About 25–37% of resectable pancreatic ductal adenocarcinoma (PDAC) had a great chance of early recurrence after radical resection, which is mainly due to preoperative micrometastasis. We herein demonstrated the profiles of ctDNA in resectable PDAC and use of ctDNA to identify patients with potential micrometastasis.A total of 113 and 44 resectable PDACs were enrolled in discovery and validation cohorts, separately. A panel containing 50 genes was used to screen ctDNA by an NGS-based assessment with high specificity.In the discovery cohort, the overall detection rate was 38.05% (43/113). Among positive ctDNA, KRAS mutation had the highest detection rate , while the others were <5%. Survival analysis showed that plasma KRAS mutations, especially KRAS G12D mutation, had significant association with OS and RFS of resectable PDAC. Plasma KRAS G12D mutation showed a strong correlation with early distant metastasis. In the validation cohort, survival analysis showed similar association between plasma KRAS G12D mutation and poor outcomes.This study demonstrated that plasma KRAS mutations, especially KRAS G12D mutation, served as a useful predictive biomarker for prognosis of resectable PDAC. More importantly, due to high correlation with micrometastasis, preoperative detection of plasma KRAS G12D mutation helps in optimising surgical selection of resectable PDAC. The US Food and Drug Administration (FDA) has approved the use of CTC as a biomarker for prognostic evaluation in three types of cancers.11 Cell-free circulating tumour DNA (ctDNA) can be effectively distinguished from normal cell-free circulating DNA (cfDNA) by specific cancer-related mutations.13 Several studies have proved a high detection rate and potential prognostic value of ctDNA in PDAC.17 However, the data regarding the complexity of exosome isolation invalidated it in other centres, and heterogeneity as well as rarity of CTCs and ctDNA restrained the application mainly in advanced patients.18 Therefore, more stable methods and more validations are necessary before clinical application, especially for patients with early-stage PDAC who are commonly considered as resectable.Liquid biopsy biomarkers are gaining interest in cancer research due to their advantages of convenient and non-invasive nature.19 For NGS techniques, random errors usually happened during the exponential amplification process, leading to high false-positive rate, and this has been one of the most important defects.20 However, some high-fidelity linear amplification sequencing methods and optimised analytical methods may help to improve.21 Hence, in this study, a highly specific ctDNA detection method was used to explore the clinical value of hotspot mutations in resectable PDAC patients and hoped that a new ctDNA biomarker can be used to optimise the surgical selection of PDAC patients and eventually improve the survival time of PDAC.In recent years, different procedures for plasma ctDNA processing, library construction and analytical methods have led to incomparable results and confusion in clinical application.Patients with resectable PDAC were collected from Changhai prospective database . All patients underwent curative surgery for tumour resection and were histologically confirmed with PDAC. Patients who died of surgical complications within 1 month after surgery have been excluded. All patients provided written informed consent to use their clinical data. The study was conducted in accordance with the national guidelines, and acquired the approval of the Ethics Committee at Changhai Hospital.3 tumour tissue was removed from the centre of the resected lesions, and then was rapidly maintained in liquid nitrogen until DNA extraction. In total, 10 mL of preoperative venous blood was collected from patients in ethylenediaminetetraacetic acid tubes . Within 2 h of collection, the blood was centrifuged at 1600g for 10 min at 4 °C to obtain plasma and a buffy coat layer (containing WBCs). The plasma was then further centrifuged at 16,000g for 10 min at 4 °C to pellet any remaining cells. Both plasma and the buffy coat layer were collected and stored at −80 °C. Patients were neither provided with the results of ctDNA sequencing nor any treatment decisions made based on ctDNA results.Approximately 0.5–1 cmBefore tumour tissue DNA extraction, at least one frozen resection of each sample was assessed by two specialist pathologists to confirm the tumour cellularity ≥20%  was used for capturing. The captured products were purified and sequenced on an Illumina Hi-Seq 2500 . The average coverage depth for all probes of NGS library was at least 500×. Sequencing results were aligned according to the human reference sequence hg19/GRCh37, and the background noise introduced by random NGS error was removed. The true variants were then identified, and the allele frequency was calculated by comparing the number of unique reads containing a variant to the total number of sequencing reads that mapped according to the position of the variant. Germline variants were identified from WBC genomic DNA sequencing, and were removed from tumour genomic DNA total variants to determine somatic mutations.The genomic NGS library of tumour tissue and WBC was constructed by using KAPA sequencing library construction kit . A panel consisting of all exons from 50 genes that are associated with pancreatic cancer . ctDNA was circularised using CircLigase II single-strand DNA ligase  with 10–30 ng of DNA per 20 μL of reaction. After ligation, the samples were treated with exonuclease  to remove the uncircularised DNA. The circularised DNA was then amplified in a rolling circle amplification reaction using Phi29 DNA polymerase , and the exonuclease-resistant random primers by using the manufacturer's instructions with slight modifications. The amplification product was then purified by AMPure beads  and sonicated to short fragments that are suitable for NGS library 7/20 construction by using a Covaris sonicator. NGS sequencing libraries were generated from 100 ng of amplified ctDNA using the KAPA sequencing library construction kit  according to the manufacturer’s instructions. After library construction, a panel consisting of all exons from 50 genes associated with pancreatic cancer was used for capturing. The captured products were then purified, sequenced on an Illumina Hi-Seq 2500  and the unique sequencing reads were determined by using an AccuraGen proprietary algorithm. The average coverage depth for all probes of Firefly NGS library was approximately 7000×. The sequencing results were aligned to the human reference sequence hg19/GRCh37, and the background noise introduced by random NGS error was removed by AccuraGen proprietary algorithms. The true variants were then identified, and the allele frequency was calculated by comparing the number of unique reads containing a variant to the total number of sequencing reads that mapped according to the position of the variant. Germline variants were identified from WBC genomic DNA sequencing and were removed from ctDNA total variants to determine somatic mutations.The QX200 system  was utilised for ddPCR experiments. Multiplex KRAS products for ddPCR were utilised, which included six KRAS mutations (KRAS G12D/G12V/G12R/G12C/G12A/G12S). For conducting the experiments, a reaction volume of 20 µl consisting of cfDNA ranging 4–80 ng (8 µL of cfDNA input in volume) was used. Emulsified PCR reactions were run on a 96-well plate on a MJ thermal cycler  following the manufacturer’s instructions. Plates were read on a Bio-Rad QX200 droplet reader using QuantaSoft version 1.4.0 software  to assess the number of droplets that was positive for wild-type KRAS and/or KRAS mutations. A sample without plasma ctDNA was used as a negative control. Three positive events were regularly used as a threshold for false positivity.The preoperative clinical variables included age, sex, smoking history, drinking history, status of diabetes mellitus, the first clinical symptom , carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), surgical procedure and radiologic tumour size. Postoperative variables included the eighth edition of American Joint Committee on Cancer (AJCC) TNM classification, differentiation degree, perineural invasion, pathological margin status, nodal involvement and treatment with adjuvant chemotherapy. Overall survival (OS) was defined as the interval from the date of surgery till the date of patient death or the last follow-up visit after 1 month post surgery. Recurrence-free survival (RFS) was defined as the time interval from the date of surgery till the date of local or regional recurrence, distant metastases, death or the last follow-up visit after 1 month post surgery. The status of recurrence was recorded as no, local, distant and both.P-values less than 0.05 were considered to be statistically significant. Statistical analysis was performed by using SPSS 22.0 for Windows . Curves for OS and RFS were generated using the R statistical package v.3.5.1 (http://www.r-project.org/), and figures were created using GraphPad Prism .Discrete variables were presented as number and/or percentage, whereas the continuous variables were presented as median and/or mean. Fisher’s exact test was used to report variable association of plasma KRAS mutations. Spearman’s rank correlation coefficient was used to determine the association between ddPCR and Firefly NGS in plasma KRAS G12D allele frequency (AF). Univariate and multivariate Cox regression analysis was used to assess the impact of prognostic factors on OS and RFS. From August 2014 to August 2015, a total of 130 consecutive patients diagnosed with resectable PDAC who underwent curative pancreatectomy were enrolled from the Changhai prospective database . Interestingly, except for KRAS, the other genes detected commonly had a pretty low positive rate of <5%. Plasma KRAS mutations were detected in 26 patients, while 87 patients did not have KRAS mutations in their plasma . We compared the AF of KRAS mutation between ddPCR and Firefly NGS. The results showed that the detection of plasma KRAS mutations by ddPCR and Firefly NGS technique was largely equivalent  and RFS  among patients with detectable KRAS mutations in ctDNA (n = 26) when compared with those without KRAS mutations (n = 87)  Fig. . At 12 mn = 13) showed a significantly reduced OS  and RFS  relative to those without plasma KRAS G12D mutations (n = 100)   Fig. . No