Synovial fluid contains cytokines, growth factors and resident mesenchymal stem cells (MSCs). The present study aimed to (1) determine the effects of autologous and allogeneic synovial fluid on viability, proliferation and chondrogenesis of equine bone marrow MSCs (BMMSCs) and (2) compare the immunomodulatory properties of equine synovial fluid MSCs (SFMSCs) and BMMSCs after stimulation with interferon gamma (INF-γ). To meet the first aim of the study, the proliferation and viability of MSCs were evaluated by MTS and calcein AM staining assays. To induce chondrogenesis, MSCs were cultured in a medium containing TGF-β1 or different concentrations of synovial fluid. To meet the second aim, SFMSCs and BMMSCs were stimulated with IFN-γ. The concentration of indoleamine-2,3-dioxygenase (IDO) and nitric oxide (NO) were examined. Our results show that MSCs cultured in autologous or allogeneic synovial fluid could maintain proliferation and viability activities. Synovial fluid affected chondrocyte differentiation significantly, as indicated by increased glycosaminoglycan contents, compared to the chondrogenic medium containing 5 ng/mL TGF-β1. After culturing with IFN-γ, the conditioned media of both BMMSCs and SFMSCs showed increased concentrations of IDO, but not NO. Stimulating MSCs with synovial fluid or IFN-γ could enhance chondrogenesis and anti-inflammatory activity, respectively, suggesting that the joint environment is suitable for chondrogenesis. The native minimal regeneration of the articular cartilage is a primary challenge for cartilage healing. The shortage of successful repair also contributes to the prevalent degeneration of the joint related to osteoarthritis (OA). The current approaches used to regenerate the articular cartilage commonly result in the formation of fibrocartilage tissue which is of low-grade quality compared to the intrinsic hyaline articular cartilage ,2. RecenIntra-articular implantation of chondrogenic-generated stem cells (MSCs induced in vitro towards chondrogenic lineage) to regenerate damaged cartilage has been used in previous studies . Based oSynovial fluid has a key role in nourishing articular cartilage, lubricating the articular joint and absorbing shock. In addition, synovial fluid contains growth factors, such as the transforming growth factor (TGF-β1) . In a stIt has been proven that synovial fluid from normal and osteoarthritic joints has a resident MSC population . Advancen = 3) used in previous studies. In this study we reconfirmed the identity of the previously characterized MSCs using cell morphology and colony forming unit assay. Equine BMMSCs and SFMSCs showed a large cluster of stellate cells and growth in a fibroblast-like phenotype  and at day 4 . However, different concentrations of allogeneic synovial fluid could only maintain proliferation and did not show any difference compared to the regular medium containing DMEM-F12, 10% FBS and 1% penicillin/streptomycin  at days 5 and 14. Adding 100% concentration of synovial fluid exhibited significantly higher GAG content in BMMSCs compared to 50% (p < 0.001) at days 5 and 14. Moreover, the 50% and 100% concentrations of synovial fluid induced a high amount of GAG compared to the regular chondrogenic medium (p < 0.001) at day 14  at day 5, but not compared to the regular chondrogenic medium. Moreover, the 50% and 100% concentrations of synovial fluid and regular chondrogenic medium induced a high amount of GAG compared to undifferentiated MSCs at day 14. The 100% concentration of allogeneic synovial fluid showed higher GAG contents than the 50% concentration and regular chondrogenic medium (p < 0.001) at day 14.Allogeneic synovial fluid (50% and 100% concentrations) showed a significant increase in chondrogenesis compared to undifferentiated cells  compared to their respective controls (non-stimulated MSCs)  A. In moned MSCs) B.Because of the inferior self-repair ability of cartilage, cartilage engineering is a new treatment approach to treat osteoarthritic or traumatic cartilage defects. Based on their chondrogenic and immunosuppressive capacity, a cell-based therapeutic strategy using MSCs has been developed for cartilage regeneration. Because of the prevalence of intra-articular injections of BMMSCs in equine regenerative medicine, we decided to use these cells in our study as a comparison with SFMSCs . FurtherAn earlier study demonstrated a significant increase in the cellularity of chondrocytes by supplement with synovial fluid, as indicated by DNA content . AdditioMany reports show chondrogenic differentiation of MSCs in vitro depending on the use of chondrogenic factors. The growth factors of the TGF-β superfamily prompt chondrogenesis of MSCs in vitro . HoweverIn veterinary practice, MSCs have many advantages for clinical applications, such as differentiating into multiple tissue types. In addition, MSCs have a potent anti-inflammatory/immunomodulatory property to treat inflammation ,45. It hAll animal procedures were carried out in accordance with the protocols approved by the University of Tennessee Institutional Animal Care and Use Committee . Six healthy horses (aged 8–12 years) were enrolled in this study.2, adherent cells were harvested at 80% confluency with 0.25% trypsin-EDTA .Isolation, characterization and expansion of BMMSCs were carried out as described previously  from 3 p2 vented tissue culture flasks and incubated at 37 °C in 5% CO2 and 95% relative humidity. This DMEM-F12 medium is the regular growth medium. Adherent cells were harvested with 0.25% trypsin-EDTA for 1 min at 37 °C and cryopreserved at 2nd–4th passages of culture for the in vitro studies.Collection of synovial fluid from normal joints and isolation of SFMSCs were performed according to our previous studies ,32. To c2) were seeded on 10 cm2 diameter dishes  and cultured using DMEM-F12 regular growth medium. When clusters of colonies were formed, at day 7, cells were fixed and stained with 0.5% crystal violet .The colony forming unit assay was conducted with slight modification, according to traditional methods . Briefly4 cells) was evaluated in the presence of autologous and/or allogenic synovial fluid with varying concentrations  at days 1 and 4 using the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay  in 24-well plates. For the assay, 100 µL of the MTS reagent was added to each well, which contained cells in 500 µL of DMEM-F12, regular growth medium, and then incubated for 3 h at 37 °C/5% CO2. Regular growth media without cells served as controls. Optical densities were measured on a microplate reader at 490 nm .Proliferation of expanded BMMSCs .Cell adhesion and viability were also assessed microscopically after days 1 and 4 by means of calcein AM  and propidium iodide  staining. MSCs were seeded at a density of 2.0 × 104 cells per well in 24 well plates (Thermo Scientific) containing DMEM-F12 regular growth medium. Cells were allowed to adhere to the tissue culture surface. At 70–80% confluency, the medium was replaced with either chondrogenic differentiation medium, or media containing either 50% or 100% of autologous and allogenic synovial fluid. The chondrogenic differentiation medium, i.e., regular medium supplemented with 100 nmol/L dexamethasone, 0.25 mmol/L ascorbic acid  and 5 ng/mL transforming growth factor beta (TGF)-β1 , was used as positive control. BMMSCs without treatment were kept as negative control. The chondrogenic medium and/or synovial fluid supplementations were changed every 3 days for a 14-day culture period. At days 5 and 14, cells were fixed with 4% paraformaldehyde for 10 min at room temperature (RT) and stained with 0.2% Alcian blue  for 1 h at RT. Photomicrographs were examined with the NIS-Elements imaging software . A total of 500 μL of 6 M guanidine/HCl  was added to the stained cells overnight at RT with shaking to extract Alcian blue. The optical density of the extracted Alcian blue was measured on a microplate reader  at 620 nm and the values were used as a measure of the GAG content.These experiments were carried out using optimized conditions as described earlier . BMMSCs 5 cells per well onto a 24-well plate for 48 h to adhere. Cells were then stimulated with IFN-γ  [BMMSCs and SFMSCs (P2–P4) were seeded at a density of 1 × 10NJ, USA)  for 48 hThe biological activity of IDO was assessed by measuring the level of the tryptophan degradation resulting in L-kynurenine accumulation in the conditioned medium of stimulated and non-stimulated MSCs after 48 h, according to the previous study, with slight modification . BrieflyNitric oxide production was verified by measuring its stable end product, nitrite, using a Griess reagent  in the conditioned medium of stimulated and non-stimulated MSCs after 48 h of treatment, according to manufacturer’s protocol. For the assay, 50 μL of the conditioned medium was added to a 96-well plate, followed by 50 μL of sulphanilamide and 50 μL N-1-napthylethylenediamine dihydrochloride (NED). Absorbance at 540 nm was measured by a microplate reader and nitrite concentrations were calculated using a standard nitrite curve.All the results are expressed as the means ± standard deviation. The data were analyzed statistically using the Student’s t-test or one-way analysis of variance (ANOVA), with Tukey’s comparison test as a posttest, using SPSS 25.0 .While we did not recognize the definitive factors, we concluded that synovial fluid is a proper substrate to differentiate MSCs into chondrocytes and proved the concept that the application of MSCs in the joint environment is suitable. SFMSCs demonstrated immunosuppressive properties under inflammatory conditions, which is another hallmark for cartilage regeneration and supports the use of SF as a delivery vehicle for MSCs. Further investigations, however, are required to examine the hyaline cartilage markers (both at the mRNA and at the protein levels) in the differentiated MSCs supplemented with synovial fluid. These studies will help understand the signaling mechanisms that are triggered by SF. Moreover, further in vivo studies to transplant the aggregate differentiated BMMSCs, in presence of synovial fluid, into cartilage defects will be critical for future clinical setting.


