Betula pubescens ssp. tortuosa) is characterized by great intraspecific variation. We explored spatio‐temporal patterns of the first two mountain birch generations on a homogeneous, early successional glacial outwash plain in SE Iceland that was the recipient of spatially extensive long‐distance dispersal ca. 30 years ago. We evaluated the decadal progress of the young population by remeasuring in 2018, tree density and growth form, plant size, and reproductive effort on 30 transects (150 m2) established in 2008 at four sites on the plain and two adjacent sites ca. 10 km away. All measured variables showed positive increases, but contrary to our predictions of converging dynamics among sites, they had significantly diverged. Thus, two of the sites (only 500 m apart) could not be distinguished in 2008, but by 2018, one of them had much faster growth rates than the other, a higher growth form index reflecting more upright tree stature, greater reproductive effort, and much greater second‐generation seedling recruitment. We discuss two hypotheses that may explain the diverging dynamics, site‐scale environmental heterogeneity, and legacies of intraspecific priority effects.Most of the Earth's surface has now been modified by humans. In many countries, natural and semi‐natural ecosystems mostly occur as islands, isolated by land converted for agriculture and a variety of other land‐uses. In this fragmented state, long‐distance dispersal may be the only option for species to adapt their ranges in response to changing climate. The order of arrival of species may leave a lasting imprint on community assembly. Although mostly studied at and above the species level, such priority effects also apply at the intraspecific level. We suggest that this may be particularly important in subarctic and arctic ecosystems. Mountain birch ( Betula pubescens ssp. tortuosa) on four sites on an outwash plain in SE Iceland. Contrary to predictions of converging dynamics, sites showed significant divergence from 2008 to 2018, and we discuss two hypotheses that may explain this, site‐scale environmental heterogeneity and legacies of intraspecific priority effects. The massive long‐distance (≥10 km) dispersal, spatially extensive colonization (>35 km2 area from ca. 1990–2016), and high, although spatially variable, recruitment of the second generation, all illustrate the mountain birch's ability to rapidly adjust its range in a shifting environment.We explored the spatio‐temporal dynamics of the first two generations of mountain birch ( We studied the early dynamics following a sudden, large‐scale establishment of mountain birch through LDD onto a sparsely vegetated glacial outwash plain in sub‐arctic Iceland. We report on decadal‐scale spatio‐temporal patterns of density, growth, fecundity, and first local seedling recruitment of the young population, and compare it to two neighboring mountain birch sites. Specifically, we explored whether early demographics of the first generation gave insights into later emerging patterns. Our predictions were that the population characteristics would converge across the flat and apparently homogeneous outwash plain.Mountain birch  is a subspecies of B. pubescens native to Fennoscandia  glacial outwash plain , despite the nearest seed source being >10 km away. Age distributions based on dendrochronology indicate that mountain birch colonized the area around 1990 . Other common species included shrubs and dwarf shrubs , graminoids , and Stereocaulon lichens  to compare the SKS population to its nearest neighboring mountain birch stands. Site VM is in Morsárdalur valley Figure , where mwww.vedur.is). Six years of data (2014–2019) are available for a temperature station on SKS itself, 5–11 km from S1–S4  transects were established at 100 m intervals southwards, until the whole north–south spread of mountain birch had been covered, or to a maximum distance of 1000 m from the first transect. At S4, two adjacent N–S series of transects were established to increase the sample size. In total, 40 transects were established in 2008, of which 30 were resampled in 2018  transects, oriented perpendicular to dry riverbanks  in our sample. For the remaining plants in the 2018 resampling, plant size only was measured for plants between 1 and 5 cm . Due to their large numbers, we counted but did not measure ≤1 cm plants , and the size of all was assigned 1 cm. No first‐year seedlings (plants with cotyledons) were quantified.In 2008, maximum plant height, length of the longest shoot, and the number of female catkins were recorded for all mountain birch plants within each transect. Since male catkins were not counted, 2 quadrats, placed at 33 cm intervals (widthwise) at every other metre (lengthwise) along each transect (n = 100 per transect). Estimated total number of S‐seedlings was then extrapolated for each of those transects. Due to VM's small sample size of trees , size and height of 40 additional randomly chosen trees (mean size = 110 cm) were measured in June 2019 and added to the 2018 dataset.At the three northernmost transects at S4, the number of S‐seedlings was so great that we counted a subsample in four 0.25 m2.5For each transect, total density was calculated as the number of all individuals divided by area. Similarly, tree density was calculated using the number of individuals ≥20 cm, flowering adult density by using individuals with catkins, and finally, catkin density by using the total number of catkins. Plant height and the length of its longest shoot are interchangeable for upright trees, but since some plants in our study were prostrate or grew at an angle, we used the latter as a main measure of size. For the same reason, the ratio between plant height and shoot length was used as an index to study spatio‐temporal variation in growth form.We assessed the decadal‐scale progress of the SKS population by comparing its status in 2008 and 2018 and exploring between‐site variation. We also investigated between‐site variation for 2008 separately on one hand, and for 2018 on the other, including the two VNP sites in the latter case. The following response variables were examined: density of all plants, trees, flowering adults, and catkins, presence and abundance of catkins per plant , plant size , and plant growth form index (S‐/L‐seedlings excluded due to missing height data). The additional trees measured at VM in 2019 were only used in analyses of plant size and growth form Sections  and 3.4.R v3.6.2.  as a random effect  with ct Table . Site di2.5.2transect (nested within sites) as a random effect was used to explore how the presence and abundance of catkins related to plant size and differed between sites and years  with rs Table . A hurdlrs Table .site, plant size, year, and interaction between the two latter variables as fixed effects  as random effect and year, site, and their interaction as fixed effects .Spatio‐temporal variation in tree size within SKS was studied using LMM with ts Table . The modyear, site, and their interaction as explanatory variables  as a random effect .For growth form comparisons on SKS in 2008 and 2018, we used LMM on ct Table . Differe33.1Catkin production of the Skeiðarársandur (SKS) mountain birch greatly increased between 2008 and 2018, and all density‐related variables had elevated values Tables . Signifi3.2year and plant size  and S4 . In 2018, the difference between sites was also significant  and S3 . In 2018, however, plants were most upright at S4 and VM, but least upright at site VS, which had significantly lower index values than all other sites, except S2 , only 10 flowering adults were recorded (3% of the sample), with a total of 106 catkins, or 0.007 catkins/m2. Using these data for comparison, catkin density had increased 150‐fold by 2008, to 1.0 catkin/m2, and almost 700‐fold by 2018, to 4.7 catkins/m2.Mountain birch density and catkin production on Skeiðarársandur greatly increased during the study period, although temporal patterns were mostly site‐specific Table . Like mo2), no first‐year seedlings and only 25 S‐seedlings (6% of sample) were recorded. However, in 2018, S‐seedlings were estimated to be over 6000 (83% of the sample), and first‐year seedlings were in the thousands. As we did not detect exceptional weather events or growing season trends through the study period , we propose that the recent surge in the seedling establishment is linked to the previously described surge in seed production, indicating greater limitation by seed than microsite early on in our study. If most of the newly established seedlings have a local origin, then this signals a turning point in the population's development since the persistence of a population requires that colonizers leave descendants in the new range  greater mean tree size and density of plants, trees, flowering trees, and catkins  than at S4 in 2018 . Soil nutrients are therefore unlikely to explain the difference in catkin production of flowering trees between the two sites in 2018, and to elucidate the reproduction dynamics of the young population, more research is needed on temporal variation in flowering and possible explanations.The number of flowers produced is generally positively correlated with plant size , and high, although spatially variable, recruitment of the second generation, all illustrate the mountain birch's ability to rapidly adjust its range in a shifting environment.In the context of distribution shifts due to global climate change, our study may provide lessons. Although directly (climate warming) and indirectly (glacier retreat) mediated by climate change, the colonization of mountain birch on Skeiðarársandur was a natural process ; investigation ; visualization (lead); writing – original draft (lead); writing – review and editing (lead). Thora Ellen Thorhalsdottir: Conceptualization ; data curation ; funding acquisition ; investigation ; methodology ; supervision ; visualization (supporting); writing – original draft (lead); writing – review and editing (lead). Anna Helga Jónsdóttir: Formal analysis ; visualization (supporting); writing – original draft (supporting); writing – review and editing (supporting). Hulda Margrét Birkisdóttir: Investigation ; writing – original draft (supporting); writing – review and editing (supporting). Kristín Svavarsdóttir: Conceptualization ; data curation ; funding acquisition ; investigation ; methodology ; supervision ; visualization (supporting); writing – original draft (lead); writing – review and editing (lead).The authors have no conflict of interest to declare.Tables S1–S4Click here for additional data file.