suchAfter that, whether the following patient characteristics were risk factors for OS and RFS were investigated: age, sex, smoking history, drinking history, the first clinical symptom , diabetes mellitus, surgery procedure, CEA, CA19-9, radiologic tumour size, pathological stage , differentiation degree, perineural invasion, pathological margin status, nodal involvement and the use of adjuvant chemotherapy. Univariate analysis demonstrated radiologic tumour size, pathological stage , nodal involvement, KRAS mutations and KRAS G12D mutation in ctDNA as considerable risk factors for RFS and OS Table .Table 2USurvival analysis suggested that patients with KRAS G12D mutation in ctDNA showed significant trend of early recurrence, the median RFS of whom was only 6 months Fig. . Then a According to the same standard with the discovery cohort, the clinical data of 47 consecutive patients from the Changhai prospective database during August 2016 and December 2016 were collected . These results were similar to the discovery cohort, where only plasma KRAS mutations had a detection rate of >5% Table , which w27 Currently, the clinical utility of ctDNA is only approved in metastatic non-small-cell lung cancer (NSCLC) by FDA as a companion diagnostic,29 while studies on many other cancers have also shown good prospects.36 The fact that even little variability in process and analysis will result in completely different results restrains the further clinical utility of ctDNA.37 In order to solve these problems, some projects, such as the European CANCER-ID, European Liquid Biopsy Academy (ELBA) and European Liquid Biopsy Society (ELBS) networks or the US-based BloodPAC, have been initiated.38 In this study, we tried to use a detection method of high specificity to address whether ctDNA in patients with resectable PDAC could possibly serve as a decisive factor for neoadjuvant therapy.As a component of liquid biopsy, ctDNA has the most potential clinical application value due to its non-invasive, real-time and highly specific genomic result.40 but the main disadvantages included sequential testing of mutations and detection of previously unknown mutations is impossible.18 In contrast, NGS-targeted sequencing method utilises enrichment of recurrently altered loci by PCR amplification or hybrid capture, allowing de novo identification of molecular alterations, but this inevitably introduces random errors in the amplification process.20 In our research, we used the ‘firefly’ method, circularised library and linear amplification to maximise the accuracy and specificity, as the false positive in clinical practice renders the patients to lose the chance to be cured by radical resection.The most popular methods such as ddPCR and NGS-based techniques have their own advantages and disadvantages, separately. ddPCR is associated with high analytical sensitivity down to single molecules and has lower turnaround time than NGS-based approaches when testing single mutations only,41 cfDNA in plasma is highly fragmented DNA that is mainly derived from the apoptotic cells, perhaps predominantly from the apoptotic leukocytes (e.g. neutrophils).42 During the early stages of cancer, the total amount of ctDNA is usually <0.01% of the total cfDNA, while >10% concentration of ctDNA is believed to be in the clinical metastatic stages, which can be easily detected by imaging examination or tumour marker testing.18 The concentration of cfDNA between 0.1% and 10% is thought to be related to micrometastasis of primary tumours, which cannot be undetectable by conventional techniques and critical for surgical outcome and long-time survival.18 Therefore, based on Firefly NGS platform, we choose 0.1% as the threshold for ctDNA positive. The combination of a reasonable cut-off value and an accurate detection method with high specificity might be helpful for the stratification of resectable PDAC patients and further provides a proper individualised treatment.Different ctDNA concentrations reflect different stages of tumour development and progression (metastatic vs non-metastatic recurrence).43 Moreover, several studies have reported that the correlation of ctDNA with tumour stage, metastatic burden and increased ctDNA mutant allele frequency was observed at the time of recurrence or progression.46 So, detection of credible tumour-driving gene mutations from ctDNA during the early-stage PDAC might reflect the formation of micrometastasis. In this study, we sequenced the whole exons of 50 genes, including KRAS, P53, SMAD4, CDKN2A and so on. However, only KRAS mutations were identified in 20–25% of patients with early-stage PDAC, while the mutation detection rates of the remaining genes, including driver genes,48 were significantly lower than that of KRAS, <5%. There might be two reasons: first, the alteration rates of these genes in PDAC tissue are correspondingly lower than KRAS;49 second, mutation type of suppressor genes like SMAD4 is usually deletion, which makes it difficult to detect in ctDNA.49 Survival analysis of the discovery cohort demonstrated that KRAS mutation, especially KRAS G12D mutation, in ctDNA was strongly correlated with poor OS and RFS, and this has been proven in tissue also.50 Further recurrence analysis showed that patients with plasma KRAS G12D mutation were more likely to develop distant metastasis, especially liver metastasis, in 6 months after surgery, and this highly suggested the existence of micrometastasis before surgery. Importantly, we found similar poor outcome in the validation cohort. Only a 6-month RFS strongly indicated that these patients should receive neoadjuvant therapy rather than upfront surgery, despite resectable PDAC in radiology.Driver gene mutations represent both primary and metastatic genetic variation during the process of tumour progression. It has been proved that identical mutations in known driver genes, especially KRAS, were present in every metastatic lesion in all PDAC patients, and the genetic similarity among the cells of metastases was higher than that expected for any two random cells taken from a normal tissue.17 However, this is due to the impairment caused by the threshold of 0.1% AF, which we chose to avoid situations, like ageing, benign tumours and preneoplastic lesions, and better reflect the systematic tumour burden of the patients.18 In fact, the Firefly NGS technique allowed a detection of 0.02% mutated DNA,21 which suggested that negative results in this study did not mean no detectable ctDNA in preoperative plasma. By maximising the specificity of our method, we could identify the resectable PDAC patients who are most suitable for neoadjuvant therapy instead of upfront surgery. It is more practical in clinical work due to the slim chance of making resectable PDAC patients lose the opportunity of radical surgery.Patients who did not harbour KRAS mutations in ctDNA but had a KRAS mutation in tissue accounted for 74.7% (74/99) -associated invasive adenocarcinoma52 and might serve as an alternative oncogene in KRAS wild-type PDAC,49 were not included and needed to be paid more attention in further study.However, our study still has limitations. Firstly, we performed a retrospective study, in which the PDAC patients were enrolled from a single institution. The conclusions we made from this research still need to be verified in a prospective study. Secondly, the panel we used in this study was established based on a research about the mutational landscape of pancreatic cancer. Genes like GNAS, which was reported to be closely related to the development of pancreatic cystIn summary, a study screening ctDNA by Firefly NGS in resectable PDAC was performed, and the results showed that KRAS mutations, especially KRAS G12D mutation, in preoperative ctDNA were significantly associated with the outcomes of resectable PDAC. The presence of plasma KRAS G12D mutation showed a strong correlation with early distant metastasis, indicating that these patients had a high probability of preoperative micrometastasis and should possibly receive neoadjuvant therapy first instead of undergoing an upfront surgery. The preoperative detection of plasma KRAS G12D mutation will help in optimising surgical selection of resectable PDAC patients and improving survival time.Supplementary material


Transcobalamin deficiency is a rare autosomal recessive inborn error of cobalamin transport  which clinically manifests in early infancy.12 metabolism. Family history revealed a sister who died at the age of 3 months with a similar clinical syndrome and with pancytopenia. She was started on empirical intramuscular (IM) cobalamin supplements  and several transfusions of washed and concentrated red blood cells. With these treatments a clear improvement in symptoms was observed, with the disappearance of vomiting, diarrhea and normalization of the full blood count. At 8 years of age injections were stopped for about two and a half months causing the appearance of pancytopenia. IM hydroxocobalamin was then restarted sine die. The definitive diagnosis could only be established at 29 years of age when a genetic evaluation revealed the homozygous c.1115_1116delCA mutation of TCN2 gene (p.Q373GfsX38).We describe the case of a 31 years old woman who at the age of 30 days presented with the classical clinical and laboratory signs of an inborn error of vitamin BCurrently she is healthy and she is taking 1 mg of IM hydroxocobalamin once a week.Our case report highlights that early detection of TC deficiency and early initiation of aggressive IM treatment is likely associated with disease control and an overall favorable outcome. In the enterocyte, the intrinsic factor - cobalamin complex dissociates and cobalamin reaches the portal circulation where it is bound to transcobalamin  [12 is freed from the vector by lysosomal acid hydrolases and reduced by cytoplasmic enzymes [Vitamin B12 undergoes two separate metabolic processes to be converted into the two co-enzymatically active forms: in the cytosol, in the form of methyl-cobalamin, it becomes part of the catalytic process of the enzyme methionine synthase; in the mitochondrion undergoes an adenosylation process that forms adenosyl-cobalamin .At this point the reduced BCongenital errors of cobalamin metabolism can affect any stage of absorption, blood transport and intracellular use.The only congenital absorption deficit is due to a genetic alteration of the enterocytic receptor (Imerslund-Grasbeck