balance,8. In fact, an array of tissues are capable to express relevant genes encoding hydroxylation enzymes for calcitriol synthesis and elimination in mammals,9. Thus, it turns out to be equally important for growth–13, immunity,15, oxidative status,17, reproductive capacity,19 and progeny performance. In pig feeding, legislation limits the Vit D supply to 1000–2000 μg/kg diet, which corresponds to 25–50 µg/kg feed,22, while 25(OH)D3 in combination with Vit D3 is allowed up to doses of 50 µg/kg feed. However, the most potent form of Vit D in terms of nutritional benefits remains unclear, largely due to divergent functional demands of tissues and cell types,9. Given the background, this review summarises the available literature for molecular and phenotypic outcomes of Vit D3 in comparison with 25(OH)D3 supplements in terms of reproductive capacities, growth performance, immunity and bone development in pigs.The secosteroid hormone Vit D is critical for the maintenance of serum Ca and P homeostasis for optimum bone development, as it modulates the active uptake of minerals through the intestine3 and 25(OH)D3 in pigs published from 1 January 2000 to 23 May 2022. The search strategy included appropriate MeSH terms  without any language restriction. In total, 454 articles from the Web of Science and 241 articles from PubMed were identified using the appropriate search query. Of these, a total of thirty-five articles were selected for review, paying attention to relevance, overlap and experimental design .We conducted a systematic query in Web of Science and PubMed to retrieve all the articles dealing with Vit D3 and 25(OH)D3 plays an important role in fertility and maturation. The objective of this section was to identify arguments for the more effective form of Vit D in the diet to improve sow reproductive capacities and growth performance of the piglets D3 on sow reproductive capacity, muscle fibre morphometry and subsequent growth performance of piglets. For this purpose, sixty-nine sows were randomly allocated to one of three dietary groups comprising (i) Vit D3 as control (37·5 µg/kg), (ii) Vit D3 and 25(OH)D3 at low levels D3) or (iii) Vit D3 and 25(OH)D3 at high levels D3). On the other hand, the piglets (n 216) from these sixty-nine sows were treated similarly to their mothers from birth until day 59 of life. No significant effect was observed on the reproductive performance of sows regardless of the dietary Vit D regimens. At the time of birth, piglets from DH-fed sows showed a significant increase in the number of primary muscle fibres in contrast to piglets from control sows at birth. However, this effect was no longer present when weaning piglets were investigated. During the nursery period, there was no effect of dietary Vit D sources on the growth performance, except for feed efficiency from day 28 to 59 and day 0 to 59. During this period, piglets from DH-fed sows showed a significant increase in feed efficiency, i.e., the body weight gain to feed intake ratio, in contrast to piglets from sows assigned to DL. Overall, dietary intake of 25(OH)D3 resulted in a significant increase in the number of primary muscle fibres at birth, but the total number of muscle fibres did not improve at birth or weaning.Thayer et al.,20 conducted two different studies to compare the effects of maternal Vit D3 and 25(OH)D3 supplementations on sow reproduction and offspring growth performance. In one study, forty-eight multiparous sows were provided a basal diet containing Vit D3  or 25(OH)D3 D3) depending on their body weight and expected farrowing date. At weaning, eighty piglets each from CON and TRT sows were fed diets containing 62·5 µg/kg Vit D3 (weaning diet) and 43·75 µg/kg Vit D3 (growing-finishing diet) with or without 25(OH)D3 (50 µg/kg) for 140 d. Unlike Vit D3, the maternal intake of 25(OH)D3 significantly improved the average daily weight gain (ADWG) and feed efficiency of the piglets at the early stage of the nursery period. However, in the later phase of the nursery period, maternal ingestion of either source of Vit D demonstrated a similar effect on the growth performance of the piglets. In this period, the piglets alone supplemented with 25(OH)D3 showed significant improvement in ADWG, feed efficiency and average daily feed intake. Moreover, in contrast to Vit D3, 25(OH)D3 significantly increased the water-holding capacity and reduced pork drip loss. Feeding 25(OH)D3 to sows and their progenies had a significant impact on the expression of candidate genes associated with muscle formation. After dietary supplementation to sows and their offspring (post-weaning diet) with 25(OH)D3, the up-regulation of myogenic markers MYOD1 (myogenic differentiation 1), MYF5 (myogenic factor 5) and down-regulation of MSTN (myostatin) were observed, suggesting the importance of 25(OH)D3 for muscle development. MYOD1 and MYF5 are involved in the regulation of myoblast proliferation and differentiation and critically determine the survival of muscle progenitor cells as well,27. On the contrary, MSTN is the negative regulator of muscle development. In the second study, a total of forty-eight multiparous sows received either a basal diet fortified with Vit D3  or a control diet containing 25(OH)D3  based on their body weight and expected farrowing date. The sows fed 25(OH)D3 demonstrated a significant increase in body weight gain and body condition score during the suckling period of the piglets. Unlike Vit D3, piglets from sows fed 25(OH)D3 also had significantly higher survival, ADWG and weaning weight than piglets from sows fed the control diet. These results indicate that dietary supplementation with 25(OH)D3 significantly enhanced growth performance in both sows and their offspring.Upadhaya et al. performed their study into two parts. First, 227 primi- and multiparous sows received a basal diet containing Vit D3  and 25(OH)D3  from mating to day 110 of gestation. 25(OH)D3-supplemented sows exhibited a significant positive impact on total litter weight and total weaning weight of the offspring compared with sows that received Vit D3. The intrauterine development of the embryos was also positively correlated with the maternal serum levels of 25(OH)D3. For the second experiment, thirty-nine sows received basal diets fortified with Vit D3  and 25(OH)D3  from the day of the mating and the treatments were continued for four reproductive cycles. Following the intake, the two dietary sources of Vit D exhibited similar impacts on sow reproductive performance. However, a significant increase in weight gain between birth and weaning was observed in the offspring of the DN-fed sows, in contrast to the offspring of DL- and DH-fed sows. In summary, the authors concluded that sows fed 25(OH)D3 showed a significant improvement in the birth weight of their offspring. But sows provided with either form of Vit D for more than one reproductive cycle may not show any noticeable impact on the growth performance of their progeny.To compare the two dietary forms of Vit D in terms of reproductive performance, Weber et al. conducted a comparative study in thirty-nine primi- and multiparous sows randomly assigned to Vit D3  and 25(OH)D3  from the day of mating to day 21 of lactation. Similar to other studies, the serum level of 25(OH)D3 increased significantly in pigs after the administration of 25(OH)D3 compared with Vit D3. DL led to a decrease in average daily feed intake and a tendency for a decrease in body weight and body weight gain compared with the other groups. Additional supplementation of HD had no significant effect. So, maternal diets supplemented with either form of Vit D (50 µg/kg) demonstrated a similar impact on the growth performance of their offspring.Witschi et al. compared Vit D3 (50 µg/kg) and 25(OH)D3 (50 µg/kg) to determine their relative effects on the reproductive capacity of twenty-four sows and the growth performance of their offspring from day 107 of gestation to day 21 of lactation. No significant effect of Vit D treatments on sow reproductive performance was observed. However, in contrast to Vit D3, offspring from 25(OH)D3-fed sows showed a significant increase in litter weight, total litter weight and piglet weight gain during the lactation period. Unlike Vit D3, ADWG and total weight gain also tended to improve in piglets from 25(OH)D3-supplemented sows. Overall, the dietary supplementation of 25(OH)D3 in pregnant sows significantly improved piglet growth performance (5·3 % increase in weight gain on average).Zhang et al. studied the effects of maternal supplementation of Vit D3 or 25(OH)D3 on piglet growth and muscle development. They provided forty sows with a control diet containing Vit D3 (62·5 µg/kg) and an experimental diet fortified with 25(OH)D3 D3) beginning 43 d before artificial insemination through day 90 of gestation. Both diets contained 12·5 µg/kg of Vit D3 to avoid possible Vit D deficiency. In comparison with Vit D3, foetuses from 25(OH)D3-fed dams showed a significant increase in the number of longissimus dorsi muscle fibres by 9·3 %. However, there were no significant effects of the maternal diet on the cross-sectional area of the muscles in piglets. Piglets from 25(OH)D3-fed sows had more Pax7+ myoblast (72 and 96 h of post-plating) in the longissimus dorsi muscle than piglets from sows fed Vit D3. Foetuses from 25(OH)D3-fed sows also had increased total myoblast proliferative capacity. Thus, a maternal diet containing 25(OH)D3 exhibited a significant positive impact on foetal muscle development.Hines et al., 144 piglets were randomly assigned to three different dietary treatments with a follow-up