The aim of this study was to compare the efficacy and safety of a novel percutaneous pedicle screw fixation and vertebral reconstruction (PPSR) vs. that of open pedicle screw fixation (OPSF) in the treatment of thoracolumbar fractures.This retrospective study enrolled 153 patients who underwent PPSR and 176 patients who received OPSF. Periprocedural characteristics, radiographic parameters, and clinical outcomes were compared between the two groups.The operation duration was 93.843 ± 20.611 in PPSR group and 109.432 ± 11.903 in OPSF group; blood loss was 131.118 ± 23.673 in PPSR group and 442.163 ± 149.701 in OPSF group, incision length was 7.280 ± 1.289 in PPSR group and 14.527 ± 2.893 in OPSF group, postoperative stay was 8.732 ± 1.864 in PPSR group and 15.102 ± 2.117 in OPSF group, and total hospitalization costs were 59027.196 ± 8687.447 in PPSR group and 73144.432 ± 11747.567 in OPSF group. These results indicated that these parameters were significantly lower in PPSR compared with those in OPSF group. No significant difference was observed in the incidence of complications between the two groups. The radiographic parameters including height of the anterior vertebra, Cobb angle, and vertebral wedge angle were better in PPSR group than in OPSF group. Recovery rate of AVH was 0.449 ± 0.079 in PPSR group and 0.279 ± 0.088 in OPSF group. Analysis of clinical results revealed that during postoperative period, the VAS and ODI scores in PPSR group were lower than those in OPSF group.Collectively, these results indicated that PPSR more effectively restored the height of anterior vertebra and alleviated local kyphosis compared with OPSF. Moreover, the VAS and ODI scores in PPSR group were better than those of OPSF group. Spine injuries, especially thoracolumbar fractures caused by various factors such as accidents are on the rise. Thoracolumbar fractures account for more than 50% of all spinal fractures , and posMagerl developed and reported for the first time in 1982 a minimally invasive percutaneous pedicle screw technique combined with external fixation . This teBased on traditional percutaneous pedicle screw fixation, we devised a new technique named percutaneous pedicle screw fixation and vertebral reconstruction (PPSR). This technique can be used in the reconstruction of the anterior column by distracting the involved vertebra and providing bone grafting to promote vertebral healing. PPSR was used for treating patients who had vertebral compression fracture. This retrospective study was carried out to compare the efficacy of PPSR and the traditional OPSF. In this study, medical records of patients with thoracolumbar fractures were retrospectively reviewed. Clinical features and surgical outcomes of the two types of surgeries were compared. The results indicated that OPSF and PPSR give both short-term and long-term benefits. Other advantages include shorter operating time, reduced financial burden, and preservation of anterior vertebral height and the local vertebral Cobb angle. Therefore, the novel PPSR is a reliable method for treating thoracolumbar fractures and offers many benefits to patients.This was a retrospective cohort study that was approved by the Ethics Committee of the Wujin Hospital of Traditional Chinese Medicine (KY-S-2019002). This study has been revised according to the STROBE checklist which is provided in the supplemental file . A totalThe procedures for PPSR are presented in Data collected from the medical records included age, gender, Body Mass Index (BMI), hypertension, diabetes, operative duration, blood loss, total incision length, postoperative stay, total hospitalization costs, fracture segment, the back and leg VAS score (0–10) and the Oswestry Disability Index (ODI) (0%–100%). VAS and ODI were collected at the time of admission, and at 1 week, 3 months, and 12 months after surgery through subsequent visit or telephone follow-up. All the clinical parameters were assessed by two junior attending physicians, and if their findings differed, a second opinion was sought from a senior physician.To evaluate the restoration of vertebral height and spinal curvature, the recovery rates of AVH, vertebral wedge angle (VWA), and Cobb angle were measured. The imaging findings were interpreted by two spine surgeons who had more than 10 years of clinical practice. If results from the two surgeons were inconsistent, the imaging findings would be evaluated by the professor with higher seniority in our team. Before surgery and 12 months after operation, all patients were subjected to radiographs, CT scans, and MRIs of the spine and the AVH, VWA, and Cobb angle were assessed. Recovery rates of AVH (%) = (postoperative AVH of the injured vertebra—pre-operative AVH of the injured vertebra)/[(AVH of the superior vertebra + AVH of the inferior vertebra)/2] . VWA wasχ2 test. Normality was checked using the Shapiro–Wilk normality test. A two-sample t-test was used for normally distributed data, while Mann-Whitney U test was used for non-normally distributed data. A P < 0.05 was considered statistically significant.All the data were measured by at least two surgeons. Data in this study were analyzed by using SPSS (version 25.0) and Graphpad prism (version 8). Continuous variables were expressed as mean ± SD, and the enumeration data were was expressed as percentage. Independent sample t test was used for group comparisons for data, including BMI, operative duration, blood loss, incision length, total hospitalization costs, recovery rates of AVH, local Cobb angle, and VWA. Categorical variables were investigated by the The demographic and baseline characteristics of the two groups were shown in P < 0.001). Volume of intraoperative blood loss was 131.118 ± 23.673 ml in PPSR group and 442.163 ± 149.701 ml in OPSF group (P < 0.001). The total length of skin incision was 7.280 ± 1.289 cm and 14.527 ± 2.893 cm in the PPSR group and the OPSF group (P < 0.001), respectively. The postoperative hospital stay of the patients was 8.732 ± 1.864 days in the PPSR and 15.102 ± 2.117 days in the OPSF group (P < 0.001). The cost of hospitalization in the PPSR group (59027.196 ± 8687.447 yuan) was significantly lower than that in the OPSF group (73144.432 ± 11747.567 yuan). Complications that were observed between the two groups did not differ considerably. Furthermore, no patients with single-level thoracolumbar fracture who received PPSR or OPSF experienced pulmonary embolism or bone material leakage after surgery.Perioperative data are shown in P = 0.100). The results of the AVH at 12-month after surgery demonstrated that the AVH in the PPSR group (2.713 ± 0.176 cm) was significantly higher than that in the OPSF group . In addition, the recovery rate of AVH in patients that underwent PPSR (0.449 ± 0.079) was notably better than in patients who underwent OPSF . In addition, the degree of spinal kyphosis was evaluated by local Cobb angle. The preoperative Cobb angles between the two groups were statistically consistent (P > 0.05). The local Cobb angle at the 12-month after surgery between the PPSR group (7.570 ± 1.422°) and the OPSF group (12.631 ± 1.421°) was statistically significant (P < 0.001). Moreover, the VWA was also measured to further assess the efficacy of the surgeries used for the vertebral reconstruction. The preoperative VWAs of the two groups did not differ. The results showed that the VWA in the PPSR group (6.747 ± 1.323°) was greater than that in the OPSF group . This finding demonstrated that PPSR are more effective than conventional OPSF in reconstructing fractured vertebra.The radiographic results of the PPSR group and the OPSF group were presented in the P < 0.001), 3-month was PPSR, 2.33 ± 0.78; OPSF, 4.88 ± 1.37 (P < 0.001), and 12-month was PPSR, 0.92 ± 0.82; OPSF, 3.36 ± 1.45 (P < 0.001). The VAS and ODI scores in the PPSR group were notably lower than that in the OPSF group on follow-up (P < 0.001), 3-month , and 12-month  after surgery (The clinical outcomes were measured with VAS score and ODI score. There was no difference between the two groups at baseline in VAS score and ODI score. The mean VAS scores at the 3-day was PPSR, 3.88 ± 1.07; OPSF, 6.89 ± 1.13 (ollow-up . This di surgery . The resvia longitude traction of the titanium rod. However, OPSF cannot effectively reduce the vertebrae fracture. These disadvantages have limited the wide use of OPSF in thoracolumbar fractures.Spine fracture injuries are quite common, with most occuring at thoracolumbar junction. According to an international statistic report, approximately 5% of spine fractures, and 54.9% thoracolumbar fractures inflict substantial financial burden , 10. GenOver the last decades, minimally invasive spinal surgery has received increased attention. In 1984, Magerl firstly described the concept of percutaneous pedicle screw fixation . This teTo address these concerns, PPSR, a novel minimally invasive internal fixation system was developed based on the conventional percutaneous transpedicular screw fixation system. PPSR was developed to restore and maintain the height of the injured vertebra and correct the spinal kyphosis resulting from vertebra fracture. PPSR can reconstruct the anterior column of the fractured vertebra by distracting the vertebra and transplant bone into the fractured vertebra. There are apparent advantages of using PPSR for treating thoracolumbar fractures without neurologic deficits. Firstly, the peroperative preoperative data in this study indicated that the operation duration, blood loss, postoperative stay, and the total costs of hospitalization in the PPSR group were notably lower than that in the OPSF group . The ratBesides PPSR, there is a traditional kyphoplasty with percutaneous screw fixation that has long been used in treating thoracolumbar fractures . AlthougInitially, fixing of the spinal column involved the whole spine. With the development of concepts and technologies has led to focusing of fixation on the adjacent vertebrae of the involved segments rather than the whole spinal column. Then, to achieve a more precise therapeutic effect, Denis proposed the concept that the spinal column could be divided into three parts: anterior column, middle column, and posterior column . The devNevertheless, there are also some limitations in this study. Cases included in this study were patients with single-segment fracture, and therefore at present the role of PPSR in treating more complicated thoracolumbar fractures has not been elucidated. In addition, the evidence level of this retrospective study was quite low. A prospective, multicenter randomized clinical trial should be carried out. Moreover, other variable that may influence the therapeutic effect of PPSR, such as the causes of fracture and other types of fracture were not considered in the study. Also, the one-year follow-up period of this study was relatively brief. A longer follow-up period would have helped our team to obtain more precise results.In conclusion, the novel PPSR has a safety profile similar to OPSF. It is worth noting that PPSR restores the vertebral height and the spinal kyphotic angle better than the conventional open internal fixation. Thus, PPSR is a reliable option for treating thoracolumbar fractures.