syndrome), which is characterized by low serum cobalamin levels, a normal intrinsic factor and megaloblastic anemia .12 [12 [TC deficiency is a typical transport error characterized by megaloblastic anemia with normal serum levels of B12 . In this12 [12 .12 (ca. 10–20%), while the methylcobalamin deficiency blocks the methylation of homocysteine, thus causing massive homocystinuria [Isolated deficiencies of the specific metabolic pathways of adenosylcobalamin and methylcobalamin lead, respectively, to methylmalonic aciduria and homocystinuria. In the first case there will be the forms of methylmalonic acidemia due to coenzyme deficiency, which respond in part to therapy with Bstinuria . The actIn particular, in 5–10, methylentetrahydrofolate reductase deficit, there is a lack of synthesis of 5 methyltetrahydrofolate which is the direct donor of the methyl group to homocysteine for the synthesis of methionine .The blockade of methionine synthetase activity causes massive homocystinuria but megaloblastic anemia has never been found.12 there is also a folate malabsorption which is characterized by severe megaloblastic anemia with very low serum folate levels. Albeit rare there are also congenital disorders of folate metabolism, such as the functional deficit of dihydrofolate-reductase and the cellular uptake defect of folate, characterized by severe megaloblastic anemia and pancytopenia. These disorders are sensitive to therapy with folate at high doses, in case of normal serum levels of folic acid. In the few cases described it is not clear whether the pathogenesis is due to a primitive metabolic deficit of folate; in any case, aminoaciduria has never been found.As for BBlood cobalamin levels are not usually low because most of serum cobalamin is bound to HC and not to TC.TC deficiency (OMIM #275350) is a rare autosomal recessive disorder: the most frequent mutations are deletions or insertions in the TCN2 gene (Cytogenetic location: 22q12.2), resulting in frame shifts that predict protein truncation , 9.Currently about 60 cases of TC deficiency have been reported in the literature  with difTwenty-five pathogenic mutations in TCN2 gene have been identified . NonsensOur patient was born to Italian non consanguineous parents. She was born from eutocic birth with a weight of 3300 g (50th percentile), head circumference 34.5 cm (50th percentile), length 50 cm (50th percentile).Neonatal screening for Phenylketonuria, Congenital Hypothyroidism and Cystic Fibrosis was negative. Neonatal screening for inborn errors of metabolism was not performed, because this screening in Italy was not present: it only started after a Ministerial Decree of 13 October 2016.She was first evaluated at the age of 30 days when she presented with recurrent vomiting with every meal (exclusive breast feed), diarrhea and weight loss (< 25% percentile). Neurological evaluation was normal.Family history revealed a sister who died at the age of 3 months with a similar clinical syndrome and with pancytopenia. This sibling was diagnosed an acute myeloid leukemia, but the diagnosis was not confirmed at post-mortem examination.RBC) 2,040,000/μL, Hemoglobin (Hb) 7.5 g/dL, Mean Corpuscular Volume (MCV) 115.2 fL, White Blood Cell Count (WBC) 5400/μL, platelets count 165,000/μL. Peripheral blood film showed hypochromic macrocytic RBC and hyper-segmented neutrophils, with no reticulocytosis (corrected reticulocyte count was 5%). Serum bilirubin was 7 g/dl (< 9.5).Complete blood count (CBC) revealed megaloblastic anemia . Screening test for pathological amino acids revealed homocystinuria.Urinary spot methylmalonic acid   was not Direct x-ray abdomen, digestive system after barium administration and Chest X-ray were normal.The clinical picture, including family history, megaloblastic anemia and laboratory findings that demonstrate a normal B12 and folate level suggested the diagnostic suspect of deficiency of TC.Therefore, treatment was started at 45 days with several transfusions of washed and concentrated RBC and intramuscular (IM) injections of hydroxocobalamin (1 mg/ day for 1 week and then 1 mg twice a week). The clinical picture progressively improved with disappearance of vomiting and diarrhea, normalization of blood tests and body growth.At the age of 5 months, following the aforementioned treatment of IM injections of hydroxocobalamin, the infant had a normal growth: weight 6950 g (50th percentile), head circumference 45 cm (50th percentile), length 61.5 cm (50th percentile). Blood analysis: RBC Count 4,270,000/μL, WBC count 7900/μL, MCV 85 fl.Neurological examination and Psychomotor development revealed normal findings.In the clinical follow-up we could observe normal phases of psychomotor development, linguistic development, cognitive development and relational and socialization skills.When she was 8 years old an attempt to suspend the treatment for about two and a half months was performed, since the parents asked to do it under clinical supervision.This caused the appearance of pancytopenia . Therefore, IM hydroxocobalamin needed to be restarted sine die.Menarche started at the age of 10 years and half. In the last pediatric examination at the age of 16 years, normal growth and development were observed .Cardiovascular and neurological examinations, including Electrocardiogram (ECG) and Electroencephalogram (EEG), were normal. Dual-energy X-ray absorptiometry was within normal limits. Blood tests showed a persistent normalization of the CBC and related indices: RBC 4,570,000/μL, Hb 14.8 g/dL, MCV 88 fL,WBC 6600/μL, platelets 261,000/μL.To clearly demonstrate the TC deficiency, we analyzed TCN2 gene to assess possible mutations. At the age of 29 years the patient underwent genetic diagnosis. The TCN2 gene (NM_000355.3) was analysed by Sanger sequencing using genomic DNA purified from patient blood. A deletion, designed c.1115_1116delCA (rs1355421014) was identified in homozygosity in exon 8. At protein level, this variation is predicted to cause the frameshift p.Q373GfsX38 resulting in a truncated protein. The mutation has a frequence of 0.000008  or 0.0000  and it is scored as pathogenic in ClinVar.Patient parents screened for the mutation were found both at the heterozygous status.At present time in 2021 the patient, who got a university degree, is a 31-years old healthy lady taking 1 mg of IM hydroxocobalamin once a week.The timeline of events of the case report is summarized in Table The majority of genes that could explain variation in vitamin B12 concentrations are reported from Caucasian population studies .TC deficiency is a rare and potentially lethal autosomal recessive disease with an early infantile onset and the following clinical and laboratory features: failure to thrive, weakness, diarrhoea, pallor, anemia, pancytopenia, agammaglobulinemia.This syndrome may resemble a neonatal leukemia or a severe combined immunodeficiency disease , 16. DifTC deficiency patients usually present with megaloblastic anemia of variable severity. A delay in diagnosis and treatment are associated with life threatening neurological and hematopoietic complications, which can also be fatal , 17.Patients might show isolated elevation in methylmalonic aciduria (MMA), whereas others show combined increase in circulating MMA and homocysteine .12, malabsorption of folate). Aminoaciduria highlighted the presence of homocysteine and the absence of methylmalonic acid, leading to the exclusion of a deficiency of methylmalonyl-CoA mutase. These findings and the presence of anemia, that is not caused by homocisteinuria alone, lead us to hypothesize the diagnostic suspicion of TC deficiency initially described in 1971 [In our patient there was a family history of a similar syndrome that included pancytopenia. The blood concentrations of folate and cobalamin were normal excluding these kinds of deficits, where they are low  together with c.501_503delCCA [She reportedly reached 32 years of age and had a normal life, but she encountered difficulties in her studies and at the age of 6, 7, and 13 years her intelligence quotient (IQ) was of 62 in Wechsler scale full. She presented progressive blurred vision and at 21 years retinopathy with partial blindness intellectual disability and depression at the age of 32 years  .In our patient, the TC deficiency suspicion, before genetic screening that was performed when she was 29 years old, was indirectly confirmed by the clear response to empiric treatment with IM hydroxycobalamin once a week starting at 45 days, that provoked disappearance of vomiting, diarrhea and normalization of the hematological picture.12 supplement to use , the dose, the administration method , the frequency (weekly vs monthly) and the duration of administration, the treatment monitoring period and follow-up.At present there are no guidelines on the treatment of TC deficiency regarding the form of BIM administration of 1 mg of hydroxocobalamin or cyanocobalamin once a week for a lifetime appears to be the most suitable treatment regime, according to the observational data reported in a series of 30 patients , that waex adiuvantibus in our patient by the observed appearance of pancytopenia, following the attempted suspension of IM hydroxycobalamin at 8 years of age. The timely resumption of treatment  normalized the hematological picture.The working hypothesis that such a treatment regime can be correct is confirmed , when treated aggressively, appears to be associated with an overall favourable outcome.TC deficiency is a rare autosomal recessive inborn error of metabolism , whichIn conclusion, our case report highlights that early detection of TC deficiency and early initiation of aggressive IM with hydroxocobalamin treatment is likely associated with disease control and a favorable outcome, the temporary suspension of the therapy lead to the appearance of pancytopenia. The timely reintroduction of the treatment after about two and a half months of suspension was necessary for the renormalization of CBC. Therefore, continuing treatment sine die appears to be beneficial.Finally, genetic counseling should be provided to affected families. Neonatal screening could be very useful in the early diagnosis of the syndrome: some studies suggest that prenatal diagnosis of TC deficiency is possible by means of measuring TC production in amniotic-fluid cells .The treatment is easily administered, does not give adverse events and overall is well tolerated and safe.