Osteosarcoma is a rare, primary tumour of bone. Curative treatment consists of multi-agent chemotherapy and complete surgical resection. Despite the use of multi-agent chemotherapy, the risk of recurrence is high. Survival outcomes for patients with osteosarcoma have not changed since the 1980′s. Based on biologic rationale, there has been interest in adding immunotherapies to upfront curative intent chemotherapy, including mifamurtide (a macrophage activator) and interferon. However, results to date have been disappointing. In the metastatic setting, checkpoint inhibitors alone have not proven effective. Ongoing translational work is needed to further understand which patients may benefit from immune-oncology approaches with standard cytotoxic chemotherapy. Osteosarcoma, Ewing Sarcoma and chondrosarcoma are the most common subtypes. While difficult to estimate, incidence rate per 100,000 person years in the Netherlands has been reported as 0.27 for osteosarcoma, 0.16 for chondrosarcoma and 0.15 for Ewing Sarcoma based on a national retrospective registry It is well recognized that the standard treatment for patients with resectable osteosarcoma is multi-agent chemotherapy cisplatin (C)-doxorubicin (D) +/- high dose methotrexate (HD-MTX) (MAP) followed by oncologic resection 2et al. performed genome-wide mRNA expression profiling on pre-treatment biopsies of patients who did and did not develop metastasis CD14 and HLA-DRA in tumour samples were independently associated with metastasis free survival within the cohort Compared to epithelial tumours, tumour associated macrophages (TAM) compose a significant amount of the osteosarcoma immune microenvironment 3in vitro and in vivo to induce monocyte and macrophage activation. This is thought to be via nucleotide-binding oligomerization domain (Nod) 2 leading to nuclear factor-κβ activation and secretion of other downstream pro-inflammatory cytokines In vitro, mifamurtide has been shown to have anti-tumour activity via macrophage activation, direct cytotoxicity and release of tumoricidal factors Mifamurtide’s active ingredient is muramyl tripeptide ethanolamine (MTP-PE), which is a synthetic derivative of muramyl dipeptide (MDP) In a double-blind placebo controlled trial of adjuvant mifamurtide for dogs with localised osteosarcoma, mifamurtide showed an improved EFS 4Beyond macrophage activation, there was interest in interferon as a novel addition to standard treatment of osteosarcoma. Interferons are cytokines with a multitude of roles within the body. Type I interferons, which include interferon α and interferon β, bind to the type I interferon receptor (IFNAR) and activate downstream signalling pathways through the activation of Janus activated kinases (JAKs) tyrosine kinase 2 (TYK2) and JAK1 in vivo studies where interferons were found to inhibit the growth of transplanted tumours of various origins in mice in vitro and in vivo to directly inhibit growth of human osteosarcoma cell lines and patient derived xenograft models in mice Initially discovered for their anti-viral effects 5Programmed death receptor-1 (PD-1) is an inhibitory receptor expressed on the surface of activated T cells and other immune cells Osteosarcoma cell lines and tumour samples have been shown to express PD-L1 in vivo to accelerate progression of metastatic osteosarcoma in preclinical mouse models in vitro, suggesting a therapeutic potential for the treatment of osteosarcoma et al. treated 60 patients with metastatic osteosarcoma previously treated with chemotherapy with nivolumab q2weeks and injected individual patient derived cultured TILs from fresh tumour biopsies. The ORR by response evaluation criteria in solid tumours (RECIST) 1.1 was 36.7%  and 20 partial response (PR)), with the median progression free (mPFS) and overall survival (mOS) 5.6 months and 13.6 months respectively for the entire population Exhaustion of tumour infiltrating lymphocytes (TILs) has been shown to occur over time in the tumour microenvironment in vivo in a carcinoma rat model, and subsequently in patients with a range of metastatic tumours to transiently reduce accumulation of immune-suppressive regulatory T cells Cyclophosphamide is an alkylating agent used alone or in combination with other therapies to treat various cancers 6in vitro and in vivo in several B7-H3 expressing cancer types, B7-H3 targeting monoclonal antibodies (enoblituzumab) and chimeric antigen receptor (CAR)-T cell based therapies are now being tested in early phase trials B7-H3 is a T cell regulator involved in cell mediated immune response through both co-activation and co-inhibition 7There has been interest in exploring cellular therapies based on preliminary results of using genetically modified T cells in other sarcomas in vitro despite low expression levels et al. showed that GD2 expressing CAR-T cells were effective at killing GD2 positive osteosarcoma cell lines in vitro, but had minimal anti-tumour effect against an in vivo osteosarcoma xenograft mouse model when administered alone Human epidermal growth factor receptor 2 (HER2) is expressed in up to 60% of osteosarcomas 8MYC,CCNE1, RAD21, VEGFA, AURKB, CDK4, TP53, RB1, PTEN, with no clear signature or sub-grouping of patients emerging. However, it was possible to group patients by somatic copy number alteration (SCNA) and subsequently pair available therapies to SCNAs . All other authors have no conflicts of interest to declare.Alannah Smrke: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. Yuen B. Tam: Conceptualization, Data curation, Formal analysis, Writing – review & editing. Peter M. Anderson: Conceptualization, Formal analysis, Writing – review & editing. Robin L. Jones: Conceptualization, Data curation, Formal analysis, Writing – review & editing. Paul H. Huang: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The cohorts were similar in gestational age and birth weight, but antenatal steroid exposure was more frequent in the second era. Although fluid management resulted in a cumulative difference in the total fluid intake over the first week of 87 mL/kg (p < 0.001), this was not reflected in a mean weight loss  or mean peak plasma sodium . The incidences of hypernatremia (>145 and >150 mmol/L), PDA ligation, bronchopulmonary dysplasia, and IVH were also similar. We conclude that in this cohort of extremely preterm infants a more liberal vs. a restricted fluid allowance during the first week had no clinically important influence on early changes in body weight, sodium homeostasis, or hospital morbidities.The optimal fluid requirements for extremely preterm infants are not fully known. We examined retrospectively the fluid intakes during the first week of life in two cohorts of extremely preterm infants born at 22–26 weeks of gestation before ( The evidence base to guide initial fluid management for extremely preterm infants is limited. A few randomized controlled trials ,2,3,4,5 The investigation evaluated an implemented increase in the total starting fluid volume allowance to extremely preterm infants at a tertiary care neonatal intensive care unit .n = 204) extremely preterm infants with a gestational age (GA) of 22–26 weeks, during a 6-year period (2002–2007). By design this included a 3-year period of lower fluid volumes (RESTRICTED) before the change in fluid allowance, and a 3-year period after the implementation of the higher fluid volume allowance (LIBERAL). Infants who died or were transferred to other units within the 1st week were excluded, leaving a total of 175 infants for further analysis  with a mean GA of 251/7 (range 221/7–266/7) weeks  weeks .p < 0.05, p = 0.0696).The groups were similar in GA and birth weight . The admn = 41) born after October 2005 received an amino acid flush solution in their umbilical artery catheter [All infants were cared for in closed intensive care incubators set at a high relative humidity of 85% during the first week, after which a setting of 50% was used. In brief, the institutional fluid prescription guidelines specified a starting total fluid volume and a standard flush solution (NaCl 9 mg/mL at 0.5 mL/h) for umbilical arterial lines. Enteral feeds  were to be started within 2 h with a volume of approximately 20 mL/kg/d and with a recommended increase of approximately 20 mL/kg/d until full feeds 170 mL/kg/d) were reached. Parenteral nutrition  were also started at birth. Supplementation of sodium was not to be provided until weight loss and a drop in serum sodium had occurred. In the RESTRICTED group an initial total fluid volume of 65 mL/kg/day was recommended with a scheduled daily stepwise increase of 10 mL/kg/day up to 170 mL/kg/day. In the LIBERAL group total fluids were started at 95 mL/kg/day (GA 22–24 weeks) and 85 mL/kg/d (GA 25–26 weeks), respectively. In the LIBERAL era the recommended daily increase in fluid volume allowance was also 10 mL/kg/d to be adjusted at the discretion of the attending neonatologist, depending on changes in body weight (determined daily), urinary output, blood biochemistry values (determined q 6 h until stable), and clinical status. The LIBERAL era guidelines advocated for fluid management also to be guided mainly by the serial determinations of body weight to allow a weight loss of up to 3–4% per day and a total maximum weight loss of 10–15%. During the study period a subset of infants , transfusion volumes, postnatal changes in body weight, and blood biochemistry values. Hypernatremia was categorized as moderate (P-Na > 145 mmol/L) or severe (P-Na > 150 mmol/L), and hyponatremia was defined as a P-Na of <130 mmol/L. Further, data on selected short-term outcomes considered relevant to early fluid provision were collected; including the incidence of intraventricular hemorrhage (IVH), surgical ligation of persistent ductus arteriosus (PDA), and bronchopulmonary dysplasia (BPD) .t-test on unpaired observations and the Fisher’s exact test were used to test for statistical significance, and p < 0.05 was considered statistically significant.The values are presented as mean ± SD or median (range), the Student’s p < 0.001) in LIBERAL vs. RESTRICTED  infants before and after an implemented change in the total fluid volume allowance during the first week of life. The two different levels of fluid volume provided had no discernible impact on weight loss, plasma sodium (P-Na), or the incidence of either hyper- or hyponatremia. In addition, there were no significant differences in the incidences of the selected hospital outcomes between the groups.The results are consistent with several randomized trials with a large variation in design and outcome measures, but which have all included preterm infants . Two preIn parallel with recently presented data from infants born at 22 and 23 weeks at our institution , the incThe present investigation is limited by its retrospective nature, and albeit being from a single center with an explicitly guideline-driven fluid management and standardized care environment we cannot exclude that changes in the clinical management over time might have influenced our findings. Regrettably, we do not have access to detailed calculations of sodium intake that might have influenced the findings, and it is conceivable that some infants in the LIBERAL group could have received a higher amount of sodium since the increase in fluids was from parenteral nutrition containing sodium. In addition, the hospital outcomes chosen are at best crude representations of perturbations in fluid balance and/or cardiorespiratory function. Nevertheless, we strongly believe that the cohort and its management to be relevant to the care currently provided to this group of highly vulnerable infants. In this context, it may be reassuring to conclude that there seems to be an interval of fluid volumes that may well support near-optimal fluid homeostasis.In a cohort of extremely preterm infants the implementation of a more liberal fluid volume allowance had no influence on early changes in body weight, plasma sodium concentrations, the incidence of hypernatremia, short-term growth, or the occurrence of IVH, PDA ligation, and BPD. As long as early sodium load is limited, the implementation of a higher starting fluid volume allowance to achieve better nutrient/caloric intakes might be a feasible approach.