The leukemic phase of ALK‐positive anaplastic large cell lymphoma (ALCL) is reported to have a poor prognosis. We, herein, report a rare case of the common type of ALK‐positive ALCL complicated by lactic acidosis. The present case indicates that the leukemic phase of ALCL should be considered in a peripheral blood smear when abnormal lymphocytes are detected, as urgent chemotherapy may rescue life‐threatening LA in the leukemic phase of ALK‐positive ALCL. These findings suggested the leukemic phase of malignant lymphoma complicated by lactic acidosis (LA). Computed tomography showed neither lymphadenopathy nor hepatosplenomegaly. She was hospitalized the same day and started on continuous hemodialysis. However, her laboratory findings of the next day of admission were WBC count, 42,540/μL; abnormal lymphocytes, 29.0%; LDH, 5719 U/L; blood pH, 7.317; HCO3, 10.8 mmol/L; and lactic acid, 88 mg/dL. She started chemotherapy consisting of etoposide, doxorubicin, cyclophosphamide, vincristine, and prednisone (EPOCH) under continuous hemodiafiltration. LA improved after the first cycle of EPOCH therapy.A 47‐year‐old woman visited our hospital with a chief complaint of a low‐grade fever and an inability to move due to fatigue. Her laboratory findings were white blood cell (WBC) count, 21,370/μL; abnormal lymphocytes, 12.0%; lactate dehydrogenase (LDH), 2836 U/L; blood pH, 7.295; HCOMay‐Giemsa staining of a peripheral blood smear revealed small to medium‐sized abnormal lymphoid cells Figure . The cytThe patient underwent chemotherapy consisting of brentuximab vedotin, doxorubicin, cyclophosphamide, and prednisone after the first cycle of EPOCH therapy. However, severe headache developed, and the central nervous infiltration of her lymphoma was detected by a lumbar puncture. Papanicolaou staining and May‐Giemsa staining of a cerebrospinal fluid smear revealed abnormal lymphoid cells, and the phenotype was found to be positive for CD4 and CD30. She was started on alectinib and, thereafter, her central nervous symptoms rapidly improved.Although ALK‐positive ALCL is associated with a favorable prognosis among peripheral T‐cell lymphoma, the leukemic phase of ALCL is reported to have a poor prognosis, especially due to early death.Noriharu Nakagawa: Writing – original draft. Masahide Yamazaki: Writing – review and editing.The authors received no specific funding for this work.The authors declare no conflicts of interest in association with the present study.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


Healthy gut microbiota is important for prognosis in cow's milk allergy (CMA). The application of synbiotics (specific pre‐ and probiotics) in extensively hydrolyzed formulae (eHFs) is a relatively new concept.To evaluate a synbiotic‐containing, whey‐based eHF (SeHF) with galacto‐oligosaccharides, fructo‐oligosaccharides, and bifidobacterium breve M‐16V in infants with CMA.A 31‐day one‐arm pilot study in 29 infants with CMA (mean age 30.8 weeks [SD 11]) was undertaken, with outcomes including gastrointestinal tolerance, atopic dermatitis symptoms, dietary intake, growth, SeHF acceptability, caregiver quality of life, and hospital‐related healthcare use.p < .05) in the severity of abdominal pain (in 57%), burping (in 46%), flatulence (in 79%), constipation (in 14%), rhinitis (41%), and itchy eyes (73%), as well as atopic dermatitis in those with severe baseline symptoms (PO‐SCORAD© reduction: 34.7–18.2 (p = .003), n = 6) were observed over time. Growth and caregiver quality of life scores significantly increased  over time. Hospital visits and medications significantly reduced  in the 6 months after SeHF initiation.Significant improvements  in extensively hydrolyzed formula (SeHF) is a relatively new concept. This study investigated the use of SeHF in 29 infants with non‐IgE mediated Cow's Milk Allergy and showed improvements in gastrointestinal and atopic symptoms, caregiver quality of life, and reduced healthcare use. The use of SeHF enhanced the management of infants with non‐IgE mediated cow's milk allergy Prebiotics are defined as a nondigestible food ingredient such as galacto‐oligosaccharide (GOS) or fructo‐oligosaccharide (FOS), that beneficially and selectively colonize and stimulate the activity of one or more of a limited number of beneficial, health‐promoting, gut microbiota species.As mounting evidence suggests that healthy gut microbiota can help improve the “allergic march,” focus around “active” management of food allergy has become of greater interest, as opposed to avoiding food allergens alone.The presence of dysbiosis has been confirmed in infants with cow's milk allergy (CMA) which is the most common type of food allergy.Lactobacillus GG) may lead to the earlier acquisition of tolerance to cow's milk, whilst the use of synbiotic‐containing‐AAF (prebiotics and Bifidobacterium breve M‐16V) may lead to fewer infections, medications, hospital admissions, and subsequent cost savings, as the composition of gut microbiota is improved, analogous to that of breastfed infants.Bifidobacterium breve, Lactobacillus Rhamnosus, and L. reteri and Lactobacillus GG) in specialized infant formula has shown significant improvements in gut levels of Bifidobacterium, rates of infections, gastrointestinal symptoms, allergic symptoms and AD in allergic infants or those at high risk of atopy, after long term follow‐up.A considerable evidence base and current guidelines for formula‐fed infants with CMA recommend the use of extensively hydrolyzed formulae (eHF) which are nutritionally complete as the first‐line choice.Bifidobacterium breve M‐16V, originally isolated from breastmilk, was the most effective strain at reducing inflammation.Bifidobacterium breve M‐16V forms part of a specific synbiotic blend with short‐chain GOS, long‐chain FOS in a specialized infant AAF, which has been shown to significantly improve beneficial gut microbiota species and gut health biomarkers, significantly reduce the number of infections and medication use for infections, and gastrointestinal issues, compared to controls (AAF without synbiotics), in several RCTs in infants with CMA.Bifidobacterium breve M‐16V strain demonstrates efficacy as well as safety in healthy infants and those with atopy.As multiple probiotic strains exist, evidence comparing various strains in the allergic response identified that Bifidobacterium breve M‐16V has been assessed in various clinical studies in infants with AD demonstrating similar results, with improvements in the balance of beneficial and pathogenic gut microbiota species, atopic and gastrointestinal symptoms.A similar synbiotic‐containing eHF (SeHF), with a specific combination of short‐chain GOS, long‐chain FOS and Bifidobacterium breve M‐16V (probiotics). These pre‐ and probiotic strains have been expressly selected due to their ability to work together in combination as synbiotics, the lack of cow's milk used at source or in processing/production, and the nonallergenic properties of the ingredients, specifically Bifidobacterium breve M‐16V. As previously mentioned, this synbiotic combination has been extensively tested for hypoallergenicity, safety, and growth.The aforementioned SeHF is a whey‐based, specialized infant, hypoallergenic formula intended for the dietary management of CMA in infants. The extensively hydrolyzed formulation contains short‐chain GOS and long‐chain FOS (prebiotics), and 22.1The study was a prospective, single‐arm, longitudinal, interventional, multicentre, 31‐day study. After the baseline visit, infants undertook a 3‐day baseline period whilst continuing their current formula, before the 28‐day intervention period. However, for infants with a clinical requirement to immediately start the study product (SeHF) after the baseline visit , the 3‐day baseline period was omitted, and infants commenced the SeHF for the 28‐day intervention period. Those who completed the intervention were invited to participate in the follow‐up phase of the study, in which hospital visits and hospital prescriptions of medications were retrospectively collected from hospital medical records and caregivers, during the 6‐months before, and 6‐months after SeHF initiation.Trials.gov (Identifier: NCT03874104), conducted in line with Good Clinical Practice and the Declaration of Helsinki, and all caregivers of patients recruited were required to provide written informed consent.The study was undertaken between October 2017 and November 2020 in nine specialist healthcare centers for the management of CMA, across the UK. Infants were recruited from pediatric nutrition and dietetic outpatient hospital clinics or community services. Ethical approval was obtained from the UK National Health Service (NHS) Research Ethics Committee  and local NHS Research & Development departments reviewed and approved the study for local conduct. The study was registered on Clinical 2.2Infants were deemed eligible to take part in the study if they were aged <13 months; currently using or requiring an eHF for the dietary management of CMA and expected to receive at least 25% of their energy intake from the SeHF. Infants were excluded from the study if they had severe CMA which required management with an AAF; were exclusively breastfed; were born <37 weeks gestation and of less than 1 month corrected age at time of screening; had primary lactose intolerance; had a history of poor tolerance to whey‐based eHFs; had major hepatic or renal dysfunction; required parenteral nutrition; were receiving postpyloric enteral tube feeding; participated in other clinical intervention studies within 1 month of recruitment, or; if the investigator had concern around the willingness/ability of the caregiver to comply with the protocol and/or study requirements. Written informed consent was obtained from the caregiver of the infant. Eligibility for the follow‐up study phase included infants who had completed the 28‐day intervention with SeHF. Infants who had been subsequently identified as requiring management with AAF, and infants for whom healthcare use data 6‐months before, and after SeHF initiation was unable to be extracted from the hospital medical records, were excluded.2.38 CFU/g of Bifidobacterium breve M‐16V. Investigators provided each infant an individualized daily minimum target volume of the SeHF, with the recognition that intake would be variable, particularly for infants mixed‐fed (breastmilk and formula) and/or approaching complementary feeding.All infants received the SeHF  for the 28‐day intervention period. The SeHF was a hypoallergenic and nutritionally complete eHF, intended for the dietary management of CMA in infants from birth either as a sole source of nutrition or as a supplement alongside breastfeeding and/or complementary foods as appropriate. The SeHF provided 66 kcal, 1.6 g protein, 0.8 g short‐chain GOS, and long‐chain FOS per 100 ml (when reconstituted at the recommended concentration (13.8% w/v)) and 1.2 × 1033.13.1.1Gastrointestinal tolerance was recorded at baseline and end of intervention using a standardized questionnaire, to indicate severity  of vomiting, nausea, abdominal pain/discomfort, bloating, burping, flatulence, diarrhea, and constipation. In addition, daily stool frequency (number per day) and stool consistency The presence and severity of AD symptoms were recorded using a validated assessment tool  were assessed via a standardized, validated questionnaire, Food Allergy Quality of Life‐Parental Burden (FAQL‐PB),3.2.3Caregivers were asked to complete an acceptability questionnaire at baseline and at the end of intervention. This questionnaire used 5‐point Likert scales (from strongly agree (1), agree (2) don't know (3), disagree (4) or strongly disagree (5)) to assess ease of preparation and use, infant enjoyment, and overall liking of their baseline formula and SeHF.3.2.4Dietary intake was recorded at baseline and end of intervention, using comprehensive 24‐h dietary recall conducted by the investigators. Dietary intake data were analyzed using a dietary analysis program (Nutritics v5.026 (Research Edition), Dublin) to estimate energy and macronutrient intakes. Due to the difficulty in