This finding indicates that, rather than increasing total energy intake, nurses redistribute energy consumed during nightshifts as a behavioural response to consecutive nightshifts. This finding informs our understanding of how the intake of energy during the biological night can influence adverse health outcomes of nightshift work.Nightshift work is associated with adverse health outcomes, which may be related to eating during the biological night, when circadian rhythms and food intake are misaligned. Nurses often undertake nightshift work, and we aimed to investigate patterns of energy distribution and dietary intake across 14 days in 20 UK National Health Service (NHS) nurses working rotational shifts. We hypothesised that the proportion of daily energy consumed during the nightshift would increase over consecutive nights. Primary and secondary outcome measures included intakes of energy and macronutrients. Our results show that nurses consumed the same total daily energy on nightshifts and non-nightshifts, but redistributed energy to the nightshift period in increasing proportions with a significant difference between Night 1 and 2 in the proportion of total daily energy consumed (26.0 ± 15.7% vs. 33.5 ± 20.2%, mean ± SD;  Substantial evidence now exists for the adverse effects of nightshift work in promoting weight gain and cardiometabolic risk ,2,3,4. HThere is evidence to suggest that the observations of adverse metabolic effects of nightshift work may be explained, in part, by discordance between food intake during the biological night and endogenous circadian rhythms, which are entrained to the 24 h light/dark cycle, and optimised for nutrient intake during the daytime . CircadiCumulatively, these data suggest that the increase in cardiometabolic risk from nightshift work reflects the time-of-day consumption of energy intake, driven by alterations in the patterns and timing of energy intake and diet composition. However, despite extensive literature on energy intake during the nightshift period itself, there is little research on patterns and timing of energy intake, and how these patterns are altered over consecutive nightshifts . Here, wParticipants were recruited from eight NHS Hospital Trusts in Southern England, who provided recruitment material to their staff. Eligibility criteria included female hospital nurses and midwives (hereafter defined as “nurses”) aged 30–55 years, who were engaged in rotating shifts of no less than 29 h per week. All the participants were mothers with at least one child aged 8–18 years. Their work schedules included day shifts and a minimum of two consecutive night shifts of at least 8 h between 20:00 and 08:00 h. Exclusion criteria included use of sleep medications and lack of English fluency. The present study was a secondary analysis of data collected during the course of previous research (October 2006–September 2008) into the impact of nightshift work on nurses and their families, at the University of Surrey ,15.Ethical approval for the study was obtained from the Surrey NHS Research Ethics Committee (REC Ref 06/Q1909/17), and the University of Surrey Ethics Committee (EC/2006/60/SOCIO), and research governance obtained from the eight participating NHS Hospital Trusts. Written informed consent was obtained from all participants.The aim of the study was to examine the impact of rotating night work on women, working as qualified NHS nurses, and their families. The study used a mixed methods approach, with endpoint measures of the quality and amount of sleep, mood ratings, and dietary intake, the latter of which was recorded by each participant in a 14-day diet diary. The findings in relation to sleep quality, cortisol and mood ratings have been previously published [n = 12) were analysed together (as “3-Night Nurses Group”), while nurses who worked two consecutive nights only (n = 8) were grouped and analysed together (as “2-Night Nurses Group”). Because highly significant differences were observed in energy and macronutrient intake between the first and second nightshifts, all nurses were also analysed as a combined group (as “All 2-Night Group”) for differences in these variables.Primary and secondary outcomes were the intake of energy and macronutrients, respectively. An additional secondary outcome was the mean number of eating occasions during the nightshift. To test for differences in energy intake over sequential nightshifts, nurses were separated by number of consecutive nights: nurses who worked three consecutive nights . The results of the analyses are presented as means and standard deviations (SD), with The study consisted of 20 nurses/midwives aged 30–54 years , who completed 14-day dietary records and were included in the analysis. A summary of dietary intake is presented in p = 0.055). However, the proportion (%) of TDEI consumed during the evening period was significantly greater during the non-nightshifts than during nightshifts (p < 0.001) than the mean energy intake (126.8 ± 102.0) for the same time period (21:00–07:00 h) during non-nightshifts  was not significantly different between nightshifts and non-nightshifts . Mean evhtshifts . Mean enhtshifts , Table 1p = 0.276) (p = 0.037) in the 2-Night Nurses Group  . Howeveres Group .p < 0.0001) in energy consumption between sequential night shifts (p = 0.004) in the proportion of daily energy distributed to the nightshift (The 3-Night Nurses Group showed a significant within-participant factor difference (t shifts . There wghtshift .p = 0.138), there was a significant difference in the proportion of total daily energy consumed during the nightshift between Nights 1 and 2 (p = 0.008)  to compare the difference in total energy and proportion of daily energy consumed over the first two nightshifts . While t= 0.008) .Changes in macronutrient distribution are also shown in Examination of the number and distribution of eating occasions across the nightshifts showed there to be no significant difference in the number of eating occasions during the nightshift between Nights 1 and 2. The distribution of eating occasions across Nights 1 and 2 is shown in There was no difference in the intake of total daily energy between night and day shifts in the nurses on rotational shifts. However, the nurses redistributed their daily intake of energy into the nightshift period in increasing proportions, with a significant increase on Night 2 in the proportion of total daily energy consumed, as compared to Night 1.The lack of difference in total daily energy intake is consistent with evidence for there being no difference in energy intake between day and nightshift workers from the wider literature ,5,6,7. OThe redistribution of total daily energy to the nightshift period over sequential nights is relevant to the associations between increased cardiometabolic risk and nightshift work, independent of any observed increase in total energy intake. Of particular relevance is the fact that the metabolic effects of one meal are not independent of energy intake at previous or subsequent meals . The conIn the present study, nurses consumed a greater proportion of energy in the evening 17:00–21:00 h) on non-nightshifts compared to nightshifts, though this did not reach statistical significance. In a study comparing fixed-day workers to shift workers working three consecutive nightshifts , both th:00 h on Limitations of our study include the dietary recall records in shift workers being prone to both under and over-reporting . The avaIn conclusion, our findings have demonstrated that during rotational nightshift work, energy intake is redistributed into the nightshift, while total daily energy remains unchanged. These data provide novel insight into the dynamic patterns of energy redistribution during consecutive nightshifts.


The SBRT plans for the EDGE and CK were generated in the Eclipse and Multiplan systems respectively with the same contouring and dose constrains for PTV and organs at risk (OARs). Dose distributions in PTV were evaluated in terms of coverage, conformity index (CI), new conformity index (nCI), homogeneity index (HI), and gradient index (GI). OARs, including spinal cord, bowel, stomach, duodenum and kidneys were statistically evaluated by different dose-volume metrics and equivalent uniform dose (EUD). The volume covered by the different isodose lines (ISDL) ranging from 10 to 100% for normal tissue were also analyzed. All SBRT plans for EDGE and CK met the dose constraints for PTV and OARs. For the PTV, the dosimetric metrics in EDGE plans were lower than that in CK, except that D99 and GI were slightly higher. The EDGE plans with lower CI, nCI and HI were superior to generate more conformal and homogeneous dose distribution for PTV. For the normal tissue, the CK plans were better at OARs sparing. The radiobiological indices EUD of spinal cord, duodenum, stomach, and kidneys were lower for CK plans, except that liver were higher. The volumes of normal tissue covered by medium ISDLs (with range of 20–70%) were lower for CK plans while that covered by high and low ISDLs were lower for EDGE plans. This study indicated that both EDGE and CK generated equivalent plan quality, and both systems can be considered as beneficial techniques for SBRT of pancreatic cancer. EDGE plans offered more conformal and homogeneous dose distribution for PTV, while the CK plans could minimize the exposure of OARs.To perform a comparison of the different stereotactic body radiotherapy (SBRT) plans between the Varian EDGE and CyberKnife (CK) systems for locally advanced unresectable pancreatic cancer. Fifteen patients with pancreatic cancer were selected in this study. The median planning target volume (PTV) was 28.688 cm For localized disease, surgery with complete resection represents the only potential treatment option associated with any substantive chance of cure4. However, due to non-specific early symptoms and aggressive behavior of pancreatic cancer, most patients were diagnosed at relatively late stages5. Most studies have demonstrated that chemotherapy combined with radiation therapy is more effective than single-modality therapy, despite continuous controversies about the role of radiation therapy exist due to conflicting clinical outcomes9. Surrounded by many important and radiosensitive gastrointestinal organs,Pancreatic cancer is the fourth leading cause of cancer-related mortality worldwide with a 5-year survival rate approximately 20%7.such as duodenum and stomach, the conventional radiotherapy for pancreatic cancer seems not to