Few clinical trials have investigated the prevention of radiographic progression in children with juvenile idiopathic arthritis treated with antirheumatic drugs. This study aimed to investigate radiographic progression in patients with systemic juvenile idiopathic arthritis (sJIA) and patients with polyarticular-course juvenile idiopathic arthritis (pcJIA) treated with the anti–interleukin-6 receptor antibody tocilizumab for 2 years in the TENDER and CHERISH randomized controlled trials, respectively.Standard radiographs of both wrists and both hands in the posteroanterior view were obtained within 4 weeks of baseline and were repeated at weeks 52 ± 4 and 104 ± 4 in both trials. All films were scored by two independent readers using the adapted Sharp–van der Heijde (aSH) and Poznanski scoring methods. Although the Poznanski score indicates bone growth limitation or cartilage growth decrease, which are not the same as joint space narrowing in rheumatoid arthritis, its change reflects damage to cartilage. Therefore, impairment in the Poznanski score as well as the aSH score was considered as a measure of structural joint damage. Radiographic progression was defined as worsening of radiographic scores beyond the smallest detectable difference.Poznanski and aSH scores were available at baseline and at one or more postbaseline time points for 33 and 47 of 112 sJIA patients and 61 and 87 of 188 pcJIA patients, respectively, providing a representative subset of the study populations. The inter-reader and intra-reader agreement intra-class correlation coefficient was > 0.8. Median baseline Poznanski and aSH scores, respectively, were − 2.4 and 24.6 for sJIA patients and − 1.5 and 8.0 for pcJIA patients. Compared with baseline, aSH scores remained stable for all sJIA patients at week 52, whereas 9.4% of sJIA patients had radiographic progression according to Poznanski scores at week 52; at 104 weeks, radiographic progression according to aSH and Poznanski scores was observed in 5.4% and 11.5%, respectively. In pcJIA patients, radiographic progression from baseline at 52 weeks and at 104 weeks was 12.5% and 2.9%, respectively, using aSH scoring and 6.5% and 4%, respectively, using Poznanski scoring.Tocilizumab may delay radiographic progression in children with sJIA and children with pcJIA.NCT00642460 ; CHERISH, NCT00988221 Trial registration numbers and dates: TENDER,  Juvenile idiopathic arthritis (JIA) encompasses a heterogeneous group of immune-mediated, chronic, noninfectious joint diseases commencing before patients are 16 years of age . Joint sTocilizumab is a humanized anti–human interleukin-6 (IL-6) receptor-alpha antibody that inhibits IL-6 signaling , 18. BasThe present analysis was conducted to investigate the effect of tocilizumab on radiographic progression for up to 2 years in patients with sJIA or pcJIA from the TENDER and CHERISH phase 3 randomized controlled trials.Eligibility criteria have been published , 20. BriThe sJIA trial was conducted across 43 centers in 17 countries, and the pcJIA trial was conducted at 58 centers in 15 countries. All centers are part of the Paediatric Rheumatology International Trials Organisation (PRINTO)  or the PStandard radiographs of both wrists and both hands in the posteroanterior view were obtained for a subset of patients who consented to X-ray within 4 weeks of baseline and again at week 52 (± 4 weeks) and at week 104 (± 4 weeks). All radiographs were scored independently by two pediatric rheumatologists  with > 10 and > 20 years of experience, respectively, in clinical and radiographic assessment of children with JIA using the adapted Sharp–van der Heijde (aSH)  and PoznCalculation of aSH scores was based on assessment of 15 areas for joint space narrowing (JSN) and 21 areas for erosion in each hand and wrist . For eacThe Poznanski method is based on measurements of the radial metacarpal (RM) width, which is the distance from the base of the third metacarpal bone to the midpoint of the distal growth plate of the radius, and the maximum length of the second metacarpal bone (M2). Although the Poznanski score indicates bone growth limitation or cartilage growth decrease, which are not the same as joint space narrowing in rheumatoid arthritis, its change reflects damage to cartilage. Therefore, impairment in the Poznanski score as well as the aSH score was considered as a measure of structural joint damage. For each wrist, the number of standard deviations between the expected and the observed RM width for the measured M2 length was calculated according to the formulae of Poznanski et al. . The RM/Importantly, the radiographic scores obtained with the two methods used in our study go in opposite directions. With the Poznanski score, a decrease is abnormal, whereas with the aSH score an increase is abnormal.Key radiographic end points were change from baseline in radiographic scores, proportion of patients without radiographic progression from baseline to weeks 52 or 104, and relationship between radiographic progression and clinical response.Radiographic progression at 1 and 2 years was determined by subtracting baseline scores from week 52 and 104 scores. Radiographic progression for aSH and Poznanski scores was defined using the smallest detectable difference (SDD) and the zero value  28]. Po. Po28]. Change from baseline to week 104 in aSH score was analyzed for patients in the pcJIA radiographic population by stratifying patients according to baseline methotrexate use (yes/no), baseline glucocorticoid use (yes/no), previous biologic use (yes/no), disease duration (< 2 years/≥ 2 years), and rheumatoid factor (positive/negative/missing).Radiographic end points were evaluated for the entire radiographic population of the sJIA trial, whereas analysis of the pcJIA trial focused on the subgroup of radiographic patients who received tocilizumab continuously throughout the study (those randomly assigned to receive tocilizumab during the double-blind period [continuous TCZ population]). Some radiographic analyses were performed for the entire radiographic population . The aSH population included all patients who received a dose of tocilizumab and had a baseline and ≥ 1 postbaseline (week 52 or week 104) score, with data at weeks 52 and 104 summarized for patients who received tocilizumab continuously. The Poznanski population included all patients who received ≥ 1 dose of tocilizumab and had ≥ 1 postbaseline (week 52 or week 104) score, with data at weeks 52 and 104 summarized for patients who received tocilizumab continuously.Missing readings of areas for JSN or erosion in the aSH score, because of either absence of a particular area due to incomplete ossification  or technical inadequacy of the X-ray film, were imputed using average readings from other readable areas. If readings from an entire hand or wrist were missing, erosion, JSN, and total adapted aSH scores were set to missing. No imputation was used for missing Poznanski score data or other analyses.Changes from baseline in aSH and Poznanski scores were analyzed using the Wilcoxon signed rank test. Correlation between aSH and Posnanski scores was assessed using Pearson and Spearman correlations. Annualized rates of progression were calculated as the change from baseline of each follow-up visit, divided by the number of days between the two assessments, and multiplied by 365.25.In total, 112 patients were enrolled in the sJIA trial and received ≥ 1 dose of tocilizumab. Baseline and ≥ 1 postbaseline aSH scores and Poznanski scores were assessed for 47 and 33 patients, respectively, from 25 of 43 (58%) centers in 14 of 17 (82%) countries. The aSH population included 45 and 37 patients at weeks 52 and 104, respectively, and the Poznanski population included 32 and 26 patients at weeks 52 and 104, respectively..In total, 188 patients were enrolled in the pcJIA trial and received ≥ 1 dose of tocilizumab. Baseline and ≥ 1 postbaseline aSH scores and Poznanski scores were assessed for 87 and 61 patients, respectively, from 25 of 43 (58%) centers in 14 of 17 (82%) countries. In the pcJIA study, 45 patients in the aSH population and 35 patients in the Poznanski population received tocilizumab continuously (continuous TCZ population). The aSH population included 40 and 35 patients at weeks 52 and 104, respectively, and the Poznanski population included 31 and 25 patients at weeks 52 and 104, respectivelyFor both studies, baseline demographics and disease characteristics of patients who underwent radiographic assessments were similar to those of the total populations , 20 Tab.Table 1Notably, the Poznanski score could not be assessed in patients with advanced carpometacarpal erosions that made it difficult to define the bone ends or in older children who had apparent radiographic closure of the second metacarpal growth plate. These phenomena did not preclude assessment of the aSH score, which explains why patients with available aSH scores outnumber those who had the Poznanski score calculated.Inter-reader and intra-reader agreement, as assessed by intra-class correlation coefficient (ICC) for radiographic scores, was good for both JIA subtypes and for both scoring methods, with all ICCs > 0.8 and most > 0.9 (results not shown). Agreement between readers was confirmed by Bland and Altman plots Figs.  and 2.FP = 0.302) and 104   and 104  or in JSN scores at weeks 52  and 104  (Table P = 0.003), but change from baseline to week 104 was not significant  ; The SDD for aSH score progression was 23.6 at week 52 and 27.4 at week 104. Using the SDD of 23.6, 100% and 94.6% of tocilizumab-treated patients showed no aSH progression at weeks 52 and 104, respectively  response criteria , did notP = 0.004) in Poznanski score was observed from baseline to week 104, and a positive, though not significant, change of 0.26 (P = 0.077) was detected from baseline to week 52 (Fig. There were nonsignificant changes in total, erosion, and JSN aSH scores from baseline to weeks 52 and 104 (Fig. Based on week 104 data, SDD values were 9.76 and − 0.88 for aSH and Poznanski progression, respectively. Using these SDDs, 87.5% and 97.1% of patients in the continuous TCZ population experienced no aSH progression at weeks 52 and 104, respectively, and one patient experienced progression after 2 years of tocilizumab treatment (Fig. Using the SDD of − 0.88, 96.0% of patients in the continuous TCZ population experienced no Poznanski progression at week 104 compared with 93.5% at week 52 (Fig. Mean annualized progression rates from baseline to week 104 were calculated in the all TCZ population. Consistent with the continuous TCZ population, annualized progression rates for total, erosion, and JSN aSH scores were − 0.75, 0.31, and − 0.95, respectively, and the Poznanski score was 0.19, indicating a lack of radiographic progression in the entire radiographic population over 2 years. The JIA ACR response rate was higher in patients without radiographic progression than in the entire study population at weeks 52 and 104 (Fig. zero value methods. There was little change in radiographic scores of structural joint damage during 2 years of treatment. This is the first time that two separate phase 3 randomized controlled trials in patients with sJIA or pcJIA have been evaluated for X-ray progression within the framework of a pediatric investigation plan for the European Medicines Agency and a pediatric study plan for the US Food and Drug Administration.TENDER and CHERISH are the first trials in JIA to include radiologic analysis. Most patients with sJIA and pcJIA treated with tocilizumab experienced no radiographic progression assessed by SDD and The present analysis was not controlled; therefore, the possibility that patients had less aggressive illness than those who were not included in the analysis cannot be excluded. However, all JIA outcome measures at the start of treatment were comparable between patients included and excluded from the analysis. Furthermore, all patients had severe, long-standing systemic or polyarticular-course disease that was not controlled despite previous use of at least one traditional second-line agent, and all had bilateral wrist involvement. Patients with sJIA and pcJIA are more likely to develop destructive disease –32, and The observed changes in aSH score in pcJIA patients and the change in Poznanski score in sJIA and pcJIA patients suggest that at least some patients experienced improvement of articular damage. Altogether, these findings indicate that tocilizumab is potentially capable of halting the progression of radiographic joint damage in children with JIA. Improvement in the rate of radiographic progression in children with JIA is not surprising because the regenerative capacity of articular cartilage is better in growing children than in adults .These observations corroborate previous reports showing amelioration of radiographic joint changes with tocilizumab treatment in children with systemic JIA , 25. CouGiven the high degree of concomitant methotrexate use among patients in the sJIA and the pcJIA studies, our data do not establish whether combination with methotrexate could enhance the effectiveness of tocilizumab. Literature is inconclusive on the effects of methotrexate on joint destruction, although two studies have suggested that this medication might have disease-modifying potential , 14, 35.We used aSH and Poznanski scores to measure radiographic progression in the present analysis because these methods were specifically developed and validated for use in patients with JIA , 6, 15. Limitations of the present analysis include the small number of patients in the radiographic populations of both trials. Limited availability of radiographs in the patient sample was due to nonparticipation of some study investigators in the radiographic study or lack of consent for radiographic assessments. Both scoring systems used to quantify radiographic damage evaluated wrist and hand joints, and it is unclear whether changes in these joints sufficiently reflect damage to large weight-bearing joints . Some raIn conclusion, this analysis of radiographic data from the TENDER and CHERISH trials suggests that tocilizumab may prevent radiographic progression in children with sJIA and children with pcJIA. However, because the natural course of arthritis in children is heterogeneous and a control group not exposed to tocilizumab was not available, we cannot draw definitive conclusions regarding the ability of tocilizumab to halt or diminish radiographic progression in JIA, and our findings should be confirmed in future studies.Additional file 1 Supplemental Methods. Inter-reader reliability methods for assessment of radiographs. Radiographic progression using cutoff of zero.


