A P value of .05 or less was considered to be statistically significant (two-tailed). Thirty-seven patients were enrolled in our study. Seventeen patients were divided into the case group treated by 3D printing model-assisted preoperative planning, and 20 patients were divided into the control group treated by conventional surgery. Patients treated with the 3D printing model had significantly shorter operation times, less blood loss, and shorter fluoroscopy times. Patients in the case group also showed better pain relief according to visual analog scale scores. However, the elevations in pelvis and hip joint functional outcomes were similar between the 2 groups, and no significant difference was shown in the radiological result. The usage of 3D printing techniques in patients with inveterate pelvic and acetabular fractures is of great importance in preoperative preparation and optimization of surgery but cannot improve postoperative function compared with conventional treatment.Treatment of deformed pelvic and acetabular fractures is a considerable challenge for orthopedic surgeons. The aim of this study was to assess the availability of a three-dimensional (3D) printing model used in patients with inveterate pelvic and acetabular fractures by comparing 3D printing technology with conventional surgery. We conducted a retrospective review of patients with inveterate pelvic and acetabular fractures treated in our department between January 2008 and June 2020. The patients were divided into 2 groups according to their willingness. Perioperative data and clinical outcomes were compared to evaluate clinical efficacy. The  These injuries are usually caused by high-kinetic energy forces, and the common mechanisms include motor vehicle accidents and falling from a height. The patients usually arrive at the emergency department with unstable hemodynamics and multiple organ injuries. Inveterate pelvic and acetabular fractures usually refer to fractures with a course of more than 3 weeks and are usually due to delayed surgery or misdiagnosis on account of the urgency of maintaining stable vital signs and limited insight provided by X-plains. The porosis and sophisticated anatomy of the pelvis and acetabulum make it difficult to achieve anatomical reduction. Common plain X-ray and computed tomography (CT) images provide limited insight into the physical configuration of fracture components, especially for complex acetabular fractures. Accurate osteotomy and deformity correction are significant challenges, even for experienced orthopedic surgeons. The treatment of inveterate pelvic and acetabular fractures usually involves extended operation time and increased blood loss compared with the treatment of normal fractures and leads to unsatisfactory results.Pelvic fractures account for 1% to 3% of all types of fractures In this study, we used 3D printing to help improve the accuracy of osteotomy and prepare pre-contoured fixation plates according to fracture segments. Three-dimensional printing techniques have been presented in complex orthopedic surgery including that of the spine, pelvis, and joints. Previous studies have demonstrated that it is an effective and reliable method for treating anterior pelvic ring and acetabular fractures,5,7–9 and have shown an advantage for elective surgery in cases of severe acetabular bone loss. However, the use of 3D printing techniques in the preoperative planning of inveterate pelvic and acetabular fractures, to our knowledge, has not been reported previously. We hypothesized that a 3D printing technique would optimize the surgery process and improve the clinical outcomes. The aim of this study was to assess the availability of 3D printing used in patients with inveterate pelvic and acetabular fractures compared with conventional radiographic planning.Advances in the three-dimensional (3D) printing technique provide surgeons with detailed models showing the precise anatomical morphology of fracture configurations.We analyzed the data of all patients with inveterate pelvic and acetabular fractures treated in our department between January 2008 and June 2020. The generic surgical indications were the presence of clinical signs including neurological pain, limb discrepancy, and vertical or rotational displacement of the pelvic ring observed on radiography. The inclusion criteria were (1) injury time > 3 weeks, (2) undergoing open reduction and internal fixation, (3) undergoing only one surgery, and (4) 18 years < age < 60 years. The exclusion criteria were (1) injury time < 3 weeks, (2) undergoing conservative treatment, (3) fractures of tumorous or osteoporotic bone. The patients were divided into 2 groups according to their willingness. Patients in the case group underwent 3D printing model-assisted preoperative planning. Patients in the control group underwent conventional surgery assisted only by plain X-rays and CT radiographs. Permission for this study was obtained from the Medical Ethics Committee at the authors’ institution, and the patients and/or their families were informed that data from the research would be submitted for publication, and gave their consent.All surgeries were performed under general anesthesia by one surgeon.In the case group, the patients were required to undergo 1.0-mm CT  of the thin pelvic layer. The data were converted to DICOM images and uploaded to the medical image processing software . A 3D image of the isolated malformed pelvis was created by subtracting the bilateral femurs using the splitting process Fig. D. The osDuring the surgery, the ilioinguinal and/or Kocher–Langenbeck approaches were used based on osteotomy position. Osteotomy was performed based on the surgery guide. The transplanted bone block was from the iliac ala and molded in accordance with the quantified module. The bone grafts were fixed with 3.5-mm pre-contoured reconstruction plate . The residual displacement of the pelvis was graded using the method of Tornetta and Matta. The reduction in the acetabulum was assessed using the Matta system. Excellent and good results were considered satisfactory results. Radiographs were assessed by 2 experienced musculoskeletal radiologists who did not participate in the surgeries. Both observers achieved exact agreement in evaluating all images (weighted kappa was 1 for the pelvis and acetabulum).Pelvic fracture types were categorized according to the Tile classification,t-test. Fisher exact test was used for nominal data. Multiple logistic regression analysis was performed in order to identify variables that were independently associated with pelvic outcomes. A P value of .05 or less was considered to be statistically significant (two-tailed).Statistical analysis was conducted using SPSS version 16.0 for Windows . The results are presented as mean ± standard deviation. Continuous variables that were normally distributed between the groups were compared using the Fifty-five patients with inveterate pelvic and acetabular fractures were identified, and 18 patients were excluded. Thirty-seven patients were enrolled in this study. Seventeen patients were divided into the case group, and twenty patients were divided into the control group. Demographic information of these patients is listed in Table The fracture types are listed in Table P > .05, Table Clinical data including age, sex, mechanism of injury, time from injury to operation, and fracture type were not significantly different between the 2 groups (P < .001). The patients in the case group also experienced less blood loss with a volume of 1222.1 ± 374.9 mL  versus 1752.3 ± 521.5 mL  in the control group (P = .004). Furthermore, there was a greater need for blood transfusion in the control group with 10.9 ± 3.4 units of packed red blood cells , compared with 7.4 ± 2.3 units in the case group  (P = .003). In the case group, the mean fluoroscopy time was 15.3 ± 5.1 s , but in the control group, the time was significantly higher with an average of 22.2 ± 7.0 s  (P < .002).Peri-operative data are shown in Table P > .05, Table P > .05, Table P = .009, Table The average follow-up times in the case and control groups were 19.4 ± 4.6 months  and 21.4 ± 5.3 months , respectively. There was no significant difference in preoperative functional parameters including Majeed, Merle D’Aubigné and Postel, and VAS scores (P = .33). The rate of satisfactory results of acetabular reduction was also higher in the case group compared with the control group (76.92% versus 57.14%). However, no significant difference was shown in either (P = .276).The radiological outcomes are shown in Table Sciatic nerve injury occurred in 2 patients in the control group. One patient in each group had venous thrombosis in the lower limb. One patient developed femoral head necrosis which was in the control group, and one patient suffered hip osteoarthritis after surgery which was in the case group. However, all of them refused surgical treatment during the follow-up period. Nonunion or mechanical complications such as instrumentation failure did not occur. The segmented bones or fragments presented in a 3D model can be manipulated in a virtual simulation and therefore help better determine the reduction procedures. Three-dimensional printing techniques have been widely used in pelvic and acetabular surgeries, and surgeons have concluded that they can optimize surgeries, improve outcomes, and reduce complications.–10,18–23The development of 3D printing techniques provides an accurate 3D model of fracture and facilitates surgical simulation. A previous study suggested that 3D printing of a fracture model was of positive significance, as it could improve the preoperative plan and reduce difficulties for clinicians by shortening the learning curve. Three-dimensional models help improve the strategies of osteotomy and bone grafting and make it possible to prepare a pre-contoured plate and predefined number and length of screws by surgical simulation and reduce the surgical time. In this study, the average operation time in the case group was significantly less than that in the control group. The surgical simulation using 3D models may offer the surgeon a better understanding of the morphology preoperatively to then perform fracture reduction more easily. The decreased instrumentation time in the 3D printing group than in the traditional method group because of the utilization of pre-contoured plates is also a critical factor.,26 Another advantage of 3D printing shown in our results was that the patients in the case group showed significantly reduced intraoperative blood loss and need of transfusion. The surgery is performed with smaller incisions and less soft tissue injury with the help of 3D printing models.,27 The decreased operation time is in favor of reduced blood loss. Reduced operation time, blood loss, and fluoroscopy time have been reported in different studies concerning 3D printing models in pelvic, acetabular, joint, and upper limb fractures.,23,28–30 Our results are consistent with previous findings, and we believe that the application of 3D models is in favor of a smoother execution of operations, making intraoperative reduction easier. The patients benefitted from surgical simulation with reduced blood loss, soft tissue injury, and radiation.Surgeries for inveterate pelvic and acetabular fractures usually take long durations of an average of more than 6 hours and are associated with excessive bleeding.,8,20 However, others declare that although a higher quality of reduction is observed in cases involving preoperative 3D printing plans compared with traditional surgery, the