The occurrence of pancreatic metastases, especially isolated pancreatic metastases (PM) in metastatic renal cell carcinoma, is associated with unusual clinical features—such as favourable prognosis and ineffectiveness of tumour volume and growth rate dependent risk factors. On the other hand, it was hypothesised that the entity of isolated pancreatic metastases should be considered as a pattern of “seed and soil” mechanism. An extensive literature search was conducted to investigate whether and what mechanism links these two observations. The result of the study shows that an unusually strong seed and soil mechanism (SSM), which allows metastases settlement only in the pancreas, inevitably leads to the ineffectiveness of these risk factors in isolated PM. The hypothesis that the entity of isolated PM in renal cell carcinoma (RCC) is to be regarded as a paradigm for an SSM is thus further supported, and the interaction with the two-phased cancer evolutionary model is discussed.In metastatic renal cell carcinoma, pancreatic metastases can appear in two clinical manifestations: (a) very rarely as isolated pancreatic metastases and (b) in the context with multi-organ metastatic disease. Both courses are characterised by rare, unusual clinical features. For isolated pancreatic metastases, the literature shows no effect on survival in all 11 publications that examined the effect of singular versus multiple pancreatic metastases; a lack of effect on survival time was also present in all 8 studies on pancreatic metastases size, in 7 of 8 studies on the influence of disease-free interval (DFI), and in 6 of 7 studies on the influence of synchronous versus metachronous metastases. In multi-organ site metastases observations, on the other hand, all five available references showed significantly better results in patients with concurrent pancreatic metastases compared to those without pancreatic metastases, although the total number of affected organs in the pancreatic metastases cohort was larger. Tumour volume-dependent risk factors thus remain surprisingly ineffective in both groups, which contradicts the usual behaviour of solid tumours. The reasons for this unusual behaviour and possible relations to tumour evolution and the hypothesis of an influence of a seed and soil mechanism in the occurrence of pancreatic metastases in metastatic renal cell carcinoma are discussed. The continuous evolution of life is triggered by two mechanisms: on the one hand by genetic alterations occurring from generation to generation and on the other hand by the proving or non-proving of the resulting altered biological properties of the organisms in confrontation with their respective environment. Genetics thus provides for larger or smaller variations in living beings and the environment for the selection of the best adapted organisms. Malignant tumours—i.e., newly emerged cell clones that are genetically in some aspects different from the organism of origin and independent of its control mechanisms—can also be regarded as “new organisms” within an organism in a somewhat exaggerated way ,3,4,5,6.At the beginning of metastasis development, there is an intravasation of genetically different cells with different biological properties from the primary tumour into the vascular system. Subsequently, a multistep cascade process requires numerous matching interactions of embolised tumour cells and host organs for successful tumour cell colonisation. Thus, the clinical properties of metastases are the common end product of tumour and host biology. Since the properties of the circulating tumour cells vary according to the primary tumour, as do the properties of the various host organs, this results in a selection of suitable tumour cells for metastasis settlement and in a non-random distribution of the metastases in various host organs.Paget  first reSince neither each embolised tumour cell nor each host organ can provide all the necessary biological properties required for successful interaction, this leads to a tumour cell selection in the early onset of metastatic colonisation with a subsequent organotropism. Studies in recent decades have elucidated this early metastatic process and demonstrated it as a multi-stage, cascade-like evolutionary process  that takes place through a variety of successful interactions of tumour cell and host organ properties ,14,15,16Metastatic cancer to the pancreas from other primary sites is rare. In autopsy studies, the frequency is estimated at 1.6–11% ,24,25,26n = 1034) covering the years 1952 to 2019 [IsPMRCC are rare observations in which singular or multiple, synchronous or metachronous to the RCC distant metastases occur definitely or over a longer period exclusively in the pancreas. Since the last literature overview  a long interval between primary RCC therapy and the occurrence of PM. An interval of 9.6 (SD 6.8) years was calculated from 470 individually documented observations ,52,53,55Since these data have already been published ,51,82 thTo clarify the question of a greater significance of a systemic or a local MP, epidemiological investigations are suitable . Since tn = 45) that were diagnosed and successfully operated before the occurrence of PM  institutional reports and compilations [In older literature there are reports that with synchronous and especially multiple isPMRCC a surgical therapy is not meaningful, since these—analogous to the experience with other solid tumours—were regarded as a negative prognosis criterion with a poor OS ,125,134.ilations ,72,111 iThe influence of single vs. multiple PM on the OS was calculated in 11 studies, all of which yielded a negative result. The influence of tumour size was documented in eight publications, all of which were also negative; the disease-free interval (DFI) between RCC and PM surgery was analysed eight times, with one exception always with a negative result. Finally, the influence of synchronous vs. metachronous PM was investigated in seven papers, six of which yielded a negative result. Of a total of 34 analyses investigating the influence of clinical risk factors such as multiple PM, size, synchronous PM, and DFI, 94.1% thus produced a negative result. The analysis of peripancreatic lymph node involvement revealed a contrary result. Investigated in seven publications, an impact on the OS was identified four times. These LN metastases apparently indicate the beginning of the transition from isPMRCC to multiple organ site metastases with poor prognosis in a relevant proportion of observations. Finally, a possible influence of staging and grading of the primary tumour has been investigated so far only by Di Franco , who repIn summary, the literature review confirms that clinical prognostic factors that reflect tumour burden and tumour growth rate, such as size, occurrence of multiple metastases, DFI, or synchronous occurrence of PM, which are generally relevant in metastasis surgery of solid carcinomas ,137,138,In early studies, PM in multi-organ metastases of RCC were often classified as terminal events, with poor forecast due to the lack of an effective drug therapy . Only adp = 0.0004), an observation made by Yuasa [p < 0.01), although the total number of affected organs was greater in the collective with PM (p < 0.001). Chrom [p = 0.022) but did not prove to be an independent variable in multivariate analysis. In a recent study, Singla [p < 0.001), 5-year survival rates 88% vs. 31%, p < 0.001). There was also a high sensitivity of the cohort with PM to antiangiogenetic agents with a disease-free survival of 26.9 vs. 8.3 months (p = 0.007) and a simultaneous resistance to immune checkpoint inhibitors with more rapid progress of PM observations (p = 0.034). Finally, within the PM cohort, Singla noted an independence (p = 0.684) of survival from the established IMDC score, which is not based on pathological–anatomical parameters. This observation, however, is in contrast to Yuasa [p = 0. 013). As a summary, all five investigations carried out so far consistently show that the presence of PM exerts a positive influence on the OS in mRCC.In 2013, Grassi  reportedby Yuasa  in 2015 by Yuasa  confirme). Chrom  also obs, Singla  comparedto Yuasa , who obsp < 0.001). Thus, the paradox is that in the mRCC, the presence of PM is associated with more favourable OS, although the total number of affected metastatic organ sites is greater. Therefore, an unconscious selection bias of favourable cases with lower number of metastatic organ sites in the PM group has to be excluded as the cause of the more favourable prognosis. The better prognosis, despite the higher rate of affected organs in the PM group, indicates that the occurrence of PM in the mRCC is associated with tumour cell properties, which trigger a more favourable course [However, in the three publications ,139,142 e course ,58,142.Over the last two decades, systemic treatment of metastatic RCC has evolved and improved dramatically. With molecular targeted therapies such as multi tyrosine kinase (MTK) and mTor inhibitors and immune checkpoint inhibitors (ICI), an effective therapy of mRCC is available with lengthened progression-free survival and OS ,148,149.IsPMRCC are an exquisitely rare presentation of mRCC. Until 2000, only 97 cases were documented , thus onn = 666), which could be confirmed here in a larger group (n = 1207). This hypothetical SSM is triggered by an interplay of host organ characteristics with specific tumour cell properties acquired during the course of tumour evolution. This SSM manifests itself in two interrelated peculiarities: (1) it allows the embolised tumour cells only in the pancreas to successfully settle and mature into clinically manifest metastases, and (2) in all other organs, however, the settlement and growth of tumour cells to metastases is either made impossible at all, or a dormant state of embolised tumour cells is forced for years [The impressive total number of observations now available allows for meaningful investigations. An increasing number of institutional and casuistic reports supplies evidence for a diffuse distribution pattern of isPMRCC within the pancreas and especially its independence of the site of the kidney affected by the tumour. This observation contradicts a high importance of a local metastatic pathway but highlights the strong impact of a systemic haematogenic MP in this entity. However, if, despite systemically haematogenic tumour cell spread, the settlement of metastases is successful only in one organ, the pancreas, then this is undoubtedly a non-random distribution. According to our best knowledge, a non-random distribution pattern of metastasis signals the effect of an SSM due to tumour cell selection resulting from interactions between tumour cells and host organs. This hypothesis of the impact of an SSM in isPMRCC was made for the first time in 2018 [or years ,20,153. n ≥ 20) publications. A total of 34 detailed analyses showed the impressive result that in 94% these risk factors remain ineffective in isPMRCC. This uniformity of results is a clear indication that the