effectively spare these organs while delivering high dose to target10. Goto and Colbert12 had performed IMRT and 3DCRT for local pancreatic cancer, and compared dosimetry and clinical outcomes. They verified.Compared with three dimensional conformal radiotherapy (3DCRT), intensity modulated radiotherapy (IMRT) can reduce the dose of normal organs surrounding tumor, and also minimize the toxicity of gastrointestinal organs13 also demonstrated that with the technology of IMRT, the prescription dose could be increased to 64.8 Gy, while maintained dose limits of OARs including spinal cord, liver, kidneys, and small bowel.that IMRT offered better dose constrains for target and organs at risk (OAR) compared to 3DCRT. Brown and Coworkers14. As a new technique born of the synthesis of all of the above-mentioned advances, stereotactic body radiotherapy(SBRT) is becoming more widespread, and it is probably known as a promising method of radiotherapy for pancreatic cancer with greater normal tissue sparing18. With higher dose per fraction, the dose gradient of SBRT plans is steeper than other conventional radiation, and has better sparing of normal structures19. Lin et al. demonstrated the SBRT have the advantage of improving the local control for pancreatic cancer compared to the IMRT20. Kumar et al.21 similarly made a dosimetric analysis of the SBRT plans with duodenal sparing using volumetric-modulated arc therapy (VMAT) and IMRT in locally advanced pancreatic cancer. SBRT plans usually applied non-coplanar field arrangement, especially for the CyberKnife  system22. With 6D robotic arm and accurate tracking techniques, the CyberKnife, a 6 MV linear accelerator, has high precision for dose delivery with a large degree of freedom, and the capability of real-time tumor positioning and correction.Although the IMRT provided a probability of better tumor control for locally pancreatic cancer, the organ motion and patient set-up errors during the treatment may affect the radiation dose of organs due to the complex site of OAR surrounding tumor23. This machine is equipped with flattening filter (FF) and flattening filter free (FFF) beams, and the high resolution multi-leaf collimators (MLC) of 120 leaves with 2.5 mm widths at the iso-center26. Thus it could deliver higher dose rates more effectively and accurately while improving the conformity of dose distribution to the target simultaneously25.As a culmination in the field of radiosurgery, EDGE  has advantages of safety, noninvasive, comfortable radiosurgery in the treatment of new experience. The general application of EDGE is the intracranial SRS technique, which can eliminate small lesions of intracranial accurately. Another application is the SBRT technique of real-time tracking and dynamic irradiation technology, focusing on body dynamic target area constantlyCurrently, there is no study directly comparing dose distributions of SBRT plans between the CyberKnife(CK) and EDGE systems. In our study, two series of SBRT plans were generated using CK and EDGE platforms, respectively. We evaluated the different dosimetric metrics for target and normal tissue, as well as analyzing the radiobiological indices to reflect the response of radiation therapy.This study performed a retrospective analysis of patients with pancreatic cancer who had undergone SBRT. 15 patients with locally advanced unresectable pancreatic cancer were included in this study. The inclusion criteria were as follows: (1) diagnose confirmed by pathological examinations; (2) locally advanced pancreatic cancer; (3) unresectable cancer intolerant of surgical resections; (3) age ranging from 18 to 75 years; (4) receiving the prescription dose of 6.5, 7.0 and 7.5 Gy × F; (5) ≥ 95% of PTV covered by prescription dose. The following exclusion criteria were used: (1) patients with a history of radiotherapy prior to the SBRT; metastatic pancreatic cancer.3 (ranged from 2.67 to 34.73), and that of the PTV was 28.688 cm3 (ranged from 5.736 to 49.246). The critical normal tissue include spinal cord, bowel, stomach, duodenum, left kidney, right kidney and spleen were outlined according the Radiation Therapy Oncology Group (RTOG) for pancreatic cancer27. All methods were carried out in accordance with relevant guidelines and regulations. Informed Consent was obtained from the patients for study participation. Consents for publication of data have been obtained from all patients. All the patients included in this study are above 18 years old.Fiducial implantation will be done under endoscopic ultrasonography guidance. The number of implanted fiducials is 3 (at least 1) which is preferable to be close to, but not in the tumor. A time-period of 4–7 days between implantation and treatment planning CT-scan is applied.CT simulation was performed with head first supine position on a Brilliance Big Bore 16-slice CT scanner  with a slice thickness of 1.5 mm. Gross target volume (GTV) and critical structures including spinal cord, bowel, stomach, duodenum and kidneys were contoured jointly by oncologist and radiologist based on the fusion of CT and magnetic resonance (MR) images on the MultiPlan system . Planning target volume (PTV) were defined by expanding the GTV with 2 or 3 mm margin in all directions. The median of GTV was 18.79 cm30, as shown in Table According to the different prescribed dose, fifteen patients were divided into three groups, and each group consisted of five patients. The prescription dose for the three groups were of 37.5 Gy/5F, 35.0 Gy/5F, 32.5 Gy/5F, respectively. The dose was prescribed to ~ 70% isodose line relative to maximum dose of PTV. After importing all image data of 15 patients into two systems, CyberKnife (CK) and EDGE, different SBRT treatment plans were designed by the same medical physicists The dose constrains of targets and normal tissue were set to meet the criteria of the RTOG 0848 and the report of AAPM Task Group No. 101 (AAPM TG-101)The CK plans were designed for G4 system with Multiplan TPS (version 4.0.2). The 6 MV FFF photon beam was applied and dose rate was set to 800 MU/min with one or two cones with size of 10–30 mm. Beside the dosimetric constraints listed in Table The plans for EDGE system were generated with the Varian Eclipse system . A VMAT plan for each case was generated using two 360°arcs with the same iso-centre at the geometric centre of PTV. The 10 MV FFF photon beam was chosen with a high dose rate of 2400 MU/min. All VMAT plans were optimized using the progressive resolution optimizer (PRO) and analytical anisotropic algorithm (AAA) with a grid size of 1.5 mm were applied in dose calculation. In order to make the plan comparisons valid, both CK and EDGE plans were nornalized to ensure ≥ 95% of PTV covered by prescription dose.mean) of PTV, as well as doses covered 99%, 95%, 5% and 1% of PTV  of PTV were categorized for plan evaluation. Meanwhile, the conformity index (CI), new conformity index (nCI), homogeneity index (HI), and gradient index (GI) were also used to quatify the plan quality. CI and nCI quatifying the target coverage and healthy tissue sparing were defined as follow31:As were listed in Tables 32 was defined as Eq.  and mean dose (Dmean) of all the contoured OARs were accessed. Moreover, organ specialized DVH metrics, for instance D0.25cc and D1.2cc of spinal cord, were also evaluated according to AAPM TG-101. The details of OAR evaluation metrics were listed in Table 34:vi is the percentage of voxels receiving dose Di. The vi and Di values are acquired from the DVHs and the sum of vi over all voxels equals 1. And parameter ‘a’ denotes the seriality property for different organs, and is usually set to a positive value for OARs. In reference35 a parameter n = 1/a was used. The EQD is calculated as follows, which is defined as biologically equivalent dose of 2 Gy per fraction:n denotes the number of fractions, and α/β is a parameter from the issue-specific Linear Quadratic (LQ) model of the certain organ, determining the fractionation sensitivity. The values of parameters a and a = b were listed in Table 35.The maximum dose (Dx percent of prescription isodose lines (Vx) ranging from 100 to 10% with intervals of 10% were compared between CK and EDGE plans. Ratios between volumes of normal tissue (Vx) and PTV (VPTV) were also calculated to minimize the effect resulted from different PTV volumes. Meanwhile, effective distance Vx and VPTV, which were calculated based on sphere volume formula V = 4πR3/3.To analyze the details of dose distribution outside PTV, the absolute volumes of normal tissue that covered by P value < 0.05, it was consider to have the statistical significance. All datas were listed in terms of mean value ± standard deviation (SD).For those 15 patients with two different SBRT plans in EDGE and CK systems, a paired t-test statistical analysis were performed using the IBM SPSS statistical software version 21  to determine the difference, and if The study was approved by the institutional review board of National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital. We confirm that all methods were carried out in accordance with relevant guidelines and regulations.The consents for publication of data have been obtained from patients.3 (5.736–49.246 cm3). All of CK and EDGE plans were normalized to ensure at least 95% of PTV covered by prescription dose.In total, a retrospective analysis of 15 patients with pancreas cancer was performed. The treatment plans of SBRT were designed in CK and EDGE systems, respectively. Plans generated in both platforms could meet the clinical criteria of PTV coverage and OAR sparing. The median volume of tumor was 28.688cmThe comparison of isodose lines from 30 to 100% of the prescription dose for a selected case was illustrated in Fig. The average DVHs of PTV for CK and EDGD plans are shown in Fig. 100, Dmean, D99, D95, D5 and D1 are displayed in Fig. 100) is slightly higher for CK, which may results from different normalization methods. Dosimetric metrics including Dmean, D99, D95, D5 and D1 are smaller for EDGE except that D99 is a little higher compared with those of CK plans. This is consistent with Fig. The dosimetric metrics of PTV including Vmax, D0.25cc, D1.2cc were slightly higher for EDGE plans with significant statistical differences, which indicates the decreased sparing of spinal cord with EDGE. From Table 5cc of bowel and the mean dose of bowel, stomach, liver, and kidneys are slightly lower for EDGE plans with statistic difference (p < 0.001), but other dose-volume metrics shows no difference.The average