difference is insignificant.,26,29 In our cases, according to the Matta radiological accession system, patients in the case group showed a better rate of satisfactory results for the pelvis (70.6%) and acetabulum (76.9%) compared with the control group (55% for the pelvis and 57.1% for the acetabulum), but there was no statistical significance between the 2 groups. The small sample size probably leads to the statistically insignificant result.The patterns in inveterate pelvic and acetabular fractures differed significantly in different patients. The complexity of fractures and malunion with callus formation made it difficult to achieve anatomical reduction. Although a more intuitive understanding of the fracture morphology can be provided by 3D printing models, whether an improved quality of reduction can be achieved remains unclear. Some reports have suggested that improved reduction could be acquired for patients with pelvic and acetabular fractures with the help of 3D models. Indications for surgery include pelvic instability and clinical problems related to pelvic deformity. Most patients with inveterate pelvic fractures are in stable condition and visit the hospital because of unbearable symptoms. Common symptoms include pain, malformation, and abnormal gait. Function improvement is the main reason for secondary treatment. In our patients, both groups showed obvious improvement of pelvic and hip function after the surgery. However, no significant difference was shown between the 2 groups (P > .05). This insignificant function improvement has also been demonstrated in other studies.,23,28,29 The compression of adjacent nerves caused by ectopic pelvis may also give rise to dysfunction of the bowel and urogenital system along with pelvic pain. Lindahl et al reported 39% of patients with pelvic malunion that presented with obvious pain in the pelvic region. Among our patients, the postoperative VAS pain score was significantly lower in the case group (P = .009). Given that there was no evident difference in function outcomes, we believe the better pain alleviation in the case group was in part due to the advantage of 3D printing model in the preoperative plan, which decreased surgical invasiveness and improved the accuracy of reduction. Our results demonstrated that patients who undergo 3D printing model-assisted preoperative planning show no significant functional improvement, but acquire better pain relief.Inveterate pelvic and acetabular fractures usually have a course of more than 3 weeks. In this study, venous thrombosis in the lower limb occurred in one patient in each group and was treated with anticoagulation therapy. Two patient experienced sciatic nerve injury in control group and was treated with neural nutrients. Complete recovery of this patient was observed at the 6-month follow-up after the surgery. No surgery-related neurovascular complications occurred in the case group. Previous studies have shown a reduced rate of postoperative complications following 3D model-aided surgery in patients with pelvic and acetabular fractures.,9,27 Since surgical simulation make surgeries less invasive and significantly reduce the operation time, we believe that the usage of 3D printing techniques may reduce the rate of complications, but more cases are needed in future studies.The decision to proceed with surgery should be made only after careful assessment in consideration of the reconstruction of pelvic malunion being associated with a high incidence of complications.The application of 3D printing technology in orthopedics is widely reported. However, to our knowledge, this is the first study that focuses on its use in the treatment of inveterate pelvic and acetabular fractures. There are still several limitations in this study. This was a retrospective study, and the use of 3D printing mainly depended on patients’ willingness. The duration from injury to surgery differed greatly in patients from 37 to 279 days. The number of patients included in this study was relatively small, and a larger sample size is needed for a more exact conclusion of the impact of 3D printing techniques on clinical outcomes in a future study.Compared with conventional radiographic planning, the application of 3D printing techniques can reduce the operative time and blood loss and lower the requirement of fluoroscopy. Patients who undergo 3D model-aided surgery gain better pain relief, but do not show significantly better function outcomes or higher quality of reduction. Our results demonstrated that for patients with inveterate pelvic and acetabular fractures, the usage of 3D printing techniques is of great importance in preoperative preparation and optimization of surgery.Conceptualization: Fulin Tao, Lin Li, Dawei Wang, Jinlei Dong, Dongsheng Zhou, Wenhao Song.Data curation: Fulin Tao, Lin Li, Dawei Wang, Jinlei Dong, Dongsheng Zhou, Wenhao Song.Formal analysis: Fulin Tao, Lin Li, Dawei Wang, Jinlei Dong, Dongsheng Zhou, Wenhao Song.Funding acquisition: Dawei Wang, Dongsheng Zhou, Wenhao Song.Investigation: Fulin Tao, Lin Li, Dawei Wang, Jinlei Dong, Dongsheng Zhou, Wenhao Song.Methodology: Fulin Tao, Lin Li, Jinlei Dong, Dongsheng Zhou, Wenhao Song.Project administration: Fulin Tao, Lin Li, Dawei Wang, Jinlei Dong, Wenhao Song.Resources: Lin Li, Dawei Wang, Jinlei Dong, Dongsheng Zhou, Wenhao Song.Software: Lin Li, Wenhao Song.Supervision: Fulin Tao, Lin Li, Dawei Wang, Wenhao Song.Validation: Fulin Tao, Lin Li, Jinlei Dong, Dongsheng Zhou, Wenhao Song.Visualization: Fulin Tao, Lin Li, Dawei Wang, Jinlei Dong, Dongsheng Zhou, Wenhao Song.Writing – original draft: Lin Li, Wenhao Song.Writing – review & editing: Fulin Tao, Lin Li, Dawei Wang, Jinlei Dong, Dongsheng Zhou, Wenhao Song.


TEK or PIK3CA. Based on these findings, targeted treatment using the PI3K inhibitor alpelisib was started with regular check-ups, therapy duration varied from 6 to 31 months. In all patients, marked improvement in quality of life was observed. We observed radiological improvement in fourteen patients (two of them being on combination with either propranolol or sirolimus), stable disease in 2 patients. For 2 patients, an MRI scan was not available as they were shortly on treatment, however, a clinically visible response in size reduction or structure regression, together with pain relief was observed. In patients with elevated D-dimer levels before alpelisib administration, a major improvement was noted, suggesting its biomarker role. We observed overall very good tolerance of the treatment, documenting a single patient with grade 3 hyperglycemia. Patients with size reduction were offered local therapies wherever possible. Our report presents a promising approach for the treatment of VMs harboring different targetable TEK and PIK3CA gene mutations with a low toxicity profile and high efficacy.This was a prospective cohort study of eighteen patients with large and debilitating vascular malformations with one or more major systemic complications. In all patients, we discovered activating alterations in either  According to the ISSVA 2018 classification , they comprise two major categories, vascular tumors and vascular malformations, which can be further divided into several subgroups. Vascular malformations (VMs) can be categorized into simple, combined, vascular malformations of major named vessels and vascular malformations associated with other anomalies.Vascular anomalies (VA) are a large and diverse group of diseases characterized by abnormal growth and development of blood or lymphatic vessels. They are associated with diverse symptomatology and often disabling conditions and remain both diagnostic and therapeutic challenges for medical professionals all over the world3. They can present as localized or diffuse lesions, and the symptomatology depends on the localization, extension, and involved anatomical structures. The appearance and symptoms are not static and can often progress during growth spurts and puberty2. Because of this varied symptomatology, multidisciplinary cooperation is of vital importance. For extensive lesions with vital organ and body part involvement, surgical procedures are very complicated because of their size and/or location. In these patients, other therapeutic options should be considered.The overall incidence of congenital VMs in the general population is 1.5%, and approximately two-thirds of cases are of venous predominance6. This opens the possibility that drugs/inhibitors currently being used in cancer patients may be used to treat patients with VMs7.Mutations in genes that are involved in two significant intracellular signaling pathways, RAS/MAPK and PI3K/AKT, have been implicated in the pathophysiology of the majority of vascular malformationsPIK3CA-mutated, hormone receptor-negative advanced breast cancer, showing very promising results8. Several authors have already demonstrated its effect in patients with PIK3CA-related overgrowth syndromes or PIK3CA-altered lymphatic malformations, and as a result of the EPIK-P1 clinical study9, it has recently been approved by the FDA for adult and pediatric patients with severe manifestations of the PIK3CA-related overgrowth spectrum13. In addition to patients with activating PIK3CA mutations, alpelisib treatment might also be beneficial for patients with activating TEK alterations, as the PI3K/AKT pathway is considered to be a central part of signaling through the TIE2 receptor encoded by this gene14. This was recently demonstrated in work published by Remy et al., in which the authors described the efficacy and pharmacokinetics of alpelisib in 3 patients with VMs harboring TEK mutation15.In this prospective observation report, we focused on the effect of the PI3K inhibitor alpelisib, which was recently approved by the FDA and EMA for the treatment of TEK or PIK3CA were found. In 12/18 patients, TEK exon 17 mutation was found, with TEK p.L914F being the most prevalent (9/12). In one of these patients, we found a TEK mutation of germline origin (p.Y897C). One patient harbored a truncating mutation in exon 23 of the TEK gene. Five patients harbored PIK3CA hotspot mutations, all of which have already been observed in patients with VM. In one patient, in addition to the somatic TEK mutation found in VA biopsy, a germline PTPN11 mutation causing Noonan syndrome was detected. The identified variants and the testing methods for each patient are listed in Table In each patient, activating mutations in either The total duration of alpelisib treatment varied from 6 to 31 months as of May 2023.In all patients, marked improvement in the quality of life (QoL) was observed. In 12 patients, radiological improvement was documented; in 2 patients, the anomaly size remained stable; in 2 patients, radiological improvement was documented after combining alpelisib with either sirolimus (No. 6) or propranolol (No. 9). For 2 patients (Nos. 16 and 17), an MRI scan was not available due to short period of alpelisib administration. However, rapid clinical response with subjective improvement in QoL was documented. Effect of the treatment for every single patient is summarized in Table TEK (9 patients) and PIK3CA (5 patients) mutation, better radiological response was observed in PIK3CA mutation group. For TEK mutation group the average volume reduction was 21% and median value was 19%. For PIK3CA mutation group the average volume reduction was 53% and median value was 45%. Moreover, both patients with stable disease harbored TEK mutation.When comparing patients with In patients with elevated D-dimer levels before alpelisib administration, a major