A lower bound for the system’s adhesion strength, based on limited measurements of interfacial separation between PAN and Cu using mechanically applied strain, is 0.48 J/m2.Electrospun polymer fibers can be used as templates for the stabilization of metallic nanostructures, but metallic species and polymer macromolecules generally exhibit weak interfacial adhesion. We have investigated the adhesion of model copper nanocubes on chemically treated aligned electrospun polyacrylonitrile (PAN) fibers based on the introduction of interfacial shear strains through mechanical deformation. The composite structures were subjected to distinct macroscopic tensile strain levels of 7%, 11%, and 14%. The fibers exhibited peculiar deformation behaviors that underscored their disparate strain transfer mechanisms depending on fiber size; nanofibers exhibited multiple necking phenomena, while microfiber deformation proceeded through localized dilatation that resulted in craze (and microcrack) formation. The copper nanocubes exhibited strong adhesion on both fibrous structures at all strain levels tested. Raman spectroscopy suggests chemisorption as the main adhesion mechanism. The interfacial adhesion energy of Cu on these treated PAN nanofibers was estimated using the Gibbs–Wulff–Kaischew shape theory giving a first order approximation of about 1 J/m The filamentary polymer architectures traditionally engendered by the electrospinning process have enabled the development of functional material systems and devices that are underpinned by design manipulations at micron and nanometric scales. Leveraging the relative ease of polymer chemistry modification, a high surface-area-to-volume ratio as well as process flexibility , electroMetallic nanostructures are known to possess striking magnetic, thermal, electronic, and surface properties markedly distinct from those of the bulk . HoweverIn principle, the contrast between the intrinsic strong cohesive energy of metallic species and the weak van der Waals forces that hold together polymer macromolecules strongly influences interfacial interaction, creating weak adhesion in most metal–polymer systems . As a reIn this paper, we utilize a bottom-up method to grow well-defined, discretely distributed copper nanocubes on aligned polyacrylonitrile (PAN) nanofibers and microfibers based on electroless deposition from aqueous solutions. The interesting mix of hydrophilicity, chemical stability, and strong mechanical performance of electrospun PAN fibers has enabled their widespread adoption and adaptability as functional scaffolds for diverse application designs ,18. FurtW = 150,000, Sigma-Aldrich, St. Louis, MI, USA)/1 wt.% acetone  solution in N,N-dimethylformamide  using a Spraybase vertical electrospinner configuration with parallel electrodes. The solution was dispensed through a 24-gauge blunt needle at a flowrate of 0.20 mL/h towards grounded electrodes in a configuration, as demonstrated in Nanometric aligned PAN fibers were electrospun from a 10 wt.% PAN (M2CO3) for three minutes. Next, samples were immersed in 1 M solution of sodium hydroxide (NaOH) at a temperature between 45–50 °C for fifteen minutes. The aligned fibers were seeded with silver species to catalyze subsequent copper deposition. An amount of 200 µL of ammonia solution (NH4OH) was added to a 10 mL solution of 0.01 M AgNO3 under constant stirring. Lastly, a 5 mL solution containing 10 wt.% glucose was added to the solution and stirred for 1 min. To prevent possible photocatalytic reduction of silver, the reaction vessel was wrapped with aluminum foil. Samples were immersed in the silver baths for 1 min and subsequently rinsed with a copious amount of deionized water. There was no apparent change in color or translucence of the samples (due to low number density of fibers). A fresh silver bath was prepared for each sample. All seeding baths were operated at room temperature under quiescent conditions. The chemicals used were reagent grade.The fibers were permanently affixed to the nylon washers (22 mm × 14 mm × 1.2 mm) with epoxy glue and allowed to cure for 24 h. To mitigate or eliminate handling-induced fiber deformation during the deposition procedure, samples were affixed to L-shaped strips wrapped with carbon adhesive tape that enabled easy transportation between baths. The deposition protocol is shown schematically in 4.5H20) and 0.6 g of disodium ethylene diamine tetraacetate (Na2H2EDTA) were dissolved and mixed in 20 mL of deionized water. An amount of 280 μL of formaldehyde (HCHO) was added into the solution. Drops of sodium hydroxide solution (0.92 g NaOH + 20 mL H2O) were pipetted into the solution to adjust the pH of the electroless bath to ∼12.5, measured using a pH meter . The silver-seeded fibers were immersed for 15 min at room temperature also under quiescent conditions. The clear blue color of the bath gradually turned pale green, which subsequently became deep green with attendant turbidity, signifying homogenous precipitation of copper in the solution. Afterwards, samples were rinsed in deionized water and air-dried. A fresh bath was prepared for each sample.0.1 g of copper salt  at a cross head speed of 0.5 mm/min in displacement control. The samples (the washers and fiber mats) were gripped by specialized serrated jaws to prevent slippage during tensile loading. Nominal gage length of the samples was 14 mm. Interfacial adhesion under distinct strain levels of 7%, 11% and 14% strain was systematically investigated. After each test, samples were extracted and prepared for imaging in a methodical manner.Post mortem imaging was carried out on the FEI Nova NanoSEM scanning electron microscope equipped with an Everhart-Thornley detector (ETD) operated in secondary electron detection mode. Prior to imaging, all samples were sputter-coated with a thin (≈2 nm) layer of platinum using a Cressington (208HR) sputter coater at a plasma current of 40 mA for 60 s. Fiber dimensions and orientation distribution were measured using ImageJ . Energy dispersive spectroscopy (EDS) was performed on the same microscope using an Oxford INCA energy system with a 30 mm window. Raman spectroscopy was conducted to provide insights into modifications in fiber chemistry and its implications for interfacial adhesion between the nanocubes and PAN fibers; Raman shifts were collected using a Renishaw inVia Raman microscope with a 532 nm laser (at 1% power) with a grating of 2400 L/mm and an objective lens of 100×.Both electrospinning configurations for fiber alignment, that is, parallel electrode and rotating drum configurations, yielded good directionality as shown in microstructures, Both fiber cross-sections were uniform along fiber length, with no occurrence of beads. Average fiber diameters and standard deviation for the nanofibers and microfibers were 336 ± 46 nm and 1.086 ± 0.103 μm, respectively. The distribution’s sharp peak about the median angles  for both microfibers and nanofibers is indicative of good alignment. Taking into consideration these angular offsets, approximately 66% of the nanofibers and 97% of the microfibers were within ± 10° of their major axial orientation. These estimates were made based on corresponding area fractions in the distribution curve. The greater deviation in alignment observed in the nanofibers can be attributed to a low flexural resistance, which is a function of their thin cross-sections, inducing much greater angular offsets than seen in the microfibers; indeed, global alignment is apparent, but local curvatures in individual fibers impede full rectilinear lengths as displayed in the microfibers.p-value > 0.05—see Metastable electroless solutions offer flexibility in compositional control, operational parameters, morphology, and size modulation of nanocrystals in metal deposition procedures. In addition, the necessary immersion of substrates into the solution aids homogenous deposition—of discrete nanoparticles or consolidated nanoparticle films—for a non-planar substrate geometry as presented by electrospun polymer fibers. Although the nanocubes predominantly evolved with a cube face contacting the supporting PAN fibers, the intrinsic cylindrical shape of the fibers altered the original planar geometry of a cubic facet, introducing commensurate curvature  was performed for elemental identification. Traditionally, when a metal film is supported by a polymer substrate, and the resulting composite structure is subjected to a tensile load, the more compliant polymer material can suppress strain localization, a prelude to delamination, in the metal film, ultimately facilitating a congruous deformation behavior . AlternaDifferences in polymer macromolecule configuration in PAN microfibers and nanofibers influences overall micromechanical deformation behavior or features with respect to stress/strain transmission during tensile loading. Chiefly, because their higher aspect ratio makes them more amenable to applied electrostatic drawing effects during processing, nanofibers possess an enhanced chain orientation or alignment along the fiber axis to a greater extent than microfibers ,33,34. FWith respect to fiber deformation, the microfibers did not exhibit necking phenomena during tensile loading, in contrast to observations of the nanofibers. Rather, craze formation  and atteGiven the strong interfacial adhesion of the nanocubes on both fiber architectures, Raman spectroscopy was used to probe possible modifications in fiber surface chemistry after copper nanocube deposition. The obtained spectra are shown in −1. This is also an IR active band, indicative of cyano functional groups [−1 and 1580 cm−1 can be seen, respectively classified as the D and G bands of carbonaceous materials [2sp bonded atoms in ring and chain configuration on the fiber surface, suggesting a conversion of initial linear or aliphatic PAN chain segments to cyclical structures; while the D-band is indicative of the presence of structural disorganization or the existence of foreign atoms (or molecular entities) in surface PAN macromolecules [D/IGI), for the nanofibers and microfibers is 1.15 and 1.30, respectively, indicating that the PAN macromolecules on the microfiber surface possess a slightly higher degree of disorganization than the nanofibers.The spectrum for pristine fibers is shown in l groups . Spectraaterials . At the olecules ,39,40. FAltogether, these chemical and structural changes imply the existence of strong chemical bonds at the nanocube-fiber interface, facilitating the strong adhesion observed in both nanofibers and microfibers. It is also plausible that the bottom-up synthesis approach for the cubic nanostructure formation helped optimize chemical interaction between the treated fiber surface and evolving crystals during growth.The elastic mismatch between a stiff material affixed on flexible substrate imposes shear stress states at the interface that can induce the nucleation and propagation of cracks. Based on the mechanics of interfacial fracture, a resultant crack length upon debonding can be used to approximate the adhesion energy following an analytical model developed by Sun  for the First, the energy release rate of a crack represents the total energy released during crack propagation per unit increase in crack size . In othe2, where the standard deviation represents uncertainties resulting from cube and crack dimension measurements. The limitations of identifying individual particles that fit this geometry at any given strain make reproducible measurements of this method challenging.An estimate of the adhesion energy was made based on microstructural evidence for nanocube delamination at an applied global strain of 11% as provided by i, and β), depicted in j* taken to be parallel to a crystal plane j, which in turn holds a parallel relationship with the top equilibrium face i. j*. Consequently, as the adhesion energy increases, crystal truncation increases and vice versa [j-th plane in the cubic nanostructures. Hence, in our analysis, given the relatively shallow