DVHs of organs at risk including spinal cord, bowel, stomach, duodenum, Liner, left kidney, right kidney and spleen are displayed in Fig. In order to further compare the dosimetric parameters of organ at risk for EDGE and CK, we calculated the radiobiological parameter EUD by the Eqs. –6) acco acco6) ap values are listed in Table The average volume of normal tissue covered by different prescription isodose lines are displayed in Fig. 5cc of bowel and the mean dose of bowel, stomach, liver, and kidneys are slightly lower for EDGE plans, the rest dose-volume metrics, as well as EUD were all lower for CK plans. When investigating the details of dose distribution outside PTV, it was obtained that the volumes covered by intermediate ISDL (ranging from 20 to 70%) were much lower for CK plans, while the EDGE plans indicated superior sparing for lower and higher dose region.In this study, we made a plan quality comparison in terms of various dosimetric metrics for pancreatic cancer SBRT between Varian CyberKnife and EDGE systems. Both of the two techniques had the capability of producing clinically acceptable plans with adequate PTV coverage and OAR sparing. These results showed that EDGE plans offered more conformal and homogeneous dose distribution for PTV, while CK plans had slightly better dose coverage of PTV and the steeper dose fall-off gradient. For OARs, except D23, which may made the better conformity and homogeneity of PTV for EDGE simultaneously, as shown in Figs. Our data indicated that the EDGE plans were more conformal and homogeneous compared to the CK plans. This may be related to the field arrangement and delivery techniques for different platforms. On the one hand, hundreds of non-coplanar field were used for CK plans while only two coplaner 360° arcs were applied for EDGE plans. This results in that the entire dose being deposited within the plane of the arcs for EDGE plans, while the radiation dose was concentrated in the center of the target area with much bigger degree of freedom for beam directions. At the same time, the hot spot in CK may be a litter larger than that of EDGE. On the other hand, the collimators of the two systems are also very different. CK plans only adopted 1–2 circular cones for beam shaping, but for EDGE system is equiped with high definition HD120 MLCs with spatial resolution of 2.5 mm25. With the 10 MV FFF modes, it could result in the relatively lower radiation dose exposure for OARs, as well as for the integrated body. However, in this study, the EDGE plans did not show any superiority for OAR sparing. Our previous study regarding to localized prostate cancer showed that EDGE plans not only provided more conformal and homogeneous dose distribution for PTV, but also steeper dose fall-off gradient and superior OAR sparing. The inconsistent results may partly due to the different shapes of PTV that affect the dose distributions. The shape of pancreatic cancer had a relatively regular shape, approximately ellipsoidal, so that both of the two series of plans were made to meet the dose constrains of PTV easily. In the Multiplan system, five ‘shells’ were applied to limit the dose outside PTV, which may lead to better normal tissue sparing. The delivery efficiency of beam is one of the most significant differences between the CK and EDGE systems. The average treatment time of per fraction was 2–3 min approximately for the EDGE, and 40–50 min approximately for the CK according to our clinical experience. On the one hand, the reduction of average delivery time can alleviate the discomfort of patients during radiotherapy. On the other hand, the effects of intra-fractional organs motion would be reduced by decreasing the treatment time for EDGE38.For the two series of plans, the CK plans used the 6 MV FFF beams, while the EDGE chose the 10 MV FFF beams. When removing the flattening filter, it can offer increasing dose rate and make the beam profile more forward at the central axis. At the same time, there are other advantages for FFF beams, such as reduction of the scattered radiation and treatment head leakage38, 39]. Whether patient specialized PTV margins could be obtain for different platforms, and how much the margins would affect the dose distribution for surrounding normal tissue will be the next tissue for our further study.Our results did show that a dose escalation of SBRT for pancreatic cancer in EDGE and CK systems both could reach the clinical criteria. But there are still some lmitations for this study. This study is a retrospective analysis and the SBRT plans for EDGE were not applied in clinical practice. Further studies were warranted to assess the clinical utility and radiobiological responses. Another limitations is that there is no consistent results for PTV margins and the organs motion[A comparative quantitative assessment of the dosimetric and radiobiological indices of SBRT plans for 15 patients with pancreatic cancer between CK and EDGE systems.We confirm that radiotherapy systems with different characteristics should be investigated and utilized to help radiation oncologists choose a proper SBRT method for each individual patient to get better therapeutic effects. Although the CK system indicate better OAR sparing, the EDGE system can be regarded as an alternative option for SBRT of pancreatic cancer, especially for patients who cannot remain lying in bed for a long time.


The first core tip of this study is that we summarized the experience of conversion therapy of intrahepatic cholangiocarcinoma (IHCC) in a single center and found conversion therapy could provide a significant improvement in the overall survival of patients with IHCC. Previous studies mainly focused on case reports and case series, and our research provided more evidence for the efficacy of conversion therapy in IHCC. The second is that we established the organoid of IHCC for drug screening and firstly verified the results of drug screening tests in cancer organoid were consistent with the levels of efficacy observed in the patient from whom it was derived. Cancer organoid is a promising technology for conversion therapy according to our study but more organoids are still needed.p = 0.017), and had a similar overall survival to that of initially resectable patients (p = 0.965). The IHCC organoid was successfully established from one of two obtained tissues. Routine hematoxylin and eosin staining and immunohistological staining found the organoid retained the histopathological characteristics of the original tissues. Whole exome sequencing results indicated the IHCC organoid retained appropriately 87% of the variants in the original tissue. Gemcitabine and paclitaxel exhibited the strongest inhibitory effects on the cancer organoid as determined using drug screening tests, consistent with the levels of efficacy observed in the patient from whom it was derived. This study indicates that conversion therapy could improve the survival of patients with IHCC despite its low success rate, and it may be directed by cancer organoids though this is merely a proof of feasibility.This study was performed to determine the efficacy of conversion therapy in intrahepatic cholangiocarcinoma (IHCC) and explore the feasibility of cancer organoid to direct the conversion therapy of IHCC. Patient data were retrospectively reviewed in this study and cancer organoids were established using tissues obtained from two patients. A total of 42 patients with IHCC received conversion therapy, 9 of whom were downstaged successfully, and another 157 patients were initially resectable. Kaplan–Meier curves showed that the successfully downstaged patients had a significantly improved overall survival compared to those in whom downstaging was unsuccessful ( Intrahepatic cholangiocarcinoma (IHCC) is a rare and highly aggressive epithelial cancer accounting for 10–15% of all primary malignant liver tumors . To dateTo improve overall survival, several novel approaches have been proposed in recent decades. The concept of conversion therapy was proposed by Bismuth H. et al. in 1996, which demonstrated that initially unresectable malignant tumors can be downstaged using systemic or local treatment, such that patients could likely undergo surgical resection . This teAppropriate in vitro models of cancer have been explored for over 100 years. Conventional two-dimensional (2D) culture systems force cancer cells to adhere to an artificial substrate, which restricts the formation of a multi-dimensional structure. The formation of a monolayer morphology and the lack of contact with other normal cells makes it nearly impossible to engineer cancer cells to mimic the important characteristics of cancer tissues in vivo . A novelThis study was undertaken to determine the efficacy of conversion therapy in patients with IHCC based on a retrospective review. Moreover, a cancer organoid was established to determine the feasibility of directing the conversion therapy of IHCC.Patients who were diagnosed with IHCC between January 2013 and June 2020, in the Second Affiliated Hospital, Zhejiang University, School of Medicine, were retrospectively reviewed. The following patients were excluded from this study: (1) those with remote lymph node metastases, large vessel invasion, or diffuse multiple lesions, (2) those not receiving any specific anti-tumor treatment, (3) those suffering from several types of cancers, and (4) those who had received anti-tumor treatment at other medical centers. This study was performed in compliance with the principles of the Declaration of Helsinki and was approved by the ethics committee of the Second Affiliated Hospital, Zhejiang University, School of Medicine (No. 2019-408). Informed consent from the included patients was not obtained owing to the retrospective nature of this study.To analyze the relationship between treatment patterns and prognosis in patients with IHCC, the demographics (age and gender), details on liver cirrhosis, number of lesions, tumor size, vascular invasion, perineural invasion, histological grade, AJCC TNM stage, residual tumor after surgical resection, perioperative adjuvant treatment, systemic treatment, and imaging examination date of the included patients were collected at admission. Patients were divided into initially resectable group, successfully downstaged group, and unsuccessfully downstaged group. All data were collected by two independent investigators and conflicts, if any, were resolved through consultation.Considering the relatively short survival time of patients with IHCC, all included