improvement was noted. The D-dimer levels continually decreased, as shown in Fig. Notable individual cases are further described in more detail below.16, we noted grade 3 toxicity of hyperglycemia requiring insulin treatment in one patient and grade 2 toxicity in 4 patients. Those specifically included mucositis in two patients, hyperglycemia requiring oral hypoglycemic medication and recurrent abdominal pain in one. The rest of the reported side effects were only grade 1, most commonly temporary abdominal discomfort, headaches, nausea, hyperglycemia, or liver enzyme elevation. Apart from the patient with hyperglycemia on insulin treatment, none of the patients required admission for treatment of adverse events, and all were managed in an outpatient setting.We observed overall very good tolerance of the treatment. According to the CTCAE v5.0One of the first patients No. 2) who was administered alpelisib treatment was an 18-year-old woman with severe and debilitating venous malformation of the right lower limb, pelvis, and genitalia. Prior to the start of alpelisib in November 2020, she underwent several surgical procedures over the years with only temporary effects. After 5 months of alpelisib treatment, we observed a rapid size reduction  and a somatic TEK gene. After starting alpelisib treatment, we observed rapid clinical effects in QoL and size reduction of the lesions, which was later confirmed with MRI imaging.The oldest patient was a 36-year-old woman who underwent a series of 25 surgeries and multiple nontargeted treatments for her VeM of a left lower limb starting in childhood before genetic testing discovered a pathogenic mutation of the Patient no. 12, with VeM of the left lower limb with muscular involvement, started the treatment due to potentially disabling surgery. Six months after the alpelisib administration, we achieved size reduction allowing safe total resection of the lesion without collateral damage to the healthy tissue. Six months after the procedure, the patient remains asymptomatic without any treatment See Fig. .Figure 417, PI3K inhibitors11, AKT inhibitors18, MEK inhibitors19, and VEGF inhibitors7; however, some of them show only limited efficacy, possibly because they are administered without prior knowledge of the driving gene mutation.The use of targeted therapeutics for the management of VMs represents a new off-label therapeutic strategy, which could be used as an addition to classic local treatment approaches following the paradigm of precision medicine. Drugs that are currently being used in this setting include mTOR inhibitorsPIK3CA and TEK genes were discovered in the lesions of 18 patients. Molecular analyses were performed using various sequencing methods ranging from Sanger sequencing to next-generation sequencing of targeted gene panels or WES. These different methods were selected based on current availability and the clinical picture suggesting a particular mutation in a given patient. The best diagnostic algorithm for the future, reflecting both economic and medical needs, remains to be established and is the subject of a future project.In this observational study, activating mutations in the 20.Obtaining a representative piece of the lesion for subsequent analyses can become the first major obstacle due to the high risk of severe bleeding. A multidisciplinary team of specialists is of vital importance in facing such challenges. In patients with a difficult surgical approach, liquid biopsy could potentially help to discover driving mutations and may serve as a potential diagnostic marker; however, further investigation is needed2 BSA). Our study used a 20% dose reduction (138.6 mg/m2/day) with high efficacy. Thus, for patients with insufficient response, the dose can be increased. In addition, for one of our patients who suffered an adverse event, restarting the drug with a 50% reduction was sufficient to maintain efficacy with a response measurable clinically and in D-dimer levels. In patient with hyperglycemia grade 3 toxicity we observed regrowth of the lesion after alpelisib cessation, restarting drug in 25% of the original dose led to suboptimal clinical and D-dimer response, however, at 37% of the original dose there was a very good both clinical and laboratory effect with only low dose of insulin therapy. Management of alpelisib induced diabetes mellitus was recently described in case report by Pia Peris et al.21.The starting dose of alpelisib was selected based on BSA-modified dosing schemes for the treatment of patients with breast cancer for whom a single dose of 300 mg is recommended 13. In our study, we also report the efficacy of alpelisib on VMs in patients without germline mutations but with somatic mutations, and moreover, this report describes alpelisib efficacy in the largest cohort of patients with somatic mutations in the TEK gene so far, thus following up on work of Remy et al.15.To evaluate the therapeutic effect, patients underwent regular check-ups consisting of current history taking, clinical investigation, and laboratory testing. With the more recent patients, we also performed MRI scans before and 6 months after the start of the treatment. A measurable response was observed in 14 out of 18 patients on MRI, and a visual clinical response was observed in all patients, proving that alpelisib is highly effective. In all patients, we observed a striking effect on clinical symptomatology within the first few months of the treatment. Similar efficacy was reported by other authors describing their experience in a cohort of 19 patients and two case reports of patients with TEK and PIK3CA mutations, both the average volume reduction and median value were higher in patients harboring PIK3CA mutation, however larger group would be needed for stronger results. The response in QoL parameters and coagulation markers were similar for both groups.When compared the radiological response in patients with In patients who originally suffered from chronic coagulopathy, alpelisib treatment resulted in a decrease and even normalization of D-dimer levels, which seems to be a very useful marker of treatment success. Furthermore, we observed pain relief with quality-of-life improvement as the most important therapeutic goal. We have not observed any signs of cumulative toxicity comparing the first and second 6 months of drug exposure thus far.After treatment discontinuation in two patients, we observed both clinical and paraclinical deterioration with a rapid rise of D-dimer levels, suggesting the need for prolonged treatment exposure. However, the optimal treatment duration and minimal effective dose remain unknown, as does the long-term toxicity profile in young patients. Different dosing and scheduling schemes have yet to be tested. Another open question is identifying the so-called “best achievable response” and reintroducing different local therapeutic options. Subjects who achieve size reduction after alpelisib treatment can be recommended for local treatments with a much lower risk of mutilation or complications such as bleeding or anatomical structure destruction. In our cohort, one patient (No. 12) experienced a 87% volume reduction of initially unresectable lesions on alpelisib, and the residual lesions were resected without bleeding or functional deficits of the thigh.This prospective observational study contributes informations about a new treatment option that is well tolerated and shows objective responses with highly appreciated quality of life improvement.TEK exon 17 by Sanger’s method. The analyses were performed on lesion biopsy specimens. DNA was extracted from FFPE tissue using QIAmp FFPE Tissue Kit  and treated with NEBNext FFPE DNA Repair Mix . For WES, both lesion and matched normal genomic DNA were used. Libraries were prepared using TruSeq DNA Exome  according to the manufacturer’s instructions and sequenced on the NextSeq500 platform using NextSeq 500/550 Mid Output Kit v2.5 (150 Cycles) (Illumina). Libraries for targeted NGS were prepared using QIAseq Targeted DNA Panel—Human Actionable Solid Tumor (Qiagen) according to the manufacturer’s instructions and sequenced on the NextSeq500 platform using NextSeq 500/550 Mid Output Kit v2.5 (300 Cycles) (Illumina). For direct sequencing of exon 17 of the TEK gene, custom-made PCR primers  were used to amplify the target sequence . Sequencing analysis of a purified and labeled PCR product was performed on the ABI 3130xl device . Patients with confirmed mutations in PIK3CA and TEK were recruited for alpelisib treatment and efficacy analysis.All patients or their legal guardians signed informed consent with molecular genetic testing and off-label treatment using alpelisib. A total of 18 patients with large, debilitating or opioid requiring VMs with one or more major systemic complications  underwent molecular genetic analysis using either whole-exome sequencing (WES), a targeted next-generation sequencing (NGS) panel, or direct sequencing of All patients included in our study were Caucasian. Prior to the start of alpelisib treatment, a thorough patient history, clinical examination, baseline imaging, and laboratory tests, including D-dimer levels, were obtained. Initial clinical symptoms with basic demographic data, class of VM according to ISSVA  and discovered mutation types are summarized in Table 2/day. As our center was not part of the EPIK-P1 study, the dosing of alpelisib in our patients differs from the dosing schedule in EPIK-P1, which led to the FDA approval of alpelisib for PIK3CA-related Overgrowth Spectrum patients in April 202222.Based on molecular findings and clinical symptoms, all 18 patients began orally administered alpelisib treatment. The dosage used for our patients was derived from the breast cancer guidelines and individually adjusted to body surface area (BSA) and varied from 50 to 300 mg per dose. The average starting dose was 138.6 mg/mRegular check-ups were performed to evaluate the efficacy of the treatment. Each patient was examined in an outpatient clinic with laboratory and clinical examinations every 4 weeks and imaging, mostly MRI scan, every 3 months ± 2 weeks.Quality of life (QoL) was assessed with a revised patient control outcome questionnaire which included an assessment of both pain and functional impairment resulting in a four-step semiquantitative scale: worse, stable, improved, and marked improvement.Adverse events were classified according to CTCAE v5.0.Discontinuation of the alpelisib treatment was allowed at the patient´s request due to adverse events and at the discretion of the treating physician.All methods were carried out in accordance with relevant guidelines and regulations.TEK or PIK3CA gene alterations when local treatment methods have only partial or temporary effect or when the lesion is inoperable because of its location. This prospective observational study contributes information about this new treatment option, which is well tolerated documenting only 1 grade 3 toxicity, objective responses, and highly appreciated quality of life improvements.Novel PI3K inhibitors such as alpelisib represent a promising therapeutic option for patients with confirmed Several questions still need to be answered, as this is an off-label, agnostic, and experimental treatment. Although we did not observe any side effect requiring permanent treatment discontinuation, long-term toxicity remains to be established. The optimal treatment duration, dosing, and scheduling are also a matter of further investigation, as well as the optimal position of this systemic treatment in the complex management of vascular malformations.