assessing the nutritional composition of breastmilk, this was excluded from the nutritional analysis. Intake of infants' baseline formula and the SeHF were reported daily (in ml) throughout the study by the caregivers. At the end of the study, this was compared to the daily minimum target volume prescribed by the investigators to assess compliance, and overall achievement of expected intake of the SeHF was also recorded. Dietetic goal(s) related to the introduction of the SeHF over the intervention period, were set for each infant by investigators at baseline. These could relate to symptom management, intake, growth, GI tolerance, or any other relevant outcome. Achievement of these goals was recorded at the end of study.3.2.5z‐scores were calculated using LMS growth macros for excel (based on UK‐WHO 2006 growth reference data for children 0–4 years old).At baseline and end of the intervention, standardized methods for supine length (recorded to nearest 0.1 cm), weight  recorded to nearest 0.1 kg), and head circumference  were used to measure infant growth by investigators. These measures were used to determine growth centiles (plotted on UK‐WHO growth charts 0–4 years) and 3.2.6During the study, serious adverse events were monitored and recorded by investigators, indicating severity and relatedness to the use of the SeHF.3.2.71)Hospital visits (elective and emergency admissions and attendances including outpatients).2)Hospital medication prescriptions .Of the infants included in the follow‐up phase, the following data were retrospectively extracted from hospital medical records, 6 months before and 6 months after SeHF initiation:To capture hospital visits and medication prescriptions outside of the study site where the infant was recruited , caregivers were asked to recall their child's healthcare use outside of the study site. This data was added to the data extracted from the medical records. For each 6‐month time period, hospital visits and medication prescriptions were recorded as the mean total number across the sample and the % of infants with ≥1 hospital visit or medication prescription.3.3p < .05. For continuous data , normal distribution was verified with the use of the Kolmogorov–Smirnov normality test before statistical analysis. Paired samples t tests were used for comparisons of two‐time points (baseline vs. end of study) for growth, compliance, nutrient intake, and caregiver quality of life (FAQL‐PB scores). FAQL‐PB scores were also analyzed per question  to understand if there were any specific differences. Nonparametric tests (Wilcoxon signed‐rank and Friedman tests) were used to analyse ordinal data , atopic symptoms, acceptability, and hospital‐related healthcare usage). AD severity was also analyzed based on baseline PO‐SCORAD© score quartiles . Patients with baseline score of 0 (i.e. no signs of AD) were excluded from this analysis. For all outcomes, analysis was also performed by comparison to historic formula use at baseline .Data were analyzed per the protocol and statistical procedures were performed using a statistical analysis package . Descriptive data  are provided where applicable. Significance was assumed at an α level of ClinicalTrials.gov Identifier: NCT02915510) showing mean 86.2% (SD 11.6) absence of gastro‐intestinal symptoms; assuming the power of 80% and an α level of .05, for noninferiority, the minimum detectable effect of 5 (noninferiority margin 0), which provided a sample size of 34. Therefore, a sample size of 40 patients was deemed suitable for this pilot study, allowing for dropouts.The sample size was calculated based on results from a similar study ) and were included in this analysis. Four patients dropped out of the study within the first 2 weeks due to: GI disturbance ; other suspected IgE reaction ; reduced oral intake of SeHF   at baseline was relatively low (mean 11.3 [SD 17.2]), as the majority of infants were already receiving an eHF, however, severity varied substantially (range 0–86.3). Most infants had no growth concerns at baseline, however, three were identified with faltering growth. Baseline formula was eHF (n = 27) of which, n = 13 were probiotic‐containing, n = 12 were prebiotic‐containing and n = 2 were standard eHF (i.e. no pre‐ or probiotics). Mean time on eHF at baseline was 15.6 weeks ([SD 11.8] [range 4.7–49.3]). Overall, only two infants were mixed‐fed receiving at least some breastmilk as well as formula at baseline. All infants were orally fed, of which 69% had started complementary feeding at baseline (n = 20). Seventeen infants were invited to participate in the follow‐up phase  of infants , with absence of abdominal pain increasing by 39% at the end of study. Burping improved significantly in 46% (n = 13) of infants by the end of study  with no severe ratings recorded. Flatulence improved significantly in 79% of infants , where severe symptoms resolved, and the majority of symptoms were rated as mild (reduced from moderate in 31%) by end of study. Constipation improved significantly in 14% of infants , which although largely absent in the majority of patients, further improved by 7% at the end of study .By the end of the study, incidence and severity of abdominal pain, flatulence, burping, and constipation significantly improved in infants whilst taking SeHF, and no severe symptoms were recorded at all   as did consistency of stools  and end of study: 4.7 (SD 1.6), p = .097, NS). Furthermore, investigators either strongly agreed or agreed that the SeHF was well tolerated in 85% of infants by the end of study. Analysis by baseline eHF type found no differences in gastrointestinal tolerance of SeHF based on the historic use of pre‐ or probiotic‐containing eHF at baseline.Stool frequency remained relatively stable with the use of SeHF (mean stool frequency baseline: 2.4 (SD 1.2) and end of study: 1.9 (SD 1.1), 4.2.2©: 11.2 (SD 16.9) ; n = 6), demonstrated a significant reduction in AD severity by the end of the study (mean PO‐SCORAD©: 18.2 (SD 22.7); p = .03) and improvements were seen as early as intervention Day 7 (mean PO‐SCORAD©: 27.1 (SD 27.4); p = .02)  however improvements were detected as early as intervention Day 7 (mean: 18.2 (SD 25.1); p = .019 vs. baseline). Severity of itchy eyes also reduced significantly by 73% at the end of study (baseline mean score: 19.0 (SD 30.4); end of study mean score: 5.2 (SD 11.4); p = .021). Severity of crying (baseline mean score: 24.5 (SD 17)), poor sleep (baseline mean score: 33.7 (SD 24.4), and wheeze (baseline mean score: 9.8 (SD 15.1)) were relatively low and remained stable at end of study . Analysis of atopic symptom severity did not detect any differences based on historic formula use.Severity of AD was relatively low at baseline (mean PO‐SCORAD4.2.3p = .004). There were no differences following analysis according to baseline eHF type, nor by scores to each specific question in the FAQL‐PB questionnaire.Caregivers reported significant improvements in quality of life by the end of study after use of SeHF. Scores from the FAQL‐PB questionnaire demonstrated significant reduction by 26.7% from baseline (mean: 30.9 (SD 23.1)) to end of study mean: 22.6 (SD 19.3), 4.2.4p = .02). Overall, 92% of caregivers strongly agreed or agreed the SeHF was acceptable compared to baseline formula . No differences in the ease of preparation and use of formula were reported between baseline formula and SeHF by the end of study . Furthermore, analysis of those on prebiotic‐containing eHF at baseline (requiring different preparation to probiotic‐containing formulae) confirmed that there were no differences in ease of preparation and use of SeHF .Perceived enjoyment significantly improved at the end of the study with SeHF when compared to baseline formula . Percentage energy from fat and carbohydrate from SeHF was similar to baseline formula. There was a difference in percentage energy from protein (p = .046), which was likely due to the differences in protein content (mean 1.78 g/100 ml baseline formula vs. 1.6 g/100 ml SeHF) however overall protein intakes were unchanged during the study . Mean intake of SeHF during the study was 615 ml/d (SD 223) and was maintained at levels similar to the intake of baseline formula (mean 637 ml/d (SD 253), p = .375, NS). Mean daily intake of SeHF was 120% (SD 56) of patient's target minimum daily volume, demonstrating good compliance to the SeHF overall. Investigators reported that 26 (96%) patients achieved expected intake . All investigators set one or more dietetic goals for each infant on the study which were related to symptoms of CMA, growth, intake of SeHF, obviating need for AAF, and provision of synbitoics to the gut microbiome. By the end of the study 90% of infants (n = 26) met all their dietetic goals and 7% (n = 2) met at least one. Furthermore, 80% of infants remained on the SeHF following study completion.Mean total energy and macronutrient intake from formula and the complementary diet remained stable between baseline and the end of study . Furthermore, there were significant increases in measures of relative growth (centiles and z scores) for all measurements , despite a stable energy and protein intake over the study. Weight for length centiles and z‐scores improved, although non‐significantly, by the end of the study (NS)  was observed Table ; n = 28.4.2.7n = 5]; GI disturbance [n = 2]; facial rash [n = 1]; loose stools and teething [n = 1]; teething [n = 1]). The majority of adverse events (9 out of 10) were reported as mild‐moderate, and only 3 out of 10 were deemed probably related to the SeHF. Of these, two were reported in the same infant which involved loose stools and teething, however, this infant completed the study with no issues and continued with the SeHF after intervention. The remaining adverse event was in one infant who terminated the study due to pain in the gut, loose stools with mucus, crying, and sneezing.No serious adverse events were reported over the duration of the study and no safety concerns were reported with the use of SeHF. Overall, 10 adverse events were recorded in 7 infants (infection/virus [4.2.8p = .019). A reduction in the incidence of any type of hospital visits was also observed, although this did not reach significance (p = .083). The incidence and mean number of overall hospital medication prescriptions were significantly lower amongst infants in the 6‐month after SeHF initiation compared with the 6 months prior (Table p = .025).Significant reductions in the mean number of overall hospital visits required by infants were observed in the 6 months after SeHF initiation compared with the 6 months prior Table . The laror Table . This wa5The primary aim of this small, single‐arm, pilot study was to evaluate the impact of SeHF over 4 weeks in infants with CMA on clinically meaningful outcomes. Findings included significant improvements in GI tolerance over time (primary outcome), reductions in atopic symptoms and AD in those most severely affected at baseline, growth, high caregiver acceptability, and improved parental quality of life.Bifidobacterium breve M‐16V, which is in line with results in other similar studies.Bifidobacterium breve M‐16V) is supported by a wealth of GI tolerance data in both healthy and atopic infants receiving an AAF with the same probiotic strain.Primary outcome results demonstrated that infants, historically taking a nonsynbiotic eHF (before study), had significant improvements in the incidence and severity of abdominal pain, burping, flatulence, and constipation over time whilst taking SeHF. The mean duration over which infants had been receiving an eHF at baseline in this study was 15.6 weeks , which is longer than the average time required for symptom resolution reported in the literature (range 4.8–13.6 weeks).