ineffectiveness of these risk factors cannot be attributed to chance due to the still small number of cases but is another characteristic of isPMRCC. This unexpected lack of prognostic significance of tumour volume and growth rate-dependent risk factors in isPMRCC cannot only be explained by the hypothesis of a strongly effective SSM, but this SSM virtually enforces this behaviour: in principle these risk factors are only an expression of the likelihood that after PM resection of occult micrometastases in other organs, a generalisation stage will take place later [The influence of tumour volume and growth rate dependent risk factors on the OS of isPMRCC was presented in 12 high volume  of occult, extrapancreatic tumour cell nests also co-explains the favourable treatment results of the isPMRCC with a 71% 5-year OS rate. These favourable results are matched by the fact that no further metastases were observed later on in 57% of the patients, while the remaining 43% showed subsequent tumour progression, 15.3% of which  appearedThe unanimously in five publications confirmed result of better treatment outcomes for patients with multi-organ site mRCC and concomitant PM compared to those without PM must also be regarded as surprising. This phenomenon cannot be due to an unconscious selection of more oligometastatic cases in the PM group, since in the publications reported so far, the number of affected organs in the PM collective was even greater ,139,142.Besides the long observational period with important advancements of surgical and medical therapies and the overall small individual sub-collectives, limitations include the retrospective nature of the analysed papers and the possibility of a selection bias.p = 0.0489). In the same year, Singla [After the genome of clear cell RCC (ccRCC) was elucidated in 2013 , studies, Singla  presente, Singla ,158,159 The reduced aggressiveness of these cell clones also explains the good prognosis of isPMRCC. However, the low aggressiveness alone cannot explain the exclusive occurrence of metastases in the pancreas in the absence of metastases in all other organs. This selective behaviour rather suggests an additional influence of a selection mechanism: an SSM, the occurrence of which in isPMRCC finds support in the epidemiological data presented. The inevitable necessary trigger for the formation of isPMRCC would of course be the evolution of tumour cells into clones with special and unusual characteristics: on the one hand these cells are endowed with a reduced aggressiveness determined by proven genetic alterations. On the other hand, in the course of tumour evolution these cells have also taken on properties that trigger in different host organ a highly specific SSM, which allows metastasis settlement only in the pancreas.Due to their exquisite rarity, no studies regarding the underlying pathomechanisms for SSM in isPMRCC have been presented so far. Therefore, at present, only analogical conclusions of more frequent and better studied large tumour entities can serve as a substitute.The effect of an SSM brings about that those tumours will gain an advantage in metastasis settlement that produce a variety of cells with different metastatic potentials, as this increases the likelihood that a suitable tumour cell reaches a suitable host organ . RCC is In principle, non-random distribution among distant organs, i.e., organotropism, will occur during metastases colonisation if the metastasis process requires successful interactions between properties of the tumour cells and those of the host organ . SeveralThe term pmN describes the influence of a primary tumour on potential host organs that occurs prior to the arrival of circulating tumour cells to create a “fertile soil” that supports seeding, colonisation, survival, and outgrowth of metastatic tumour cells ,180,181.The impact of immune defense in RCC was first recognised by cases of spontaneous metastasis regression in RCC, at that time interpreted as the effect of enhanced immune defense ,186. TheA successful interaction between a CK receptor on the tumour cell surface and a suitable ligand on the host organ is a prerequisite for the activation of numerous signal transducing pathways which are critical in the early metastatic process . Since tOrganotropism can also be caused by the necessity for the metastasizing tumour cells to adapt to different energy sources available in individual organs. Each host organ poses various challenges to colonizing tumour cells in terms of availability of energy sources and oxygen or oxidative stress ,194. ThiThus, several biological processes are known that can trigger an SSM by seed and soil properties. Therefore, in metastatic disease, the clinical behaviour is the end product of tumour and host biology , both ofThe occurrence of PM in metastatic RCC—both isPMRCC and PM in multiple organ site mRCC—is coupled to clinical features that differ in some aspects from those common in solid tumour metastases, e.g., the possibility of isolated occurrence (isPMRCC), ineffectiveness of volume and growth-rate related risk factors, and better prognosis despite a larger number of affected organs. In the case of isPMRCC in particular, three special features can be detected that differ from the behaviour generally observed in extrapancreatic metastases of mRCC: (1) Genetic alterations associated with lower tumour aggressiveness; 2) The lack of influence of tumour volume and growth rate dependent prognostic factors; and (3) Immunotherapy, which is very effective in mRCC, remains ineffective in isPMRCC. Of course, it is conceivable that these three properties are completely independent of each other. However, this leads to the unsatisfactory explanation that these embolised RCC cells are equipped with an accumulation of unusual biological properties. The data presented, however, can just as well be explained by the coincidence of special RCC cells with a postulated highly specific SSM in isPMRCC. At the beginning of the process there is undoubtedly the evolution of tumour cells that leads to a multiplicity of different cell species with different cell genomes (according to the cancer evolutionary model, this cell diversity is the result of a phase of genome chaos  The lack,8,203 wiWhich biochemical alterations of the tumour cells and/or of the host organs trigger such an SSM in isPMRCC is currently unknown and reserved for future investigations. Though isolated PM of the RCC are very rare, they deserve increased investigations as there are indications that clinical course is determined—in addition to genetic changes— to an unusually high degree by an SSM too. An elucidation of the biochemical changes that control this SSM could lead to a deeper understanding of the processes that promote or prevent the colonisation of metastatic tumour cells.


Digestive tumours, a common kind of malignancy worldwide, have recently led to the most tumour-related deaths. Angiogenesis, the process of forming novel blood vessels from pre-existing vessels, is involved in various physiological and pathological processes in the body. Many studies suggest that abnormal angiogenesis plays an important role in the growth, progression, and metastasis of digestive tumours. Therefore, anti-angiogenic therapy is considered a promising target for improving therapeutic efficacy. Traditional strategies such as bevacizumab and regorafenib can target and block the activity of proangiogenic factors to treat digestive tumours. However, due to resistance and some limitations, such as poor pharmacokinetics, their efficacy is not always satisfactory. In recent years, nanotechnology-based anti-angiogenic therapies have emerged as a new way to treat digestive tumours. Compared with commonly used drugs, nanoparticles show great potential in tumour targeted delivery, controlled drug release, prolonged cycle time, and increased drug bioavailability. Therefore, anti-angiogenic nanoparticles may be an effective complementary therapy to treat digestive tumours. In this review, we outline the different mechanisms of angiogenesis, the effects of nanoparticles on angiogenesis, and their biomedical applications in various kinds of digestive tumours. In addition, the opportunities and challenges are briefly discussed. The human digestive system consists of digestive gland organs  and digestive tubes . Digestive tumours, principally hepatocellular carcinoma, pancreatic cancer, oesophageal cancer, gastric cancer, and colorectal cancer, lead to the greatest number of tumour-related deaths worldwide , 2. More3 in the absence of angiogenesis and would then enter a dormant state, thus raising the possibility of using anti-angiogenic antibodies for the treatment of cancers  could avoid immune clearance, further prolonging their half-life and specifically accumulating in tumour tissues, called the enhanced permeability and retention (EPR) effect –24. ThusAngiogenesis primarily consists of four sequential steps: I) dissolution of extracellular matrix components surrounding blood vessels like basement membrane glycoproteins by proteolytic enzymes; II) activation and migration of endothelial cell; III) proliferation of endothelial cell; and IV) formation of capillary tubes . However3 without vascular supply because of insufficient oxygen and nutrition supply and poor clearance of metabolic waste, which would further cause hypoxia or acidosis  signal pathway, further activating mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) cascades, phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, and so on to promote tumourigenesis, progression, and drug resistance –52[y3-y6For example, the anti-angiogenic efficacy of bevacizumab might be greatly weakened by the alternative pro-angiogenic signals generated during tumour proliferation and metastasis . The hypWith the development of science and technology, the field of nanobiology has attracted increasing attention in recent years. Nanotechnology-based medicine, also known as nanomedicine, has promoted tremendous advances in cancer treatment, especially in the areas of targeted drug delivery and medical imaging. Meanwhile, nanomedicine has made remarkable achievements in the research and development of drug development for clinical tumour treatment. Among them, many nanobased drugs have been used in the clinical chemotherapy of multiple gastrointestinal tumours, such as colorectal cancer, including doxorubicin liposomes, paclitaxel liposomes, and albumin-bound paclitaxel . The druGenerally, nanomedicine is defined as a technology used to study the properties and potential applications of materials sized 20–200 nm . ComparePassive-targeting nanomaterials, which can be transported into tumour tissues through the EPR effect, are also the most widely studied nanoparticle drug delivery systems at present. To ensure that the tumour tissues receive sufficient nutrients and oxygen to facilitate rapid tumour growth, the blood vessels of most solid tumours have structural defects and produce a large number of vascular permeability factors so that most tumours exhibit high vascular