patients were followed up for at least three months. The recurrence and metastasis of IHCC were recorded during follow up.Two tissue samples of resected IHCC were collected from two independent patients whose tumors were downstaged successfully after conversion therapy. The tissues were used to establish patient-derived cancer organoids to determine their effectiveness in directing the treatment pattern in conversion therapy. The experimental procedures were approved by the ethics committee of the Second Affiliated Hospital, Zhejiang University, School of Medicine, and informed consent from patients was obtained before tissue collection.3) and incubated in 2 mg/mL of collagenase D  for 2 h at 37 °C. The isolated cells were passed through a 100-µM cell strainer , pelleted using centrifugation at 300× g for 5 min at room temperature, and embedded in a mixture of Matrigel matrix  and culture medium on ice in a 1:1 ratio.The obtained tissues were shipped on ice within 4 h. The tumor tissues were cut into small pieces  after the plates were coated using 200 μL of the Matrigel matrix mixture. Then, cells were overlaid with 500 µL of culture medium. The culture medium contained advanced Dulbecco’s modified Eagle medium/F12  supplemented with 1× penicillin/streptomycin , 1× Glutamax , 10 mM N-2-Hydroxyethylpiperazine-N-2-Ethane Sulfonic Acid , 1× B27 supplement , 1× N2 supplement , 10 mM nicotinamide , 1.25 mM N-acetyl-L-cysteine , 10 nM gastrin , 5 μM A83-01 , 50 ng/mL recombinant human epidermal growth factor , 100 ng/mL recombinant human fibroblast growth factor 10 , 10 µM Y-27632 , 500 ng/mL recombinant human R-Spondin1 , 10% Afamin/Wnt3a CM , and 100 ng/mL recombinant human Noggin . The culture medium was replaced every 4–5 days.Appropriately 2−5 × 10g for 5 min at room temperature and fixed in 10% neutral-buffered formalin for 2 h. Next, the organoids were embedded into 4% low-melting agarose after centrifugation, and the agarose blocks were embedded into paraffin. Sections of primary tissues and organoids (4-μM thick) were prepared and subjected to routine hematoxylin and eosin (H&E) staining and immunohistological staining. For immunohistological staining, mouse monoclonal antibodies against cytokeratin-7  and epithelial cell adhesion molecule  were used.Primary tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin blocks using a standard procedure. Organoids were released from the Matrigel Matrix by incubating them in the Cell Recovery Solution  for 1 h, according to the manufacturer’s instructions. Organoids were centrifuged at 300× Genomic DNA of primary tissues and organoids were extracted using GenElute Mammalian Genomic DNA miniprep kits . The quality of the extracted genomic DNA was verified by monitoring DNA degradation using 1% agarose gels and measuring the DNA concentration using Qubit DNA Assay kit . The exome sequences were enriched from 0.4 μg of genomic DNA using an Agilent liquid capture system (Agilent SureSelect Human All Exon V6) according to the manufacturer’s guidelines. Clustering was performed using a cBot Cluster Generation System and an Illumina PE Cluster kit  according to the manufacturer’s guidelines. Sequencing was performed using an Illumina platform and 150 bp paired-end reads were generated.The cancer organoids were released from the Matrigel matrix by incubating in the Cell Recovery Solution  for 1 h followed by the incubation in 0.25% trypsin-ethylene diamine tetraacetic acid solution to allow its dissociation into single cells. These single cells were resuspended in Matrigel Matrix mixture on ice and approximately 500–1000 cells were embedded in a 384-well plate. The cells were left undisturbed to recover for 2 days. Then, the culture medium was replaced with fresh medium containing 0.01, 0.1, 1, 10, or 50 µM of gemcitabine, 5-fluorouracil (5-FU), cisplatin, paclitaxel, infigratinib, or ivosidenib. After incubation with different drugs for 72 h, the viability of cancer cells was determined using a CellTiter-Glo 3D Cell-Viability assay . The viability of cancer cells incubated in a culture medium with phosphate-buffered saline served as a negative control. The screening of each drug was performed in triplicate.p-value of <0.05 was considered statistically significant.Continuous variables in this study are expressed as medians with interquartile ranges and compared using the Mann–Whitney U test. Categorical variables are expressed as a number using percentages and compared using the Chi-squared test. The Kaplan–Meier method and log-rank test were used to compare differences in overall survival among successfully downstaged, unsuccessfully downstaged, and initially resectable patients. SPSS Statistics 22.0  was used for statistical analysis and a two-tailed Patients with IHCC admitted to our hospital between January 2013 and June 2020 were retrospectively reviewed as depicted in the flow chart . A totalThe data of patients receiving conversion therapy are listed in The detailed characteristics of patients who were downstaged successfully are presented in On the other hand, detailed conversion strategies in 33 unsuccessfully downstaged patients were as follows: 13 patients receiving gemcitabine-based chemotherapy, 5 patients receiving transcatheter arterial chemoembolization, 4 patients receiving radiofrequency ablation, 4 patients receiving 5-fluorouracil monotherapy, 2 patients receiving radiotherapy, 2 patients receiving immunotherapy, 1 patient receiving targeted therapy, and 2 patients receiving immunotherapy combined with targeted therapy.p = 0.017). Moreover, successfully downstaged patients had an overall survival similar to that of initially resectable patients (p = 0.965).All patients were followed up for at least 3 months. Two patients succumbed during the follow-up period, one of whom succumbed 39 months after diagnosis owing to lung metastasis of cancer, while the other succumbed to postoperative complications. An overall survival curve was generated based on the Kaplan–Meier method and log-rank test as shown in To determine the effectiveness of cancer organoids in developing a treatment pattern in IHCC conversion therapy, a cancer tissue was collected from patient 8 and patient 9 to establish cancer organoids. However, one of the cancer organoids from patient 9 was not established successfully owing to the extent of necrosis in the collected sample. Gross specimen and H&E staining results of the other IHCC tissue are shown in WES was performed by extracting the total DNA of the cancer organoid and original tissue to determine if the organoid retained the gene expression profiles of the original tissue. The circos plots of the original tissues and cancer organoids depicted in The established organoid was used for drug screening and tested with the chemotherapeutic agents, gemcitabine, 5-FU, cisplatin, paclitaxel; and the drugs used for targeted therapies, namely, infigratinib and ivosidenib. Patient 8 received gemcitabine plus albumin-bound paclitaxel treatment as conversion therapy for three months. Magnetic resonance images A,B indicIn this study, we evaluated the present situation of IHCC conversion therapy in a single study center. The results confirmed the effectiveness of conversion therapy using IHCC; however, the success rate of downstaging using conversion therapy in patients was relatively low (21.4%), and thereby, warranted an in vitro cell model to direct the treatment pattern. We established a cancer organoid of IHCC and determined that it could mimic the structural characteristics and retain the gene expression profiles of the original IHCC tissues. Moreover, the results obtained after drug screening using the cancer organoid was consistent with the clinical responses of patients, indicating the ability and effectiveness of the organoid to design a treatment pattern for conversion therapy.Advanced IHCC was previously considered unsuitable for surgical treatment; however, conversion therapy improved the prognosis of patients to some extent. As reported in a systematic review, 27 of the 132 patients across 10 studies were downstaged successfully using conversion therapy and 23 patients were reported to be alive at the last follow-up that was conducted . The surThe concept of using cancer organoids was proposed several decades ago and has been used as a model for drug screening in recent years. Ganesh K. et al. found that the drug sensitivity of the cancer organoid-based platform is positively correlated with clinical response . Some otA more suitable approach could likely entail the collection of tumor tissues using needle biopsies for organoid establishment in patients with initially unresectable cancer, followed by comparison with the clinical responses obtained based on drug screening. Nuciforo S. et al. attempted this approach in liver cancers and obtained hepatocellular carcinoma organoids and IHCC organoids at relatively low success rates of 33.3% and 60%, respectively . In thisTargeted therapy for IHCC has been explored for several decades. FGFR and IDH1 inhibitors are the two most studied class of compounds in recent times. FGFR variants are often observed in several cancer types including IHCC. FGFR-containing fusions caused by chromosomal translocations have been detected in 10–15% of the cases of IHCC and are known to play a vital role in the proliferation and metastasis of IHCC ,32. It hOur study had several limitations. First, it comprised only a small sample size of 42 patients receiving conversion therapy and the period of follow up was relatively short, which may lead to bias in data analysis. Moreover, many IHCC patients admitted to our hospital were diagnosed as metastatic stage and refused any treatment due to financial pressure or other reasons, which may result in some referral bias. A multi-center study would have been a better choice considering the low incidence of IHCC. Secondly, the cancer organoid established in the current study was relatively small compared to that in our previous study, which posed several challenges while performing immunohistochemistry and immunofluorescence analyses ,24. TherTo conclude, conversion therapy, as a novel treatment modality in IHCC, can result in an improvement in overall survival and better prognosis of patients. The low success rate of downstaging in conversion therapy continues to pose a challenge. The therapeutic approach using cancer organoids could likely play a vital role in directing conversion therapy owing to its ability to retain the original genetic expression profile and drug sensitivity.