©) did not change in the total study group, those with the most severe symptoms (n = 12) observed significant improvements over time. The severity of the atopic symptoms rhinitis and itchy eyes also improved significantly over time, but other symptoms did not show any changes. Considering that symptom severity was low at the beginning of the study, most likely due to established non‐synbiotic containing eHF use, the further reduction in AD symptoms is interesting, although difficult to interpret without a control group. Similar results have also been found in other studies on GOS, FOS and Bifidobacterium breve M‐16V demonstrating improved AD severity in non‐CMA infants taking the same SeHF used in this study.Although AD severity . Furthermore, subgroup analysis of those on non‐probiotic (prebiotic‐containing) eHF at baseline confirmed that there were no differences in ease of use with SeHF  which supports the practicality of using the synbiotic‐containing formula. Clinical guidelines recognize palatability as an important consideration when choosing suitable formula for the dietary management of CMA,The SeHF was found be to highly acceptable by caregivers of infants although it required different preparation than nonprobiotic‐containing eHF (due to the presence of probiotic bacteria). In this study, 100% of caregivers found the SeHF as easy to use as baseline formula , and whilst all were following a cow's milk protein‐free diet, four infants were also following a soya free diet . Existing evidence suggests the presence of soya may risk cross‐reactivity in those with CMA and whilst most guidelines do not suggest soya formula first line, even fewer discuss routine exclusion of soya in complementary foods.6Bifidobacterium breve M‐16V enhances the dietary management of CMA infants already well established on eHF, although the results of this study are difficult to interpret without a control group. By the end of the study, significant improvements were observed in abdominal pain, burping, flatulence, constipation, rhinitis, itchy eyes, AD severity in those with most severe symptoms, and growth, whilst caregiver quality of life was also improved. Furthermore, SeHF was associated with reduced hospital‐related healthcare use in the 6 months after initiation in this cohort. This study adds to the evidence highlighting and supporting the clinical and health‐economic benefits of using synbiotic‐containing hypoallergenic infant formula for the dietary management of CMA, although the results of this study are difficult to interpret without a control group. The results in this study are specific to the synbiotic short‐chain GOS, long‐chain FOS, and Bifidobacterium breve M‐16V and cannot be extrapolated to other types of synbiotic formulae. Furthermore, controlled, robust research to explore the longer‐term benefits of synbiotics, specifically the blend used in this study, for the clinical management of infants with CMA is required.Overall, the results of this small, single‐arm, pilot study demonstrate that SeHF containing GOS, FOS, and Conceptualization and methodology prospective study phase: Robert M. Browne, Gary P. Hubbard, and Rebecca J. Stratton. Conceptualization and methodology follow‐up study phase: Fiona J. Kinnear and Abbie L. Cawood. Chief investigator: Rebecca J. Stratton. Investigation and acquisition of data: Robert M. Browne, Kiranjit Atwal, Fiona J. Kinnear, Lynne Graham, Sankara Narayanan, Lisa Cooke, Catherine Casewell, Sally‐Ann Denton, Joan Gavin, Ailsa J McHardy, Debbie Evans, Rachel Vallis, Devasmitha Venkataraman, Sarah Donohoe, Victoria Steele, Sonia Armstrong, and Gary P. Hubbard. Formal analysis, Kiranjit Atwal, Robert M. Browne, Fiona J. Kinnear, and G.P.H; Writing—original draft preparation, Kiranjit Atwal. Writing—review and editing, Kiranjit Atwal, Gary P. Hubbard, Fiona J. Kinnear, and Rebecca J. Stratton. Visualization, Robert M. Browne, Kiranjit Atwal, Gary P. Hubbard, and Rebecca J. Stratton. Supervision: Kiranjit AtwalA, Gary P. Hubbard, and Rebecca J. Stratton. Project administration, Robert M. Browne, Kiranjit Atwal, and Gary P. Hubbard. Funding acquisition, Gary P. Hubbard and Rebecca J. Stratton.K.A, G.P.H, R.M.B, A.L.C, F.K., and R.J.S. are employees of Nutricia Ltd; L.C, has received payment from Nutricia for previous writing activities; L.C, S. Denton, have received educational grants from Nutricia Ltd. for conference attendance; L.C, S. Denton, have received honorarium from Nutricia Ltd. for conference presentation; have provided scientific advice and expertise for Nutricia Advisory Boards; L.G, C.C, S.N, J.G, A.J.M, D.E, R.V, D.V, S. Donohoe, V.S, and S.A. declare no competing interests with the study funder.Trials.gov (Identifier: NCT03874104) and conducted in line with Good Clinical Practice and the Declaration of Helsinki. Informed consent was obtained from all parents/carers involved in the study.The study was approved by the UK National Health Service (NHS) Research Ethics Committee  and local NHS Research & Development departments reviewed and approved the study for local conduct. The study was registered on Clinical


Prostate cancer (PCa) is a complex disease. An ongoing accumulation of mutations results in increased genetic diversity, with the tumor acquiring distinct subclones. However, non‐genetic intra‐tumoral heterogeneity, the cellular differentiation state and the interplay between subclonal evolution and transcriptional heterogeneity are poorly understood. Here, the authors perform single‐cell RNA sequencing from 14 untreated PCa patients. They create an extensive cell atlas of the PCa patients and mapped developmental states onto tumor subclonal evolution. They identify distinct subclones across PCa patients and then stratify tumor cells into four transcriptional subtypes, EMT‐like (subtype 0), luminal A‐like (subtype 1), luminal B/C‐like (subtype 2), and basal‐like (subtype 3). These subtypes are hierarchically organized into stem cell‐like and differentiated status. Strikingly, multiple subclones within a single primary tumor present with distinct combinations of preferential subtypes. In addition, subclones show different communication strengths with other cell types within the tumor ecosystem, which may modulate the distinct transcriptional subtypes of the subclones. Notably, by integrating TCGA data, they discover that both tumor cell transcriptional heterogeneity and cellular ecosystem diversity correlate with features of a poor prognosis. Collectively, their study provides the analysis of subclonal and transcriptional heterogeneity and its implication for patient prognosis. During tumor evolution, distinct tumor subclones acquire preferential transcriptional subtypes which show different cellular differentiated status. In addition, these subtypes or subclones have close communication with each other or other stromal or immune cells within the tumor ecosystem. These transcriptional heterogeneity and also tumor ecosystem diversity are correlated with patient prognosis. A steady increase in advanced or metastatic prostate cancer has been observed at diagnosis in China, advocating for an improvement in treatment strategies. Tumors comprise a heterogeneous collection of cells with distinct genetic and phenotypic properties that can differentially promote progression, metastasis, and drug resistance. Thus, there is an urgent need to further our understanding of prostate cancer heterogeneity.Prostate cancer (PCa) remains the second most frequently diagnosed cancer in men, with more than 1 414 259 new diagnoses and 375 304 deaths per year globally.1 The  Moreover, cancers are hierarchically organized with a stem cell‐like population, sustaining tumor growth through self‐renewal and differentiation. The tumor microenvironment also generates intra‐tumoral heterogeneity by exerting different selective pressures in distinct regions of the tumor. These models are not mutually exclusive and act together to create a complex system with multiple layers of heterogeneity established by the distinct genetic, transcriptomic, and functional properties of different cells. Subsequently, understanding the function and effect of different cell populations on tumorigenesis, including which features promote tumor initiation, progression, or drug resistance, will also be key.Intra‐tumoral heterogeneity arises through both genetic and non‐genetic mechanisms. The stochastic accumulation of mutations through genomic instability results in increased genetic diversity, with the tumor acquiring subclones with distinct genotypes over time.7 MoreEmerging single‐cell technologies provide a new opportunity to profile individual cells within tumors and investigate what roles they play in these processes. It is likely that different tumor subclones can acquire transcriptional changes to coordinate subclones’ survival, growth, and competition during tumor evolution. Therefore, it was necessary to evaluate tumor cell transcriptional status among different tumor subclones in untreated PCa patients using single‐cell RNA sequencing technology (scRNA‐seq). Here, we performed scRNA‐seq of primary tumor tissues from 14 untreated PCa patients. On analyses of these data, we observed large degree of intra‐tumoral genomic and transcriptional heterogeneity. Subclones within a patient showed distinct plasticity of cell differentiation states and communication patterns with other cell types within the prostate cancer ecosystem, which has implications for both tumor composition analysis for diagnostics and therapeutic assignment.22.1Figure To decipher the landscape of the tumor ecosystem, we generated scRNA‐seq profiles for primary tumors from 14 treatment‐naive prostate cancer patients using a 10× Genomics‐based platform Figure . To furt2.2Figure EPCAM, KRT5, KRT8, and CDH1  in each cell inferred by inferCNV  to identify the underlying regulatory network in each tumor subtype. As expected each tumor subtype was driven by relevant TFs,17 incs Figure . Moreoves Figure . Thus, t2.5 a tool that is able to quantitatively infer and analyze intercellular communication networks among ligands, receptors, and their cofactors from scRNA‐seq data. We found that endothelial cells, T cells, macrophages, and fibroblasts were the dominant communication hubs  or intercellular communication network may drive the cell plasticity of subclones or subtypes. Thus, we applied CellChat,18 a ts Figure . We found clearly different communication strength among these cells  to assess the correlation of tumor subtype heterogeneity and cellular diversity of the tumor ecosystem. We found a positive correlation (according to Shannon's diversity index [SHDI]) between tumor subtype heterogeneity and cellular diversity in the tumor ecosystem , luminal A‐like differentiated cells (subtype 1), luminal B/C progenitor‐like cells (subtype 2), and basal‐like cells (subtype 3). Every tumor contained malignant cells corresponding to four transcriptional states at various extents. We confirmed our “EMT‐like” and “Luminal A‐ and B/C‐like” subtypes using scRNA‐seq dataset from a recently published paper, although they didn't find the “basal‐like” subtype in their dataset. This may be due to the different patient cohort because we indeed identified the basal gene highly‐expressed tumor cells in prostate cancer tissue. In addition, we found that genetic distinct subclones presented with preferential developmental subtypes. This phenomenon is consistent with a previous study in human oligodendroglioma which CNV subclones differed in relative distribution of different tumor cell subpopulations. These results suggested that non‐genetic developmental heterogeneity may super‐impose over subclonal evolution in PCa. Thus, we provided evidences at single‐cell level that PCa tumor cells displayed significant non‐genetic plasticity, supporting that transcriptional heterogeneity may also play important role in cancer progression, adaptation, and evolution within tumor ecosystem.PCa is a clinically heterogeneous disease. Many attempts have been made to unravel intra‐tumoral genetic heterogeneity in PCa.22 How Indeed, the subclone with dominant subtype 0 phenotype had weaker communication strength both within tumor cells and with other cell populations, suggesting loss of strong cell–cell contacts enabling subclones gaining mesenchymal phenotype to adapt to changing microenvironment. Furthermore, CellChat analysis enables the identification of key intercellular communication pathways, such as MK and MIF signalling pathways in PCa