permeability . The pheNanoparticles greater than 400 nm in diameter are easily swallowed up by the reticuloendothelial system as foreign matter during blood circulation. Even if they reach the blood vessels near the tumour, they cannot permeate into tumour tissues because they are larger than the blood vessels. That is why the EPR effect is best when the size of the nanoparticles is from 20 to 200 nm –67. The Stimulus-responsive nanoparticles achieve targeted delivery and controlled release of drugs by responding to the stimulation of physics, chemistry, and biomolecules . Their aIt is well known that tumour tissue has a lower pH than normal tissue because cells in normal tissue are powered by oxidative phosphorylation, while in tumour tissue, cells are powered by glycolysis, resulting in the production of large amounts of lactic acid, which is known as the Warburg effect . The pH According to the literature, cancer stromal cells actively secrete glutathione (GSH), resulting in a concentration of GSH in tumour cells (2~10 mmol/L) 100~1,000 times that in normal cells (2~20 μmol/L) and 100 times that in normal tissue, resulting in a strongly reducing environment in colorectal cancer , 73. DueActive-targeting nanomaterials, a novel approach to antitumour nanotechnology, can specifically bind to receptors overexpressed on the surface of tumours and tumour vascular endothelial cells by modifying the corresponding ligands on the surface of nanoparticles. Active targeting mainly depends on the interaction between ligand molecules and the surface receptors of tumour cells. Therefore, the ideal ligands used for active targeted delivery of antitumour drugs should be able to bind to tumour cells as much as possible but not to normal cells. A variety of ligands have been used for active antitumour drug targeted delivery, including folic acid, glucose, peptides, proteins, antibodies, and small interfering RNAs (siRNAs) , 79. TheBecause different tumour stages require corresponding treatment methods, the treatment of digestive tumours largely depends on accurate imaging diagnostic technology. CT, MRI, and PET are the most commonly used imaging techniques for diagnosing digestive tumours in the clinic. Nevertheless, these techniques are mainly based on the histomorphology and metabolic changes of tumours, which exhibit poor sensitivity in some cases, such as micrometastasis and small tumours . NanoparIn addition, new technologies such as near-infrared (NIR) fluorescence imaging can be used to observe the tissue morphology and metabolism, providing additional possibilities to detect important anatomical structures and tumour lymph node metastasis in real-time during surgery . The wavPhototherapy, a light-mediated therapy, has gradually attracted increasing attention recently because of its advantages of minimal invasiveness, spatiotemporal controllability, and low toxicity in tumour treatment. Phototherapy includes photodynamic therapy (PDT) and photothermal therapy (PTT). PDT mainly relies on photosensitizers to absorb energy under light conditions, causing a series of photochemical and photobiological reactions and producing cytotoxic substances such as reactive oxygen species (ROS), which selectively damage tumour tissues . IndocyaA synergistic system of phototherapy and chemotherapy was constructed through nanotechnology, in which the improvement of vascular permeability caused by phototherapy could increase the accumulation of nanoparticles in tumours, further enhancing the effect of chemotherapy . The thein vivo through a magnetic field and then respond to acidic tumour environment for releasing loaded drugs  nanoparticles are considered one of the most appropriate therapeutic options against tumours, and they have the advantages of chemical stability, small dimensions, low cytotoxicity, and inherent biocompatibility . Additioing mice . Based o, a thymidylate synthase inhibitor, is a commonly used chemotherapeutic drug against colorectal cancer with various side effects, such as bone marrow suppression, anorexia, and vomiting . LiszbinPEG-RGD complex. GNPPEG-RGD could not only target pancreatic cancer cells but also be used as a drug carrier and radiosensitizing agent. Moreover, the uptake of GNPPEG-RGD by cancer-associated fibroblasts (CAFs) could be 10% higher than that of pancreatic cancer cells, causing the targeted killing of CAFs and achieving an antitumour effect  plays an important role in multiple biological processes, such as oxidative metabolism, angiogenesis, tumourigenesis, metastasis, and relapse, while its imbalance can cause various diseases . A retroSilver (Ag) nanoparticles are another important therapeutic noble metal widely used in medical applications. Gurunathan et al. first reported that Ag nanoparticles could have anti-angiogenic potential by inhibiting the VEGF-induced PI3K/Akt cell survival signal in bovine retinal endothelial cells . It was 3O4 nanoparticles (MNP-Fe3O4) called GA-MNP-Fe3O4, which inhibited the migration and proliferation of pancreatic cancer cells and downregulated the downstream target gene of angiogenesis, VEGF (in vivo (Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used as targeted drug carriers with superior biocompatibility, good chemical stability, and low toxicity . Wang etis, VEGF . It has is, VEGF . Additio to deliver VEGF-targeted siRNA (siVEGF) for synergistic targeted treatment against angiogenesis. The observations in xenograft pancreatic adenocarcinoma mice indicated that such nanoparticles could significantly accumulate in tumour tissues and inhibit the growth and angiogenesis of the tumours. Moreover, low cytotoxicity, good biocompatibility, and negligible organ toxicity were observed in this study . HoweverN-deoxycholic acid-glycol chitosan (DGC) as a carrier loaded with the commonly used chemotherapeutic agent docetaxel (DCT) and the angiogenic marker peptide for gastric cancer (GX1) to obtain multifunctional vascular targeting nanoparticles (GX1-DGC-DCT). GX1 could effectively promote the uptake of nanoparticles by cells, as observed by confocal laser scanning microscopy. After intravenous injection of GX1-DGC-DCT, tumour growth in xenograft gastric cancer models showed a tumour inhibition rate of 67.05% compared with the single DCT group , and polycaprolactone (PCL) are FDA-approved commercially available biodegradable copolymers that are widely used to prepare nanoparticles for drug delivery . Apatiniin vitro and in vivo , National Key Technologies R&D Program of China (No. 2017YFC0110904), and Clinical Center for Colorectal Cancer, Capital Medical University (No. 1192070313).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We demonstrate that ASF1b is involved in regulation of cell cycle and PI3K/AKT/mTOR signaling through experiments and database analysis. Mechanistically, ASF1b promoted the proliferation, migration and invasion of GC cells. Taken together, this study highlights the role of ASF1b, which provided new insights into the underlying mechanism of progression and metastasis in GC for the first time.Gastric cancer (GC) is one of the most common malignant tumors with poor outcomes. Identification of new therapeutic targets is urgently needed. Accumulating evidence has shown that anti-silencing function 1b (ASF1b) contributes to the progression in multiple cancer types. However, detailed mechanisms of ASF1b tumorigenesis in gastric cancer remain elusive. This study showed that ASF1b was upregulated in GC tissues and remarkably correlated with TNM stage, histological grade and poor prognosis of GC. We induced down and up-regulation of ASF1b in GC cell lines and monitored the changes in their biological behavior. Furthermore, loss of ASF1b was efficient to suppress subcutaneous xenograft tumor growth  Gastric cancer (GC) is one of the most common malignancy with high cancer mortality rates worldwide. According to the International Agency for Research on Cancer (IARC) GLOBOCAN cancer statistics in 2020. There were 5.6% gastric cancer in all new cancer cases diagnosed. In addition, GC was the fifth most commonly diagnosed cancer type which responsible for 7.7% of all deaths from cancer after lung cancer 18.0%), colorectal cancer 9.4%), liver cancer 8.3%) 8.0%, col% 8.0%, c%, liver Currently, precise classification and new therapies have been widely applied in GC. With the rapid development of molecular typing and molecular diagnosis in cancer, the underlying mechanism of GC were explored in the help of big data bioinformatics, Next-generation sequencing (NGS) and drug screening in xenograft model Gene Expression Profiling Interactive Analysis (GEPIA) was proved to be an effective integrated data-mining platform for identify differential expression genes (DEGs) by investigating the transcriptional and survival data It is well-established that abnormal gene expression, teaming up alterations in DNA methylation and histone modifications, including gene overexpression or silencing in vitro to investigate the biological behavior change. Furthermore, BALB/C nude models were established using stable AAV-shRNA-ASF1b AGS cells or AAV-shRNA vector AGS cells to assess how ASF1b knockdown suppresses tumor growth. Our results show that ASF1b was closely associated with proliferation, migration and angiogenesis in GC cells and might be an independent prognostic marker and potential therapeutic target for GC.In our research, ASF1b was identified as a potential candidate oncogene in GC through analyzing data collected from both public databases and patient samples in GC. Moreover, we induced ASF1b overexpression and knockdown in GC cell lines and conducted a comprehensive study According to the histopathological results, we randomly collected 200 GC samples from the First Affiliated Hospital of Zhejiang University during February 2012 to December 2016. All the patients enrolled were non-gastroesophageal junction tumor and had not received adjuvant chemotherapy treatment or radiation therapy prior to surgery. These patients had available data, including clinicopathological features and follow-up information through inpatient chart review and telephone calls. Exclusion criteria included previous GC surgery, insulin-requiring diabetes, severe heart failure, renal and hepatic failure or dysfunction in cirrhotic. Tumor staging of the GC patients was classified according to the American Joint Committee on Cancer 8th edition (AJCC 8th edition) criteria. The survival of patients was tracked until July 2021. We developed Quantitative PCR (qPCR) of 40 GC tissue samples and matched normal adjacent tissues (NAT) to evaluating ASF1b expression in GC samples. High-throughput tissue microarray (TMA) sets for immunohistochemistry (IHC) included 160 paired GC tissue samples were applied to investigate the correlation between ASF1b and clinicopathological features. Both the univariate and multivariate Cox analyses were carried out to evaluate the prognostic significance of the ASF1b expression in overall survival (OS) and disease-free survival (DFS). This study was approved by the local hospital ethics committee , and all