period of 28 d, namely a normal Ca-P (PC), low Ca-P (NC) diet supplemented with 62·5 µg/kg Vit D3 and low Ca-P diet supplemented with 25(OH)D3 D3). The experiment was conducted for 28 d and consisted of phase 1 (days 0–14) and phase 2 (days 15–28). PC diet contained 0·81 % Ca, 0·60 % total P and 0·72 % Ca, 0·53 % total P, and NC diet contained 0·56 % Ca, 0·47 % total P, and 0·45 % Ca, 0·39 % total P in phase 1 and phase 2, respectively. Compared to NC, the body weight (day 28) and ADWG (days 15–28 and days 1–28) of the weaned piglets increased significantly after their dietary ingestion of NC + 25D. Overall, the piglets fed 25(OH)D3 showed noticeable growth performance even when no dietary source of Vit D was provided to their mothers.In contrast to the aforementioned studies, there are very few studies that have investigated the growth performance after supplementing two dietary sources of Vit D directly to piglets rather than to pregnant sows. In the study of Zhang et al., Zhao et al. also conducted feeding trials only in piglets to evaluate growth efficiencies. They supplemented a total of 240 weaned piglets  with a positive control diet , negative control diet , phytase  and 25(OH)D3 D3; Phy + HyD, NC + 37·5 µg/kg Phy + 50 µg/kg HyD). The experiment was divided into two phases (6–10 kg and 10–20 kg). Regardless of the body weight, HyD supplements showed no significant effect in average daily feed intake or feed efficiency of the weaned piglets in contrast to NC. However, ADWG of piglets (10–20 kg) increased noticeably after dietary intake of HyD compared with NC. In essence, piglets from Vit D-deprived sows demonstrated noticeable improvement in their growth after dietary ingestion of 25(OH)D3 without altering their feed intake or feed efficiency. However, data on body composition traits have not been reported in this study.Corresponding to the study of Zhang 3 is significantly better than piglets from sows fed Vit D3,33. Unlike Vit D3, feeding 25(OH)D3 to sows helps to noticeably increase offspring growth performance by improving weaning weight, ADWG, total body weight gain and body condition scores. In the absence of maternal supply of Vit D sources, supplementation of 25(OH)D3 to the offspring alone can also significantly boost their growth performance. 25(OH)D3 also outperforms Vit D3 in increasing the number of muscle fibres and improving the proliferation and differentiation ability of muscle cells.Thus, most studies show that sows fed either form of Vit D have a similar impact on their reproductive ability. However, the survivability of piglets from sows fed 25(OH)D3 and 25(OH)D3 to promote immunity in pigs D3 was more effective than Vit D3 in fortifying the body’s immune system when receiving a low Ca-P diet. In contrast to NC, dietary supplementation of 25(OH)D3 (NC + 25D) reduced the incidence of streptococcal infections by significantly increasing the serum concentrations of immunoglobulin A (IgA) and G (IgG) in piglets at weaning. Since these two types of immunoglobulins (Igs) are involved in inhibiting streptococcal and other bacterial infections,35. Furthermore, in pigs receiving the 25(OH)D3 supplement, the serum concentration of IgM was enhanced at day 28, while one of the complement components (C3) was reduced at day 14. Compared to NC, diets containing 25(OH)D3 led to a significant rise in the serum concentration of catalase (CAT) at day 28, and this might be taken as a hint toward an improved antioxidant capacity. CAT is regarded as a first-line defence antioxidant enzyme that is fundamental and vital to the overall defensive mechanisms in biological systems. However, NC and NC + 25D demonstrated similar effects on the serum status of other oxidative enzymes like super oxidase dismutase, total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px). In contrast to Vit D3, the intestinal concentration of SCFA significantly increased following the dietary intake of NC + 25D. For example, the abundance of Lachnospiraceae, which plays a crucial role in maintaining gut health, increased. The principal function of Lachnospiraceae is to break down the complex polysaccharide into SCFA, including acetate, butyrate and propionate. SCFA are involved in boosting immunity and reducing inflammatory reactions in the gut and other organs by, i.e. acetyl-CoA synthesis, suppression of histone deacetylase, signalling via G protein-coupled receptors and metabolic integration. Thus, dietary inclusion of 25(OH)D3 resulted in a significant improvement in humoral as well as innate immunity and gut immunity of the weaned piglets.In the study of Zhang et al. compared the immune competence of sows supplemented with Vit D3 and 25(OH)D3. Sows fed either source of Vit D demonstrated a similar effect on the level of Igs  in colostrum. However, diet containing 25(OH)D3 significantly increased IgG levels in milk on day 21 of lactation. The expression of genes concerning fatty acid metabolisms, i.g., ACCa (Acetyl-CoA carboxylase α) and FAS (fatty-acid synthase) increased considerably in the mammary gland in response to 25(OH)D3 enriched diets. Lipid metabolism is directly involved in the regulation of the immune system through the activation of M1 and M2 macrophages. The piglets from 25(OH)D3-fed sows demonstrated a significant elevation in the concentration of butyrate in the cecal digesta compared with piglets from sows fed Vit D3. The elevated concentration of butyrate following the breakdown of dietary fibres indicates improvement in bacterial metabolism,41 which plays a crucial role in adaptive immune response via two specific pathways: first, it acts directly on monocyte-derived dendritic cells–44 and second through its action on T lymphocytes. So, in contrast to Vit D3, the dietary inclusion of 25(OH)D3 during lactation period led to a significant increase in the concentration of milk IgG; and butyrate concentration in the cecal digesta of suckling piglets due to improved bacterial metabolism in the gut.In another study, Zhang et al. compared Vit D3 and 25(OH)D3 to evaluate the immune capacity by offering three different dietary treatments to 149 piglets weighing 5–7 kg for 14 or 21 d. The dietary supplements contained a baseline diet mixed with Vit D3 , which served as a control diet. The second regimen included the control diet mixed with an additional 50 µg/kg of Vit D3 . The third one consisted of the control diet mixed with 25(OH)D3 D3). Leucocytes are involved in both innate and humoral immune responses and play a critical role in fighting against infections and defence against foreign elements. The authors observed that supplementation of 25(OH)D3 resulted in a significant increase in total blood leucocyte  counts in piglets, accompanied by a parallel increase in serum 25(OH)D3. Compared with NC, piglets fed HyD demonstrated a significant improvement in the viability of total blood leukocytes and monocytes. The HyD-fed group also showed a significant increase in the viability of bronchoalveolar leukocytes compared with PC. However, unlike Vit D3, 25(OH)D3 did not only increase the number and viability of leucocytes but also significantly improved their phagocytic capacity. Overall, dietary supplementation of 25(OH)D3 compared with Vit D3 showed a significant positive influence on systemic blood and peripheral bronchoalveolar mucosal compartments, resulting in an increase in leukocyte count as well as the survival and phagocytic ability of the discrete leucocyte populations.Konowalchuk et al., considerably lower serum levels of interleukin-6 (IL-6) at day 42 and higher serum levels of interleukin-1 (IL-1) at day 140 were observed in growing pigs supplemented with 25(OH)D3 compared with Vit D3. IL-6 is an anti-inflammatory cytokine that contributes to host defence via stimulation of the acute phase response, haematopoiesis and immune responses. IL-1 is a strong inflammatory cytokine involved in a wide range of immunological responses. It is predominantly produced by macrophages during defensive reactions to protect the body from infection and disease through inflammation and innate and adaptive immune responses. The authors were unable to explain the underlying reason for this up- or down-regulation of the pro- and anti-inflammatory cytokines. According to Tanaka et al., the fluctuation of inflammatory cytokines is due to the maintenance of immune homoeostasis. In conclusion, the supplementation of 25(OH)D3 exhibited a more positive impact than Vit D3 on pig health.In the study of Upadhaya et al. compared two different dietary sources of Vit D to observe their effects on humoral immunity by measuring the serum concentration of IgG and lysozyme. For this purpose, 160 post-weaned piglets (weaned at 21 days of age) were divided into two groups matched for maternal origin and body weight. Each group included sixteen pens with five animals each and received the same diet, differing only in the source and doses of Vit D used. The piglets received either Vit D3 (50 µg/kg) or 25(OH)D3 (50 µg/kg) for 48 d. The serum concentration of 25(OH)D3 increased considerably after dietary supplementation of 25(OH)D3 compared to Vit D3. The increased concentration of 25(OH)D3 was associated with the positive modulation of parameters related to the humoral immune systems. In contrast to Vit D3, dietary supplementation of 25(OH)D3 led to a significant increase in the serum concentration of IgG and lysozyme. So, unlike Vit D3, the dietary supplementation of 25(OH)D3 significantly improved humoral immunity and strengthened the immune response of the piglets without compromising their health.Meuter 3, the dietary intake of 25(OH)D3 significantly improves immune status of the body. In particular, the improvement of humoral immunity is reflected in the increased concentration of serum immunoglobulins and phagocytic capacity of the macrophages in piglets, which allows the animals to respond more effectively to potential health challenges. 