patients. Notably, these highly communicated signalling pathways have been reported to be activated as novel biomarkers and therapeutic targets in PCa. This result suggests that scRNA sequencing approaches could indeed be used to address the phenotypic heterogeneity of cancer cell populations and may be of crucial importance in designing personalized therapeutic approaches.Further supporting this notion, subtype 0 cells showed the plasticity along the epithelial–mesenchymal axis. It has been showed that cells moving forward and reversibly on the epithelial–mesenchymal axis, enabling them to adapt to and appropriate new environments during tumor progression.27 Ind Whereas with limited number of cases and insufficient follow‐up period in current cohort, we further validated the role of tumor cell transcriptional plasticity in patients’ outcome in TCGA cohorts. Given that the role of tumor microenvironmental cues in shaping tumor cell plasticity, indeed we identified both a higher proportion of subtype 0 tumor cells and the higher cellular diversity of the tumor ecosystem were associated with a poorer outcome . to obtain the UMI matrix, which was further imported into R (v4.0.3) and processed with the Seurat package (v3.2.1). Cells with a detected gene number <200 or >5000 or a high mitochondrial transcript ratio (>15%) were excluded. After normalization and scaling, the batch effect between patients was then removed using the mutual nearest neighbor algorithm in Seurat. The batch‐corrected matrix was used for further analysis and visualization. The top 2000 highly variable genes were extracted to perform principal component analysis (PCA), and the top 30 principal components (PCs) were used for cluster analysis. Cell type were annotated by the SingleR package (v1.2.4) and then checked manually.Raw data generated with the 10× Genomics platform were aligned to the GRCh38 reference genome using Cell Ranger software (v4.0.2)30 to p values less than 0.05, average logFC larger than 0.25, and percentage of cells with expression higher than 0.25. Gene enrichment analysis was performed for marker genes of each cell type with Metascape (https://metascape.org/). The marker genes or signature genes for cell types were as follows: epithelial cells , B cells , endothelial cells , neutrophils , nature killer cells , T cells , macrophages , fibroblasts , and undefined cells .To identify differentially expressed marker genes for each cell type, the FindAllMarkers function in Seurat was used under default parameters. Marker genes were selected as those with adjusted  follows:33 epi Briefly, 500 non‐malignant cells, including macrophages, endothelial cells, and T cells, were randomly selected as a normal reference, and another 300 non‐malignant cells (as spike‐in cells) were randomly selected together with epithelial cells for CNV inference and hierarchical clustering as described. Then, the dendrogram was cut at the highest point at which all the spike‐in controls belonged to one cluster. The epithelial cells that clustered together with normal spike‐in controls were identified as “normal epithelial cells”, whereas the remaining cells were identified as “tumor cells”. Then, tumor cells were run with inferCNV again to infer CNV and tumor subclones. The tumor cells were re‐clustered with the Seurat package (resolution = 0.12), each subcluster was defined as a transcriptional subtype. The marker genes or signature genes for cell types or subtypes were referred: epithelial cells , luminal A cells , luminal B cells , luminal C cells , basal cells , EMT genes , and stemness genes .Copy number analysis was performed with the R package inferCNV (v1.0.6) under default parameters. To identify tumor cells from epithelial cells, the authors followed the recently published approach by Ashley Maynard et al.34 Bri under default parameters. Highly variable genes were selected by the built‐in DispersionTable function of Monocle.All tumor cell expression data were used for single‐cell pseudotime analysis using the Monocle (v2.16.0) package35 und the authors calculated the G1S scores and G2M scores for each malignant tumor cell with the CellCycleScoring function in Seurat. They defined a cell as cycle‐active as long as one score was greater than 0.1.Based on the 43 G1S genes and 54 G2M genes defined in the previous study,36 the in R. First, they inferred potential targets of each TF based on their co‐expression function indicated by GENIE3. Then, potential direct binding targets were selected according to DNA motif analysis and compared with the reference motif database downloaded from RcisTarget (https://resources.aertslab.org/cistarget/). The regulon activity of each cell was analyzed via the AUCell function. Each cell was scored for regulon activity using the function AddModuleScore in Seurat, and final visualization was implemented with the featurePlot function in Seurat.To explore the dominant transcription factor in different subclones, the authors applied SCENIC with the SCENIC package (v1.2.1)37 in  The ligand and receptor genes expressed by each cell were projected into a manually selected reference communication network and the probability of communication in each pathway was inferred by gene expression. They statistically analyzed the communication probability between subclones and other non‐malignant cells as well as the communication between different subclones. Histograms were implemented with the ggplot2 package (v3.3.2) in R.To enable a comprehensive analysis of cell–cell communication molecules, the authors applied cell–cell communication analysis based on CellChat (v1.0.0).18 The (https://cibersortx.stanford.edu/) was used to construct the cell‐type‐specific signature matrix according to the top 200 marker genes of each cell type, and then the constructed signature matrix was used to infer the fractions of different cell types and transcriptional subtypes. Bulk expression data from the TCGA PRAD project were downloaded from the GDC data portal  and all 495 patients were included in the analysis. For each patient, the information entropy contained in its expression data was calculated by using the following formula:p represents the fraction of cell type i in the patient, and X represents the collection of all cell types or transcriptional subtypes.An online tool38  as a whole; to calculate the information entropy of the transcriptional subtype, the fraction of each transcriptional subtype in all tumor cells was calculated. Then, the correlation analysis of information entropy between transcriptional subtypes and the ecosystem was performed in R (v4.0.3), and further visualization was implemented with the pheatmap (v1.0.12) and ggplot2 (v3.3.2) packages. All 495 patients were divided into two groups by hierarchical clustering for the proportion of all cell types according to the results of CIBERSORTx or by the median cell proportion for each cell type. Progression‐free survival (PFS) was used as a clinical endpoint, and the KM curve was plotted using the survminer (v0.4.8) package.As described above, the authors obtained the fractions of various cell types in the tumor ecosystem of patients in the PRAD project through the CIBERSORTx online tool.38 Allm sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for10 min and 5% BSA in PBS for 1 h. Slides were incubated overnight at 4 °C with a primary antibody, which was followed by incubation with an HRP‐linked secondary antibody  at room temperature (30 min). Diaminobenzidine (DAB) was used as chromogen, and the sections were counterstained with haematoxylin. Representative fields were selected from these slides.Primary antibodies included ZEB1, KLK3, TACSTD2, and KRT15. Formalin‐fixed and paraffin‐embedded tissue sections (5 µm) were deparaffinized and rehydrated. Antigen retrieval was carried out using 10 m (https://ngdc.cncb.ac.cn/gsa‐human/s/Bv7gBES8). For survival analysis, bulk RNA‐seq data from the following studies were used: TCGA . All other data supporting the findings of this study are available from the corresponding author on reasonable request.Data have been deposited in the Genome Sequence Archive for Human (GSA‐Human) under accession HRA00082340 (htt‐test was used to reveal the statistical difference in Figures p‐value or q‐value less than 0.05 was considered as significant difference. All statistical analyses were conducted in R4.0.3.All statistical analyses of scRNA‐seq data were performed using the Cell Ranger software, Seurat, SingleR, Metascape website, inferCNV, Monocle, SCENIC, CellChat, CIBERSORTx online tool, survival, survminer, and GSVA. Visualization of data was made using R packages ggplot2 and pheatmap. The data in Figures The authors declare no conflict of interest.G.G., Y.H., J.Z., and X.L. contributed equally to this work. W.C., H.H., L.Z., X.L. conceived the study. G.G., Y.H., J.Z., and X.L. designed the experiments. G.G., Y.H., J.Z., X.L., X.L., Y.G., Z.L., J.W., W.Z., Y.X., Y.P., J.D., and B.Z. performed the experiments and analyzed the data. W.C., G.G., Y.H., and J.Z. wrote the paper.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file.Supplemental Video 3Click here for additional data file.


Induction chemotherapy (IC) comprising docetaxel, cisplatin, and fluorouracil (TPF), combined with concurrent chemoradiotherapy (CCRT) effectively improves the survival rate of locally advanced nasopharyngeal carcinoma (LA‐NPC). Selecting patients whose risk of tumor recurrence and metastasis is high and the appropriate chemotherapy intensity is a concern. We combined tumor‐node‐metastasis staging with the load of Epstein–Barr virus (EBV) after IC to select the individualized chemotherapy strength.The clinical data and prognostic factors of patients with stage III–IV LA‐NPC treated with TPF IC combined with CCRT were analyzed retrospectively. The conventional treatment group received the standard three cycles TPF IC combined with CCRT. For the new treatment group, the cycles of IC were determined according to whether the EBV‐DNA disappeared completely after a certain course of IC, if so, subsequent IC was stopped and the chemoradiotherapy stage was entered. Propensity score matching (PSM) was performed at a ratio of 1:1 to balance baseline characteristics. Survival outcomes and adverse events between the conventional treatment group and the new method treatment group were compared.p = 0.026).The study included 256 patients, among whom 192 were matched successfully into 96 pairs. The patients were followed up for a median of 51 months. The proportions of patients receiving three, two, and one cycle of IC after PSM in the routine and new treatment cohorts were 93.8%, 3.1%, 3.1% versus 21.9%, 49.0%, 24.0%, respectively. However, their 3‐year distant metastasis‐free survival, local recurrence‐free survival, progression‐free survival, and overall survival did not differ significantly. The incidence of grade 3–4 neutropenia toxicity in CCRT decreased significantly in patients receiving the new treatment method compared with that in the conventional treatment group (Combining TNM stage and EBV‐DNA load after IC to determine the courses of IC in patients with LA‐NPC did not alter the curative effect but decreased toxicity. The cycles of induction chemotherapy IC were determined according to whether the EBV‐DNA disappeared completely after a certain course of IC. In the new method group, the cycles of induction chemotherapy were reduced by 28%, and the serious neutropenia toxicity was decreased by 10% during concurrent chemoradiotherapy, comparted with the routine strategy. However, their 3‐year distant metastasis free survival, local recurrence free survival, progression free survival, and overall survival did not differ significantly. The patients received induction chemotherapy (IC) regimens of docetaxel, cisplatin, and fluorouracil (TPF), plus two cycles cisplatin CCT from an attending doctor's team at Sun Yat‐sen University Cancer Center. Pretreatment, cfEBV‐DNA was detectable in the patients. Patients were recruited based on the following inclusion criteria: Age > 18 years old diagnosed histologically with LA‐NPC at the Union for International Cancer Control  stage