patients involved in this study have written informed consent prior to enrollment.Human GC cell lines, including HGC-27, AGS, MGC-803, MKN-45, SNU-1, GES-1  and human embryonic kidney cell line (HEK293T) were purchased from the Cell Bank of the Chinese Academy of Sciences . HGC-27, AGS, MGC-803, MKN-45 and SNU-1 were cultured in RPMI 1640  supplemented with 10% fetal bovine serum (FBS)  and 1% antibiotics. GES-1 and HEK293T were cultured in DMEM  supplemented with 10% FBS and 1% antibiotics. Cells were digested by 0.25% Trypsin/EDTA  in logarithmic growth phase. All of the cells were cultured at 37 °C in a fully humidified atmosphere of 5 % CO2.According to the manufacturer's instructions, the total RNA from the tissue samples and cells was extracted by TRIzol reagent . Then, the cDNA was synthesized with a PrimeScript RT reagent kit . Real-time qPCR analyses were performed with SYBR Green reaction system . The relative expression of ASF1b was quantified using the 2-ΔΔCT method, with GAPDH as the internal control. The specific primers sequences used were listed in Table Negative control siRNA (NC) and siRNA-targeting the human ASF1b (ASF1b-si) sequence were purchased from GenePharma company . PcDNA3.1 with ASF1b overexpression (ASF1b-OE) and empty pcDNA3.1 (vector) were provided by GENEray. Cells were transfected with Lipofectamine 3000 transfection reagent  in Opti-MEM medium . For each 6-well plate, 5 nM siRNA and 400ng plasmid were diluted in 200 μL opti-MEM medium respectively. Then, added the mixture of transfection chemicals and Lipofectamine 3000 reagent into individual well for 6 h before replacing with fresh complete medium in accordance with the manufacturer's instructions.The AAV-shRNA-ASF1b plasmid (pCDH-CMV-MCS-EF1-CopGFP-T2A-puro) was constructed by GENEray . HEK293T cells were transfected with a plasmid mix of short hairpin RNA constructs targeting ASF1b, pMD2.G and psPAX2 using Opti-MEM medium and Lipofectamine 3000. ShRNA sequences were shown in Table IHC was performed both on TMAs sections and mouse tumor sections. Paraffin-embedded sections were deparaffinized in xylene, rehydrated through graded alcohols and incubated with 3% hydrogen peroxide for 10 min. The slides were then immunostained with primary antibodies for ASF1b , vascular endothelial growth factor (VEGF)  or Ki67  at 4◦ C overnight, and incubated with secondary antibody  at room temperature for 30 min. Then the slides were stained with DAB Chromogen  and counterstained with hematoxylin. ASF1b expression was evaluated based on the percentage of positive-stained area. The percentage of the staining was categorized as 1 (0-25%); 2 (26-50%); 3 (51-75%); 4 (>75%), and the staining intensity was graded on a scale of 0 to 3, with 0 (negative); 1 (weak); 2 (moderate) or 3 (strong). IHC score were evaluated by multiplying the frequency and intensity scores.Cell viability assay was examined using the Cell Counting Kit-8  according to the manufacturer's instructions. Briefly, 2,000 cells were seeded into 96-well plates. after 0h, 24h, 48h, and 72h post-transfection, 10 μl CCK-8 solution in 100 μl complete medium was added to each well. After that, the cells were incubated for 2 h at 37 °C. Then, the absorbance was measured at 450 nm wavelength.Cells were harvested during exponential growth. Briefly, 1000 cells per well were inoculated in 6-well plates and incubated for 10-14 days in RPMI-1640 medium. The colonies were stained with a 0.05% crystal violet solution after washing with PBS. Images of colonies were captured and subsequently counted with ImageJ .4) were seeded into 24-well format transwell chambers with 8.0um pore polycarbonate filter inserts . The chambers were placed in 24-well plates containing RPMI-1640 medium and 10% FBS. For migration assays, cells were suspended in 200 μl serum-free RPMI 1640 medium and cultured in the upper chamber. The lower chambers were filled with 700 μl RPMI-1640 and 10% FBS. For invasion assays, the inserts were coated membranes with Matrigel (50 μl/well)  before adding the cells. Cells were incubated at 37°C for 24 h. Subsequently, filters were fixed using methanol for 15 min, stained with 0.1% crystal violet for 15 min, and washed twice with PBS. The number of cells were counted in five randomly selected microscopic views (100x).The tumor cells  Lysis Buffer  mixed with phenylmethanesulfonyl fluoride (PMSF)  and serine-type phosphatase inhibitor . The protein concentration was examined using the BCA Protein Assay Kit . 20 μg per sample were separated by SDS-PAGE and transferred to PVDF membrane. Membranes were blocked with 10% skim milk for 1 h at room temperature and incubated overnight with primary antibodies at 4 °C. After washed thrice with TBST (10 min each), membranes were incubated with an appropriate secondary antibody at room temperature for 1-2 h. After washed secondary antibody, membranes were imaged via enhanced chemiluminescence (ECL) solution . The quantitative changes in the luminescence were estimated by ImageJ.The primary antibodies are as follows: cyclin-dependent kinase , CDK4 (#ab137675), cyclin D1 (#ab16663), P21 (#ab109520), P27 (#ab190851), mTOR (#ab32028), p-mTOR (#ab109268), PI3K (#ab191606) ; p-PI3K (#17366), AKT (#4691), p-AKT (#4060) ; ASF1b  and GAPDH . These antibodies were diluted 1000-fold with primary antibody diluent .5 - 1× 106) were collected and then washed with PBS for thrice. Cell Cycle Staining Kit was purchased from Multi Sciences . 1 ml DNA staining solution and 10ul Permeabilization solution was added and mixed. Incubated for 30 min at room temperature avoiding light away from light. Finally, cells were analyzed on FACSCalibur (BD Bioscience) and different phase of the cell cycle were conducted by FlowJo software.To perform cell cycle assays, cells (2 × 10http://gepia2.cancer-pku.cn/#index) with p-value < 0.01 and | log2 fold changes | > 2 as the thresholds. DEGs was further screened by univariate Cox regression analysis with significant Hazard rate (HR) from OS and DFS . We used Kaplan-Meier plotter (KM plotter) , which included the GEO, European Genome-phenome Archive (EGA), TCGA database, to investigate the relationship between ASF1b expression and clinicopathological features in STAD, and further verified via Oncomine (www.oncomine.org). Co-expression genes (CEGs) of ASF1b were predicted by cBioPortal  online analysis with |Spearman's r| ≥ 0.5 as the threshold. In addition, Gene Oncology (GO) terms enrichment analysis of the CEGs were elucidated with David 6.8 database, including biological processes (BP), molecular function (MF) and cellular component (CC). Meanwhile, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment was performed at KOBAS 3.0 (http://kobas.cbi.pku.edu.cn/).The DEGs in STAD were firstly explored from the GEPIA database  stably transfected with shRNA-NC and shRNA-ASF1b were subcutaneously inoculated into left flanks of mice , respectively. Tumor growth was monitored for every week. At 4 weeks after injection, mice were sacrificed, tumors were isolated from the sacrificed mice, and fixed in formalin. All animal work was performed at the First Affiliated Hospital of Zhejiang University. Animal experiments were approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University , and conducted according to the Guidelines for Care and Use of Laboratory Animals of Zhejiang University.Female BALB/c nude mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. . To evaluate the relevance of ASF1b expression to tumor growth The differences between gastric cancer tissues and paired adjacent normal tissues were assessed using a two-tailed paired Student t test. T test was used for comparison between two groups while one-way analysis of variance (ANOVA) was used to compare differences among multiple groups. The correlations between ASF1b expression and the relevant clinicopathological features of GC patients were analyzed using the Pearson χ2 test and Fisher's exact test in SPSS. Kaplan-Meier survival curves was constructed using the “Survival” package in R. The univariate and multivariate Cox proportional hazards multiple regression model was performed by “forestplot” package to investigate the prognostic significance. Statistical analysis was performed with GraphPad Prism 9 software, SPSS 26.0 software and R 4.0.4 program. A p value of < 0.05 was considered statistically significant. All cell experiments were repeated three times independently.in vitro and in vivo). Base on GEPIA database, we excavated 4548 DEGs between tumor and normal tissue in STAD including over-expressed and under-expressed. Furthermore, survival analysis shows 356 and 380 genes were significantly correlated with OS and DFS, respectively. Finally, we computed the intersection of the three gene sets to obtain 8 genes . Western blot assay was repeated three times independently, and the mean gray levels of the protein bands were quantified by using the ImageJ program C. Westerhttp://kmplot.com/analysis) database, showed that high ASF1b was significantly associated with poor OS and DFS in GC patients , respectively , was significantly inhibited in ASF1b-si group. Our research highlighted cell cycle regulation, as one of the major mechanisms of cell growth, was highly related to the promotion of cancer by ASF1b.Given these findings, we hypothesized that ASF1b is involved in the tumorigenesis of GC and progression. As is shown in Figure To explore the potential underlying mechanism of ASF1b in invasion and migration of GC, we found ASF1b may be involved in PI3K/AKT/mTOR pathway in GC. PI3K/AKT/mTOR pathway activation serves a critical role in cell growth, proliferation and cell repair, and has been observed in many types of tumors in vivo rendered our research conclusions more reliable and trustworthy.However, our study had some limitations. First, this was a single centre retrospective study which enrolled comparatively small sample size of patients with GC. Moreover, no ASF1b inhibitors had been reported, and the function of cell cycle signaling pathway during ASF1b upregulation required further investigation by corresponding inhibitors. In future, relevant genes of ASF1b needed be explored in transcription or translation. Interestingly and noteworthy, this was the first study systematically reported the role of ASF1b in GC. In our cohort, we investigated both the mRNA and IHC of ASF1b in GC samples. Additionally, the expression of ASF1b at different levels in GC cells was explored to further verify the conclusions revealed by the dataset. Lastly, the experiments In conclusion, high levels of ASF1b correlate with poor clinical outcome in GC patients. Functional and mechanistic studies suggested that ASF1b significantly induce GC cell proliferation and metastasis. In additional, ASF1b is involved in cell cycle and PI3K/AKT/mTOR pathways in GC cells. Therefore, this study defines ASF1b as a novel prognostic factor biomarker of clinical value. Future work should concentrate on leveraging these findings that highlight ASF1b as a potential therapeutic target for GC.Supplementary figures.Click here for additional data file.