Identifying cultivars of leguminous crops exhibiting drought resistance has become crucial in addressing water scarcity issues. This investigative study aimed to select soybean and cowpea cultivars with enhanced potential to grow under water restriction during the vegetative stage. Two parallel trials were conducted using seven soybean  and cowpea cultivars  under four water levels  over 21 days. Growth, water content, membrane damage, photosynthetic pigments, organic compounds, and proline levels were analyzed. Drought stress significantly impacted the growth of both crops, particularly at 45 and 30% FC for soybean and 60 and 45% FC for cowpea plants. The BÔNUS8579IPRO and TMG1180RR soybean cultivars demonstrated the highest performance under drought, a response attributed to increased amino acids and proline contents, which likely help to mitigate membrane damage. For cowpea, the superior performance of the drought-stressed Xique-xique cultivar was associated with the maintenance of water content and elevated photosynthetic pigments, which contributed to the preservation of the photosynthetic efficiency and carbohydrate levels. Our findings clearly indicate promising leguminous cultivars that grow under water restriction, serving as viable alternatives for cultivating in water-limited environments. Water scarcity has emerged as a pressing global concern, and several regions have confronted a critical shortage of water due to the combined effects of population growth, rapid urbanization, and climate change. These problems are directly reflected in detrimental consequences for agriculture, industry, and people’s health. The scarcity of water resources presents not only challenges in fulfilling fundamental human requirements but also puts ecosystems and biodiversity at risk ,2.Glycine max L. Merrill) and cowpea  stand out as a crucial component for humans and ruminant animals. Leguminous crops are also associated with the fixation of biological nitrogen due to their abilities in mutualistic association, thus reducing costs of industrially manufactured chemical fertilizers in supplying nitrogen to plants.In face of populational growth in recent years, leguminous crops constitute an important solution to ensure food and nutritional security, with a key socioeconomic role ,3. AmongSoybean is among the most explored crops worldwide, and Brazil has become the world’s leading producer of grains, followed by the United States and China ,5. Never2 availability for chloroplasts, which impairs the net photosynthesis and carbohydrate biosynthesis . For soybean trials, the cultivars AS3810 IPRO, M8644 IPRO, TMG1180 RR, NS8338 IPRO, BMX81I81 IPRO, M8808 IPRO, and BÔNUS8579 IPRO were investigated; while the cultivars Aracê, Novaera, Pajeú, Pitiúba, Tumucumaque, TVU, and Xique-xique were studied in the cowpea trials. In all cases, four replications per treatment (one plant per pot) were employed, totaling 112 experimental units per experiment.Two independent experiments were conducted in a randomized complete design, in 7 × 4 factorial schemes composed of seven soybean  at the Federal University of Ceará (UFC), in Fortaleza, Ceará, Brazil. Detailed information about the soybean and cowpea cultivars are outlined in The soybean and cowpea cultivars were chosen based on their performance, production stability, and wide adaptability across the agricultural regions of the Brazilian Cerrado and semiarid areas. These regions are characterized by rainfed cultivation and subjected to periods of extended dry spells. The soybean seeds were acquired through Celeiro Sementes farm, a specialized company in soybean seed propagation. Cowpea seeds were obtained from the Active Germplasm Bank . Subsequently, the plant material was immediately frozen and lyophilized to measure the dry mass. The index of relative tolerance to drought (RToler) was calculated by comparing the total dry mass of drought-stressed plants to the total dry mass of control plants . SpecifiTo estimate the relative water content (RWC), leaf discs were initially weighed to obtain the fresh weight (FW). Subsequently, the samples were immersed in deionized water for a period of 6 h at room temperature to achieve full turgidity, and the weight was recorded as the turgid weight (TW). Afterward, the samples were subjected to oven drying at 65 °C for 24 h and weighed to determine the dry weight (DW). The RWC was estimated by using the formula RWC (%) = [(FW – DW)/(TW – DW)] × 100.Electrolyte leakage was employed as a measure of cell membrane damage, utilizing a conductivity meter. Leaf discs and root samples weighing 0.1 g were placed in sealed vials filled with deionized water, and then incubated at room temperature on a rotary shaker for 12 h. Thereafter, the electrical conductivity of the solution (EC1) was measured. Later, the homogenate was subjected to incubation at 100 °C for 15 min, and the conductivity was measured once more (EC2). The percentage of membrane damage (%) was calculated as EC1/C2 × 100.3 at room temperature in the dark for 72 h. The contents of chlorophyll a (Chl a), chlorophyll a (Chl b), chlorophyll total , and carotenoids were estimated through spectrophotometry readings at wavelengths of 480, 649, and 665 nm, following the equations defined by Wellburn method [The photosynthetic pigments were extracted by incubating leaf discs in dimethyl sulfoxide (DMSO) saturated with CaCOn method .g for 10 min at 4 °C, and the resulting supernatant was collected. This extraction process was repeated twice on the remaining precipitate. Subsequently, all the collected supernatants were combined and adjusted to a final volume of 25 mL with 80% ethanol. The content of soluble carbohydrates was estimated by readings at 490 nm, using anhydrous D-glucose as a standard [Soluble carbohydrates and free amino acids were extracted after homogenizing 10 mg of leaf lyophilized samples in 5 mL of 80% aqueous ethanol at 75 °C for 1.0 h. Then, the homogenate was centrifuged at 3000× standard . Yet, thstandard . g for 10 min at room temperature. The supernatant was collected and used to measure the proline content by absorbance at 520 nm, with L-proline serving as the standard.The proline content was assessed using the ninhydrin method as outlined by the Bates method . Aqueousp ≤ 0.05) with the Sisvar software [For each experiment, the data underwent analysis of variance (ANOVA) using the F test at a significance level of 5%. Post hoc comparisons of means were conducted using Scott–Knott’s test (software . The clusoftware .Our findings reveal insights into innovative agricultural practices, underscoring the remarkable efficiency of soybean in managing water requirements, not only in terms of water-saving potential in agricultural production but also in terms of enhanced production strategies. TMG1180 RR and BÔNUS8579 IPRO are the most drought-resistant soybean cultivars, highlighting defense pathways to avoid tissue desiccation. Xique-xique emerged as the most drought-resistant cowpea cultivar, characterized by a high ability to maintain water content, proline, and photosynthetic pigments, which contributed to the preservation of soluble carbohydrates under water restriction. The data might help plant breeders and farmers in mitigating drought-related damages in general, providing suitable information regarding tolerant soybean/cowpea cultivars for advancing plant breeding efforts, and exploring viable alternatives for cultivating leguminous crops in arid and semiarid regions toward sustainable and resilient agriculture.