III or IVA; an Eastern Collaborative Oncology Group performance status of 0 to 1; sufficient bone marrow and organ function; and pretreatment EBV DNA >0 copies/ml.The patients were treated with two treatment schemes: The conventional treatment group was treated with standard three‐cycle TPF IC combined with CCRT. In the new treatment group, the cycles of IC were further determined according to whether the EBV‐DNA disappeared completely after a certain course of IC. Once the post‐IC EBV‐DNA dropped to 0, the subsequent IC was stopped and the chemoradiotherapy phase was entered.Propensity score matching (PSM) at a ratio of 1:1 was performed to balance the baseline characteristics. For the two groups, survival and adverse events were compared.2.2The patients received a pretreatment physical examination. The patients were then subjected to imaging, including tomography or radiography of the chest, computed tomography or sonography of the abdomen, a whole‐body bone scan, and nasopharynx and neck magnetic resonance imaging (MRI). If indicated clinically, positron emission tomography‐computed tomography (PET‐CT) was carried out. Staging of the patients was carried out according to the International Union against Cancer/American Joint Committee on Cancer (UICC/AJCC) staging system manual (8th edition).2.3Bam HI‐W region. cfEBV‐DNA analysis was performed at various time points: Pretreatment (2 weeks before IC initiation), after each cycle of IC (post‐IC), and within and during 1 week of CCRT completion (post‐CCRT).Quantitative real‐time polymerase chain reaction (qPCR) was carried out to measure the pretreatment level of EBV‐DNAs. The qPCR system initially detected the 2.42 on day 1, cisplatin at 65 mg/m2 on day 1, and 5‐ fluorouracil at 550 mg/m2 on days 1–5, repeated every 3 weeks. Concurrent CT comprised cisplatin at 80 mg/m2 on day 1, repeated every 3 weeks. The chemotherapy dose setting was based on the results of our previously published dose climbing trial from 2009 to 2010.9/L, agranulocytosis fever, granulocyte deficiency infection, platelets <25 × 109/L, alanine aminotransferase/aspartate aminotransferase > 2.5 to <5 × the upper limit of normal, grade 2 neurotoxicity and nephrotoxicity, grade 3–4 mucositis and diarrhea, for the first time during chemotherapy, the administration was continued after reducing the dose by 20%. If the toxicity lasted for more than 2 weeks, permanent drug withdrawal was considered according to the judgment of the doctor.The patients were treated using two treatment schemes: The conventional treatment group was treated with standard three‐cycle TPF IC plus CCRT. In the new treatment group, once the post‐ICT EBV‐DNA load dropped to 0, these patient did not receive further IC and were treated with CCRT directly. The administration of IC was carried out as follows: docetaxel at 60 mg/m2.5Radical IMRT was delivered to all patients. The prescribed radiation doses comprised 66–70 Gy to the involved neck area and 70 Gy to the primary tumor. All bilateral cervical lymphatics and potential local infiltration sites were irradiated at 54 Gy or greater. All patients received 33 fractions over 6–7 weeks at five daily fractions per week.2.6Follow‐up of patients using imaging methods was carried out every 3 months in the first 2 years, every 6 months in years 3–5, and yearly thereafter. Follow‐up duration was calculated from first treatment day to the last visit or death. Disease‐free survival  was the primary endpoint. Overall survival , distant metastasis‐free survival , and locoregional relapse‐free survival  comprised the other endpoints. Routine plasma EBV‐DNA determination, radiography of the chest, ultrasound of the abdomen, neck, and nasopharynx contrast‐enhanced MRI, and nasopharyngoscopy were carried out. PET/CT was performed only if considered necessary. Adverse events were graded using The Common Terminology Criteria for Adverse Events (version 5).The late toxicity of salivary glands, subcutaneous tissue, and skin were assessed using the Radiation Therapy Oncology Group and European Organization for Research and Treatment of Cancer Late Radiation Morbidity Scoring Criteria.2.7t‐test was carried out to compare continuous variables.R software version 4.0.233.1Between July 2011 and June 2019, 256 patients with stage III–IV NPC were recruited and gave consent. The conventional treatment group comprised 150 patients and the new treatment group comprised 106 patients. Following PSM analysis, we selected 192 patients (96 pairs). The patients' clinical characteristics were well balanced between the routine and new treatment groups  of cisplatin (N = 245 (95.7%); 11 (4.3%) received CCD < 160 mg/m2). The baseline characteristics of the patients and the treatment details pre‐ and post‐PSM analysis are shown in Table Cisplatin was used in combination with radiotherapy. Most of the patients in the cohort were treated with ≥160 mg/m3.2For the whole and matched cohorts, the median durations of follow‐up were 54 months (range: 4–89 months) and 49 months (range: 6–89 months). In the whole cohort, we recorded 16 (6.3%) locoregional recurrences, 31 (12.1%) distant metastases, 5 (2.0%) synchronous locoregional and metastatic recurrences, and 52 (20.3%) disease progress. In the convention therapy group and new treatment group, 8 (5.3%), 20 (13.3%), 4 (2.7%), and 32 (21.3%) versus 8 (7.5%), 11 (10.4%), 1 (1.0%), and 20 (18.9%) patients, respectively, experienced locoregional recurrence, distant metastasis, synchronous locoregional and metastatic recurrences, and disease progress. In the matched cohort, the proportions of locoregional recurrence, distant metastasis, synchronous locoregional and metastatic recurrence, and disease progress were 5 (5.2%), 9 (9.4%), 3 (3.1%), and 17 (17.7%) in the convention therapy group. In the new treatment group, 8 (8.3%), 10 (10.4%), 1 (1.0%), and 19 (19.8%) patients, respectively, developed locoregional recurrence, distant metastasis, synchronous locoregional and metastatic recurrences, and disease progress.p = 0.532), , , and , respectively. For the matched cohorts, the 3‐year LRFS, DMFS, OS, and DFS rates for the routine and new treatment cohorts were , , , and , respectively. The survival curves in Figures For the whole cohort, the 3‐year LRFS, DMFS, OS, and DFS rates for the routine and new treatment cohorts were , the proportion of neutrophil lymphocytes, C‐reactive protein, and total treatment time, the intensity of chemotherapy, tumor regression, and adverse events. In the univariate analysis, BMI index (≥18.5 < 23.9 and ≥23.9 < 27), hemoglobin states (≥110 < 150 and >150 g/L), LDH > 245 (μ/L), pretreatment cfEBV‐DNA ≥4000, and cervical lymph nodes obtained partial remission after IC were associated significantly with failure‐free survival (FFS). Adjustment for various prognostic factors was achieved by Cox multivariate analysis of FFS including the five significant factors from the univariate analysis. The above indicators were ultimately identified as independent prognostic makers for FFS. A poorer FFS was associated significantly with a low BMI index, elevated LDH, Pre‐DNA ≥ 4000, and poor regression of cervical lymph nodes after IC chemotherapy , AJCC status, the intensity of IC and concurrent CT , total treatment time, and the GAP between the beginning of IC and CCRT, routine blood and biochemical tests, the detailed tumor invasion site of each patient for 134 anatomical structures, and treatment failure status. We observed that TPF cycles correlated positively with N stage, pretreatment cfEBV DNA load, skull base, parapharyngeal invasion, and cervical region 2 lymph metastasis . However, there was no correlation with post‐IC nasopharynx and neck regression response . Pretreatment cfEBV DNA load correlated positively with N stage and AJCC stage .The variables studied included T and N categories, pretreatment cfEBV‐DNA load . Meanwhile, the cycles of IC were reduced by 28% and the overall treatment time was shortened to a certain degree.Accumulating evidence supports the use of IC before CCRT in patients with LA‐NPC; therefore, clinicians are paying more attention to the differentiation strength of IC.EBV DNA is verified biomarker for nasopharyngeal carcinoma, demonstrating clinical utility for disease surveillance, risk stratification, and population screening.Analysis of the trend of longitudinal cfEBV DNA over the IC and CCRT treatment phases allowed Lv et al. to defined four cfEBV DNA response phenotypes according to their sensitivity to treatment .p < 0.010). The proportions of patients who received one cycle, two cycles, and three cycles of IC in the new treatment cohorts of our study were 27.%, 44.3%, and 22.6%, respectively. The results of the two research schemes are not consistent, because Lv′s study included a variety of schemes of IC, such as triplet TPF IC, docetaxel–cisplatin (TP), cisplatin–fluorouracil (PF), and gemcitabine–cisplatin (GP). Our study further confirmed that for patients showing complete regression of cfEBV DNA after IC, de‐intensification of IC might be advisable considering this subgroup's superior survival and the reduced toxicity. Beyond our clinical studies, to investigate the potential of post‐treatment EBV DNA as a biomarker to guide the further use of adjuvant chemotherapy in patients with LA‐NPC, the NRG Oncology cooperative group of the National Clinical Trial Network (NCTN) has initiated the NRG‐HN001 clinical trial (NCT02135042).In Lv′s research, a biological CR (cBR) was achieved by 63.2% of patients in the IC phase; by 36.4% of patients after one cycle of IC; by 20.7% of patients after the second cycle of IC, and by 6.1% of patients post‐IC3–4. The clusters demonstrated significantly different clinical endpoints between the cBR post‐IC1 group and the cBR post‐IC2–4 group (3‐year DFS = 94.3% vs. 78.0% p = 0.029).Peng et al. found that patients with NPC with the N2–3 category who received two cycles of IC had better OS than those received 3–4 cycles (92.4% vs. 80.8%, In a 2016 studySome limitations ought to be highlighted. In the present study, we attempted to avoid treatment heterogeneity resulting from physician bias: The cohort comprised patients treated at a single institution by a team under a single attending doctor. Next, during CCRT, there were inconsistencies in the timing and frequency of the assessment of tumor markers. Finally, although EBV DNA PCR‐based assays are known to be vulnerable to marked inter‐laboratory variations, our cohort were treated at a single institution and thus the EBV DNA measurements may not have been affected by inter‐laboratory variation.In summary, we investigated the effect of de‐intensifying chemotherapy in favorable LA‐NPC. We identified that more courses of IC in complete responder patients might not be effective to improve prognosis. Combining TNM stage and EBV‐DNA load after IC to determine the courses of IC in patients with LA‐NPC did not alter the curative effect, but decreased neutropenia toxicity during CCRT. We will identify these treatment‐resistant subgroups to investigate the role of chemotherapy intensification in future research.Study concepts: Qun Zhang, Zhen‐Wei Peng, Zhuo‐Sheng Gu, Wei Luo, and Yong‐Yu Mei. Study design: Wei Luo and Yong‐Yu Mei. Acquisition of data: Qun Zhang and Zhuo‐Sheng Gu. Data analysis and interpretation: Zhen‐Wei Peng, Yan Wang, Fang‐He, and Wen‐Bing Zhao. Drafting and revising the article: Qun Zhang, Zhen‐Wei Peng, and Zhuo‐Sheng Gu. Reviewing the article: Wei Luo and Yong‐Yu Mei. All authors gave final approval of the version to be published. All authors have agreed on the journal to which the article has been submitted and agree to be accountable for all aspects of the work.This work was supported by a grant from the Medical Science and Technology Research Projects of Health Commission of Guangdong Province [grant number A2020606].No conflicts declared.This study was approved by the Research Ethics Committee of Sun Yat‐sen University Cancer Center. Written informed consent was obtained from each subject before recruitment.All authors have read and approved the final version to be published and signed the author disclosure form.