Arabidopsis mutant male meiotic restitution 1 (mmr1), which produces a small subpopulation of diploid or polyploid pollen grains. Cytological analysis revealed that mmr1 produces dyads, triads, and monads indicative of male meiotic restitution. Both homologous chromosomes and sister chromatids in mmr1 are separated normally, but chromosome condensation at metaphase I is slightly affected. The mmr1 mutant displayed incomplete meiotic cytokinesis. Supportively, immunostaining of the microtubular cytoskeleton showed that the spindle organization at anaphase II and mini-phragmoplast formation at telophase II are aberrant. The causative mutation in mmr1 was mapped to chromosome 1 at the chromatin regulator Male Meiocyte Death 1 (MMD1/DUET) locus. mmr1 contains a C-to-T transition at the third exon of MMD1/DUET at the genomic position 2168 bp from the start codon, which causes an amino acid change G618D that locates in the conserved PHD-finger domain of histone binding proteins. The F1 progenies of mmr1 crossing with knockout mmd1/duet mutant exhibited same meiotic defects and similar meiotic restitution rate as mmr1. Taken together, we here report a hypomorphic mmd1/duet allele that typically shows defects in microtubule organization and cytokinesis.Meiosis drives reciprocal genetic exchanges and produces gametes with halved chromosome number, which is important for the genetic diversity, plant viability, and ploidy consistency of flowering plants. Alterations in chromosome dynamics and/or cytokinesis during meiosis may lead to meiotic restitution and the formation of unreduced microspores. In this study, we isolated an  Most higher plants, especially for angiosperms, have undergone at least one round of whole genome duplication (WGD) in evolution ,2. FormaArabidopsis, at least 23 genes have been identified, which, in case of dysfunction, cause meiotic restitution  and jason mutants, which results in partial or complete fusion of the spindles . A. Ammr1 mmmr1 that consistently produces a small fraction (around 5%–10%) of unreduced pollen grains. The phenotype is caused by a single amino acid change in the PHD domain of MMD1/DUET, a chromatin regulator that is specifically expressed in male meiocytes and functions in male meiosis [MMD1/DUET lead to MMD1/DUET null mutants, which display strong male sterility due to various defects in meiosis that culminate into meiocyte cell death [MMD1/DUET is initiated normally, forming meiocytes that progress up to pachytene as in the wild-type. During the subsequent steps in the meiosis program, two cycles of chromosome condensation take place. In mmd1/duet, diakinesis chromosomes are paired into bivalents and appear less compact and show inter-bivalent interactions [mmr1. In mmr1, an alteration in chromosome structure was observed at metaphase I, when bivalents are under tension from kinetochore microtubule pulling forces. In the wild-type, the kinetochores are slightly pulled apart, which is evident from the thin DAPI-stained threads perpendicular to the equatorial plane. These extensions are also absent, or at least far less pronounced, in null mmd1/duet [mmr1, the altered chromosome condensation in metaphase I is not as severe as in the null mmd1/duet mutant, suggesting that mmr1 is hypomorphic. The reduction in fertility of mmr1 is very mild compared to mmd1/duet, in line with the occurrence of cytological defects in only a subpopulation of the meiocytes and microspores. The mmr1 mutation therefore has a minor impact on the functioning of MMD1/DUET.In this study, we reported the identification of a recessive mutant  meiosis ,25. Ds tll death ,25. Maleractions ,25,47, wmd1/duet . In mmr1mmr1 is located within the PHD domain of MMD1/DUET, which forms an aromatic cage, required for the specific binding to H3K4me2 [mmr1 allele introduces a negative charge in the PHD finger, modifying its electrostatic balance and likely changing the strength and/or specificity of histone binding. As mmr1 displays a hypomorphic phenotype, we speculate that the mmr1 allele has the same histone specificity but with slightly weaker binding affinity. The PHD domain is of critical importance to MMD1/DUET function, as PHD deletion mutants and selected PHD amino acid changes show the same sterility phenotype as a knockout line [mmr1 [mmr1, suggesting that the G618D amino acid change interferes with MMD1/DUET functioning during both meiosis I and II. The mild alteration in chromosome condensation at metaphase I in mmr1 suggests that the point mutation G618D is more related to MMD1/DUET function in phragmoplast organization.The G618D amino acid change in  H3K4me2 ,23,47. Mout line  reportedne [mmr1 . The repne [mmr1 . ChromosTDM and the meiosis spindle II organizer JASON is severely reduced in duet, in line with the defects in spindle position and cytokinesis [CAP-D2, CAP-D3, CAP-H, and CAP-H2. The cap-d3 mutants show a male meiotic chromosome condensation phenotype such as mmd1/duet, indicating that the prophase I chromosome condensation phenotype is due the misexpression of CAP-D3 in MMD1/DUET [mmr1 shows a very mild phenotype in metaphase I chromosome condensation and a low frequency of meiotic restitution, we speculate that a reduction in the expression of CAP-D3 and Jason is minor and only occasionally drops below a threshold to generate the observed defects in chromosome condensation and cytokinesis. The isolated mmr1 mutant in this report thus provides a tool to analyze the function of regions within the PHD domain in MMD1/DUET with a specific correlation to telophase II and cytokinesis.MMD1/DUET has been shown to profoundly modulate gene expression in anthers and seems to do this differentially during early stage and late stage of meiosis . Expressokinesis . MMD1/DUMD1/DUET . Since m


Oligonucleotides fluorescence in situ hybridization (Oligo-FISH) is an emerging technology and is an important tool in research areas such as detection of chromosome variation, identification of allopolyploid, and deciphering of three-dimensional (3D) genome structures. Based on the demand for highly efficient oligo probes for oligo-FISH experiments, increasing numbers of tools have been developed for probe design in recent years. Obsolete oligonucleotide design tools have been adapted for oligo-FISH probe design because of their similar considerations. With the development of DNA sequencing and large-scale synthesis, novel tools have been designed to increase the specificity of designed oligo probes and enable genome-scale oligo probe design, which has greatly improved the application of single copy oligo-FISH. Despite this, few studies have introduced the development of the oligo-FISH probe design tools and their application in FISH experiments systematically. Besides, a comprehensive comparison and evaluation is lacking for the available tools. In this review, we provide an overview of the oligo-FISH probe design process, summarize the development and application of the available tools, evaluate several state-of-art tools, and eventually provide guidance for single copy oligo-FISH probe design. Fluorescent in situ hybridization (FISH) is a powerful technique to detect and locate a specific DNA sequence on a chromosome. Since the emergence of in situ hybridization in 1969 and the subsequent development of FISH ,3, this Oligo-FISH probes offer many advantages compared with conventional probes derived from ribosomal DNA (rDNA), bacterial artificial chromosome (BAC) sequences, or repetitive sequences. Although rDNA and many other tandemly repeated sequences can generate strong FISH signals, they are not suitable for comparative cytogenetic studies because of their prevalence and diversity among different species ,10,11. PRecently, many advances have been made regarding FISH assays; however, little progress has been made in developing computational tools that support the design of FISH probes, especially for oligo-FISH. Most of these tools are only used to design oligo probes for bacterial rDNA or microarrays, which might be not suitable for oligo-FISH. Although repetitive sequences-based oligo-FISH methods like nondenaturing FISH (ND-FISH) show the advantage in identifying chromosomes or specific segments in genome ,15,16,17A successful oligo-FISH experiment requires the synthesis of massive amounts of single-copy oligos as hybridization probes from the genome a. The piThe stringency, hybridization stability, and efficiency of an oligonucleotide are directly associated with its length . In theoMelting temperature (Tm) is an important factor that affects the hybridization of a DNA probe. The Tm is the temperature at which half of the oligonucleotides are paired with their complementary sequence to forming a duplex c 26]. I. I26]. IOther important factors that need to be considered when designing oligo probes are the formation of secondary structures and dimers. If single-stranded oligonucleotides fold and form secondary structures, such as hairpin loops, they may fail to hybridize to the target sites c 40,41],4140,41]The most important aspect that determines the success of a FISH experiment based on oligos is the specificity of the probes. Oligo probes should only bind to their intended target DNA. Oligo probes with multiple target sites in genome lead to high background signals, and eventually fail to produce the correct signals in a FISH experiment d. To desWith the development of FISH technology, oligonucleotide-based probes have gradually become the new-generation FISH probes in animals, plants and bacteria ,52,53. Hhttps://oligopaints.hms.harvard.edu/; accessed on 4 May 2021) to design and synthesize FISH probes [C. elegans, D. melanogaster, Arabidopsis, mouse, zebrafish, and human) [OligoArray is a popular program that allows the design of oligonucleotides at the genomic scale. It was first developed in 2002  and furtH probes . This plH probes  to desigH probes . Oligopad human) .PROBER is specialized for FISH oligo probes design . It firsmathFISH  and webFWith the requirement for high-resolution FISH and the development of synthesis technology, oligonucleotide-based FISH has been greatly improved. The length of oligo probes has become shorter (<100 bp) and the design of oligo probes has entered the high-throughput era. Massive parallel synthesis of probes becomes prevalent. In addition, more and more plant researchers have applied oligo-FISH technology for their cytogenetic studies.http://www.repeatmasker.org; accessed on 4 May 2021) for repetitive sequences filtering and utilizes BLAT [In 2015, the Jiang laboratory developed a bioinformatic pipeline called Chorus, to design and select oligo probes for FISH in plants on a genome-wide scale . The pipzes BLAT  to identzes BLAT  based onzes BLAT . In 2021zes BLAT . Chorus2zes BLAT . In addizes BLAT . Both mezes BLAT ,73,74,75To simplify the design process and improve the specificity of oligonucleotides, Hendling et al. developed a web-based design tool called Oli2go . The webhttps://paintshop.io/; accessed on 4 May 2021) for the design of genome-scale oligonucleotide for FISH experiments [Beliveau and his colleagues focused on the visualization of chromosome structures and chromatin dynamics. They developed a versatile pipeline, named OligoMiner, for the genome wide design of oligo probes for FISH . OligoMieriments . This plhttp://ifish4u.org; accessed on 4 May 2021) was also developed.iFISH is a platform designed specifically for FISH probes selection . It usesProbeDealer is a recently developed tool to design oligo-based FISH probes . It utilAegilops speltoides [Kmasker plants is a tool designed to assess complex plant genome sequences . It alsoeltoides . The webThere are some other platforms that enable oligo probe design, such as commercial probe design platforms from Arbor Biosciences or LGC Biosearch Technologies; however, these tools are not in the scope of this article.To fulfill the requirement of different experiments using oligo-FISH, more and more computational tools or platforms for oligonucleotide probe design have appeared and are freely available. Although these tools are implemented to design probes, they show marked differences in their pipelines and functions. In