Familial exudative vitreoretinopathy (FEVR) is a rare congenital disorder of retinal vascular development. We aimed to study the vascular characteristics around the optic disc in neonates with FEVR and the relationship with disease severity.A retrospective, case-control study including 43 (58 eyes) newborn patients with FEVR at stages 1 to 3 and 30 (53 eyes) age-matched normal full-term newborns was conducted. The peripapillary vessel tortuosity (VT), vessel width (VW) and vessel density (VD) were quantified by computer technology. The t-distributed stochastic neighbor embedding (t-SNE) algorithm was used to visualize the relationship between the severity of FEVR and the characteristics of perioptic disc vascular parameters.P < 0.05). Subgroup analysis showed that VW and VD increased significantly with progressing FEVR stage (P < 0.05). And only VT in stage 3 FEVR was significantly increased compared with stage 1 and stage 2 (P < 0.05). After controlling the confounders, ordinal logistic regression analysis indicated that the VW  and VD  were significantly independent correlated with the FEVR stage, but VT  was not correlated with FEVR staging. Visual analysis based on the t-SNE algorithm showed that peri-optic disc vascular parameters had a continuity along the direction of FEVR severity.The peripapillary VT, VW and VD were significantly increased in the FEVR group compared with the control group (In the neonatal population, there were significant differences in peripapillary vascular parameters between patients with FEVR and normal subjects. Quantitative measurement of vascular parameters around the optic disc can be used as one of the indicators to assess the severity of FEVR. Familial exudative vitreoretinopathy (FEVR), first described by Criswick and Schepens in 1969, is a rare disorder of retinal vascular development that primarily affects retinal angiogenesis, leading to hypervascularization and poor vascular differentiation in the peripheral retina –4. Mild Peripheral retinal abnormalities have always been the focus of FEVR research. With the development of fundus imaging technology, more and more evidence show that retinal vascular development failure caused by FEVR not only affects the peripheral retina, but also affects the macula. Yonekawa et al. described various macular structural abnormalities including vitreomacular traction, diminished foveal contour and cystoid macular edema . Chen etTo date, few studies have been reported on the characteristics of the optic disc and its surrounding vessels in patients with FEVR. Yuan et al. determined that patients with asymptomatic FEVR had more blood vessels radiating from the optic disc at the posterior pole, a significantly larger disc-to-macular distance as well as a remarkably smaller optic disc with a decreased horizontal diameter , 11, 12.Therefore, this study aimed to quantitatively analyze the vascular characteristics around the optic disc, including vascular tortuosity, width and density, based on the fundus images of neonatal FEVR patients taken by a wide-field fundus imaging system (RetCam 3) with the aid of computer technology. At the same time, the relationship between the peri-optic disc vascular parameters and the severity of FEVR was further analyzed. The quantitative evaluation of vascular parameters in the posterior pole of FEVR patients can further reveal some disease characteristics that are difficult to detect with the naked eye, which is of great significance for a comprehensive insight into the pathogenesis of FEVR.This was a retrospective, case-control study. Patients were full-term newborns who underwent routine fundus screening within 7 days of birth at Anhui Province Maternity and Child Health Hospital between September 01, 2017 and May 31, 2021. A total of 43 (58 eyes) FEVR patients from stage 1 to 3 were included in this study, and 30 (53 eyes) age-matched normal full-term neonates were included as controls. All participants underwent fundus examination using a wide-field fundus imaging system RetCam 3 , and patients suspected of FEVR were included after confirmation of diagnosis by genetic testing (high-throughput next-generation sequencing technology). The study adhered to the principles of the Declaration of Helsinki and was approved by Ethics Committee of the Anhui Province Maternity and Child Health Hospital of Anhui Medical University.FZD4, LRP5, NDP, TSPN12 and ZNF408; (3) newborns who underwent fundus examination within 7 days of birth; (4) normal full-term newborns with no history of oxygenation. Exclusion criteria: (1) FEVR patients with retinal detachment involving the macula or total retinal detachment; (2) FEVR patients with retinal folds or vitreous hemorrhage; (3) patients with non-FEVR vitreoretinal pathology  were also excluded. Patients were staged based on the FEVR clinical staging system suggested by Pendergast [To ensure the reliability of the diagnosis of FEVR patients, we adopted strict inclusion criteria as follows: 1) the presence of avascular areas in the peripheral retina of both eyes as confirmed by a wide field fundus imaging system (RetCam 3); (2) genetic test results indicating the presence of at least one pathogenic mutation in 5 genes, including ndergast : stage 1 the presr) were obtained using the Hough circle detection algorithm. We delineated the region of interest (ROI) with the center of the OD, as shown by the red circle in Fig. The fundus images of the subjects were acquired using a wide-field fundus imaging system (RetCam 3), and then the characteristics of blood vessels (BV) around the optic disc (OD) were analyzed by computer technology. All fundus images in this experiment meet screening criteria as follows: (1) visible optic disc, blood vessels and macula; 2) clear quality standards for assessing the disease. Figure  clear quhttps://github.com/orobix/retina-unet). For BV segmentation, all fundus images in this study were resized to a common resolution of 640 × 480 pixels and the U-Net was trained on 500,000 image patches (48 × 48 pixels) extracted from 200 retinal images, with manually labeled images of blood vessels used as ground truth. The manual segmentation method has been published in a previous paper [In this study, there are two U-Net segmentation models: BV segmentation and OD segmentation . The codus paper . The traus paper  was emplAccording to the study , the typIn Fig. Based on the results of vessel segmentation in retinal images, we examined the morphological characteristics of the vessels surrounding the OD, as shown in Fig. where To obtain the average width of vessels within the ROI region, we utilized the Euclidean distance transform  to evaluwhere Following the paper , retinalwhere t-distributed stochastic neighbor embedding (t-SNE) is a statistical method that is used for reducing dimensionality and visualization . The opeIn this research, we employed the t-SNE method to visualize the vessel features  proposed in this paper, revealing the potential relationship between these features and disease severity.P values less than 0.05 were defined as statistically significant.Statistical analysis of data was performed using SAS Statistics software version 8.02 . The measurement data were expressed as mean ± standard deviation. According to the normality test, comparisons of means between two groups were performed using the unpaired t-test or Wilcoxon-Mann-Whitney test. And comparisons among groups were performed using the Kruskal-Wallis test or one-way ANOVA. An ordinal logistic regression model was used to determine the correlation between perioptic disc vascular parameters and different stages of FEVR patients, with adjusted odds ratios (aOR) and 95% confidence intervals (CI) estimated to minimize confounding factors. Variables entered into the model included VT, VW, VD, sex, age, gestational age at birth, and birth weight. Table 4 cm− 3) and 70.80 ± 4.05 (104 cm− 3) (P = 0.0266), respectively  around the optic disc in the FEVR group was significantly increased compared with the control group, 72.43 ± 3.58 (10P = 0.7975); ;  (see Table The average peripapillary vascular width (VW) in the FEVR patients was significantly increased compared with the control group. Quantitative analysis showed that the mean VW around the optic disc in the FEVR group was significantly increased compared with the control group, 66.42 ± 3.52(µm) and 63.46 ± 3.30 (µm), respectively  (see Table P = 0.0290); ; . The VD in the 2-stage and 3-stage FEVR groups was significantly higher than that in the control group, and the VD in the 1-stage FEVR group was not significantly different from the control group ; ;  (see Table The perioptic disc vascular density in the FEVR patients was significantly increased compared with the control group. The results indicated that the peripapillary vascular density (VD) in the FEVR group was significantly higher than that in the control group, 5.29 ± 1.13(%) and 4.59 ± 0.69(%), respectively (P = 0.0002), VD  was significantly independent associated with FEVR staging, after adjusting for confounders such as sex, age, gestational age, and birth weight, and VT  was not correlated with FEVR staging . After controlling for confounding factors such as sex, age, gestational age, and birth weight, the perioptic disc VW was significantly independently correlated with the severity of FEVR, but the correlation between VT and the severity of FEVR was not significant. The results of logistic regression models also indicated that the risk of FEVR staging progression increased by 55.71% for every 1 μm increase in perioptic disc VW. Based on the above results, we can see that the increased diameter and tortuosity of the vessels around the optic disc may be one of the pathological features of FEVR.Due to defects in retinal vascular development, patients with FEVR exhibit a high degree of venous-venous and arteriovenous shunts and a high degree of nonperfusion in peripheral capillaries , 27, 28.Our findings showed that FEVR patients had higher peripapillary VD compared with controls, which increased with progressing stage. Furthermore, logistic regression models demonstrated a significant correlation between peri-optic disc VD and FEVR severity. After controlling for confounders such as sex, age, gestational age at birth, and birth weight, VD around the optic disc was significantly independently associated with the severity of FEVR. The results of logistic regression models also indicated that, each 1% increase in peripapillary VD was correlated with an 88.03% increase in the risk of FEVR staging progression. Thus, we can find that increased peripapillary VD may also be another pathological feature of FEVR. In this study, we used tic disc , 11, 12.Lrp5−/−, Ndpy−/−, Fzd4−/− and Tspan12−/− all showed delayed primary retinal angiogenesis, a sparser density of blood vessels on the retinal vitreous surface, and a complete absence of a secondary and tertiary vascular system [In the present study, FEVR patients showed a significant increase in perioptic disc VD. In addition, Chen et al. indicated that the VD in the superficial and deep layers of the fovea decreased in patients with FEVR, and in humans, the disappearance of the superficial and deep retinal capillary systems was not observed –10. Hower system , 26, 29.In order to further explain the different retinal vascular phenotypes produced by the same gene mutation in mice and humans, based on published research results , 30 and To further evaluate the possible value of peripapillary vascular parameters in monitoring changes in FEVR, we used the t-SNE algorithm to visualize FEVR severity and the vascular characteristics  analyzed in this paper. The visualization results show that the vascular parameters have the characteristics of continuity along the direction of FEVR severity. This study used the 5-classification system proposed by Pendergast et al. to describe the staging of FEVR . FEVR ocAdmittedly, our study has some limitations. Firstly, the sample size is relatively small, and it is necessary to expand the sample size to repeat the results of this research. In addition, neonates did not undergo FAG due to the risks of general anesthesia and allergic reaction to sodium fluorescein. So, the possibility of overlap between phase 1 and phase 2 FEVR cannot be completely ruled out. Moreover, Although the causative gene was identified by genetic testing in all enrolled FEVR patients, for the FEVR population, the causative gene could only be found in less than 50% of FEVR patients , 32, whiIn conclusion, we found that the increased tortuosity, width and density of the vessels around the optic disc may be one of the pathological features of FEVR. Quantitative measurement of peripapillary vascular parameters can be used as one of the indicators to assess the severity of FEVR.