The present study aimed to examine attentional biases' components and processes toward the interpersonal evaluation information among athletes after state thwarting need for relatedness. 51 athletes completed a visual dot-probe task while their eye-movements were tracking. Results indicated athletes showed different attentional bias pattern. Acceptance information is early orientation ; early acceleration detection; sustained to late attention maintenance (difficulty in disengaging). Rejection information is early orientation ; early accelerated detection; continuous attention to maintenance (attention avoidance); late attention to maintenance (difficulty in disengaging). That is to say, they had motivation to seek acceptance toward the accepted interpersonal evaluation information and to avoid rejection information toward the rejected one. Therefore, it is suggested that the coaches provide more interpersonal communicating opportunities, so as to help them to restore their demands toward interpersonal communication, and provide the customized attentional bias trainings to improve their coping response after state thwarted need for relatedness. When it comes to the scene that Zhu Ting and Yuan Xinyue were hand in hand casually while entering the court, they admired the teamwork in this volleyball team as if they are beloved family. It is apparent that vital role dose interpersonal relationship play among athletes. The relationship between athletes and coaches is significant as well. After witnessing conflict between Sun Yang and his coach Zhu Zhigen as well as the blood shed of Wang Meng, we could conclude that how to deal with terrible relationship deserves more reflection. The need for relatedness is a concept in self-determination theory, which means an individual is eager to gain understanding, care, and support from interacting with others and experience a sense of belonging [After Chinese Women's Volleyball Team won the World Cup, Japanese media made compliment in elonging . Early selonging . When laelonging . The conelonging , increaselonging .According to self-determination theory, when individual's relatedness needs are thwarted, their internal psychological needs balance will be broken up consequently. Aimed at recovering the initial balance, individual will generate restoration motive and try to reconnect relatedness need. This process is about relatedness need restoration , 6. The Existing researches discovered that individuals with state relatedness need thwarting would take priority to process interpersonal clues and possessed attentional biases toward interpersonal evaluation information , 12, 13.Relatedness need thwarting was not discriminated between trait and state in previous studies , 12, 13.What is more, previous paradigms  on studying attentional biases are all spatial. These paradigms predict cognitive processing before the appearance of probe point on the basis of behavioral reaction that occurred after appearance of probe point . There aBased on previous study, the goals of present research are twofold. First, we would like to distinguish state from trait of relatedness need thwarting. Second, we wished to investigate attentional bias components and time course toward interpersonal evaluation information of athletes with state relatedness need thwarting through combining the eye-movement tracking technique with dot-probe paradigm. Faced with admitted interpersonal evaluation information, we hypothesize that attentional pattern of athletes with state relatedness thwarting is early orientation ; early acceleration detection; sustained to late attention maintenance (difficulty in disengaging). While facing rejected one, those participants show early orientation ; early accelerated detection; sustained to late attention maintenance (attention avoidance).M = 21.94, SD = 3.20) who are in service with ages ranging from 14 to 29 years old. There are 43 male athletes and 8 female athletes with exercise experience  ranging from 1 to 11 years. These athletes' titles are national first-class or above (n = 10), second-class (n = 20), and third-class (n = 21). Their training sports include wrestling, swimming, and basketball. The study was approved by the university's research ethics committee and volunteers provided written informed consent prior to participation.Participants are made up of 51 athletes  ×2 . Relationship clues within subjects were tested. Dependent variables are consistency effect, RT bias score, difficulty in attention disengagement index, direction bias score, first fixation latency bias score, first fixation duration bias score, and overall gaze duration bias score.Cyberball is a virtual ball-tossing computer game designed to create the experience of state thwarting need for relatedness via participant interactions with two on-screen avatars. The standard version of the task has two separate blocks, an initial inclusion block and a subsequent exclusion one. At the start of the task participants were told that they had been assigned to the Blue Team and would be playing a ball-tossing game with members of a different colored team . This manipulation was implemented to reduce in-group versus out-group uncertainty present in the use of neutral avatars and to potentially increase the real-world validity of the situation, as sudden total exclusion by an out-group is more likely than by ones in group. At the start of the inclusion block, one of the avatars passes the ball to the player who then makes a selection, via a button box, to pass that ball to either one of the two avatars. In the initial inclusion block, upon receiving the ball the selected avatar would then either (1) pass the ball directly back to the player (50% chance), (2) pass the ball to the second avatar who would then pass the ball directly back to the player (40% chance), or (3) pass the ball to the second avatar who would then pass the ball back to the first avatar who would then pass the ball directly back to the player (10% chance). This sequence of events was repeated for a total of 40 passes, 26 of which were target trials which involved the player monitoring another avatar. After these 40 exchanges had accrued, the exclusion block began after a 10 s rest break. In this block, the first 8 passes proceeded in the same manner as in the preceding inclusion block after which the exclusion trials began. After this point the avatars ceased passing the ball to the player and proceeded to pass the ball back and forth between themselves, never passing the ball to the player, until a total of 24 passes had been made. At this point the participant was told that the task had been completed.α = 0.88.The questionnaire of state relatedness need thwarting for operating and checking was adapted , to moniα = 0.75.This research adopted PANAS scale , measuriα = 0.85.This paper adopted the Basic Psychological Needs Scale (BPNS) compiled by Richer, which is translated and revised by domestic scholar Kong . The preThis research was conducted by means of TobiiT60XL tracker. The participants need to be seated approximately 65 cm in front of a 24-inch, 60-Hz monitor with resolution of 1920 ∗ 1600 pixel in a low sun-shining environment.A) when the dots were located on the left side of the screen and another key (L) when the dots were on the right side as quickly as possible. Each probe appeared until a response was made, followed by a blank screen for 200 ms. In total, 108 trials were performed in this experiment  = 5.21, p = 0.000, cohen's d = 1.459). Besides, the Cyberball task could not cause variance between positive emotions  = 1.53, p = 0.13, cohen's d = 0.428) and negative emotions  = -1.57, p = 0.12, cohend's = −0.440).The independent variable is group; scores of operable examination questionnaire and positive as well as negative emotions are dependent variable. The independent sample F  = 0.549, p = .459, ηp2 = .001, that is to say, no inhibition of return or consensus facilitating effect. The results demonstrated a significant main effect for groups, F  = 16.702, p = .040, ηp2 = .021, and other main effects as well as interaction effects were not significant. In the dimension of RT bias score, main effect of facial types was significant, F  = 1.134, p = .289, ηp2 = .011. Besides, the post hoc test revealed that the bias scores of angry faces were greater than those of happy faces. The analysis of the difficulty in attention disengagement index showed a significant main effect in groups, F  = 9.558, p = .003, ηp2 = .089. In addition, the post hoc test demonstrated that the difficulty in attention disengagement index of groups of state relatedness need thwarting was more significant than control groups  = 2.079, p = 0.050, cohend's = 5.444 (scores are above 50). In first fixation latency score, t（1, 20） = -5.941, p < 0.001, cohend's = −1.267 (scores are less than 0). In first fixation duration bias score, t  = 2.921, p = 0.008, cohend's = 0.623 (scores are above 0). In overall gaze duration bias score, t  = 18.651, p < 0.001, cohend's = 3.976 (scores are above 50).The one-sample t  = 3.512, p = 0.002 cohend's = 5.817(scores are above 50). In first fixation latency score, t  = −7.082, p < 0.001, cohend's = −1.510 (scores are less than 0). In first fixation duration bias score, t  = −3.356, p = 0.003, cohend's = −0.716 (scores are less than 0). In overall gaze duration bias score, t  = 20.088, p < 0.001, cohend's = 4.283 (scores are above 50).The attention biases of participants with state relatedness need thwarting toward angry faces were attentional facilitation; continuous attention to maintenance (attention avoidance); difficulty in attention disengagement. In first fixation direction bias score, We used 2  ×2  repeated measures covariance analysis of the four EM indices with the trait thwarting need for relatedness as covariate. Slope homogeneity test resulted in that interaction effects among all variables and the trait thwarting need for relatedness were not significant. Therefore, the analysis of covariance could be conducted.F  = 9.497, p = 0.003, ηp2 = 0.102. The post hoc test showed that the first fixation direction bias scores of group of state relatedness need thwarting were greater than those of control group. The results in first fixation latency score revealed a significant main effect for group, F  = 19.339, p = 0.000, ηp2 = .195. The post hoc test showed that first fixation latency scores of group of state relatedness need thwarting were more significant than those of control group. The main effect for facial type was significant, F  = 6.307, p = 0.014, ηp2 = 0.073, presenting that absolute value of first fixation latency scores of angry faces was more significant than those of happy ones. In first fixation duration bias score, main effect for facial type was significant, F  = 10.236, p = 0.002, ηp2 = 0.113, manifesting that absolute value of first fixation duration bias scores of angry faces was more significant than those of happy ones. In addition, interaction effect was significant, F  = 12.106, p = 0.001, ηp2 = 0.131. Simple effect analysis found that first fixation duration bias score of state relatedness need thwarting group toward angry faces was more significant than control group, p = 0.002; that is to say, the initial attentional time of state relatedness need thwarting group toward angry faces was greater than control group. Besides, first fixation duration bias scores of happy faces in group of state relatedness need thwarting were more significant than those of angry faces, p = 0.000. The results in overall gaze duration bias score demonstrated a significant main effect for group, F = 28.264, p = 0.000, ηp2 = 0.261. The post hoc test showed that overall gaze duration bias scores of group of state relatedness need thwarting were more significant than those of control group  Attention pattern of happy faces turned out to be early orientation ; early acceleration detection; sustained to late attention maintenance (difficulty in disengaging), (ii) whereas attention pattern of angry faces was early orientation ; early accelerated detection; continuous attention to maintenance (attention avoidance); late attention to maintenance (difficulty in disengaging). The attention pattern of accepted interpersonal information is consistent with original hypothesis; when it comes to the pattern of rejected one, the hypothesis is confirmed partly. This result is in line with Chen et al.'s research ; that isAthletes with need thwarting would adopt various strategies in attentional biases processing procedure. Firstly, after experiencing state relatedness need thwarting, participants spent more time on locations of emotion-related stimuli. Secondly, when spatial vision disappeared without picture stimuli from emotional faces, thes athletes were more prone to return to where stimuli occurred, thus resulting in more RT . TraditiOverall, this study balanced trait relatedness need thwarting; athletes paid more attention on accepted information rather than rejection information. In overall gaze duration bias, scores of state relatedness need thwarting group toward interpersonal evaluation information were more significant than control group. And when faced with happy and angry faces, the overall gaze duration bias scores were all greater than 50. However, there was a significant interaction effect in first fixation duration bias score. Facing happy faces, first fixation duration bias score was greater than 0, whereas facing angry ones, score was less than 0. All in all, participants with state relatedness need thwarting had attentional maintenance no matter facing accepted information or rejection one. But, difficulty in attention disengagement existed in the process of accepting accepted information all the way. There were both difficulty in attention disengagement and attentional avoidance in rejecting rejection information.Based on the goal conflict model , when stIn addition, on the basis of dual-system model, participants' processing of rejection information was influenced by reflective and impulsive systems , 41. ImpDifferent interpersonal evaluation information has various impact on attentional biases and influences how athletes deal with state relatedness need thwarting. Therefore, coaches could construct platforms for athletes to recover state relatedness need, provide more opportunities for team members to communicate, and set up common objectives so as to strengthen mutual trust and raise team recognition. Coaches could also draw up targeted plan to train attentional biases in order to enhance athletes' capability to cope state relatedness need thwarting.When athletes' state relatedness need thwarted, they are more prone to generate more attentional biases toward accepted information compared to rejection information. They would generate motivation to accept acceptance of accepted information and generate motivation to reject rejection information.