25(OH)D3 also positively affects the serum concentration of inflammatory cytokines to maintain immune homoeostasis. Moreover, 25(OH)D3 is superior over Vit D3 in modulating the systemic and mucosal antimicrobial responses, suggested by increased numbers of leucocytes and their survival and phagocytic ability in blood and bronchoalveolar compartments. Compared to Vit D3, the significant expression of genes related to fatty acid metabolism due to dietary intake of 25(OH)D3 is also noteworthy. Fatty acids positively affect immune cell functions through a variety of complex mechanisms by improving phagocytosis, T-cell signalling and antigen presentation capability. In contrast to Vit D3, the role of 25(OH)D3 in improving gut immunity is also noteworthy. Dietary supplementation of 25(OH)D3 alters the gut microbiota and thus might promote specific metabolic processes, which plays a crucial role in maintaining intestinal health.Thus, unlike Vit D3 and 25(OH)D3 on mineral status and bone development D3 in improving serum mineral status. In contrast to Vit D3 (NC), the dietary inclusion of 25(OH)D3 (NC + 25D) significantly normalised the serum levels of Ca, bone-specific alkaline phosphatase and osteocalcin (OC) in low Ca-P fed pigs depending on the duration of the dietary supplementation. The increased serum concentration of Ca indicates the compensatory efforts of 25(OH)D3 to maintain Ca-P homoeostasis in pigs fed with low Ca-P diets. Serum level of bone-specific alkaline phosphatase (synthesised by osteoblasts) positively correlates with bone formation. OC is also considered as a serum marker of osteoblastic bone formation which acts in the bone matrix to regulate bone mineralisation via resorption. Unlike OC, the supplementation of piglets with both forms of Vit D exhibited a similar impact on the serum status of bone resorption markers like tartrate-resistant acid phosphatase (TRAP) and pyridinoline (PYD). TRAPs perform bone resorption by catalysing the hydrolysis of various phosphates and anhydrides in an acidic environment, and PYD is synthesised by the reaction at the side chain of the collagen residues during trimerisation in skeletal development. Overall, 25(OH)D3-enriched diets proved to be more effective in improving bone integrity in low Ca-P-fed pigs compared with Vit D3, as indicated by increased serum bone formation biomarkers to balance bone mineralisation and its homoeostasis.Zhang et al., Zhao et al. also demonstrated the superiority of dietary 25(OH)D3 over Vit D3 in restoring bone mineral status in pigs supplemented with low Ca-P. Unlike Vit D3 (NC), the serum level of Ca increased significantly in piglets (10–20 kg) after dietary ingestion of 25(OH)D3 (HyD). Serum Ca needs to be regulated in narrow physiological ranges and serves as an important marker of bone turnover and osteoblast functions,57. Its elevation in piglets fed low Ca-P indicates the body’s compensatory effort to balance the level of Ca and P to achieve optimal mineralisation. However, no major dietary effect on the serum status of Ca was observed in 6–10 kg piglets. Dietary supplementation of Vit D had also no discernible effect on the serum levels of P. This could be due to the body’s ability to maintain serum P levels in pigs fed low P,58. Thus, the supplementation of dietary 25(OH)D3 led to a significant impact on increasing the serum Ca to improve the mineral homeostasis in pigs receiving low Ca-P diet.Similar to the study of Zhang et al. performed a similar experiment, but with a different dosage of 25(OH)D3 compared with the aforementioned studies. For the mineral balance study, twenty-four finishing boars (13 weeks of age) with an initial live weight of 42 kg were used and fed the following diets containing low P , T1 + Phytase , T1 + 25(OH)D3 D3) or T1 + Phytase + 25(OH)D3 D3) for 66 d. Accordingly, in contrast to Vit D3 (T1), dietary inclusion of 25(OH)D3 (T3) significantly improved Ca digestibility, and retention in low Ca-P fed pigs, suggesting the same remedial endeavour as reported in the above studies to balance mineral homeostasis. Ca digestibility and P retention are closely associated with bone mineralisation and growth performance. However, the dietary intervention did not affect P metabolism, serum mineral  status, bone ash and bone strength.Doherty et al. compared the effectiveness of Vit D3 and 25(OH)D3 to determine the plasma mineral status following the supplementation of sows with basal diets containing Vit D3 and 25(OH)D3. Dietary intake of Vit D3 and 25(OH)D3 did not consistently affect the plasma status of Ca, P and OC associated with skeletal development and resorption throughout the reproduction cycle. However, the concentration of CrossLaps (β-CTx) increased significantly before parturition in both treatments and was the highest at the end of lactation. This observation suggests the mobilisation of Ca from the sow’s skeleton to the developing foetuses and piglets for their optimum bone development. Thus, in healthy pigs, Vit D3 and 25(OH)D3 resulted in similar impacts on the concentrations of bone mineralisation markers.Weber et al., thirty-nine sows (thirteen in each treatment) were assigned to Vit D3 and 25(OH)D3 supplementation from the day of mating until day 21 of lactation. Rosenberg et al. used six-week-old piglets (n 40) supplemented with Vit D3  and 25(OH)D3 (50 or 250 or 500 µg/kg) for 42 d. Witschi et al. and Rosenberg et al. reported no significant difference in ash%, weight, length, mineral content and mineral density of bone in response to the dietary intakes of Vit D3 and 25(OH)D3. However, low dietary intake of Vit D3 (200 μg) was associated with reduced bone-breaking strength, cortical bone mineral content and density at the tibial midshaft of the piglets. Thus, both forms of Vit D implied a similar effect on bone formation in healthy pigs.In the study of Witschi 3 outperforms Vit D3 to improve the serum mineral status associated with bone mineralisation in pigs fed low Ca-P diet. Unlike Vit D3, the serum concentrations of Ca, bone-specific alkaline phosphatase and OC increase significantly in pigs receiving basal diets containing 25(OH)D3 and low Ca-P. Therefore, the upregulation of serum markers associated with bone turnover indicates a noticeable positive influence of 25(OH)D3 to balance mineral homeostasis in pigs experiencing Ca-P deficiency. The comparative efficacy of Vit D3 and 25(OH)D3 on mineralisation in healthy pigs appears to be similar.In summary, 25(OH)DVitamin D metabolism is attributed to endogenous adaptive mechanisms for maintaining mineral homeostasis, which is critical for animal health. In particular, potential long-term consequences due to nutritional strategies in the early life stages need to be addressed as summarised in the concept of nutritional programming, thereby inducing resource-efficient phenotypes. However, evaluation must also consider a range of effects in peripheral tissues to account for unintended metabolic effects . With re3 and 25(OH)D3 results in similar reproductive outcomes. The growth performance of piglets and sows fed 25(OH)D3 is significantly better than those fed Vit D3. The superiority of 25(OH)D3 over Vit D3 is evident in enhancing innate and humoural immunity. Improved bone mineralisation in pigs supplemented with 25(OH)D3 on diets low in Ca and phosphorus provides an option to balance animal welfare and resource efficiency. Thus, 25(OH)D3 is a promising and potential alternative to Vit D3 for promoting growth, immunity and bone development in the pigs.In sows, dietary supplementation of Vit D


N6-methyladenosine (m6A) modification of HBV RNA, which plays complex roles in the HBV life cycle. In addition to m6A modification, 5-methylcytosine (m5C) is another major modification of eukaryotic mRNA. In this study, we explored the roles of m5C methyltransferase and demethyltransferase in the HBV life cycle. The results showed that m5C methyltransferase NSUN2 deficiency could negatively regulate the expression of HBV while m5C demethyltransferase TET2 deficiency positively regulates the expression of HBV. Subsequently, we combined both in vitro bisulfite sequencing and high-throughput bisulfite sequencing methods to determine the distribution and stoichiometry of m5C modification in HBV RNA. Two sites: C2017 and C131 with the highest-ranking methylation rates were identified, and mutations at these two sites could lead to the decreased expression and replication of HBV, while the mutation of the “fake” m5C site had no effect. Mechanistically, NSUN2-mediated m5C modification promotes the stability of HBV RNA. In addition, compared with wild-type HepG2-NTCP cells and primary human hepatocytes, the replication level of HBV after NSUN2 knockdown decreased, and the ability of the mutant virus to infect and replicate in wild-type HepG2-NTCP cells and PHHs was substantially impaired. Similar results were found in the experiments using C57BL/6JGpt-Nsun2+/- mice. Interestingly, we also found that HBV expression and core protein promoted the endogenous expression of NSUN2, which implied a positive feedback loop. In summary, our study provides an accurate and high-resolution m5C profile of HBV RNA and reveals that NSUN2-mediated m5C modification of HBV RNA positively regulates HBV replication by maintaining RNA stability.Chronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and