Corynephorus canescens and of Festuca vaginata dominance, respectively, belonging to the community Festuco vaginatae–Corynephoretum have been monitored. We aimed at (1) quantifying the diversity and biomass of the cryptogamic communities; (2) exploring the cryptogamic response to management changes; and (3) studying the effect of experimental management (fencing) on the cryptogamic assemblages. The sites have been compared in 2013 and 2018, respectively. Forty microplots per site per management have been analyzed in both years. Samples of lichens and bryophytes were hand-sorted, dried and then measured. Fencing has led to increased biomass of cryptogams within a few years. Lichens in general benefited comparatively more from exclosure than bryophytes. The increase in lichen biomass  is clearly due to the over 10-year absence of grazing. The only lichen favored by moderate grazing is the legally protected C. magyarica. Short spells of low-intensity grazing can promote the species richness of cryptogams in the community.Cryptogams, often neglected in vegetation dynamics studies, compose a large part of biomass and contribute to the biodiversity of sandy grasslands. Since the work of Verseghy (1970s), their productivity has not been analyzed in Hungary. We studied the lichen and bryophyte dynamics  at two Eastern Hungarian dry sandy grassland sites. The sites of  The diversity of terricolous lichens and bryophytes can be substantial, sometimes exceeding that of vascular plant species ,3,4,5,6.Conservation efforts in European dry grasslands focus mainly on ‘keystone species’ of vascular plants, thus leaving several taxa (among them various cryptogams) almost overlooked ,18,19,20In lowlands, dry habitats with high cryptogamic diversity occur mostly on sand. Sandy grasslands, both on acidic and on calcareous substrata, are widely distributed throughout Europe. These grasslands, as a rule, are exploited as pastures, so their conservation status is highly dependent on management through livestock grazing ,36,37,38Sandy grasslands of the Pannonic region  are thrSeveral studies, mostly from the tundra, alpine grasslands and heath communities, showed that significant changes occur in cryptogamic communities at the onset of grazing, during changes in grazing intensity ,49,50,51Festuco vaginatae–Corynephoretum and other communities) where cryptogams have not been covered [Since the pioneering works of Verseghy ,65,66,67 covered  or only  covered . Cryptog covered ,71. A re covered  where bo covered .2 to 30 m2, for lichen communities. The used plot sizes extend over five orders of magnitude, which is even more than those reported for relevés in vascular plant communities [2, respectively. The size of the largest frequently used plot types has been 1 m2 and was mostly used for terricolous lichen communities, where vascular plants were also recorded [Methods of floristic and biomass survey in cryptogamic assemblages, due to their limited extension, differ significantly from those in vascular communities. A review by Berg et al.  demonstrmunities . The mosrecorded ,75. Gülerecorded  recommenCladonia magyarica.In the present study, we aimed (1) to quantify the floristic composition, diversity and biomass of terricolous cryptogamic communities; (2) to explore the response of cryptogams to management changes (subject to different grazing pressure); and (3) to study the effect of an experimental change (fencing) of management on the cryptogamic assemblages with regard to the performance of the legally protected lichen Corynephorus canescens dominated site (CC) and 9 from the Festuca vaginata dominated site (FV), respectively) have been identified in the samples. The total number of lichen taxa tended to be higher in CC (up to nine at a given date and management) than in FV (three or four). The studied communities did not differ in the total number of bryophytes (three or four at a given date and management). In total, 15 taxa  has shown two magnitudes of variation. In both communities, the two most frequent species have been C. rangiformis  and C. rei (about one-sixth of summed lichen records). The communities are distinguished by the presence of C. foliacea in the Festuca vaginata dominated site  and C. furcata and C. magyarica in the Corynephorus canescens dominated site , respectively. Among the bryophytes, Syntrichia ruralis and Brachythecium albicans were more frequent, both having around 40%, while Polytrichum piliferum had around 20% of all bryophyte records . Of all the lichen biomass, C. rangiformis formed more than 90% (overrepresented compared to its frequency). In contrast, C. foliacea, C. furcata and C. rei have been strongly underrepresented compared to their frequencies (<1% of lichen biomass). The biomass share of C. magyarica has been similar to its frequency. Among the bryophytes, Polytrichum has been overrepresented (close to 45%), Brachythecium underrepresented, while Syntrichia had a consistent share compared to its frequency record  and bryophytes (54%) is similar to that of the overall frequency records. The averages of the biomass of the lichens, bryophytes and of all the cryptogamic taxa are shown in y record  , both in case of frequency and biomass data. When PCA was applied to the frequency records, it emphasized the dominance of small but widespread species such as Cladonia magyarica (CC) or C. rei (both sites) (Cladonia rangiformis (both sites), Brachythecium albicans (CC) or Polytrichum piliferum (FV). The effect of the contrasting treatment is also clearly visible, as the fenced and grazed sites are shown separately on both sides of the X axis. Based on the frequency data, the CC ‘grazing ceased’ samples were positioned among the fenced samples, but when their biomass data have been considered, these were ordinated among the grazed ones. Irrespective of the data type, Cladonia rangiformis proved a good indicator of fenced stands, whereas grazed samples have been indicated by Cladonia magyarica and Syntrichia ruralis  clearly separated the h sites) , while b ruralis .Corynephorus canescens and the Festuca vaginata dominated sites , irrespective of the number of categories used /b)2) and the three parameter version of the Lorentzian equation as f = a/(1 + ((x − x0)/b)2), where x is the cryptogamic biomass (g) at 0.01 m2 and f is the species number of cryptogamic species at 0.01 m2, respectively, whereas a, b and x0 are parameters calculated via iterative parameter estimate functions  than for the Corynephorus canescens dominated one (p < 0.015). The correlation coefficients, however, proved rather weak for both equations and for both sites.The Gaussian equation has yielded the most satisfactory estimates. The reliability of the parameter estimates proved similarly high according to the two equations and also for the regressions , but as a trend, the reliability of the regressions for both sites proved slightly higher for the Gaussian equation than for the Lorentzian. The regression reliabilities proved to be somewhat better for the a parameter) and its location along the x axis  have been very similarly estimated by the two equations. The estimates for the two sites differed consequently: both the maximal values and their locations proved higher in CC than in FV. The Gaussian equation, however, consequently estimated the maxima to be about 0.5% higher, irrespective of the site, while it estimated its position to be 4 to 5% higher than did the Lorentzian one  in the fenced parts for the two periods. Firstly, between the fence construction and the first sampling .  Then, secondly, from the first sampling till the second one .  We found that productivity altered during the study in such a way that its absolute value proved higher both for the lichen and bryophyte fractions in the second period. In the lichens, it has been increased from a rate of ≤10 g/mne in FV . The Shannon’s diversity values calculated for the frequency records (from 1.36 to 1.83) proved higher than those for the biomass ones (from 0.69 to 1.23). These figures represent evenness values from 0.54 to 0.77 (portion of possible maxima) for the frequency records and from 0.31 to 0.53 for the biomass, respectively .The average values of the Shannon’s diversity did not differ much between the two sites, although the averages of the evenness tended to be 6.4% (biomass) and 9.3% (frequency) higher at the FV site than at CC, respectively. For the fenced parts, the diversity scores have declined between 2013 and 2018 at both sites, whereas no congruent tendency for the unfenced parts has been detected. Decline as well as stagnating figures have been found in the grazed parts while the figures for the formerly grazed part of CC, where grazing ceased, have even increased by 2018 . The ratCladonia magyarica Vain. [Cladonia mitis Sandst., C. cariosa (Ach.) Spreng., Xanthoparmelia pokornyi (Körb.) O. Blanco, A. Crespo, Elix, D. Hawksw. & Lumbsch and X. pulvinaris (Gen.) Ahti et D. Hawksw.) have not been recorded in the vicinity of our sampling sites [All the recorded cryptogams are common to the Nyírség region  and alsoca Vain. . Those tng sites ,81,82. Cladonia species. This genus also represents a major identification challenge, and we have 10 taxa detected in the samples. It includes the morphologically similar but chemically distinct species pair C. subulata (only with