Approaches that improve performance under the two stresses are essential to sustain productivity. The objectives of this study were to (i) assess the extent of variation in tolerance to DSHTS from among the existing best drought tolerant (DT) hybrids; (ii) examine the response patterns of the hybrids to DSHTS; (iii) identify traits that contributed to better performance under DSHTS; and (iv) select the best hybrids with tolerance to DSHTS stress. We evaluated 40 DT hybrids under DSHTS, DS, and well-watered (WW) conditions for three years. Highly significant (p < 0.001) differences were found among hybrids for grain yield and other traits. Moderately to low repeatability values were detected for grain yield under DS (0.63) and under DSHTS (0.48). Grain yield under DS was not correlated with grain yield under DSHTS , but it was correlated with grain yield under WW . Grain yield was strongly correlated with ears per plant, ear and pant aspects, days to anthesis and silking under both DS and DSHTS. Tassel blast accounted for 28% of the yield reduction under DSHTS. The top five DT hybrids produced 9 to 26% more grain yields than the best commercial hybrid. Three hybrids produced high grain yields under DTHTS and DS as well as under WW. These hybrids will be tested further in collaboration with partners for possible release.Drought and high temperature are two major factors limiting maize productivity in sub-Saharan Africa. An increase in temperature above 30  Zea mays L.) is a major crop for both human consumption and animal feed in Sub-Saharan Africa (SSA). Drought (DS) and heat stress (HTS) are two major limiting factors affecting maize productivity in the tropical lowlands, where erratic rains and increased temperatures are becoming of frequent occurrence [°C reduces grain yield by 1% under optimal rain-fed condition and by 1.7% under DS [Maize  under DS thereby increasing the canopy temperature by more than 9 °C [Drought and heat stress occur simultaneously in farmers’ fields when the growing season is hot, and the distribution of rains is erratic. Heyne & Brunson  showed than 9 °C . Maize gThe breeding strategies in maize that have been adopted so far have focused on improvement of each stress separately. Preliminary results of hybrids evaluated under the Drought Tolerant Maize for Africa (DTMA) project revealed that tolerance to DS does not necessarily confer tolerance to HTS or DSHTS, which have implications to breeding for adaptation to climate change in maize production systems in SSA . UnderstThe genetic material that was used in this study consisted of 29 promising DT three-way cross maize hybrids, 10 commercial hybrid checks, and a local variety commonly grown by farmers in the area. These hybrids have been evaluated in regional trials in partnership with the National Agricultural Research System (NARS) in West and Central Africa (WCA) for over three years .°C, respectively, which were detrimental to fertilization, seed setting, and grain-filling in maize. Minimum day and night temperatures of ≤27 °C and ≤20 °C, respectively, are considered to be optimal for the growth and development of maize in the tropical lowlands.The trial under DSHTS was conducted during the dry season in 2013, 2014, and 2015. In each year, the lowest day and night temperatures were recorded in February at the time of planting and the highest in April. During evaluation of this trial, there were incidences of rains in the months of April and May, but the amounts were not sufficient to have significant effects on the intensity of drought stress under elevated temperature. In each year, the peak of high temperature occurred in April , which c−1). A compound fertilizer was applied at the rates of 60 kg N, 60 kg P, and 60 kg K ha−1 at the time of planting. An additional 60 kg N ha−1 was applied as top dressing four weeks later. Gramoxone and atraxine were applied as pre-immergence herbicides at the rates of 1.5 L gramoxone and 2.5 L atraxine in 200 L of water ha−1. Subsequent manual weeding was done to keep the trials weed free.The 40 hybrids including a local check was arranged in 8 × 5 alpha lattice design (incomplete design) with three replications evaluated for three years (2013–2015) under DSHTS and for two years (2014 and 2015) under DS and WW. Each replicate consisted of eight incomplete blocks with five plots in each block. In each year, the genetic materials, experimental design, and replications were the same. Each entry was planted in a two-row 5 m plot spaced 0.75 m apart with 0.50 m spacing between hills within a row. Three seeds were planted in a hill and thinned to two plants per hill three weeks after sowing to attain a population density of 53,000 plants per hectare  in Kano State in Nigeria, where hot and dry weather occur between February and June. The soil type is Regosols, with mainly sandy to clay loam texture, pH of 5.9, organic carbon 4.3 g kg−1, and residual nitrogen 0.24 g kg−1 [°C. This helped in conducting this trial under DSHTS, whereby water supply was carefully controlled through furrow irrigation. The flowering and grain filling periods were generally in April when rainfall incidences were negligible and relative humidity ranged from 15% to 46%, allowing for exposure of the hybrids to drought and elevated temperatures  and flowered in mid-April, the hottest period at Kadawa. The trial was initially grown under well-watered conditions prior to exposure to drought and heat stress. A gravity irrigation system was used to supply sufficient water through furrows to the crop every four days during the first 45 days after planting. Thereafter, irrigation was withdrawn in mid-April when mean day temperature ranged from 36 to 45 °520 N, 3°430 E, altitude 60 m) in Nigeria during the dry season. The experiment was conducted in two adjacent blocks separated by 50 m to prevent spill-over of irrigation water. Maize seeds were planted in mid-November in 2014 and 2015, so that flowering occurs in mid-January where incidences of rainfall were minimal. A sprinkler irrigation system was used to provide sufficient water every week during the first 35 days after planting. Thereafter, irrigation was withdrawn from DS block to induce drought stress at flowering and grain filling stages, allowing for the crop to mature on stored soil moisture. The WW block continued to receive irrigation once a week until physiological maturity.The same set of hybrids were evaluated under controlled DS and well-watered (WW) conditions at Ikenne  and barren plants (BP) were only recorded on DSHTS trial. TB was rated during flowering on a scale of 1 to 5, where 1 = few plants showing tassel blast and 5 = all plants showing severe tassel blast, whereas BP was rated on a scale of 1 to 5, where 1 = few plants without ears and 5 = more than 50% or all plants bearing no ears recorded in each plot two weeks after flowering. Anthesis to silkin interval (ASI) was computed as a difference between days to silking and anthesis. Plant and ear heights were recorded on five randomly selected plants, as distance from the ground to the base of the tassel and the upper ear, respectively. Leaf death score (leaf senescence) was rated on a scale of 1 to 10, where 1 = 10% dead leaf area and 10 = 100% dead leaf area recorded only in DS and DSHTS trials. Number of ears per plant (EPP) was computed as the proportion of the total number of ears harvested divided by the total number of plants at harvest. Plant aspect was visually scored on a scale of 1 to 5, where 1 = excellent overall phenotypic appeal and 5 = poor overall phenotypic appeal. Similarly, ear aspect was scored on a scale of 1 to 5, where 1 = clean, uniform, large, and well-filled ears and 5 = rotten, variable, small, and partially filled ears. All ears harvested from each plot were shelled and used to determine the percentage grain moisture and grain weight. Grain yield adjusted to 15% moisture was computed from the grain weight recorded form each plot.The analysis of data was performed using a mixed model procedure in SAS  version Cluster analysis using Euclidean distance with standardized values of agronomic data was computed to classify the DT hybrids into groups. A dendrogram was constructed using the Unweighted Pair Group Method with Arithmetic means (UPGMA) and the resulting tree was used to determine the association between the DT hybrids under DS and DSHTS. Phenotypic coefficients of correlations for grain yield with secondary traits were computed to further determine the secondary traits contributing to grain yield. Grain yields and TB scores were plotted on a graph to show the trend of yields of the hybrids under DSHTS conditions.p < 0.001) were found among hybrids for all of the traits measured under DSHTS. Hybrid x year interaction was significant for grain yield and seven of the other 10 traits. Repeatability values for the measured traits under DSHTS ranged from 0.13 for TB to 0.73 for plant height. Mean grain yields of the hybrids varied from 1016 kg ha−1 for a commercial hybrid CH2 to 3453 kg ha−1 for DT hybrid M1227-17. The top five DT hybrids produced 9% to 26% more grain yields than the best commercial hybrid check CH9 and out-yielded the local check by 56% to 64%. Most of the hybrids were significantly (p < 0.05) better than the local check in plant and ear aspect scores, had more EPP and fewer TB and BP.In the combined analysis of variance across years , year ha−1 for commercial hybrid CH2 to 3151 kg ha−1 for DT hybrid M1124-17. The best commercial hybrids (CH4 and CH5) were competitive with the best DT hybrids under DS. Under WW conditions, differences among the DT hybrids were significant for grain yield and most traits. Hybrid x year interaction was significant for days to silking, plant height, plant, and ear aspect scores. Repeatability values ranged from 0.28 for ASI to 0.85 for days to silking. Mean grain yields of the hybrids ranged from 1272 kg ha−1 for local check to 5126 kg ha−1 for DT hybrid M1227-5.Results of combined analysis across two years under DS  revealedr = −0.60; p < 0.0001) only under DS. A significant and negative relationship was detected between grain yield and BP and TB only under DSHTS.The correlations of grain yield with other agronomic traits measured in this study are presented in r = 0.74; p < 0.001). However, the correlation between grain yield under DSHTS and that under DS condition  as well as that under WW condition  were weak. ASI, EPP, days to anthesis, and silking under DSHTS were significantly correlated with ASI, EPP, days to anthesis, and silkiing interval under DS and WW conditions. A similar trend was observed between EPP, ASI, days to