liver cancer, despite strong prevention and treatment efforts. The study of the epigenetic modification of HBV has become a research hotspot, including the  N6-methyladenosine (m6A) modification and 5-methylcytosine (m5C). However, compared with m6A modification, fewer studies on RNA m5C modification regulating viruses have been reported. This is partly due to the low abundance of m5C modification in mRNA, and the limits of sequencing technology, which hinder the identification of accurate and repeatable m5C landscape of virus RNA. Here, we discovered that depletion of NSUN2, an m5C methyltransferase, could negatively regulate the replication of hepatitis B virus (HBV). Importantly, we combined in vitro bisulfite sequencing and high-throughput bisulfite sequencing methods to determine the distribution of HBV RNA m5C modification. Two sites modified by NSUN2 with the highest-ranking methylation rates were identified, and mutations of these two sites could lead to decreased replication of HBV. Mechanistically, m5C modification promotes the stability of HBV RNA. Taken together, we provide a high-resolution and accurate atlas of m5C in HBV RNA and reveal a functional role of m5C in viral replication, which provides potential targets for the development of antiviral drugs.The study of the epigenetic modification of viral RNA has become a research hotspot, including the  Chronic hepatitis infection due to hepatitis B virus (HBV) is still the main cause of hepatocellular carcinoma (HCC), and curing chronic hepatitis B (CHB) presents many challenges. Exploring the mechanisms behind its infection and replication has been an important research direction. HBV belongs to the Hepadnaviridae family. Its genome is a double-stranded relaxed circular DNA (rcDNA) of only 3.2kb. The coding frames and the transcriptional regulatory elements are highly overlapped. HBV genome DNA can transcribe five types of RNA: 3.6 kb preC RNA, 3.5 kb pgRNA, 2.4 kb preS1 RNA, 2.1 kb preS2/S RNA, and 0.7 kb X RNA. pgRNA/preC RNAs are longer than the genome length, because these two types of RNA contain terminal redundancy of sequences at their 5’ and 3’ ends, including the epsilon structures. All the HBV RNAs begin at different transcription start sites, but they end at the same transcription termination signal, which means they share a common region at the 3’ end . T. T42]. Tin vivo, C57BL/6Gpt-Nsun2+/- mice were generated and used for experiments after genotyping  or the transcriptional activity of HBV promoters  noncoding RNA led to RNA degradation  methionine , and 40 units of RNase inhibitor, then incubated at 37°C for 60 min, as described previously [3H-labeled products were purified using DEAE-Sephadex A-50 columns and quantitated with a liquid scintillation counting machine (PerkinElmer). No-isotopic methylated RNA fragments were similarly prepared, except using cold SAM (New England Biolabs). miR-125b mimic was used as the positive control [Reaction mixtures were prepared with reaction buffer  according to the instructions: 70°C for 5 minutes and 64°C for 45 minutes, with 3 cycles. The converted RNA was reverse-transcribed with adaptor R primers and then amplified with adaptor primers. The amplified products were connected to the T vector, the positive clones were selected through blue-white spot screening, and then sequenced with Sanger sequencing in Sangong (China). The sequencing results were analyzed with DNMAN (version 7.0). The methylation rate of single C was calculated as the ratio of the reads number of unconverted C to the total number of sequenced reads.in vitro was subjected for bisulfite conversion using an EZ RNA Methylation Kit (Zymo research) according to the instructions: 70°C for 5 minutes and 64°C for 45 minutes, with 3 cycles. After purification, the converted mRNA was used for library construction using a KAPA RNA HyperPrep kit (Illumina). Sequencing was then performed using the Illumina platform of ABlife company (China). The sequencing data were analyzed with meRanTK (version 1.2.0) after quality control to identify the methylation distribution and stoichiometry of m5C sites on the HBV genome, and the conversion rate of luciferase control was analyzed. The raw sequencing data has been deposited in the Genome Sequence Archive (GSA) in National Genomics Data Center, China National Center for Bioinformation [http://ngdc.cncb.ac.cn/gsa-human.pHBV1.3 was transfected into HepG2 and HepG2-NSUN2-KO cells. After 48 hours, TRIzol reagent was added to extract total RNA, and oligo-dT magnetic beads (New England Biolabs) were used to enrich mRNA with two cycles. The enriched 1 μg mRNA with 5 ng of luciferase RNA transcribed ormation , 55, undHepG2 or Huh7 cells were cotransfected with pHBV1.3 and pSV-β-gal. After 48 h, the supernatant was collected. The levels of HBsAg and HBeAg antigens in the culture supernatant were assessed using an enzyme-linked immunosorbent assay kit . The OD values at a 450 nm wavelength were determined with a Multifunction microplate reader (Molecular Devices) according to the manufacturer’s protocol. Cells were lysed with passive lysis buffer (Promega). The activity of β-galactosidase in cell lysates was measured with a Beta-Glo kit (Promega) to normalize the efficiency of transfection.The process of extracting HBV rcDNA from intracellular core particles was performed as previously described with a few modifications . In brieThis assay was conducted according to a protocol described previously . At the Southern blotting for HBV core-associated rcDNA and recovered DNA was conducted as described previously . All of Transfected cells were collected and lysed with NP40 lysis buffer . The mixture was separated on polyacrylamide gel with a gradient concentration, then transferred to a nitrocellulose membrane . The membrane was blocked with 5% nonfat milk and incubated overnight at 4°C with primary antibody against NSUN2 , HA , beta-actin , GAPDH , and HBc . After washing 3 times, the membrane was incubated with goat anti-rabbit secondary antibody for 1 hour. Finally, the membrane was washed 3 times and then exposed with a ChemiDoc Imaging system (Bio-rad) after adding Chemiluminescent HRP Substrate (Millipore).GAPDH) functioned as an endogenous standard. All the primers used in qRT-PCR are listed in The total RNA from cells was isolated using TRIzol reagent (Invitrogen). The cDNA was synthesized using a NovoScript 1st Strand cDNA Synthesis Kit (Novoprotein) after removing DNA. Quantitative real-time PCR (qPCR) was prepared with Hieff qPCR SYBR Green Master Mix (Yeasen) and performed with a CFX96 Real-Time PCR Detection System (Bio-rad). The mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase  was added to inhibit RNA transcription. Then, cells were collected at 0, 3, 6, and 9 hours with TRIzol reagent for RNA extraction and qPCR. All the procedures followed the instructions provided with the DIG Northern Starter Kit (Roche Diagnostics). In brief, 4 μg total RNA was separated using 1% denaturing agarose gel containing 2.2 M formaldehyde and transferred to an Amersham Hybond-N membrane  by capillary transfer. After UV cross-linking of the transferred RNA to the membrane and blocking with prehybridization solution at 60°C for 1 h, denatured DIG-labeled RNA probe was added for hybridizing overnight. Then, the membrane was blocked with blocking solution for 30 min and incubated with DIG antibody for 30 min. Finally, after adding CDP-Star (Roche), the membrane was exposed for 15 min with ChemiDoc MP (Bio-rad). The DIG-labeled plus strand-specific RNA probe corresponding to nucleotides 156–1061 of the HBV genome was used for HBV RNA detection . We usedViral infections of HBV were performed as previously described . Huh7 waTotal DNA including HBV intracellular DNA and cccDNA from infected cells was extracted using a TIANamp Genomic DNA Kit (TIANGEN Biotech). DNA samples for cccDNA qPCR were treated with 500 U/mL T5 exonuclease (NEB) at 37°C for 30 min. A cccDNA-specific primer pair listed in HBV transfected cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.2% TritonX-100. After blocking with 1% BSA, cells were stained with primary antibody against HBcAg (Dako) followed by Alexa-Fluor-labeled goat antirabbit IgG (Thermo Fisher). We added 1 μg/mL of Hoechst 33342 (Thermo Fisher) to stain the nucleus before analyzing. The cell images were captured with an Inverted Fluorescence Microscope (Leica).5C antibody  or anti-m6A antibody  in 800 μL of IPP buffer  for 2 h at 4°C. The mixture was then incubated with 30 μL proteinA/G beads (MedChemExpress) overnight at 4°C. The beads were then washed 5 times with IPP buffer, followed by RNA extraction and qPCR analysis.Total RNA from HBV transfected or infected cells was incubated with anti-mNsun2+/- mice were obtained from Gempharmatech Co.,Ltd  and housed and bred in specific pathogen-free conditions. The knockout targeting strategy is outlined in in vivo experiments, 4-week-old male mice were used and separated into four groups (six mice per group) based on genotyping results. Rapid genotyping was performed using the KAPA mouse genotyping kit (Kapa Biosystems), and the primers used for genotyping are shown in C57BL/6JGpt-P ≤ 0.05 was considered statistically significant.All experiments were repeated at least three times. The results are presented as means ± SD unless stated otherwise. The statistically significant differences were determined using Student’ s t-test for two groups and one- or two-way ANOVA analysis for more than two groups. Statistical analyses were achieved using Prism 8 software (GraphPad Software Inc.). S1 FigA) Western blot of NSUN2 protein for HepG2 and HepG2-NSUN2-KO cells. (B) ELISA results of HBV antigens after NSUN2 knockout in HepG2. (C) Northern blot (left) and gray degree (right) analysis of HBV RNA after NSUN2 knockout in HepG2. Ribosomal RNAs (28S and 18S) were used as the loading control. Relative levels of HBV RNA were quantified using Quantity One. (D) Southern blot (left) and gray degree analysis (right) of HBV core associated DNA after NSUN2 knockout in HepG2. RI, replication intermediates. (E) Western blot of NSUN2 protein for Huh7 and Huh7-NSUN2-KO cells with NSUN2 or NSUN2-I302A/321A rescue after 48 hours. Immunoblots shown are representative of three independent experiments. Graphs show the mean ± SD derived from three independent experiments and were analyzed by two-way ANOVA analysis (two targets) followed by multiple comparisons test. ns, not significant for P > 0.05, *P < 0.05, **P < 0.01, ***P< 0.001, ****P< 0.0001.