fumarprotocetraric acid) and C. rei  [As in the psammophilous grassland communities of Central Europe in general ,65,83,84ic acid) ,89,90,91C. magyarica and C. furcata have been found as codominants [Festuco vaginatae–Corynephoretum samples, C. rangiformis has been the dominant lichen species most often. Among the bryophytes, Syntrichia ruralis has been the most widespread species in the calcareous sands [S. ruralis has been confined to the grazed parts of both the CC and FV sites. In the fenced part of CC, Brachythecium albicans became dominant by 2018 while Polytrichum piliferum kept dominance among the bryophytes in the fenced part of FV. In contrast to the calcareous sands of Central Hungary with Festucetum vaginatae danubiale  where C.ominants ,65, in ous sands , while i2), it only involved 10 subsamples in contrast to the 40 in our study.The only published records of specific terricolous cryptogamic biomass from the Carpathian Basin came from IBP studies conducted on the open calcareous sandy grasslands of Central Hungary . Their s2 and the maxima from 225–285 g/m2, in Verseghy’s studies these figures were 125–205 g/m2 and 260–405 g/m2, respectively, during the 1968 sampling campaign.The value of the cryptogamic biomass, including both the lichen and bryophyte biomass, in this study series was definitely higher than that found in our own research. While in our case the minima of the cryptogamic biomass ranged from 25–40 g/m2 in both studied communities, the averages fell between 120 and 140 g/m2 in the Festucetum vaginatae danubiale community and between 115 and 165 g/m2 in the Brometum tectorum community, while the maxima reached 360 g/m2 and nearly 450 g/m2, respectively, in the order described above.During the whole sampling period (through 29 sampling dates), only the biomass of lichens in Verseghy’s studies had minima of 50–55 g/mFestuco vaginatae–Corynephoretum, however, the corresponding figures for the lichens ranged from as low as 5–15 g/m2 for the minima while the maxima only had 155 g/m2 in the Corynephorus dominated site and 225 g/m2 in the Festuca vaginata dominated one, respectively. It is not known whether these differences are attributed to differences in the physical properties between the two landscapes or other factors.In our 2 biomass in June while vascular plants had their summer peak at 150 g/m2. The cryptogamic surplus was even more pronounced in the other two communities, reaching up to 290 g/m2 biomass paralleled with only 60 g/m2 of vascular biomass during the June sampling [Few data enable a direct comparison of vascular vs. cryptogamic biomass Pannonian sandy grasslands, and these are mostly from Central Hungary . In opensampling .2 aboveground vascular phytomass has been harvested at the beginning of the experiment. It was in contrast with (95–)110–125(–145) g/m2 for the fenced plots, showing an early positive effect of grazing exclosure on vascular phytomass.In the unfenced plots of our sites, an average of (65–)80–100(–125) g/m2, somewhat higher than in our study. .) These records have been paralleled with a relatively low (0–)2.5–11(–13.5–22.5) g/m2 lichen biomass and (0–12.5–)23.5–62.5(–75–115) g/m2 bryophyte biomass, respectively.In the Nyírség region, the single grassland study where biomass both of vascular plants as well as of lichens and bryophytes (none of them identified at the species level) has been sampled has recently been conducted . In thatKCl: 5.37 ± 0.46 SE) as well as higher organic matter content (w/w%: 1.44 ± 0.54 SE) compared to ours, suggesting that most samples came from more productive grasslands  and only some from Festuco vaginatae–Corynephoretum.Soil samples from these grasslands, however, had an apparently higher pH (pH2 on soils with lower organic matter content (≤0.6%), whereas it measured (110–)150–160(–185) g/m2 on soils relatively rich (≥1.5%) in organic matter . It is very likely that above a certain amount of vascular biomass, the cryptogamic and vascular biomass are negatively correlated.Minor variations in the soil properties have similar effects on the biomass at our study sites. The aboveground vascular biomass in our sites varied around (20–)55–65(–75) g/mhttps://landmark2020.eu/glossary/livestock-unit-lsu/ accessed on 3 August 2023). This is non-experimental evidence supporting our findings concerning increased lichen biomass after grazing exclosure.As no individual records, only group averages, are presented in , direct 2 . These sites are practically devoid of perennial vascular plants and have developed a low diversity, cryptogam dominated grassland where over 98% of cryptogamic biomass came from Cladonia rangiformis . The rapid recovery of dense cryptogamic mats on calcareous sandy fallows in Hungary has also been described, where they can block the establishment of weed species and prevent the formation of their soil seed banks [In the Nyírség region, the possible maximum of the lichen biomass could in any case be somewhat higher. On fallow lands, developed after the abandonment of acidic sandy arables, the lichen biomass can also reach up to 285 g/mp values). Low correlation coefficients, even in the best-fit Gaussian and Lorentzian equations, widely accepted ones used in describing the average effect caused by a large number of factors [Selecting peak equations to describe the connection between the biomass and species richness biomass had the background logic of expecting a low species number both in the case of minimal biomass  and in the case of high biomass . In contrast, for intermediate biomass scores, neither low nor high biomass seems to limit the number of species. For most of the tested equations, however, the probability of estimates for some or all parameters proved unacceptable . Even the diversities calculated for Verseghy’s Brometum tectorum samples proved somewhat higher . In our study, the lowest H′ and evenness values have been associated with long-term grazing exclosure (in 2018), both in CC and FV  greatly exceed those of our samples from C and FV .Cladonia rangiformis from becoming monodominant. Less competitive, smaller and/or short-lived cryptogams can, however, colonize neither under heavy grazing pressure nor in the total absence of grazing. We found that the lichen diversity of the fenced parts already started to decline before the first sampling (4.5 years after exclosure) and dropped greatly till the second sampling (10 years after exclosure). These changes can be well interpreted by the ‘intermediate disturbance theory’ ,104. In Lichens and bryophytes possess a more fragile structure and have slower growth rates than most of the vascular plants . They caThe first direct effect of grazing on cryptogams is the consumption, although its importance also depends on the grazer. Consumption of lichen by reindeer has strong seasonal constraints; nevertheless, it forms an important part of the reindeers’ winter diet ,109. No In contrast to the often low impact of consumption, the decline of lichen biomass and species richness can rather be linked to trampling in various ecosystems. The effect of trampling seems important in the arctic landscape ,113 but Polytrichum hyperboreum in arctic grasslands was found to be resistant to trampling even during dry summers [C. magyarica Vain., the only lichen species favored by moderate (fragmenting) grazing then abandonment. Short spells of low-intensity grazing seem to promote this legally protected species in the studied grasslands. Some bryophytes also showed a rapid recovery after grazing exclusion, probably due to their ability to re-establish from fragments and to tolerate the shading caused by plant cover [In dry conditions, cryptogams are more sensitive to trampling . Humidit summers . Differe summers . This cant cover . Generalnt cover ,117. Furnt cover .Polytrichum species and small Cladonia species with non-branching podetia also possess apparently effective dispersal mechanisms, which enables them to colonize areas devoid of competitive large, branching Cladonia species [Grazing, however, can have an indirect impact on cryptogams, too. On the one hand, removing vascular biomass can promote the development of cryptogams via decreased shading. It was concluded that cryptogams in heavily grazed parts are typical fugitive species which cannot stand competition in more dense vegetation . Additio species . The iss species  have fou species ,47,48,49Herbertus hutchinsiae Gottsche & Rabenh. in a study from Scotland) [Cladonia [C. rangiformis, one of the tallest lichens in our study, can already effectively block smaller cryptogams and even hinder the growth of most vascular plants   or more Cladonia ,116. In r plants . In contIncreasing grazing intensity has been shown to consistently lead to a decline in cryptogams’ richness, while the number of lichens and bryophytes increased with the time since exclusion in drylands ,45,52. GAlthough there is a consensus that biomass accumulation during cryptogamic (primary) succession can be very slow, lasting for decades ,123,124,A further challenge is to investigate changes in vascular plants, bryophytes and lichens simultaneously in our grazing exclosures. Such a complex approach, covering the spatial and diversity constraints of all of these groups, is very rare in the literature ,125,126.Open sand steppes. A site of Corynephorus canescens