curvatures, we have assumed a proximate crystal plane that is tangential to the apex of the interfacial curvature. Consequently, Alternatively, an approximation of the adhesion energy can be obtained through analysis of resultant particle shapes taking into consideration that these shapes are derived from the equilibration of surface free energies with the immediate microenvironment, including the substrate. This method has the advantage of having many particles, which can be evaluated under identical deposition conditions. Firstly, at a constant volume, the final particle shape should be derived from minimization of the total surface free energy . Accordice versa . A usefuFor the computation of the adhesion energy, the approximate elevation profiles of copper nanocubes exhibiting distinct levels of truncation on the nanofibers are shown in 2 [Previous micrographs have provided strong evidence to infer that the equilibrium shape of the copper nanostructures, if isolated or unattached, is a cubic structure. However, in a stricter sense, accurate structural derivation of the equilibrium shape of the free particle from the particle shape as modified by the substrate can only be made if it contains a Wulff point that also represents a center of inversion symmetry . Otherwis  copper facet as obtained with molecular dynamics (MD) simulations (0.51 ± 0.02 J/m2) [2) using a four-point bend testing [2) using in-situ tensile tests inside a scanning electron microscope [For the Gibbs-Wulff-Kaischew shape theory, inaccuracies in the measurement of copper nanocube dimensions represent the major source of uncertainties in the adhesion energy quantification. Nevertheless, with the aforementioned factors as qualifications, the adhesion energy as predicted by the strain energy release model is in good agreement with values predicted for styrene-02 J/m2) . In addi testing  and for croscope . Altoget2 and a lower bound of 0.48 J/m2, respectively, for copper nanocubes on PAN nanofibers with diameters on the order of 300 nm.Discrete nanostructures on electrospun fibers are essentially consolidated structures of two distinct material classes. Ascertaining the integrity of interfacial adhesion is pivotal for a sustained functional performance. For the purpose of studying interfacial adhesion, we have synthesized well-defined copper nanocubes on PAN nanofibers and microfibers via a solution-phase, bottom-up synthesis method. The nanocube morphology represents a model nanostructure for the study of interfacial adhesion of metallic nanostructures on electrospun fibers, as it affords a definite contact area for microstructural assessment. Micromechanical deformation of these composite structures has revealed the robust adhesion of copper nanocubes on the nanofibers and microfibers. Raman shifts provide strong evidence for chemisorption as the primary anchoring mechanism, and this is believed to be optimized by the fact that crystal growth was based on a solution-based deposition protocol. Finally, the adhesion energy computation using the Gibbs–Wulff–Kaischew shape theory and energy release rate model give a useful first order approximation value of about 1J/m


Current guidelines pertaining to diagnosing macrocytic anemia in association with vitamin B12 and folate deficiency recommend that vitamin B12, folate, homocysteine, and methylmalonic acid assays should be assessed concurrently due to their close relationship in metabolism. We aimed to investigate the completion of these assays in local clinics and hospitals without in‐house clinical laboratories in Korea.We retrospectively reviewed data from the laboratory information system between September 25, 2017, and June 30, 2019, to investigate usage rates of vitamin B12, folate, homocysteine, and methylmalonic acid assays in patients with macrocytic anemia.During the study period, 14 894 Korean adults among 109 524 (13.6%) total hemoglobin‐tested subjects underwent concurrent erythrocyte mean corpuscular volume (MCV) tests. Among these 14,894 adults, 265 (1.2%) from 94 local clinics or hospitals without in‐house clinical laboratories in Korea had macrocytic anemia. Furthermore, among these 265 adults, only one woman underwent serum vitamin B12 and folate assay and one man underwent serum homocysteine testing during the study period. No patients among the 265 individuals with macrocytic anemia received erythrocyte folate or methylmalonic acid testing .The results of this study provide basic information regarding utilization rates of assays in association with vitamin B12 and folate deficiency. Making more data available is expected to improve rates of testing in patients with macrocytic anemia in local clinics and hospitals without in‐house clinical laboratories in Korea. Statistical analyses were executed using Microsoft Excel 2019 and MedCalc version 19.1.5 .We retrospectively reviewed the test results from Korean adults who underwent hemoglobin (Hb) testing between September 25, 2017, and June 30, 2019, through the laboratory information system of Green Cross Laboratories. Green Cross Laboratories, one of the largest referral clinical laboratories in South Korea, provides clinical specimen analysis services including serum folate, erythrocyte folate, serum homocysteine, and methylmalonic acid  tests; complete blood count findings including Hb; and erythrocyte mean corpuscular volume (MCV) to clinics and hospitals nationwide. Missing data for age or sex were excluded. All data were anonymized before being adopted for statistical analysis. The protocol of this study was approved by the institutional review board (IRB) of Green Cross Laboratories (IRB GCL‐2020‐1014‐01). A waiver of informed consent was approved by the IRB since the use of a waiver would not adversely affect the rights or welfare of the study subjects because the study was retrospective and involved no more than minimal risk to the subjects. The study was conducted in accordance with the Declaration of Helsinki. Hb and MCV were analyzed using Sysmex XN 9000 analyzers. The reference intervals of Hb and MCV for adults were as follows: 11.0 to 15.0 g/dL of Hb in women, 13.0 to 17.0 g/dL of Hb in men, and 80 to 102 fL for MCV in all adults. Serum and erythrocyte folate were determined using the Elecsys Folate assay traceable to World Health Organization (WHO) International Standard (IS) 03/178 on Cobas 8000 e801 analyzers . Serum homocysteine level was determined using the AutoLab Homocysteine assay  on Cobas 8000 c702 analyzers  traceable to standard reference material 1955. Methylmalonic acid concentration in serum, plasma, random urine, and 24‐hour collected urine samples was measured using gas chromatography‐mass spectrometry analyzers  with isotope‐labeled internal standards, which were developed and validated by Green Cross Laboratories (detailed information of the method available upon reasonable request to the corresponding authors). The accuracy of the assays during the study period was maintained using the Proficiency Testing/Quality Management program of the College of American Pathologists survey in the United States. Age among the overall subjects was not normally distributed. The numbers and percentages of each test considered were presented in this study. Mann‐Whitney U test when appropriate was adopted to compare age in sex groups. All 3P > .05). Among 265 patients with macrocytic anemia, only one woman underwent both serum vitamin B12  and folate  assays. Separately, one man from a different clinic underwent serum homocysteine testing during the study period  and his results were within the reference interval. No patients among 265 patients with macrocytic anemia underwent folate in erythrocytes or methylmalonic acid testing  received concurrent erythrocyte MCV tests. Among these 14 894 adults, 265 adults  aged 23.9 to 95.4 years (median: 68.8 years) had macrocytic anemia, showing Hb findings below the lower limit of the reference interval for each sex and gender and MCV results of greater than 102 fL, from among 94 local clinics or hospitals without in‐house clinical laboratories in Korea. Age was not significantly different among the patients with macrocytic anemia  and 1.1% (1/94 clinic each) for total clinics and hospitals while those rates of erythrocyte folate and methylmalonic acid tests in varied specimen types were 0.0%. During the study period, methylmalonic acid assays using varied specimen types were conducted by university and hospitals with own clinical laboratories. Although the scope of this study was to examine test utilization in local clinics without their own clinical laboratories, during the study period, 259 patients were tested for methylmalonic acid using 24‐hour urine and 13 of them presented elevated results , 2,361 patients were tested for methylmalonic acid by random urine and 472 of them had elevated results , 144 patients were tested for methylmalonic acid in plasma and 20 of them had elevated results , and 151 patients were tested for methylmalonic acid in serum and 28 had elevated results  from university hospitals and other hospitals with their own clinical laboratories. In South Korea, the test utilization committee of the Korean Society for Laboratory Medicine developed and published guidelines for test utilization in 2020 that mainly focus on testing under hospital conditions.Although the exact number of cases of macrocytic anemia was not available, using the Korean Classification of Disease code on the Healthcare Bigdata Hub developed by the Health Insurance Review and Assessment Service in South Korea, about 5,000 patients/year with vitamin B12 deficiency anemia , about 3,500 patients/year with folate deficiency anemia (D52), and about 5,500 patients/year with other nutritional anemia (D53) were cared for from 2017 to 2019. When reviewing results pertaining the D531 code for other megaloblastic anemia (not otherwise specified), more than 1,700 patients/year visited clinics and hospitals from 2017 to 2019. Considering the underutilization of vitamin B12, folate, homocysteine, and methylmalonic acid assays in association with vitamin B12 and folate deficiency in adult patients with macrocytic anemia in local clinics and hospitals identified in this study, the disease burden of vitamin B12 and folate deficiency may be altered when tests became more widely adopted.The limitation of this study is that certain clinical data including a detailed history, physical examination, and other laboratory and image studies associated with macrocytic anemia or vitamin B12 or folate deficiency were not gathered. However, this study provides valuable information about rates of test utilization in local clinics and hospitals without in‐house clinical laboratories in South Korea. Further, the results of this study offer a basic overview regarding conducting assays in association with vitamin B12 and folate deficiency to improve test utilization in patients with macrocytic anemia in local clinics and hospitals without in‐house clinical laboratories in Korea. Considering that the most significant clinical effect of properly performed laboratory tests is improved patient outcomes, future studies are needed to clarify the clinical impact of appropriate test utilization of vitamin deficiency assays.The authors declare no conflict of interest.All authors contributed to article preparation. Rihwa Choi contributed to conceptualization; Rihwa Choi contributed to data curation; Rihwa Choi contributed to formal analysis; Sang Gon Lee and Eun Hee Lee contributed to funding acquisition; Rihwa Choi, Youngju Oh, Mi‐Jung Park, Youngrae Kim, and Serim Kim contributed to investigation; Rihwa Choi, Youngju Oh, Mi‐Jung Park, Youngrae Kim, and Serim Kim contributed to methodology; Sang Gon Lee contributed to resources; Sang Gon Lee and Eun Hee Lee contributed to supervision; Rihwa Choi contributed to validation; Rihwa Choi contributed to writing—original draft; Rihwa Choi, Sang Gon Lee, and Eun Hee Lee contributed to writing—review and editing. All authors read and approved the final article.