(A-D) pHBV1.3 and pSV-β-gal were transfected into HepG2 and HepG2-NSUN2-KO cells. At 48 hours later, supernatant was collected for ELISA. Cells were harvested for RNA or rcDNA extraction. β-galactosidase activity in cell lysates was measured to normalize efficiency of transfection for ELISA. ((TIF)Click here for additional data file.S2 Figin vitro methylation was used as the negative control. The sequence at the bottom indicated with red arrow represented the original or reference sequence and was not included in the total read numbers.Multiple alignments of bisulfite-converted sequences of sites C2017, C2268, and C131 with Sanger sequencing. Ratios of unconverted reads to total reads are indicated. Fragment of HBe-2 without (TIF)Click here for additional data file.S3 FigA) IGV alignments of the regions containing sites C131, C2017, and C2268 from two biological replicates of high-throughput bisulfite sequencing of HBV RNA. Reads were aligned to the bisulfite-converted HBV genome sequence (NC_003977.2). Red in bar chart indicates original Cs from reference genome were converted to Ts, and blue in bar chart indicates m5C methylated Cs were not converted. Conversion rates (methylation rate) are indicated with percentages. Y-axis indicates read depth. (B) Sequence alignment of the regions containing sites C131, C2017, and C2268 from different HBV genotypes, with GenBank accession numbers. Red boxes indicate site positions.((TIF)Click here for additional data file.S4 FigA) Schematic diagram of the prcccDNA/pCMV-Cre system, which utilizes the Cre recombinase to generate a surrogate for cccDNA known as rcccDNA. C2017A and C2268T mutations were introduced into the corresponding regions, respectively. (B and C) prcccDNA and pCMV-Cre were transfected into HepG2 cells. At 48 hours later, supernatant was collected for ELISA. Cells were harvested for RNA, rcccDNA, and rcDNA extraction. (B) Southern blot result of rcccDNA (top), Northern blot result (medium) of HBV RNA, and Southern blot (bottom) result of HBV rcDNA. RI, replication intermediates. (C) ELISA results of HBV antigens after site mutations. Graphs show the mean ± SD derived from three independent experiments and were analyzed by two-way ANOVA analysis (two targets) followed by multiple comparisons test. ns, not significant for P > 0.05, ****P< 0.0001.((TIF)Click here for additional data file.S5 FigA) Half-life of HBV preC/pgRNA after TET2 depletion in Huh7. The expression of TET2 in Huh7 was depleted with siRNA, followed by transfection with pHBV1.3. At 36 hours later, actinomycin D  was added, and then cells were harvested after 0, 4, 8, 12, and 16 hours for qPCR with GAPDH as the control. The TET2 mRNA level was also determined with qPCR for assessing knockdown efficiency. (B) MeRIP-qPCR of m5C methylated HBV transcripts in Huh7 after TET2 depletion. FAM129b and HPRT1 serve as positive and negative controls, respectively. (C) Northern blot (left) and gray degree analysis (right) of HBV genomic and subgenomic RNA after NSUN2 knockout in Huh7. Graphs show the mean ± SD derived from three independent experiments and were analyzed by unpaired Student’s t test. ns, not significant for P > 0.05, **P < 0.01, ***P< 0.001, ****P< 0.0001.((TIF)Click here for additional data file.S6 FigA) Nsun2-202 transcript was used for sgRNA designing, and exon 4 - exon 9 was the targeting region with 8954 bp fragment deleted. (B) Two sets of primers were used for genotyping. (C) The size of targeted band is shown. For primer set F1/R1, if the WT band is too large, it may not be possible to obtain a WT band. According to all the genotyping results, no homozygote mice was detected. (D) Sequence alignment of Nsun2-202 transcript and PCR product using primer F1/R1 from Nsun2 heterozygote mice.((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


FGFR-driven urothelial cancer, resistance to selective FGFR inhibitors is largely explained by the emergence of FGFR kinase domain mutations and alterations in the PI3K–mTOR pathway.In patients with  FGFR-driven urothelial cancer, and molecular mechanisms of resistance leading to patient relapses have not been fully explored. We identified 21 patients with FGFR-driven urothelial cancer treated with selective FGFR inhibitors and analyzed postprogression tissue and/or circulating tumor DNA (ctDNA). We detected single mutations in the FGFR tyrosine kinase domain in seven (33%) patients  and multiple mutations in one (5%) case . Using Ba/F3 cells, we defined their spectrum of resistance/sensitivity to multiple selective FGFR inhibitors. Eleven (52%) patients harbored alterations in the PI3K–mTOR pathway . In patient-derived models, erdafitinib was synergistic with pictilisib in the presence of PIK3CA E545K, whereas erdafitinib–gefitinib combination was able to overcome bypass resistance mediated by EGFR activation.Several fibroblast growth factor receptor (FGFR) inhibitors are approved or in clinical development for the treatment of In the largest study on the topic thus far, we detected a high frequency of FGFR kinase domain mutations responsible for resistance to FGFR inhibitors in urothelial cancer. Off-target resistance mechanisms involved primarily the PI3K–mTOR pathway. Our findings provide preclinical evidence sustaining combinatorial treatment strategies to overcome bypass resistance.See related commentary by Tripathi et al., p. 1964.This article is featured in Selected Articles from This Issue, p. 1949 FGFR) gene family are frequent events in urothelial cancer. FGFR3 mutations, a hallmark of low-grade, non–muscle invasive bladder cancer, are present in 12% to 22% of high-grade, muscle-invasive tumors (FGFR3 mutations in up to 60% of high-grade cases (FGFR3 fusions (with TACC3 as the most frequent partner gene) are found in up to 2% to 3% of bladder cancer and detected in upper tract urothelial carcinoma as well  and median progression-free survival (PFS) were 40% and 5.5 months, respectively ; four out of 15 patients with FGFR3 molecular alterations treated with chemotherapy achieved disease response  samples at progression on infigratinib. In four patients, acquired mutations in the FGFR3 tyrosine kinase domain were detected  and combinatorial treatment strategies in preclinical models, providing potential future guidance for clinical application.Here we present a molecular and functional study of resistance to selective FGFR inhibitors in patients with n = 39 bladder, n = 15 upper urothelial tract, n = 2 with both localizations). Thirty-eight patients had both whole-exome sequencing (WES) and RNA sequencing (RNA-seq) performed on tissue biopsy, nine had WES only, and one had RNA-seq only; eight patients had a targeted next-generation sequencing (NGS) panel on ctDNA and/or tissue biopsy.We collected clinical and molecular data for a total of 56 patients with advanced/metastatic urothelial cancer , FGFR3 (34%), and KMT2D (34%). Taking into account the limitation of evaluating expression profiles from bulk RNA-seq analyses of tissue biopsies with variable tumor/stroma cellularity, we investigated differential expression of genes involved in the main signaling pathways in the 39 samples with RNA-seq available. No clear pattern was highlighted between FGFR3-driven and non–FGFR3-driven patient samples with the exception of FGFR3 mRNA . Analyzing FGFR family member expression levels specifically, we did indeed reveal a strong significant overexpression of FGFR3 mRNA in FGFR3-driven samples compared with FGFR3 wild-type (WT) ones . By contrast, only a slight decrease was observed for FGFR1 and FGFR4 mRNA and no difference was observed for FGFR2 expression.The assessment of the mutational features has been the main objective of seminal studies shedding light on the frequency of diseases . We therdiseases . The mosFGFR3::TACC3 fusion, and three displayed an FGFR3 Y373C mutation. One patient (MR904) harbored an FGFR2::FAM83H-AS1 rearrangement and one (MR103) an FGFR4 D276N mutation , futibatinib (n = 4), or pemigatinib (n = 3). As best objective responses, we observed one patient with a complete response (CR), 12 patients with partial responses (PR), six patients with stable disease (SD), and two patients with progressive disease (PD). Of note, all patients were previously treated for advanced disease, and the majority received the FGFR inhibitor as the second treatment line , but no clear correlation between the type of cytotoxic agents received by patients prior to FGFR inhibitors and molecular alterations could be evidenced (Supplementary Table S2).Postprogression samples were collected on erdafitinib  of these four FGFR3 mutations could not be assessed due to ctDNA amplicon size limitation.In the cohort of 19 nts 