anthesis, and silking recorded under DS with the same set of traits being measured under the WW condition. Ear and plant aspect scores under DS were strongly correlated with ear and plant aspect scores under WW.Results of association of the same traits under DSHTS and DS as well as under WW conditions are presented in −1). Most of the DT hybrids, including two commercial hybrid checks (CH8 and CH9), had low TB and produced >2000 kg ha−1 grain yields under DSHTS. This trend was also observed when grain yield was regressed on mean day temperatures during the critical flowering and grain filling stages (results not shown). For every 1 °C increase in temperature at flowering and grain filling period, grain yield dropped by 30 kg ha−1 accounting for 3.53% grain yield variation.Regression of grain yield on TB scores showed that hybrids with high TB scores had low grain yields . TB accoA dendrogram generated from the standardized data of grain yield, plant and ear aspect scores, EPP and ASI is presented in The cluster analysis classified the 40 hybrids into two major groups under DS . The fir−1 for M1124-29 to 3453 kg ha−1 for M1227-17  had longer stay-green with leaf death scores ranging from 6.5 to 6.8. Although the top 10 hybrids selected under DS produced grain yields ranging from 2757 kg ha−1 to 3151 kg ha−1, they produced low grain yields under DSHTS. However, two of the top 10 hybrids (M1124-24 and M1227-3) selected under DS were also found among the 10 best hybrids under DSHTS  and had yield advantage of 56% to 64% over the local check under DSHTS . The besWhen considering the importance of identifying maize genotypes with high levels of tolerance to combined drought and heat stress for climate change adaptation, we screened promising drought tolerant hybrids under DS and DSHTS. The significant differences found among DT hybrids for grain yield and several other agronomic traits recorded under both DS and DSHTS, indicate the presence of genotypic difference among promising DT hybrids. The significant interactions of hybrids with environments for grain yield and other agronomic traits suggest the influence of DS and DSHTS on the expression of these traits. The greatest influence of both DS and DSHTS occurred in mid-April each year, during the three weeks around flowering and grain-filling stages. These two stages are the most critical period determining yield potential in maize.The extent of yield loss is dependent on severity of drought and heat stress, field environment and the maize genotypes under study. In this study, drought stress reduced grain yield by 58%, while DSHTS reduced grain yield by 77%, agreeing with previous findings that demonstrated more yield loss from combined effects of drought and heat stress than drought stress alone ,22,23. CTriticum aestivum L.) [Previous studies in maize ,34 and wivum L.)  showed tr = 0.075) between grain yield under optimum and that under stressed conditions.The observed significant correlations between the same traits measured under DS, DSHTS as well as WW conditions suggest the presence of common genetic factors controlling expression of these traits under the three growing conditions. However, the observed weak correlations between grain yield under DS and DSHTS indicate the presence of independent genetic factors controlling yield performance under these conditions, which agrees with previous studies ,34. The The strong correlation between grain yield and TB and BP demonstrated that these traits together with other secondary traits  should be included in an index to select promising genotypes under DSHTS. Secondary traits that are strongly correlated with grain yield suggest that they function together in determining the plants’ response to combined stress . AlthougKnowledge of the relationships among DT hybrids developed from diverse genetic backgrounds can help to identify a set of specific DT hybrids that have good levels of tolerance to DS and DSHTS. Grain yield and morphological (adaptive secondary) traits were used to estimate these relationships. Under DSHTS, group G-2 clustered DT hybrids tolerant to DSHTS, including two commercial hybrids (CH6 and CH9), which were competitive with the best DT hybrid, suggesting that these hybrids had common adaptive secondary traits that were better expressed under DSHTS as predicted by dendrogram. The majority of DT hybrids and commercial hybrids grouped in G-1 showed high levels of susceptibility to DSHTS with low grain yields. However, seven of the susceptible hybrids , including a released hybrid and commercial hybrid were grouped (G-1) with best DT hybrids under DS, suggesting that some DT hybrids that are susceptible to DSHTS perform better under DS condition.Although we observed few DT hybrids with desirable traits grouped together in the dendrogram, most desirable agronomic traits were not found in a single DT hybrid, suggesting the need for multiple traits pyramiding by introgressing the desired traits into the genetic background of adapted DT hybrids. Secondary traits, like EPP, TB, ear and plant aspect scores, days to anthesis, and silking that were found to be strongly correlated with grain yield under stressed conditions can be combined with other traits, such as canopy temperature and leaf firing for field screening. Alam et al.  suggesteWe observed that some productive DT hybrids were tolerant to DSHTS as well as under WW conditions. However, some DT hybrids together with a commercial hybrid CH7 ranked among the worst yielders under DSHTS, with grain yields well below the trial mean, suggesting that not all DT hybrids are tolerant to DSHTS as found in other studies ,34,35. W°C) when the hybrids used in this study were selected under DS at Ikenne during the dry season. This agrees with Sehgal et al. [Lens culinaris Medic.) genotypes evaluated under DS, HTS, and DSHTS. Limited information is available on performance of DT maize hybrids under DSHTS, which need further investigation to understand the relationship between tolerance to DS and DSHTS and the genetic and biological mechanisms that control tolerance to DSHTS in maize. Comprehensive study by the use of a combination of conventional and molecular breeding tools is needed to understand the mode of inheritance of adaptive secondary traits that confer tolerance to DSHTS to enhance breeding scheme for DSHTS. Genotypes showing tolerance to DSHTS will be used as a source for the development of new inbred lines. In addition, stress tolerant maize germplasm introduced from diverse sources should be screened to identify source materials for developing new multiple-stress tolerant inbred lines with higher levels of tolerance to DSHTS.Although we have not designed an experiment specifically for heat stress, the level of tolerance observed in some DT hybrids under DSHTS may be attributed to the effects of the relatively high day temperatures 32–37  °C when In conclusion, our study revealed that the combined effects of drought and heat stress at flowering and grain filling stages were detrimental to maize grain yield. Large variations were observed in grain yield and other agronomic traits among the DT hybrids under DSHTS condition. The effects of DSHTS on grain yield were more on commercial hybrids than on DT hybrids. The weak correlation of grain yield under DSHTS with that under DS and WW suggest the complexity of genetic control of yield under DSHTS in maize. The strong correlations between grain yield and some secondary traits, including ear and plant aspect scores and EPP under both DS and DSHTS, suggest the existence of common genetic factors controlling these traits that may be used to enhance productivity under the two stresses. Evaluation of promising DT hybrids under DSHTS, DS, and WW allowed for the identification of three high yielding DT hybrids  with high levels of tolerance to DSHTS. The parents of these hybrids can be used as source materials for developing inbred lines with higher levels of tolerance to DSHTS. The top five hybrids under DSHTS with yield advantages of 9% to 25% over the best commercial hybrid are suitable for production in tropical lowlands where DSHTS is of common occurrence in farmers’ fields. The study highlighted the need to modify the screening techniques and the addition of more adaptive traits, including leaf canopy temperature and application of high throughput phenotyping and genotyping techniques to enhance the understanding of DSHTS mechanisms and their genetic basis of inheritance to improve the breeding scheme for tolerance to combined drought and heat stress.


Observations among Israeli pediatric rheumatologists reveal that pediatric Juvenile Spondyloarthritis (JSpA) may present differently compared to patients from the United States (US). This study is aimed to compare the demographic and clinical variables of Israeli and US JSpA patients upon presentation.We performed a retrospective, cross-sectional, multicenter comparison of JSpA patients among 3 large Israeli pediatric rheumatology centers and a large US pediatric rheumatology center. Patients with diagnosis of Juvenile Ankylosing Spondylitis (JAS) and/or Enthesitis-related Arthritis (ERA) were included. The demographic, clinical and radiologic features were compared.p < 0.05), whereas peripheral arthritis and enthesitis were significantly more prevalent among US patients . In addition, 96.7% of the Israeli patients versus 29.7% of the US patients demonstrated sacroiliitis on MRI . Less than one-third of the Israeli patients (32%) were HLA-B27 positive vs. 66.7% of US patients (p = 0.007).Overall 87 patients were included . Upon presentation, inflammatory back pain, sacroiliac joint tenderness and abnormal modified Schober test, were significantly more prevalent among Israeli patients (59% vs. 35.4, 48.7% vs. 16.7, and 41.2% vs. 21.5%, respectively, all Israeli children with JSpA presented almost exclusively with axial disease compared to US patients who were more likely to present with peripheral symptoms. HLA B27 prevalence was significantly lower in the Israeli cohort compared to the US cohort. Further studies are needed to unravel the genetic and possibly environmental factors associated with these findings.The online version contains supplementary material available at 10.1186/s12969-020-00489-8. Spondyloarthritis (SpA) represent a group of heterogeneous conditions characterized by arthritis, enthesitis and increased risk of axial disease, with similarities in the genetic susceptibility .Although SpA usually begins in the third or fourth decade of life, 10–20% of patients experience symptoms during childhood, and the diagnosis of Juvenile Spondyloarthritis, (JSpA) accounts