Introduction: The transverse myocutaneous gracilis (TMG) flap has become a popular and reliable alternative for autologous breast reconstruction. Initially described as a valuable tissue source for women with low body-mass index, indications nowadays have widely expanded. The Western civilization demographic development with its aging population and the steady growing average BMI has led to increasing breast reconstructions with TMG flaps in overweight and aged individuals. Patients and Methods: A total of 300 TMG free flaps for unilateral autologous breast reconstruction were evaluated in the form of a retrospective double center cohort study. Data extraction, study group formation and statistical analysis , Pearson’s chi-squared statistical analysis and relative risk calculation) were done specifically to evaluate age and BMI as risk factors for postoperative complications and outcome. Results: No significant differences in patients’ age and BMI in the complication groups compared to the no-complication group could be found. No significant difference regarding the occurrence of complications could be found in any of the formed risk-groups. No significant increase of minor-, major- or overall complications, flap loss or revision surgeries were found in the elderly patient groups or for patients with overweight. Conclusion: Age and overweight do not significantly increase the risk for postoperative complications after breast reconstructions with free TMG flaps. The findings of this study support the fact that microsurgical breast reconstruction with a free TMG flap should not solely be reserved for younger patients and females with a lower BMI. Today, microsurgical free tissue transfer represents a standard tool of plastic surgeons for breast reconstruction around the globe. While free flaps from the lower abdomen, such as the deep inferior epigastric perforator (DIEP) flap, are widely respected as the gold standard and first-line flap for microsurgical breast reconstruction, numerous valuable other flaps represent important alternatives.While sufficient abdominal fat tissue is a prerequisite for any abdominal-based tissue reconstruction, obesity and its comorbidities, such as diabetes or hypertension, are known risk factors for any surgical procedure, including microsurgery ,2,3,4.The thighs provide a popular alternative source for free flaps used for breast reconstruction offering numerous different angiosomes, which can be converted into various free flaps. A few years after its first description by Harii et al., the transverse musculocutaneous gracilis (TMG) flap became an increasingly popular alternative for free autologous tissue breast reconstruction ,6,7. ItsDue to an exceedingly aging population and with obesity becoming one of the most common diseases in the industrialized world ,12, we rDetailed risk stratification of the impact of higher age and overweight in patients having a breast reconstruction with TMG flaps has not been conducted to our knowledge so far. The aim of this study was to investigate potential risks and complications in a yet overlooked patient population receiving breast reconstructions with TMG flaps and identify a potential negative interrelation.Three hundred patients receiving a free TMG flap for unilateral breast reconstruction during the period of September 2010 to October 2020 were evaluated in the form of a retrospective double center cohort study . Data ex2, therefore falling into the overweight range according to the Center for Disease Control and Prevention Guidelines [Upon analysis, 69 patients had a BMI higher or equal to 25 kg/midelines . Ninety-idelines   was built and compared to younger patients with lower BMI. The relative risks for the different groups for the occurrence of complication events were calculated. Homogeneity concerning additional well-known, relevant comorbidities  within the study groups (sorted according to BMI and age) was analyzed, and no significant difference could be found in any of the groups formed (p < 0.05). All statistical analyses in this report were performed by use of Social Science Statistics  and GraphPad® . The study was conducted in accordance with the Ethical Principles for Medical Research involving Human Subjects of the Declaration of Helsinki.All patients were treated according to a standardized two-team approach of simultaneous flap harvest and recipient site dissection. The optimized surgical technique has been published recently . Both sep = 0.399), the average age of the minor complication group was 47.1 , and the average age of the overall complication group was 46.7 years  all compared to 47.7 years (SD 10) in the no complication group. ANOVA analysis also showed no significant differences in patients’ BMI of the groups. The average BMI of the major complication group was 22.8 kg/m2 , the average BMI of the minor complication group was 23.1 kg/m2 , and the average BMI of the overall complication group was 23.3 kg/m2  all compared to 22.7 (SD 2.9) in the no complication group. The average age for patients who had a flap loss was 46.7 years (SD 9.1), and the average BMI was 23.9 kg/m2 (SD 3.13). No significant differences compared to the “no flap-loss” group were observed (p(age) = 0.819, p(BMI) = 0.170). Average age and BMI for the patients, who required a revision surgery was 46.35 kg/m2 (SD 12.36) years and 23.21 (SD 3.01), compared to 47.5 and 22.92 in the “no revision surgery” group ((p(age) = 0.426, p(BMI) = 0.480). The overall complication rate was calculated to be 49% (147/300). One hundred fifty-three patients (51.00%) had no complication, 83 patients (27.67%) had a major complication, and 70 patients (23.33%) had a minor complication (minor and major complications were assessed separately—double count possible). Flap loss was observed in 19 cases (6%). Revision surgery was required in 72 patients (24%). The average follow-up was 2.12 years.Patient characteristics are summarized in 2, and 133 patients (44.33%) were older than 50 years. Age groups were formed by sorting into over 40, over 50 and over 60 and compared to the younger patients regarding major complications, minor complications, overall complications, flap loss and revision surgery (p = 0.267 and p = 0.408). For an example representing the elderly patient group, see Crosstabulation tables were formed analyzing specific groups of risk according to the hypothesis. In general, 69 patients (23%) had a BMI higher or equal to 25 kg/m surgery . No sign2) (p = 0.460).Complications were also categorized according to patient BMI (over and below BMI of 25 kg/m2) . Regardip = 0.349).Evaluation and assessment of potential risks and complications of microsurgical breast reconstruction procedures with free flaps have been conducted for over 20 years due to the high increase of these operations. Early on, being overweight or suffering from obesity, as well as older age, were identified as potential risk factors for complications. First, Chang et al. observed significantly higher rates of complications in overweight and obese patients having a breast reconstruction with free TRAM flaps . BesidesA study published by Torabi et al. in 2018 found advanced age to be an independent risk factor after microsurgical breast reconstruction with DIEP flaps while also reporting disadvantages for wound healing and for flap loss in patients with higher age . ContrarAbout 20 years ago, the senior authors of this article introduced the TMG flap as a standardized and valuable free flap alternative for microvascular breast reconstruction ,29. A fep = 0.02). The described mean difference of age in the described study, however, was only five years, which represents a limitation and likely points out a structural bias of the statistical analysis. Other studies  but did do not observe any significant correlations of higher complications and age. However, these studies conclude that patients with a higher maximum body mass index before their massive weight loss had significant (p < 0.01) more complications. A direct comparison of the examined massive weight loss patient collective and patients receiving a breast reconstruction, who never had comparable massive weight alterations, is therefore conflicting and should be dismissed. The comparison of thigh lifts and free TMG flap donor sites are furthermore flawed by the fact that the evaluated thigh lifts were performed in a wise pattern fashion, while the free TMG flap donor sites were always closed in a horizontal thigh lift fashion. Therefore, although thigh lift surgeries are well-known to have high risks of postoperative hematoseromas, infections, wound healing disturbances and dehiscence, with an overall 68 percent of patients experiencing at least one complication [During our literature search, we were able to identify several studies, which evaluated a correlation of complications and advanced age in cosmetic thigh-lift surgeries, which in many ways represent a similar donor site wound as a free TMG flap does. In a study by Kühn et al. , age shoy et al. , Losco ey et al. , Arthrusy et al. ) on cosmlication , these cp = 0.267 and p = 0.408). Regarding overweight, the highest relative risk calculated was 1.21 for major complications for patients with BMI over 25 kg/m2 (p = 0.460). We also formed a risk group, combining both risk factors according to the hypothesis and carried out a comparison against the most favorable patient group (<25 BMI (kg/m2) and < 50 years vs. ≥25 BMI (kg/m2) and ≥ 50 years). The highest relative risk calculation was observed for the major complications  in this comparison, but not no significant difference could be found.Although prior studies evaluated various risk factors for perioperative complications and were able to point out potential pitfalls and risks in a smaller patient sample size, a specific analysis of the risk stratification of age and increased BMI in breast reconstructions with the TMG flap has not been conducted so far. Our study group analyzed a larger-sized patient cohort of 300 TMG free flaps for breast reconstruction during the last 10 years and did an extensive evaluation to answer the question of any impact of age and overweight on complication risk. Our data analysis could not prove any significant relation of higher age and overweight to an increased rate of complications. The highest relative risks for revision surgery were observed in patients over 50 (RR 1.26) and for minor complications for patients over 60 (RR 1.5) , the overall number of patients over 70 years of age and with BMI higher than 30 (kg/m2) was relatively low for a profound statistical analysis. Thus, clear data on not only overweight (BMI >25 (kg/m2)) but obesity (BMI >30) could not be evaluated, and the highest age group was represented by sexagenarians only. Further studies with even larger sample sizes are needed to generate more powerful data in these patient groups. Furthermore, we need to point out the likely presence of a certain inclusion bias since the majority of patients qualifying for breast reconstruction with a TMG flap, in general, have a lower BMI compared to patients suitable for breast reconstruction with abdominal-based free flaps. Therefore, although the analyzed patient cohort is very large compared to most studies, the true amount of overweight patients assessed is not extensively high. With all limitations aside, we believe that this study underlines the important fact that microsurgical breast reconstructions with TMG flaps should not only be offered to younger and lower BMI patients but can safely be performed in patients with higher age and increased BMI. Although lower-abdominal-based free flaps, such as the DIEP flap, remain the gold standard in overweight and obese patients for autologous breast reconstruction, the free TMG flap offers a valuable and safe alternative when abdominal tissue is sparse, or an abdominal-based free flap is not feasible anymore.Overall, our study demonstrates that advanced age and overweight do not play an essential role as a risk factor for complications in breast reconstruction with the free TMG flap in comparison to the majority of complication assessment studies for abdominally based free flaps. The study has several limitations, including its retrospective character, which increases the risk for observational bias. Although the present analysis was performed on the highest number of consecutive free TMG flaps ever evaluated in a single cohort study (Age and overweight do not significantly increase the risk for postoperative complications after breast reconstructions with free TMG flaps. The findings of this study support the fact that microsurgical breast reconstruction with a free TMG flap should not solely be reserved for younger patients and females with a lower BMI.