37%; . Sequencnd E587Q . Three cnd E587Q  and B, wFGFR3 amplification and PIK3CA E726K mutation were found in the tissue biopsy only  in the postprogression liquid biopsy, together with an FGFR3 V555M mutation and a TSC2 frameshift event .In line with limited ctDNA shedding in cases of intrathoracic-only disease, the FGFR3 V553L mutation was detected only in the tissue biopsy and not in the ctDNA of patient MR406, who experienced mild, thoracic-only progression on pemigatinib . Given tThe VAF increase of both FGFR3 S249C and V555L reflected the progression during erdafitinib treatment in patient MR560 .For patient MR86, an FGFR3 N540K mutation was detected in tissue biopsy in addition to the driver mutation FGFR3 S249C and a PIK3CA E545K mutation already existing in the pre-erdafitinib sample .FGFR2 rearrangement in one patient with urothelial cancer (MR904), third-line pemigatinib resulted in a CR (FGFR2::FAM83H-AS1), as well as an FGFR2 L551F mutation (according to NM _022970.3 transcript).Additionally, upon detection of an  in a CR . Upon prFGFR3-driven urothelial carcinoma, we detected an alteration in the PI3K–mTOR pathway in postprogression and/or in baseline samples (In 11 of 19 patients (58%) with  samples .PIK3CA activating mutation was detected at progression, but in three cases the PIK3CA alteration was present before the treatment with FGFR inhibitors . Patients MR15 and MR86 achieved clinical responses on erdafitinib before progression. Patient MR86 acquired an FGFR3 N540K mutation, whereas an EGFR hyperphosphorylation [determined by a phospho–receptor tyrosine kinase (RTK) array] potentially explained the resistance for patient MR15 . By contrast, patient ST2267, who had a baseline PIK3CA H1047R tumor mutation, experienced primary resistance to futibatinib. For patient MR336, the PIK3CA E545K mutation was acquired during treatment with erdafitinib, which potentially conferred the primary resistance to futibatinib .In five samples, a PIK3CA mutations, TSC1/2 inactivating mutations were also frequently detected (5/19) in patients progressing on FGFR inhibitors .In patient MR246, who had an FGFR3 S249C-driven bladder cancer that ultimately progressed on erdafitinib, an NF2 frameshift event was detected by ctDNA together with an FGFR2 V517M mutation.Finally, a PTEN C136fs mutation was detected in a patient with primary resistance to erdafitinib (M2057).The frequency of these mutations arising in tumor suppressor genes suggests they should be considered as potential targets for overcoming treatment resistance.FGFR3::TACC3 fusion harboring either N540K, V553L, V553M, V555L, V555M, or L608V mutations. The expression levels of the FGFR3::TACC3 exogenous fusion protein were homogeneous across cell lines . Ba/F3 cells were exposed to increasing doses of the FGFR inhibitors erdafitinib, infigratinib, pemigatinib, rogaratinib, futibatinib, derazantinib, AZD4547, and zoligratinib to determine their corresponding IC50 values , suggesting the drug exerted off-target effects on proliferation. In light of this observation, and considering that LY2874455 is no longer in clinical development, we did not include it in the pharmacologic experiments.In order to evaluate the functional impact of FGFR3 kinase domain mutations on the activity of FGFR inhibitors, we generated Ba/F3 cells with an 50 values in the subnanomolar/nanomolar range against FGFR3::TACC3 WT-driven Ba/F3 cell lines. Each FGFR3 kinase domain mutation conferred a variable increase in the IC50 values to FGFR inhibitors. The IC50 against the frequent N540K mutant was >100 nmol/L for all the agents with the exception of erdafitinib and futibatinib (5 times lower). Similarly, erdafitinib and futibatinib retained low activity against the gatekeeper V555L/V555M, V553M, and L608V mutations. The V553L mutant appeared to confer a moderate degree of resistance to FGFR inhibitors, as IC50 values were close to 10 nmol/L for five inhibitors but at subnanomolar/nanomolar concentrations for erdafitinib, infigratinib, and futibatinib. Of note, this mutation emerged in a patient progressing on pemigatinib . Analysis of intracellular signaling across models confirmed the viability assays. FGFR3, AKT, and ERK phosphorylation was abrogated after treatment with low concentrations of erdafitinib in the WT model, whereas higher doses were required in the N540K, V553M, V555L, and L608V mutants  in inhibiting tumor growth in the MR15 PDX . Of note, the characteristics of the FGFR3 kinase domain at resistance are distinctly different from what has been observed in FGFR2-driven cholangiocarcinoma. In patients suffering from cholangiocarcinoma, the occurrence of polyclonal  FGFR2 kinase domain mutations is frequently reported at progression on reversible FGFR inhibitors  and gatekeeper (V555L) mutation in conferring resistance. V555L, thought to interfere with drug binding , boosts FGFR2-driven tumors. In our patient MR904, the mutation emerged at progression on pemigatinib, and Varghese and colleagues reported a similar finding at progression on infigratinib in a patient affected by an FGFR2-driven cholangiocarcinoma  availability of postprogression tissue and/or ctDNA available for molecular analyses.To be included in this study, patients had to satisfy the following criteria: (i) diagnosis of an advanced urothelial cancer; (ii) treatment with a selective FGFR inhibitor due to the detection of a molecular alteration in one of the Patients started FGFR inhibitor treatment between 2013 and 2021. Patients were treated in the setting of clinical trials or compassionate use programs allowing treatment with FGFR inhibitors on the basis of molecular selection. Disease response was measured according to RECIST, and PFS was calculated from the date of the targeted inhibitor start to the day of radiologic evidence of progression.The molecular analyses were performed within institutional studies ongoing at Gustave Roussy whose aim is the molecular characterization of tumors: MATCH-R , and are being conducted in accordance with the Declaration of Helsinki.The objectives of the MOSCATO and MATCH-R studies are to address patients to targeted treatments according to molecular alterations and to assess molecular mechanisms of resistance, respectively. These studies initially focused on molecular analysis performed on tissue biopsy, as ctDNA technologies were not commonly used and blood/plasma samples were not collected . On the other hand, CTC and STING-UNLOCK were developed to study molecular alterations from blood samples . The availability of postprogression samples relied on the precise study in which individual patients were enrolled.Postprogression tumor, when possible, underwent WES with or without concomitant RNA-seq. The main limitation of WES/RNA-seq performance was the proportion of tumor cells ≥30% in the tissue sample. In cases in which the proportion of tumor cells was between 10% and 30%, molecular analyses with targeted NGS panels  were performed. For WES, the mean coverage was 140×.FGFR genes. Briefly, extracted circulating free DNA from patient samples was processed through a library preparation that includes the addition of unique molecular identifiers, followed by a hybrid capture–based workflow and high-depth sequencing. For each patient with longitudinal ctDNA assessment, only analyses performed with the same platform were reported.ctDNA analyses were performed with Illumina or Foundation Medicine liquid biopsy panels. Importantly, all ctDNA sequencing had complete coverage of the kinase domains of FGFR3::TACC3 fusions were created using the pLenti6/V5 directional TOPO Cloning Kit  according to the manufacturer's instructions. Point mutations in the FGFR3 kinase domain of the FGFR3::TACC3 fusion were introduced using the QuickChange XL Site-Directed Mutagenesis Kit  according to the manufacturer's protocol and using the following primers:FGFR3 N540K forward (F) CAAAAACATCATCAAGCTGCTGGGCGCCTGCFGFR3 N540K reverse (R) GCAGGCGCCCAGCAGCTTGATGATGTTTTTGFGFR3 V553L F CGCAGGGCGGGCCCCTGTACTTGCTGGTGGAGTACGCGGCCFGFR3 V553L R GGCCGCGTACTCCACCAGCAAGTACAGGGGCCCGCCCTGCGFGFR3 V553M F CGCAGGGCGGGCCCCTGTACATGCTGGTGGAGTACGCGGCCFGFR3 V553M R GGCCGCGTACTCCACCAGCATGTACAGGGGCCCGCCCTGCGFGFR3 V555L F GCCCCTGTACGTGCTGCTGGAGTACGCGGCCAAFGFR3 V555L R TTGGCCGCGTACTCCAGCAGCACGTACAGGGGCFGFR3 V555M F GCCCCTGTACGTGCTGATGGAGTACGCGGCCAAFGFR3 V555M R TTGGCCGCGTACTCCATCAGCACGTACAGGGGCFGFR3 L608V F AGGTGGCCCGGGGCATGGAGTACGTGGCCTCCCAGAAGTGCATCCACFGFR3 L608V R GTGGATGCACTTCTGGGAGGCCACGTACTCCATGCCCCGGGCCACCTLentiviral vectors expressing FGFR2::BICC1, and the primers used for creating L551F mutations were as follows:FGFR2 L551F F GGGAAACACAAGAATATCATAAATTTTCTTGGAGCCTGCACACAGGATGFGFR2 L551F R CATCCTGTGTGCAGGCTCCAAGAAAATTTATGATATTCTTGTGTTTCCCThe same procedure was used to create lentiviral vectors expressing cis/trans status of mutations.To assess the allelic status of FGFR3 S249C and V553L in patient MR406, mRNA obtained from the tissue biopsy underwent retro-transcription, and a PCR spanning the two mutated residues was performed on cDNA. Amplicons were subcloned into the pCR2.1-TOPO vector (Invitrogen) according to the manufacturer's protocol. Individual cDNA was sequenced by Sanger sequencing to determine the All animal procedures and studies have been approved by the French Ministry of “Education nationale, de l'Enseignement supérieur et de la Recherche” (APAFIS#2790-2015112015055793 and APAFIS#2328-2015101914074846). Fresh tumor fragments were implanted in the subrenal capsule of 6-week-old female NOD/SCID gamma (NSG) mice obtained from Charles River Laboratories. PDX-bearing NSG mice were treated with gefitinib  or