for 15–20% of the chronic arthritis diseases in children in North America and Europe , (2) BowA single diagnostic system that is representative of the JSpA population is still lacking. In 2002, the International League against Rheumatism (ILAR) developed the currently used classification for Juvenile Idiopathic Arthritis (JIA). Enthesitis-related arthritis (ERA) which is a category of JIA, describes a disease which affects the joints and entheses and may involve the sacroiliac (SI) joints. The diagnosis of ERA requires the presence of arthritis and enthesitis, or arthritis or enthesitis with two or more minor criteria which may include sacroiliac symptoms , 9. The N = 87) met the inclusion criteria , Sheba Medical Center  and Rambam Health Care Campus . As a comparison group, we used data from the large tertiary center, Benioff Children’s Hospital ) from 1/2013 to 12/2017. The protocol for this study was reviewed and approved by each of the committees for the protection of human subjects from the four institutions. All subjects met the following inclusion criteria: (1) Diagnosis of ERA according to ILAR criteria upon presentation  OR diagnosis of unilateral or bilateral sacroiliitis, proved by MRI imaging, who did not meet the ILAR criteria for diagnosis of ERA, (2) Aged < 16 years at symptom onset. In order to ensure that all patients with axial involvement in all sites are included, we used all available codes for axial involvement. Exclusion criteria included: (1) Diagnosis with any other rheumatologic disease at the time of JSpA diagnosis or subsequently , Juvenile Psoriatic Arthritis), (2) Lack of sufficient criteria for diagnosis of ERA, JAS or sacroiliitis based on MRI imaging), and (3) Follow up documentation of less than 4 months of initial presentation. Patients from all sites were identified from the medical records using identical ICD 9 and 10 diagnostic codes ; family history of autoimmune disease in a first degree relative; clinical signs, symptoms, and physical examination findings , dactylitis, uveitis, and AAU)); laboratory findings , C-reactive protein (CRP), erythrocyte sedimentation rate (ESR); radiologic findings (magnetic resonance imaging (MRI) evidence of active or damaged SI joints), and systemic medications prescribed on presentation and upon disease course , Disease-modifying antirheumatic drugs (DMARDs), and biologic therapy with TNF blockade).Peripheral arthritis was defined as joint pain, swelling or limited range of motion plus tenderness. Enthesitis was defined as tenderness to palpation on entheses insertions. A positive modified Schober test was defined as an increase of less than 6 cm between two lines: 5 cm below and 10 cm above the line joining the dimples of Venus , when the patient is fully flexed attempting to touch his toes compared to standing position. An increased CRP level was defined as greater than 5 mg/L, and an increased ESR level was defined as greater than 20 mg/h. Positive MRI findings of active SI joints were defined as bone marrow edema and joint space enhancement, and positive MRI findings of chronic sacroiliitis were defined as erosions and/or sclerosis.As an additional quality assurance, an Inter-Center Comparison of MRI interpretations was performed by comparing the interpretations of trained musculoskeletal (MSK) radiologists for a random sample of 20 MRIs, 10 from each country (US and Israel). The 10 randomly selected MRIs from one country were interpreted blinded to the initial interpretation by a radiologist from the other country , aiming to calculate the level of agreement between the musculoskeletal radiologists in respect to the presence or absence of active and chronic findings. Inter-center comparison of MRI interpretations yielded a matching of 95% (kappa = 0.9). In more details, 11 MRIs were interpreted as positive by the US and Israeli MSK radiologists, 8 MRIs were interpreted as negative by both MSK radiologists and one MRI was interpreted as positive by the American radiologist as compared to the Israeli radiologist, who interpreted in as negative.In addition to the comparison of the clinical and demographic features between the Israeli and the US cohorts, we examined how many patients from each cohort fulfilled each condition, i.e., ERA or JAS. For that purpose, patients from each cohort were grouped according to one or more of the following phenotypes: (1) ERA as defined by ILAR criteria, (2) JAS defined as sacroiliitis visualized on MRI and symptom onset age < 16 years as accepted , 15. AddP value of ≤0.05 was considered significant.The data were analyzed using BMDP software . Fisher’N = 87). Patients were predominantly male of Caucasian ethnicity. Although there was about 20% difference in males’ fraction between the cohorts (56.4% vs. 75% in the Israeli vs. US cohort), the difference was not statistically significant (p = 0.11). As compared to the US patients, Israeli patients were significantly older upon presentation  as well as at diagnosis . A two-fold increase in prevalence of family history of HLA-B27-associated diseases was not statistically significant .Demographic features of Israeli vs. US patients are depicted in Table Once restricting the analysis to patients with axial involvement upon presentation, supported by MRI findings (26 Israeli and 6 US patients), the mean age for symptom onset and for time of diagnosis were not statistically significant .p < 0.05), whereas peripheral arthritis and enthesitis were significantly more prevalent among the US patients . Presence of the HLA-B27 was more frequently seen in US than Israeli patients (66.7% vs 32% p = 0.007). The presence of Sacroiliitis defined by MRI was significantly higher in the Israeli cohort ) as compared to the US cohort ; p < 0.001), with the presence of pelvis MRI from time of presentation being 77% for both cohorts.Axial symptoms and signs including inflammatory back pain, SIJ tenderness, and positive modified Schober test were significantly more prevalent among Israeli patients upon presentation as compared to the US patients . In contrast, Israeli patients usually met criteria for JAS (30 (76.9%) vs. 11 (22.9%), p < 0.001). Fulfillment of both ERA and JAS criteria was similar between Israeli and US cohorts .Patients were classified as ERA by ILAR criteria, JAS, or both Fig. . US patiN = 67) had MRI imaging upon presentation, 57 of which had a known HLA-B27 status. Table p = 0.07). Upon presentation, a positive HLA-B27 status was significantly more prevalent among patients without sacroiliac involvement (74.1%) as compared to merely 30% of patients who presented with sacroiliitis (p = 0.001).Overall 77% of each cohort . An additional 20.5% of Israeli vs. 8.3% of US patients were prescribed DMARDs  at the time of diagnosis and 7.7% vs. 8.3% were prescribed biologic (TNF inhibitors) agents at that time (Table Most patients were taking NSAIDs)Ibuprofen, Naproxen, Indomethacin) upon presentation to pediatric rheumatology or were prescribed NSAIDs at time of diagnosis by pediatric rheumatology, with a significantly greater proportion among the US compared to the Israeli patients . Peripheral symptoms of arthritis and enthesitis were the dominant presentation of the US group and were comparable with other reports , 17–21. p = 0.003). This delayed presentation of Israeli children to the rheumatologic clinic may be partially explained by our results that show a much higher percentage of enthesitis in the US cohort, which is known to be a painful symptom which usually urge patients to seek medical care. It may also be related to differences in referral patterns. Interestingly, once restricting the analysis to include only patients with axial involvement upon presentation, no significant difference was found between the age at symptom onset and diagnosis between the two cohorts. This may explain the overall differences in age of symptom onset and age of diagnosis, as the Israeli cohort is comprised from a significant percentage of patients with axial disease as compared to the US cohort.The ages at symptom onset and at diagnosis in the Israeli cohort were significantly higher compared to US cohort. The mean age at diagnosis of the Israeli cohort, 14.3 ± 2.7 years, was also higher than reported for ERA and JAS cohorts worldwide, in which the mean age at diagnosis has been reported to be 10 to 13 years –28. In rP = 0.001). Although the overall percentage of HLAB27 in the US population is 6.1% [Although a major etiologic factor in ERA and JAS is believed to be related to HLA-B27 , 23, in  is 6.1% , the Isrp = 0.004). These differences may be explained by delayed administration of NSAIDs among Israeli patients due to a different disease phenotype, who, in many cases, present to the clinic with indolent axial symptoms, such as inflammatory back pain and without an available MRI test, which is required to establish the diagnosis, and therefore, are not given any medication until the diagnosis is supported by MRI. The fact that the US cohort presented with more peripheral arthritis and enthesitis, a diagnosis that can be established clinically by a musculoskeletal exam, might explain the higher usage of NSAIDs upon the time of diagnosis. Another possible explanation to the lower percentage of NSAIDs administration at time of diagnosis in the Israeli cohort is that since more than 20% of Israeli patients were prescribed with a DMARD  and/ or biologic agents , NSAIDs were avoided.Interestingly, the use of NSAIDs upon presentation to pediatric rheumatology and time of diagnosis was significantly lower in the Israeli cohort , ANA positivity and inflammatory markers. This also made classification more difficult. Second, the study population range period varied among the cohorts due to lack of sufficient number of patients in the Israeli cohort. However, the ERA criteria were published in 2001 and has not been updated since ever, and both cohort of patients were diagnosed after December of 2004 . Third, In conclusion, this study reveals that the clinical and demographic features of children diagnosed with JSpA in Israel are different compared to a US cohort from a large US center. Israeli children are older upon presentation and time of diagnosis as compared to their US counterparts, are more likely to be HLA-B27 negative and usually present with axial symptoms rather than peripheral arthritis or enthesitis, which are both significantly more common in the US cohort. Further studies are needed to explore the genetic and environmental factors that are associated with our findings.Additional file 1: Supplementary Table 1. ICD 9 and ICD 10 codes used for medical records.