The high frequency  rainfall regime A with a short rainfall duration (RD), low rainfall amount (P), and high mean rainfall intensity (Im) yielded a lower runoff depth and higher soil loss rate. Rainfall regime B with a longer RD, and a higher P and Im, however, produced higher a runoff depth and lower soil loss rate. Terraced plots had the highest runoff and soil loss reduction efficiencies of over 96.03%. Contour tillage had comparable sediment reduction efficiency to that of the terraced plots on gentle slopes (gradient less than 11.0%), while its runoff reduction efficiency was less than 13.11%. This study implies that in the Miyun Reservoir catchment and similar regions in the world, contour tillage should be promoted on gentle slopes, and the construction of terraced plots should be given ample consideration as they could greatly reduce water quantity and cause water shortages in downstream catchments.Cultivated land plays an important role in water and soil loss in earthy/rocky mountainous regions in northern China, however, its response to soil conservation measures and rainfall characteristics are still not fully understood. In the present study, 85 erosive rainfall events in 2011–2019 were grouped into three types, and the responses of runoff and soil loss to soil conservation measures and rainfall regimes on five cultivated plots with different slopes in the upstream catchment of the Miyun Reservoir were evaluated. Results found that mean event runoff depths and soil loss rates on the five plots ranged from 0.03 mm to 7.05 mm and from 0.37 t km Soil erosion is an expansive environmental problem with significant ecological implications. It is associated with on-site land degradation, off-site sediment siltation in rivers, reservoirs, and lakes, and water resource use ,2,3. Thu−2 yr−1, representing the land use type suffering from the highest soil loss in sub-Saharan Africa [9 t yr−1, contributing to around 30% of the total soil loss in China [−2 yr−1, and under conditions of extreme rainstorms even 6.94 × 103–95.89 × 103 t km−2, which is dozens of times the rate of grass and forest lands [−2 yr−1, and soil loss from the cultivated land accounts for 60–80% of the total [It is widely recognized that land use affects soil loss. Among all the land use types, sloping cultivated land usually suffers higher soil loss and acts as an important sediment source area ,5. In thn Africa . In Chinin China . On the The Miyun Reservoir is an important source of drinking water for people in Beijing, 70% of which is provided by the Miyun Reservoir ,11,12,13In the last two decades, researches on soil erosion and its response to soil conservation measures have been done in this region. At the catchment scale, studies on the effect of land use change and soil conservation measures on runoff and sediment transport dynamics have been conducted ,21,22,23Therefore, runoff and soil erosion data from 85 erosive rainfall events over five cultivated plots were used to explore the responses of runoff and soil loss to soil conservation measures and rainfall characteristics in the upstream catchment of the Miyun Reservoir, northern China. The specific aims were to (i) compare the differences in surface runoff and soil loss under different soil conservation measures, (ii) identify their responses to different types of rainfall events, and (iii) give suggestions to implement reasonable soil conservation measures in the study area.The study area is located in the Shixia catchment (117°4′30″ E and 40°34′40″ N) , upstrea2 with elevations of 130–390 m a.s.l. It exhibits an earth-rock hilly geomorphology. Around 20.6% of the catchment has a slope gradient od less than 8.7%, 50.2% of the slopes range from 8.7% to 26.8%, and slopes larger than 36.4% only account for 16.2% of the total. The lithology is dominated by gneiss, scattered with granite and limestone. The soil cover consists of cinnamon soils, as per the Chinese soil classification system which was developed on alluvial and diluvial parent materials. The soils are around 30 cm in depth, and around a 10 cm horizon of weathered rock mixed with soil appears under the soil layer. The catchment is mostly covered by artificial Robinia pseudoacacia, Pinus tabulaeformis, and economic forest. The major crops are corn (Zea mays) and wheat (Triticum aestivum).The catchment has an area of 33 km2 in area and 5 m in width. The slope gradients range from 6.1% to 29.6% with soil thicknesses of around 30 cm. Detailed information of the plots was given in There are 22 runoff plots in the catchment . The bou−2 event−1) was obtained by using soil loss amount and plot area. Annual H (AH) and the annual SLR of each plot were obtained by summing the event-derived ones.Surface runoff and soil loss were collected and measured after each rainfall event in 2011–2019. A nine-hole diversion bucket and a tank were used to collect the runoff mixed with sediment from the plots. The runoff amount was calculated and sampled with 1000 mL flasks after each rainfall. After settling for 24 h, sediment was separated from the water, dried in an oven at a temperature of 105 °C, and subsequently weighted to determine the sediment concentration. The soil loss of each plot was calculated by multiplying the average sediment concentration and the runoff amount. The runoff depth  of the plot after each rainfall event was calculated by using the total runoff amount during an entire rainfall event period and plot area. An event SLR (t kmm), and maximum intensities at 30 min (I30) and 60 min (I60) were obtained. Annual rainfall amount (AP) was obtained by summing event P.A self-recording rain gauge and rain barrel were installed near the plots to monitor rainfall process and rainfall amount. According to the records, rainfall duration (RD), rainfall amount (P), mean rainfall intensity . In the present study, rainfall eigenvalues of P, RD, Im, I30, and I60 were employed to group the erosive rainfall events.In order to study the effect of rainfall characteristics on surface runoff and soil loss, the K-means clustering method was used to group the erosive rainfall events in 2011–2019. To determine the number of groups, many criteria were used. Normally, the classification must meet the ANOVA criterion of significance , and AHi represents the mean annual H on the ith plot (where a soil conservation measure is present). Therefore, separate ARRE for each plot were obtained. In order to evaluate the effects of contour tillage and terracing on surface runoff, their ARREs were not averaged in the present study. Similarly, the mean annual soil loss reduction efficiency (ASLRE) for each plot was calculated as follows:0 represents the mean annual SLR on plot 1, and ASLRi represents the mean annual SLR on the ith plot where a soil conservation measure is present. The calculated ASLREs were given the same treatment as the ARREs.In the present study, erosive rainfall was defined as the rainfall that induces runoff and soil loss. The mean annual runoff reduction efficiency (ARRE) of a soil conservation measure was calculated as follows:p < 0.05.Pearson correlation analysis was performed to assess the relationships between H, SLR, and their influencing factors. Fisher’s protected least significant difference (LSD) test was conducted to compare the means of Hs and SLRs on the plots. Treatments were considered significant if In 2011–2019, the mean AP was 507 mm, ranging from 410 to 579 mm. In contrast, the mean annual erosive rainfall amount was 326 mm, ranging from 249 to 434 mm. There were 85 erosive rainfall events in the study period, ranging from seven to eleven rainfall events per year a. Most e30 and I60, the Im had the largest mean square value , although Im was an efficient indicator for grouping the rainfall events  on runoff and soil loss of the cultivated plots were evaluated.m, producing a higher SLR and lower H. However, rainfall regime B with a higher RD, P, and low Im yielded a higher H and lower SLR. Terraced plots can concurrently intercept runoff and sediment, resulting in the highest ARRE  and ASLRE  on gentle slopes. Although contour tillage measures had comparable ASLREs to terraced plot 3, their ARREs were much smaller than the terracing on gentle slopes. The effects of rainfall characteristics and soil conservation measures increased with decreasing slope gradients.Responses of runoff and soil loss to the three rainfall regimes were different. The frequently occurring rainfall regime A had a short RD, low P, and high IIn the Miyun Reservoir catchment where water shortages exist, water-saving soil conservation measures should be given priority. Therefore, contour tillage should be promoted on gentle slopes, and terracing should be given further attention in both the study area and similar regions around the world, as it can intercept a large portion of surface runoff which could greatly reduce the downstream water quantity, which exacerbates water shortages and endanger the safety of drinking water in downstream areas.