erdafitinib [15 mg/kg qd in 10% HP-beta-CD (hydroxypropyl beta cyclodextrin)] or pictilisib  alone or in combination by oral gavage. Eight mice per group were treated for up to 40 days, and tumor volume and mouse weight were measured twice per week.FGFR3::TACC3 or the FGFR2::BICC1 fusions with a WT kinase domain or with the mentioned kinase domain mutations. Infected Ba/F3 cells harboring FGFR fusions were selected in the presence of blasticidin (14 mg/mL) and IL3 (0.5 ng/mL) until recovery, and a second selection by culturing the cells in the absence of IL3 was then performed. FGFR2/3 fusions and corresponding kinase domain mutations were confirmed on the established cell lines by Sanger sequencing.Parental Ba/F3 cells were purchased from DSMZ (ref. ACC 300) and cultured in DMEM 10% FBS in the presence of IL3 (0.5 ng/mL). Ba/F3 cells were infected with lentiviral constructs, as reported previously , to exprAfter several attempts, we were not able to establish IL3-independent Ba/F3 cells driven by FGFR3 S249C or FGFR3 Y373C mutations. Previous experience showed that FGFR2 cannot drive IL3-independent proliferation in the same way as FGFR1 , and we Patient-derived cell lines (MR15) were developed from PDX samples by enzymatic digestion with a tumor dissociation kit  and mechanical degradation with the gentleMACS dissociator. Cells were cultured with DMEM/F-12 GlutaMAX 10% FBS and 10% enriched with hydrocortisone 0.4 mg/mL, cholera toxin 8.4 ng/mL, adenine 24 mg/mL, and ROCK inhibitor 5 mmol/L  until a stable proliferation of tumor cells was observed, as described previously . CultureFGFR fusion and mutations by RT-PCR and Sanger sequencing. The cells were not tested for Mycoplasma contamination, but cells were not maintained in culture for more than 2 months after establishment or thawing.As for cell verification, we confirmed that engineered Ba/F3 as well as MR15 patient-derived cell line harbored the corresponding Erdafitinib, infigratinib, pemigatinib, futibatinib, derazantinib, AZD4547, zoligratinib, and LY2874455 were purchased from Selleck Chemicals. Rogaratinib was purchased from MedChemExpress.Cell viability assays were performed in 96-well plates using the CellTiter-Glo Luminescent Cell Viability Assay . For Ba/F3, we seeded 4,000 cells/well and we treated cells for 48 hours. For the MR15 patient-derived cell line, we seeded 3,000 cells/well and treated cells for 5 days.FGFR3::TACC3 fusion with a WT or a mutated kinase domain were treated for 24 hours with the corresponding doses of FGFR inhibitors in the presence of heparin 25 μg/mL  and aFGF 50 ng/mL , as previously reported , pAKT (4060S), AKT (4691S), pERK (9101S), ERK (9102S), pS6 (4858S), S6 (2217S), and BIM (2933S). Anti-FGFR3 (ab133644) and anti-pEGFR (ab5644) antibodies were purchased from Abcam, anti-EGFR (sc-03) antibody was purchased from Santa Cruz Biotechnology, and anti–β-actin (A1978) antibody was purchased from Sigma-Aldrich.https://ega-archive.org/. Any additional information required to reanalyze the data reported in this article is available upon request from the corresponding author.WES/RNA-seq raw data files from this study are deposited at the European Genome–phenome Archive (EGA) using the accession code EGAS00001007335. Access to this shared dataset is controlled by the institutional Data Access Committee, and requests for access can be sent to the corresponding author. Further information about EGA can be found at Supplementary Table 1Patient population, treatment outcomes and post-progression sample availability.Click here for additional data file.Supplementary Table 2Systemic treatment received by patients prior to selective FGFR inhibitors, according to the detection of FGFR kinase domain mutations and/or PI3K/mTOR pathway alterations.Click here for additional data file.Supplementary Table 3IC50 values of Ba/F3 cells harboring FGFR3:TACC3 mutants exposed to eight selective FGFR inhibitors.Click here for additional data file.Supplementary Figure 1RNAseq analysis from tissue biopsies of 39 advanced urothelial cancers.Click here for additional data file.Supplementary Figure 2Documentation of the allelic status in cis of FGFR3 S249C and V553L mutations from MR406 tissue biopsy.Click here for additional data file.Supplementary Figure 3Acquisition of mutations in PI3K/TSC1 pathway at progression to FGFR inhibitors.Click here for additional data file.Supplementary Figure 4Expression of FGFR3:TACC3 was homogeneous across Ba/F3 cell lines.Click here for additional data file.Supplementary Figure 5Synergic effect of gefitinib and erdafitinib in MR15 cell line.Click here for additional data file.


SF3B4 expression and immune cell infiltration.Hepatocellular carcinoma (HCC) accounts for a majority of primary liver cancer cases and related deaths. The purpose of this study was to assess the diagnostic value of splicing factor 3b subunit 4 (SF3B4) as a novel non-invasive biomarker for HCC and determine the association between SF3B4 were measured using quantitative reverse transcription PCR.An enzyme-linked immunosorbent assay (ELISA) was used to detect SF3B4 levels in plasma samples obtained from healthy controls (HCs) and patients with chronic hepatitis, liver cirrhosis, and HCC. The expression levels of autoantibodies that detect SF3B4 in the plasma samples of each group of patients were measured. Small extracellular vesicles (EVs) were isolated from patient sera, and the expression levels of EV-SF3B4 in serum EV increased but not in the buffy coat or serum of patients with HCC. Serum EV-SF3B4 displayed better diagnostic power than AFP for all stages of HCC (AUC = 0.968 vs. 0.816), including early-stage HCC (AUC = 0.960 vs. 0.842), and this was consistent in the external cohort. Single-cell RNA sequencing indicated that SF3B4 expression was correlated with myeloid-derived suppressor cells. The Tumor Immune Estimation Resource database reconfirmed the correlation between SF3B4 expression and immune cell infiltration in HCC.ELISA results confirmed that the expression levels of SF3B4 proteins and autoantibodies in the plasma of patients with HCC were higher than those in HCs. However, their diagnostic performance was not better than that of alpha-fetoprotein (AFP). The mRNA expression of SF3B4 shows potential as a novel non-invasive diagnostic biomarker of HCC.SF3B4 may be associated with tumor immune infiltration in HCC, and EV-The online version contains supplementary material available at 10.1186/s13046-023-02867-y. Liver cancer was reportedly the sixth most commonly diagnosed cancer and the third leading cause of cancer-related deaths worldwide in 2020, with approximately 906,000 new cases and 830,000 deaths . HepatocAlthough various studies have deepened our understanding of HCC carcinogenesis, HCC surveillance mainly relies on abdominal ultrasonography and blood alpha-fetoprotein (AFP) measurements in high-risk groups with hepatitis B, hepatitis C, and liver cirrhosis (LC) –5. It maKruppel-like factor 4 in HCC  plotting), and heatmap generation. Briefly, the most variable genes  across the filtered cells were selected using the FindVariableFeatures function to perform PCA on scaled data. Subsequently, the FindNeighbors and FindClusters functions were used to construct a shared nearest neighbor (SNN) graph (‘dims = 1:30’) and cluster the cells (‘resolution = 1’), respectively. The RunUMAP function was used with 50 PCA dimensions (‘dims = 1:50’) to visualize cells on a 2-D UMAP plot. Cells annotated as ‘MDSCs’ were extracted and used to generate independent Seurat objects. The same parameters were applied for the functions mentioned above except the RunUMAP function, which uses 30 PCA dimensions (‘dims = 1:30’). The enrichGO function in the R clusterProfiler package (v3.18.1) was used to perform Gene Ontology (GO) analyses of the top 100 enriched genes. Enrichment analysis of the ‘The Molecular Signatures Database (MSigDB) Hallmark 2020’ database was performed using the enrichr function in the R enrichR package (v3.0).Unique molecular identifier (UMI) counts and metadata of filtered cells (https://cistrome.shinyapps.io/timer/) contains comprehensive resources that enable investigations into the correlation between gene expression and immune cell infiltration via data pertaining to over 30 types of cancers in TCGA. The association between SF3B4 and tumor purity and the four types of immune cells  cells, regulatory T cells, and MDSC) were plotted using TIMER. In addition, the correlation between SF3B4 and biomarkers of MDSCs was explored using the ‘correlation’ module.The Tumor Immune Estimation Resource (TIMER) website . Statistical significance of differences between two groups was assessed using paired Student’s t-test or unpaired Welch’s t-test using GraphPad Prism version 10.0 . One-way analysis of variance (ANOVA) with Tukey’s post-hoc analysis was used for multiple comparisons among three groups. Kaplan–Meier survival curves were constructed to assess the significance of the prognostic power between two patient groups. Significant differences between survival curves were determined using the log-rank test. Receiver operating characteristic (ROC) and Cox proportional hazard regression analyses were performed using IBM SPSS software . ROC curves were analyzed to evaluate the sensitivity, specificity, and respective area under the curve (AUC) with 95% confidence intervals (CIs) for plasma SF3B4 protein, plasma anti-SF3B4 autoantibodies, and serum-derived EV-SF3B4 expression in tumor and non-tumor tissues using the pan-cancer database, TCGA. Among the 33 different cancer types, SF3B4 was significantly upregulated in tumor tissues compared