dominance  and another one of Festuca vaginata dominance , phytosociologically belonging to the grassland community Festuco vaginatae–Corynephoretum Soó in Aszód 1935 [(https://novenyzetiterkep.hu/node/765 accessed on 29 May 2023) and is classified by Natura 2000 habitats as ‘6260 Pannonic sand steppes’ (closely related to 2340 ‘Pannonic inland dunes’) (https://natura2000.eea.europa.eu/ accessed on 29 May 2023). We studied the dynamics of cryptogams  in two dry sandy grassland sites in East Hungary, located 15 km and 25 km east of the city of Debrecen, respectively. Both belong to the G1 class of national vegetation classification, A-NÉR zód 1935 , have beCarex stenophylla, Cynodon dactylon and Poa bulbosa. Their perennial dicots with significant cover include Chondrilla juncea, Eryngium campestre, Potentilla arenaria, Rumex acetosella, and Thymus degenianus. (The underlined species as well as Festuca vaginata at first benefited being excluded from grazing.) Further constituents of these communities are the horsetail Equisetum ramosissimum as well up to about 30 further angiosperms, mostly small annual dicots. These communities are subject to different levels of grazing from domestic herbivores (mostly beef cattle and/or sheep) and have become increasingly abandoned in the latest decades due to dropping livestock densities.In addition to their dominant grasses, providing the phytosociological name of the community, these sites host a number of further frequent monocots, such as Cryptogamic vegetation has been compared at both sites in 2013 and 2018, respectively, in an experimentally fenced part  as well as a non-fenced part subject to changing grazing pressures, respectively. The fenced and unfenced parts are located at 20 to 30 m apart and had a similar composition and dominance when fencing has taken place. Fencing was used to model the prevailing trend of falling stocking densities.At the CC site, a flock of about 140 sheep grazed during the growing season, passing through twice the area per day. Grazing, however, has been ceased in late 2015 as the forestry directorate, the owner of the area, has no longer contributed to it in order to protect new forest plantations nearby.The FV site was mainly grazed by a herd of around 40 beef cattle, less frequently by around 70 to 100 sheep. Grazing here has fluctuated, but its intensity has shown an overall increase throughout the study period. Hares and roe deer have been observed accidentally in the non-fenced parts of both communities, although the low density of their droppings suggested only a negligible grazing pressure. Therefore, the 140 cm tall fence, excluding sheep, cattle roe deer and hare, almost completely eliminated the effect of grazing. It was not possible, however, to exclude small rodents. We did not separate the effects of cattle and sheep, nor was it possible to separate the effects of grazing  and trampling (causing biomass decay).https://www.met.hu/eghajlat/ accessed on 29 May 2023).The climate of the study areas is moderately continental, with hot and dry summers (precipitation is mainly caused by thunderstorms and downpours), while winters are a few degrees colder and harsher than the national average. The average of the annual temperature is around 10.5 °C, while the annual sum of sunshine exceeds 2200 h. The average of the yearly precipitation falls between 550 and 600 mm. The average of the rainfall during the vegetation period (April−September) is between 300 and 350 mm, with extremes from 210 to 620 mm 80–120(–125) g/m2 while that of the Corynephorus canescens dominated one was (95–)100–120(–145) g/m2, respectively . Production of vascular phytomass varied yearly following weather patterns, with the highest figures in 2008 and the lowest ones in 2009, respectively. The average production in the unfenced stands has stayed at 80–100 g/m2 while that of fenced ones has been at around 120 g/m2 in first three years of exclosure, respectively. At the beginning of the experiment, the vascular phytomass in the KCl–CC: 4.44–4.45; pHKCl–FV: 4.80–4.84) and with negligible CaCO3 content (at both sites: <0.05 w/w%), low organic matter content  and low available nitrogen  as well as low phosphorous  content. Somewhat higher figures for organic matter and phosphorous in CC can be indicators of the former arable usage decades before the beginning of the experiment.The topsoil (0–5 cm) of the sites is unanimously sand (portion of the 0.05–2 mm size fraction is 93.06% in CC and 95.36 in FV) of acidic chemistry (pH2), microplots per site per management have been collected with a soil monolith between 25 February and 7 March in 2013 and between 10 and 14 October 2018, respectively. The microplots have been (1) evenly scattered in zones along the length and width of plots, so as their middle and ends as well as their center and sides closer to the bordering buffer zone are similarly represented and (2) have been centered on randomly thrown markers (within selected zones) in order to guarantee a similar representation of grass tussocks and gaps.The biomass of the cryptogams has been sampled in 2013 and in 2018 . The aboveground vegetation of 40, 10 × 10 cm size , have been collected yearly from blocks at the end of June between 2008 and 2010. This period involved a rather dry year  as well as a very wet year  and so represents a large portion of the possible weather fluctuations in the region (www.met.hu accessed on 29 May 2023).A preliminary survey of the vascular biomass of the study sites has been completed at the beginning of the exclusion experiment. Twenty samples, each of 20 × 20 cm size (0.04 mm/m%) and available phosphorus (mg/kg). In the case of the pH, we have used the potentiometric method and have measured the parameter in a potassium chloride solution suspension. The organic matter content has been determined via the Tyurin method, so we have used the titrimetric method and oxidation by chromic acid. The phosphorus content of the soil extract has been converted into heteropoly acid with ammonium molybdate and has been reduced to a molybdenum blue complex with ascorbic acid. The AL-soluble phosphorus (AL–P2O5) content of the soils was determined via spectrophotometry. The light absorption has been measured at 480 nm.Soil analyses have been carried out by the accredited laboratory of the Research Institute of Karcag of the Hungarian University of Agriculture and Life Sciences. The following three parameters were measured: pH (KCl), total organic matter content  and the public database of MycoBank . For the identification of critical taxa in the lichen genus Cladonia, standard spot tests and high-performance thin-layer chromatography (HPTLC) in solvent C  have been applied [Pleurosticta acetabulum (Neck.) Elix & Lumbsch (norstictic acid) and Leucodermia boryi (Fée) Kalb  were used as controls. After chromatographic development, the plates were examined under UV light (254 and 366 nm) then sprayed (CAMAG TLC Sprayer) with water, dried (to investigate the fatty acids), then subsequently sprayed with a 10% sulfuric acid solution and heated at 110 °C for 5–10 min. Finally, the plates were cooled to room temperature and studied under UV light (366 nm).Lichen identification has been completed using the manuals of Verseghy , Smith e applied . The latBryophytes have been identified by consulting the relevant handbooks and papers according to Erzberger and Schröder , while tPairwise and multiple comparisons have been performed using the SigmaPlot 12.0 software package. Comparisons of the species number and biomass data per site per management per date have been performed via ANOVAs on the ranks, and in case of a significant result, we used Kruskal–Wallis tests to identify a significant pairwise difference(s).Principal coordinate analysis (PCA) supported by the CANOCO and CANODRAW software packages  has beenThe possible correlation of the cryptogamic biomass and the combined species number of lichens and bryophytes has been also studied. After a preliminary evaluation of the data, we systematically tested the available peak equations supported by the SigmaPlot 12.0 software package . We used the package option ’dynamic fitting’ enabling estimation of parameters via an iterative approach so as to obtain the best possible estimates for the parameters and to achieve the best possible fit. The automatic initial parameter estimate function has been used as well as a 200 fit option, while the maximal number of iterations has also been set to 200. The accuracy (probability) of the parameter estimates, results of the analysis of variance  for the rates of regression and residual as well as the magnitude of the correlation coefficients have been evaluated.Corynephorus canescens and Festuca vaginata dominated sites by the distribution of the samples among the species numbers classes as well as among the biomass classes using chi2 tests [2, while for biomass records in three categories as <0.5, 0.5–1, ≥1 g and in five categories as <0.1, 0.1–0.5, 0.5–1, 1–5, ≥5 g dry biomass/0.01 m2. We compared the i2 tests . These tCorynephorus canescens (CC) and Festuca vaginata (FV) dominated sites. The maximal possible diversity values have been calculated using the total number of taxa recorded throughout the study at a given site and an even dominance distribution.Shannon’s diversity and evenness ,134 have