Reference centile charts are widely used for the assessment of growth and have progressed from describing height and weight to include body composition variables such as fat and lean mass. Here, we present centile charts for an index of resting energy expenditure (REE) or metabolic rate, adjusted for lean mass versus age, including both children and adults across the life course.Measurements of REE by indirect calorimetry and body composition using dual-energy X-ray absorptiometry were made in 411 healthy children and adults (age range 6 to 64 years) and serially in a patient with Resistance to Thyroid Hormone α (RTHα) between age 15 and 21 years during thyroxine therapy.NIHR Cambridge Clinical Research Facility, UK.nd and 98th centile respectively). The 50th centile of the index ranged from 0·49 units (age 6 years) to 0·34 units (age 25 years). Over six years, the REE index of the RTHα patient varied from 0·35 units (25th centile) to 0·28 units (<2nd centile), depending on changes in lean mass and adherence to treatment.The centile chart indicates substantial variability, with the REE index ranging between 0·41 and 0·59 units at age 6 years, and 0·28 and 0·40 units at age 25 years (2We have developed a reference centile chart for an index of resting metabolic rate in childhood and adults, and shown its clinical utility in assessing response to therapy of an endocrine disorder during a patient's transition from childhood to adult. For example, REE regressions by Schofield6 and Henry7 which are based on weight and height exhibit this effect. Here we present an approach to the challenge of describing REE in terms of body composition with a continuous, unsegmented relationship through the 6 to 65years age range.Describing the physiological phenotypes of uncommon genetic disorders brings the challenge of determining the extent to which characteristics differ from those of a healthy population. This becomes more challenging when the individual is both developing through age and undergoing treatment. Measurement of resting energy expenditure (REE), a physiological variable dependent on body composition, which can change in catabolic  or endocrine (e.g. hyper or hypothyroidism) states, is useful in determining nutritional, energy intake or drug therapy.8 The current United Kingdom-World Health Organization (UK-WHO) growth charts10 consist of reference data based on the British 1990 (UK90)11 and the WHO standards for weight, length, body mass index (BMI) and head circumference from 2 weeks of age up to 4 years. The UK90 data were also used to construct new charts for birth between 23 and 42 weeks gestation.8 They are valuable for defining and detecting altered growth in an individual, due to for example small for gestational age,13 childhood obesity,14 rapid weight gain,15 malnutrition16 and epidemiology of childhood health at a population level.17Reference, growth centile charts are widely used in paediatric clinical settings to assess measurements such as height, weight and head circumference through childhood.18 and UK infants and children, have emerged.19 Body composition can be tracked from infancy to childhood and into adulthood and can be related to many clinical conditions and poor health outcomes. Reference charts allow clinicians to interpret observations across a wide range of diseases, conditions and treatments22 and are therefore an invaluable tool, contributing to assessment of nutritional status and management in clinical populations.More informatively, body composition reference charts, including data for total body water, fat mass and fat-free mass (FFM) for UK children, adolescents and young adults23, with earlier diagnosis of heritable metabolic disorders, has enabled commencement of therapies in childhood. Resistance to thyroid hormone alpha (RTHα) or congenital, nongoitrous hypothyroidism 6 (OMIM 614450), due to heterozygous mutations in thyroid hormone receptor α (TRα), is characterised by childhood developmental and growth retardation and a relative hypothyroid state in hormone-resistant TRα-expressing tissues, resulting in reduced REE.25 Like conventional childhood hypothyroidism, thyroxine therapy alleviates many symptoms and can promote normal growth and development.2 However, due to feedback regulation within the pituitary-thyroid axis being mediated by a different, normal thyroid hormone receptor subtype (TRβ), circulating TSH is not a useful biomarker in assessing response to thyroxine therapy25The advent of genome sequencing2627. The method summarizes the reference data with three smooth curves: the power transformation lambda needed to normalise the data (the L curve), the median mu or 50th centile (M curve), and the coefficient of variation of the distribution sigma (the S curve). The three curves allow any required centile to be drawn. The LMS method is a special case of the Generalised Additive Models for Location Scale and Shape (gamlss) family of models28 which can be fitted using the gamlss package in R.When monitoring therapy of these patients longitudinally, defining expected REE using current methods is difficult. The LMS method fits growth references to data by assuming that the data at each age are distributed as skew normalet al.,19 used it to derive reference centiles for body composition  and four-component body composition variables from the age of 6 weeks to 20 years. They produced charts with the 25th, 50th, 75th, 91st and 98th centiles.The LMS method has been widely used to construct reference data. Wells Using this method we first sought to construct reference centiles for REE to describe the variation in REE and body composition across childhood and adulthood, providing uniformity across the age spectrum; second, we used the centile chart to plot the change in REE index of a patient with RTHα, evaluating its value in assessing the response to thyroid hormone therapy in this disorder.5. All measurements in each participant were made on a single occasion, upon waking after an overnight stay in the National Institute for Health and Care Research (NIHR) Cambridge Clinical Research Facility. All investigations were part of ethically approved protocols  or were clinically indicated and undertaken with prior, informed written consent and assent for children under the age of 16. The healthy participants were free from disease, and non-smoking: they were excluded from the study if they were pregnant or receiving any metabolism-influencing medications. Recruitment and data collection took place between 2014 and 2019. Energy expenditure was calculated from the corrected gas exchange volumes  using the derivations of Elia and Livesey.29 Body composition was assessed using dual energy X-Ray absorptiometry . Precision of the instruments have been previously reported as iDXA precision for lean mass; 0.4 % CV, REE precision 3.8% CV.30The REE measurement protocols used has been described previouslyTo present the normality of the healthy paediatric (age 6-16 years) data, height, weight and BMI were converted into centiles using the British 1990 WHO reference child data using the excel LMS function.Statistical analysis was performed using R version 4·0·5 and gamlss package version 5·3-4.Scatterplots and Pearson correlations were examined for all variables , bone mass, fat mass and lean mass) considered influential in explaining the variation in REE. Gamlss was used to model the mean and variance of REE as functions of age and body size. Preference was given to log transformed body size variables, to best represent the allometric relationships between them. Age was fitted as a penalised cubic B-spline curve, with age logged to improve the fit by stretching the age range for children relative to adults. Interactions between sex, age and body size were also tested for.31The gamlss package was used to fit the regression models, and the optimal model was identified as that minimising the Bayesian Information Criterion (BIC). This method has been described elsewhere.0.67, kJ/min/kg) was generated from the optimal model, where the centiles were spaced exactly two-thirds of an SD apart, giving the rounded centiles 2, 9, 25, 50, 75, 91 and 98.32A centile chart describing the distribution of the REE index  and adults (n = 126), from which the models were derived are presented in the The correlations between variables were greater with the variables log-transformed, with the single exception of bone mass versus lean mass (r = 0·69 versus 0·76) . Lean ma0·67 (REE Index) plotted against age. Adding interaction terms did not reduce the BIC. There was also no evidence of heteroscedasticity - the residual SD was 0·092, corresponding to a residual coefficient of variation in REE of 9·2%, with this residual not depending on age or lean mass.The optimal gamlss model predicted log REE as a combination of log lean mass and a spline curve in log age. The coefficient for log lean mass = 0·67 SE 0.02, which means that antilogged, the model was equivalent to the index REE/LMnd, 9th, 25th, 50th, 75th, 91st and 98th) and LMS values for the REE index were plotted , free thyroxine (FT4), free triiodothyronine (FT3) and creatine kinase (CK), a marker of thyroid hormone action in skeletal muscle) were measured .th and 25th centiles) and associated with thyroid biochemistry , characteristic of RTHα. Initially, thyroxine therapy (62·5 mcg daily) raised circulating FT4 and FT3 concentrations, with a concomitant increase in REE index (0·369 units) of one centile. However, from age 16 to 18 years his REE index fell sharply , in association with a marked gain (7 kg) of lean mass that was not matched by an increase in thyroxine dosage, reflected in falling circulating FT3 and rising CK levels. Subsequently, stepwise increases in thyroxine dosage till age 21·1 years, did augment his REE index , with concomitant increases in circulating FT3 followed by normalisation of raised CK. At age 21·5 years, a sharp decline in REE index (0·289 to 0·275 units) in the context of stable thyroxine dosage and lean mass, suggested that the patient was not adherent with therapy, with biochemical measurements  confirming this. Latest measurements at age 21·8 years, showing a rise in REE index (0·284 units) and improvement in biochemical indices , suggest that adherence with thyroxine therapy has been restored.At baseline (age 15 years), the patient’s REE index was subnormal  period. Further, we illustrate utility of the REE centile chart in treating a patient with RTHα, a type of congenital hypothyroidism in which this physiological variable best measures the response to thyroxine therapy.nd to the 98th centiles of an REE index, modelled using gamlss. Compared to the UK-WHO reference child, the children in our dataset were mostly representative of a healthy population, however in 7 year old girls and 9 year old boys the mean centile for BMI was >72. Our results highlight a decline in REE, adjusted for body composition, during childhood and a plateau through adulthood. At each age we observe a wide degree of variation, with a coefficient of variation of 9.2%.The centile chart consists of the 2et al.,33 have reported similar findings to the present study, such that both total energy expenditure (TEE) and basal energy expenditure when adjusted for FFM decline in childhood and reach a plateau at 20 years of age. At 60 years of age a second break point was apparent when adjusted TEE and basal energy expenditure declined further, potentially linked to a decline in fat mass and FFM.Recently, Pontzer et al.,34 who have published centile charts for TEE using the LMS method in infants from one to 12 months of age. They observed a curvilinear increase in TEE whether expressed as MJ/day or relative to body weight or FFM (MJ/day/kg) throughout this period of rapid growth. Similar to the current study they observed a large degree of variation at each age and they concluded that a large proportion of the reported variation is biological and not error of measurement.The reported rapid increase in adjusted TEE during the first year of life has been documented by Reichman 37 have found that fat mass also contributes to variation in REE. In our current work, which combines child and adult datasets , the contribution of fat mass from the adult dataset is insufficient to influence the overall REE index. Whether the centile chart is not appropriate in individuals with grossly deranged fat mass (e.g. lipodystrophy) is unclear, but it should be used with caution in such contexts.Our previous linear regression equation for predicting REE in children included lean mass and sex as contributory variables. However, in adults we and others,38 Other growth reference studies such as the Fourth Dutch Growth Study and the Cuban and First Dutch growth study ranged from 14,500 to 55,000 in sample size.31 However they were focussed on height and weight, where the measurements are relatively easy to obtain, whereas capturing REE and lean mass require much more complex technologies. Nevertheless, to address this limitation, we will continue to develop our dataset to close relative paucity of measurements within our sample.We acknowledge that our healthy participant sample is smaller than that recommended for constructing centiles for use at a population level. The WHO growth standards required at least 200 children per sex per 3 month age group for a cross-sectional study.2 illustrates its clinical utility. Thus, a decline in REE from age 16 to 18 years accompanied by an increase in lean mass of ~6.7 kg, prompted a further increase in dosage of thyroxine therapy to account for growth. Subsequently, a marked fall in REE index (at age 21.5 years) despite stable body composition and thyroxine dosage, suggested non-adherence with therapy and biochemical indices  supported this notion. Once non-adherence was rectified, both REE index and biochemical markers improved. Overall, this centile chart enables the impact of both altered body composition and thyroxine therapy on this physiological parameter (REE), to be discerned at the same time. However, when measuring this parameter as a baseline characteristic of RTHα, we suggest that, rather than REE index, deviation of measured REE from a predicted value to derive a Z-score, is a more appropriate but complementary approach.24Our application of the centile chart to RTHα, a disorder in which resting energy expenditure is a better marker of thyroid status in TRα-expressing peripheral tissues than circulating TSH measurements,In conclusion, we have produced an REE index centile chart, adjusted for body composition and age, covering children and adults. This chart enables seamless monitoring of a physiological parameter (REE) that changes with growth, in relation to age-appropriate control data from childhood into adult life. The chart also provides a useful approach to discern the effects of a therapeutic intervention which changes growth, body composition and resting energy expenditure.Appendix Content