with that in non-tumor tissues in 10 cancer types, including LIHC  using a 22 K protein chip to investigate the diagnostic potential of SF3B4 as a blood-based biomarker of HCC. All subjects were males with LC of HCC etiology involving HBV. Serial clustering analysis revealed that the SF3B4 autoantibody was positively expressed from LC to HCC groups but was undetected in the control group  to mitigate the bias associated with a single institution study. Baseline characteristics of the cohorts are presented in Tables SSF3B4 in cohort 3 and the external cohort according to the mUICC stage showed that serum EV-SF3B4 expression gradually upregulated with tumor stage progression  compared to that of AFP according to ROC curves  for early HCC (mUICC stage I). Moreover, serum EV-SF3B4 exhibited a better AUC (0.854) than AFP (0.548) in the CH/LC vs. HCC model  for early HCC (mUICC stage I). Subsequently, we executed a rigorous validation of the diagnostic proficiency of EV-SF3B4 against AFP utilizing ROC analysis in the external cohort. EV-SF3B4 manifested superior AUC values in every comparison, despite AFP displaying elevated AUC values in relative comparisons within the external cohort. The AUC values were 0.816 for AFP and 0.968 for EV-SF3B4 when compared against non-tumor (NT) conditions inclusive of HC, CH, and LC to HCC. The AUC values were 0.842 for AFP and 0.960 for EV-SF3B4 between NT and mUICC I  compared to the AUC of serum EV-SF3B4 (0.968) . The analysis was conducted on HC: 56 individuals, CH: 50 individuals, LC: 54 individuals, mUICC I: 61 individuals, mUICC II: 28 individuals, and mUICC III/IV: 43 individuals, excluding patients with missing AFP values. Analysis of the combination of AFP and EV-SF3B4 against NT and HCC, NT and mUICC I/II, and NT and mUICC I showed an AUC of 0.942, 0.954, and 0.948, respectively  derived from patients with HCC to assess the differences between SF3B4 expression levels across distinct immune cell populations. Nine cell types were identified among 50,023 QC-filtered HCC cells , 42 (48.8%), 56 (65.1%), 32 (37.2%), 46 (53.5%), and 48 (55.8%) patients, respectively. The mRNA expression levels of SF3B4 and other MDSC markers in HCC tissues of the same patient showed a high positive correlation , except for LOX  than AFP . EV-SF3B4 showed a higher positivity rate (85%) than AFP (30%) in early HCC when positivity cut-off values of 7.81 fold were used for EV-SF3B4 and 20 ng/mL for AFP. Moreover, EV-SF3B4 demonstrated a high positivity rate (84%), even in AFP-negative early HCCs. The combination of AFP and serum EV-SF3B4 exhibited an AUC of 0.942 (95% CI:0.908–0.966) in the HCC vs. non-tumor model and an AUC of 0.935 (95% CI:0.899–0.962) in the HCC vs. CH/LC model. These findings indicated that serum EV-SF3B4 alone (or combined with AFP) shows clinical potential as a diagnostic biomarker for early HCCs.SF3B4 is a subunit of the U2 small nuclear ribonucleoprotein that plays a crucial role in the splicing process, cell cycle regulation, cell differentiation, and immune deficiency-related activities . It is aSpliceosomes play a key role in cancer and immunity , 25. TheSF3B4 levels in patients with malignancies other than HCC were not measured. Consequently, we were unable to confirm the specificity of serum EV-SF3B4 levels in HCC. Furthermore, slightly different cohorts  were used rather than the same cohort when implementing various measurement methods. This was unavoidable since only a limited number of samples were provided by the Biobank.This study was affected by several limitations. Firstly, serum EV-SF3B4 levels regarding HCC diagnosis was excellent when used alone or combined with AFP. In particular, serum EV-SF3B4 may play a diagnostic role in early HCC, wherein AFP is low (< 20 ng/ml). SF3B4 is highly expressed in MDSCs in the tumor microenvironment, and the aberrant expression of SF3B4 and MDSC markers may help predict poor prognosis in patients with HCC.The performance of serum EV-Additional file 1: Supplementary Table 1. Clinicopathological characteristic of the patients in cohort 1. Supplementary Table 2. Clinicopathological characteristic of the patients in cohort 2. Supplementary Table 3. Clinicopathological characteristic of the patients in cohort 3. Supplementary Table 4. Clinicopathological characteristic of the patients in the external cohort. Supplementary Table 5. Univariate and multivariate Cox regression analyses of factors associated with overall survival and disease-free survival in TCGA LIHC dataset. Supplementary Table 6. Markers of immune cells. Supplementary Fig. 1. The correlation between the expression of SF3B4 gene and the prognosis of ten different types of cancer in TCGA.A Overall survival rate and B Disease-free survival rate, as well as the expression of SF3B4 gene in various tumors using GEPIA2 software. Supplementary Fig. 2. EV-AFP expression and its diagnostic power in HCC. A Box plot of AFP expression in blood-derived EVs from different diseases. NP, normal person; CHD, coronary heart disease; CRC, colorectal cancer; HCC, hepatocellular carcinoma; PAAD, pancreatic adenocarcinoma; TPM, transcripts per million B Expression of serum EV-AFP based on the stage of liver disease (left) and AUCs of serum EV-AFP compared to serum EV-SF3B4 for diagnosing HCC (right) in the external cohort. Statistically significant differences were determined using one-way ANOVA with Tukey’s multiple comparisons test. Compared to HC; *P <0.05, **P <0.01. Supplementary Fig. 3. The distribution of five well-known MDSC markers in whole cells. Supplementary Fig. 4. The relationship between SF3B4 Copy Number Variation and immune infiltration. *P <0.05, **P<0.01. Supplementary Fig. 5. Bar chart showing the expression of SF3B4 and MDSC markers in a set of 86 pairs of HCC tissues.


Human calprotectin  is an abundant neutrophil protein that contributes to innate immunity by sequestering nutrient metal ions in the extracellular space. This process starves invading microbial pathogens of essential metal nutrients, which can inhibit growth and colonization. Over the past decade, fundamental and clinical studies have revealed that the S100A8 and S100A9 subunits of CP exhibit a variety of post-translational modifications (PTMs). This review summarizes PTMs on the CP subunits that have been detected and highlights two recent studies that evaluated the structural and functional consequences of methionine and cysteine oxidation on CP. Collectively, these investigations indicate that the molecular speciation of extracellular CP is complex and composed of multiple proteoforms. Moreover, PTMs may impact biological function and the lifetime of the protein. It is therefore important that post-translationally modified CP species receive consideration and integration into the current working model for how CP functions in nutritional immunity. Transition metals are essential nutrients that bacterial pathogens must acquire from the host environment during infection  and a His6 motif (site 2)  binding markedly increases the transition metal affinities of both sites and enables multi-metal sequestration  and binds other M(II) with only relatively low affinity; in contrast, the His6 site is functionally versatile and sequesters Mn(II), Fe(II), Ni(II), and Zn(II) , which provides two His residues for M(II) coordination, encapsulates the M(II) in the site, and blocks solvent access -binding proteins, S100A8  and S100A9  Fig. . Each S12 heterotetramer, which displays enhanced proteolytic stability and metal-binding affinities and is capable of competing with microbial pathogens for multiple M(II)  on the human protein. In recent years, various PTMs on S100A8 and S100A9 have been detected in human specimens Table . We highπ-methylation on S100A9 detected in various murine tissues and cell in culture , sputum from cystic fibrosis (CF) patients, serum, plasma, nasal mucus, pimple pus, and kidney stones Table . Evidenc15N-labeled CP-Ser [S100A8(C42S)/S100A9(C3S) variant] immediately after collection and monitoring for Met oxidation of 15N-S100A8(C42S) and 15N-S100A9(C3S) by mass spectrometry , hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) during the oxidative burst  including superoxide -induced tetramerization, a key feature of CP that confers metal-sequestering ability and protease resistance , providing a potential explanation for the retained antibacterial activity. Further investigation revealed that oxidation of S100A9(M81) disfavored Ca(II)-induced tetramerization. Notably, S100A9(M81) is located in the tetramer interface along with a number of other hydrophobic residues on S100A8 and S100A9 -bound heterodimer is more susceptible to proteolysis than the Ca(II)-bound heterotetramer  ions revealed that the predominant species was a Ca(II)-bound heterotetramer with at least one S100A8–S100A9 “intradimer” disulfide bond. This result was striking because the possibility of a disulfide linkage within a CP heterodimer was largely overlooked in all prior work; an early study of the CP heterodimer concluded such a linkage was not possible based on a homology model  coordination sphere (Hadley et al. Beyond understanding how various PTMs affect CP structure and function, many additional questions exist. Specific to the examples described above, MetO and disulfide bond formation are reversible PTMs, but it is unknown whether methionine sulfoxide reductases and thioredoxins reverse these modifications. In addition to the oxidative PTMs highlighted in this review, other PTMs detected in murine and human samples are fascinating and require investigation. For instance, it was recently discovered that the N