addition, as we discussed above, specificity is the key consideration for a successful oligo-FISH experiment, thus the specificity of the probes designed by these tools needs to be inspected. Furthermore, the practicality and performance of these tools or platforms should also be considered. For example, tools with obscure and awkward pipelines may preclude users from implementing them, especially those users who have little bioinformatic knowledge. Therefore, we chose four commonly used and state-of-art oligo-FISH probe design tools or platforms, and comprehensively discussed and evaluated them. We aimed to provide instructions to scientists who require designed oligo probes for FISH.As oligo-FISH became more and more popular for the study of chromosome organization and gene expression, the demand for bioinformatic design utilities for oligo probes increased. Beliveau et al. firstly designed oligo probes using the OligoArray tool ,61 and bOligoMiner is equipped with python and uses standard bioinformatic file formats at each step in the probe mining process. OligoMiner provides two distinct modes for predicting probe specificity and filtering multi-copy probes. One is named Unique Mode (UM), which aligns all probe sequences to the target genome using bowtie2 and identifies uniquely mapped candidate probes. The other approach is termed Linear Discriminant Analysis Mode , which uses supervised machine learning (ML) methods from the scikit-learn package. This mode is implemented to connect probe sequence alignment scores and duplexing probabilities. LDM can identify potentially problematic DNA hybridization effectively as well as the much slower thermodynamic simulations, thus, producing better FISH results.OligoMiner enables several post-processing functions for designed probes. The kmerFilter function uses Jellyfish  to screeOligoMiner provides command-line operations that can be performed on Windows, Macintosh, or Linux systems. Additionally, OligoMiner can be directly installed via conda, an open source package and environment management system .Unlike OligoMiner, iFISH is an open-source repository that hosts genome-scale oligo-FISH probes for human . In factAlthough iFISH is not implemented for de novo oligo probe design, compared with OligoMiner, iFISH has several advantages. For example, iFISH can control the probe density in a given genomic region of interest, especially for chromosome spotting probes spaced on the same chromosome. This function is very useful for specific FISH experiments. The authors have provided a 40 mer public database, and compared these oligo probes with OligoMiner’s probe datasets. The results showed that the iFISH-40 mer dataset had a higher density (by approximately 2.6 times) than the OligoMiner Balance (OMB) dataset, which indicates that the iFISH dataset is more suited for designing oligo-FISH probes in the human genome, especially for small regions. In addition, the FISH signals produced by iFISH probes were higher than those produced by OMB probes, which proves the importance of probe density in a given region.Based on selected chromosome spotting probes from iFISH, intermingling of chromosome territories can be easily distinguished. Nevertheless, oligo probes are only designed for human using iFISH, and more probe datasets for other species are needed. In general, iFISH is a valuable platform for oligo-FISH probe design.Chorus software  was initChorus2 and OligoMiner have similar approaches to design genome-wide oligo-FISH probe. They are both built by python and use NGS alignment and k-mer-based methods to filter probes. However, Chorus2 produces more specific probes because it introduces a particular strategy called ChorusNGSfilter. The ChorusNGSfilter function utilizes shotgun sequences data to further filter potential repetitive sequences in pre-designed oligos, using Jellyfish  to calcuExcept for the genome-wide design of oligonucleotide probes, Chorus2 also allows the design of conserved probes among genetically related species. With the ChorusHomo function, users can find syntenic regions between two species and use the probes designed from these regions for FISH in both genomes. In addition, Chorus2 enables the design of probes for a species without a reference genome. The corresponding function, ChorusNoRef, takes advantage of the reference genome from a closely related species and uses shotgun sequences of the target species to build a pseudo-genome sequence. Chorus2 then designs oligos from the constructed pseudo-genome sequence. With these two functions, oligo-FISH can be applied for further researches.Chorus2 is available on any modern system and can be installed using conda. Chorus2 also provides a user-friendly GUI for users to design and select probes. Furthermore, the authors offered nine oligo probe datasets for different plant and animal species. With the assistance of the comprehensive documentations and video tutorials, Chorus2 is easily to use for both dry-lab and wet-lab scientists.The Beliveau laboratory recently developed an interactive platform for the reproducible design of oligo-FISH experiments named PaintSHOP, to further improve the design of oligo probes . PaintSHPaintSHOP is built with a dynamic web application using the Shiny framework from the R programming language. Similar to iFISH, PaintSHOP is implemented to design probes from pre-built oligo datasets. However, PaintSHOP can also de novo generate genome-wide probes. PaintSHOP inherits the probe mining process of OligoMiner, but it improves some procedures and calculates an on-target score and an off-target score for each probe. PaintSHOP utilizes a machine learning model that was built using the XGBoost library, and uses the length, GC-content, dinucleotide counts, and NGS alignment scores as features to predict probe specificity quantitatively in the whole genome. Moreover, PaintSHOP incorporates genome annotation information to identify probes in genome intervals shared by all transcript isoforms of a given gene. All these processes in the pipeline are integrated into an automated Snakemake workflow .With the improvement of the probe design pipeline, PaintSHOP designed more probes than iFISH4U probes for human, which made up for the deficiency in the OligoMiner-designed probe sets. By contrast, the PaintSHOP resources host FISH probe sets of minor species, and de novo probe design using PaintSHOP requires the knowledge of bioinformatics, such as Snakemake. In summary, PaintSHOP greatly facilitates the usage of oligo probes in transcriptome and genome-scale oligonucleotide FISH experiments.The pipeline of each tool discussed above are quite different. Thus, we first performed a comprehensive comparison of the design processes among the tools .OligoMiner requires a single sequence (such as one region in the genome or one chromosome) in the fasta format as the input, and extracts oligo sequences with a given length and thermodynamic properties. After this step, the extracted sequences are aligned to the target genome sequence using NGS aligner Bowtie2 with customized parameters. Output results are then cleaned based on their uniqueness in genome (UM) or on a machine learning model (LDM), which takes the probe length, alignment score, and certain other thermodynamic properties into consideration. Cleaned oligo probes can be further filtered by their k-mer values using Jellyfish to remove sequences with high-abundance k-mers.iFISH does not provide a pipeline for de novo probe design; however, the authors described the processes by which they built the iFISH-40 mer database. First, unique 40nt sequences from the human genome were extracted using Jellyfish and custom scripts. Then, probes with long homopolymer stretch (≥7) or extreme GC contents (<35% or >80%) were filtered out. Then, probes that had 70% or higher homology to more than one genomic location were detected by VMATCH and discarded. The secondary structure and melting temperature of each probe were calculated. Finally, all non-overlapping probes were aligned to reference genome using Bowtie to check the presence of off-target effects, and only probes with ≤10 off-target sites (mismatches ≤ 5) were retained as clean probes.dTm (hybrid Tm − hairpin Tm) > 10 °C are kept. The second process utilizes shotgun reads from the target genome to further filter potential multi-copy oligos. In this step, the distribution of the k-mer frequencies from NGS data are counted, and each oligo is assigned a k-mer score that represents their repetitiveness. The last process is to select the non-overlapping single-copy oligos. By default, oligos with a k-mer score between the 10% and 90% quantiles are retained. These selected oligo probes can be finally synthesized and maintained as permanent oligo resources.Chorus2 follows three major processes to design oligo probes. The first process is to design all oligo probes with a given length from the target genome. Jellyfish is then used to remove putative repeat sequences and filtered oligo sequences from a sliding window are further filtered by the presence of homopolymers and then mapped to target genome by BWA to examine their uniqueness in the genome. The hybrid Tm and hairpin Tm of each oligo are analyzed using Primer3-py. Only probes that mapped to exactly one genome loci and have a PaintSHOP takes advantages of the OligoMiner pipeline with slight modifications. It identifies all possible probes between 20 and 60 nucleotides in length with a Tm between 42 and 47 °C in soft-masked genome regions by default. A new automatic model selection and hyperparameter optimization is performed using TPOT, a method converged on a XGBoost regressor, to simulate the potential “probe-target” relationship. PaintSHOP calculates the on-target and off-target score of each oligo based on machine learning results. Finally, probes are filtered by their k-mer scores and annotated with reference annotation information.To obtain a more comprehensive understanding of each tool, we performed oligo-FISH probe design using these tools on a local server see , and theArabidopsis, maize and human). The tools were run using their default parameters or instructive parameters. The length of the designed oligo probes was set as 45nt for all tools. We first compared the number of oligo probes designed by each tool in the three species. In Arabidopsis genome, the four tools developed a similar number of probes, which may be due to the small size of the genome  and oligo probes in the three chromosomes from different genomes (Next, we compared the density of probes designed by the four tools in different genomes. We first counted the coverage of probes designed by the four tools in several genomic windows . Probes  genomes a. The pl genomes b–d. We c genomes e–g. This genomes c. The reWe also compared the speed and memory usage of these tools . The pipOligonucleotide-based FISH technology has been applied for various research areas, including traditional cytogenetic studies and burgeoning 3D genome researches ,72,73,80We reviewed and compared the four latest developed tools that enable genome-scaled oligo probe design comprehensively. From their documentation and our evaluation results, we offer some instructions for choosing which tool to use: OligoMiner and Chorus2 software are suitable for users who do not have high performance computers. OligoMiner is preferred for mammals and Chorus2 is preferred for plants. For wet-lab scientists, the web server OligoMinerApp is a good choice to develop probes in the 10 provided genomes. The authors of Chorus2 also provide well-rounded documentations and video tutorials to follow. iFISH is used for chromosome spotting or obtaining uniform probes in regions of interest in human. PaintSHOP is recommended for users who want to design and select oligo probes without complicated considerations. Users can select pre-designed probes from in the probe sets hosted by PaintSHOP.The present review may not cover all the tools used for oligo-FISH probe design, and many oligo design tools were not evaluated; however, we have summarized the generalized principles to design oligo probes and marked the features of each tool in