Case report: [18F]FAPI-42 PET/CT visualize primary adenoid cystic carcinoma not detected by [18F]FDG.

Adenoid cystic carcinoma (ACC) is a rare salivary gland cancer. Still, its growth and invasion progress is slow, and its hematogenous metastasis is ACC's most common distant metastasis. Because of the broad expression and low background uptake of fibroblast activation protein (FAP) in tumor stroma, FAPI is considered another potential tracer of ACC in addition to FDG. In this case, we report a patient who was diagnosed with metastatic ACC liver cancer by fine needle aspiration biopsy (FNAB) and underwent PET/CT examination of [18F]FDG and [18F]FAPI-42 to find the primary cancer lesion. Finally, the primary cancer lesion was found in the left submandibular gland and was pathologically confirmed as ACC after resection.

Three-Dimensional Cross-Linking Network Coating for the Flame Retardant of Bio-Based Polyamide 56 Fabric by Weak Bonds.

Weak bonds usually make macromolecules stronger; therefore, they are often used to enhance the mechanical strength of polymers. Not enough studies have been reported on the use of weak bonds in flame retardants. A water-soluble polyelectrolyte complex composed of polyethyleneimine (PEI), sodium tripolyphosphate (STPP) and melamine (MEL) was designed and utilized to treat bio-based polyamide 56 (PA56) by a simple three-step process. It was found that weak bonds cross-linked the three compounds to a 3D network structure with MEL on the surface of the coating under mild conditions. The thermal stability and flame retardancy of PA56 fabrics were improved by the controlled coating without losing their mechanical properties. After washing 50 times, PA56 still kept good flame retardancy. The cross-linking network structure of the flame retardant enhanced both the thermal stability and durability of the fabric. STPP acted as a catalyst for the breakage of the PA56 molecular chain, PEI facilitated the char formation and MEL released non-combustible gases. The synergistic effect of all compounds was exploited by using weak bonds. This simple method of developing structures with 3D cross-linking using weak bonds provides a new strategy for the preparation of low-cost and environmentally friendly flame retardants.

Spectrum and antibiotic sensitivity of bacterial keratitis: a retrospective analysis of eight years in a Tertiary Referral Hospital in Southwest China.

Purpose: The objective of this study was to investigate the epidemiological characteristics, distribution of isolates, prevailing patterns, and antibiotic susceptibility of bacterial keratitis (BK) in a Tertiary Referral Hospital located in Southwest China. Methods: A retrospective analysis was conducted on 660 cases of bacterial keratitis occurring between January 2015 and December 2022. The demographic data, predisposing factors, microbial findings, and antibiotic sensitivity profiles were examined. Results: Corneal trauma emerged as the most prevalent predisposing factor, accounting for 37.1% of cases. Among these cases, bacterial culture results were positive in 318 cases, 68 species of bacteria were identified. The most common Gram-Positive bacteria isolated overall was the staphylococcus epidermis and the most common Gram-Negative bacteria isolated was Pseudomonas aeruginosa. Methicillin-Resistant Staphylococci accounted for 18.1% of all Gram-Positive bacteria. The detection rate of P. aeruginosa showed an increasing trend over time (Rs=0.738, P=0.037). There was a significant decrease in the percentage of Gram-Negative microorganisms over time (Rs=0.743, P=0.035). The sensitivity of Gram-Positive bacteria to linezolid, vancomycin, tigecycline, quinupristin/dalfopristin, and rifampicin was over 98%. The sensitivity rates of Gram-Negative bacteria to amikacin, meropenem, piperacillin/tazobactam, cefoperazone sodium/sulbactam, ceftazidime, and cefepime were all above 85%. In patients with a history of vegetative trauma, the possibility of BK should be taken into account in addition to the focus on fungal keratitis. Conclusion: The microbial composition primarily consists of Gram-Positive cocci and Gram-Negative bacilli. Among the Gram-Positive bacteria, S. epidermidis and Streptococcus pneumoniae are the most frequently encountered, while P. aeruginosa is the predominant Gram-Negative bacteria. To combat Gram-Positive bacteria, vancomycin, linezolid, and rifampicin are considered excellent antimicrobial agents. When targeting Gram-Negative pathogens, third-generation cephalosporins exhibit superior sensitivity compared to first and second-generation counterparts. As an initial empirical treatment for severe cases of bacterial keratitis and those unresponsive to fourth-generation fluoroquinolones in community settings, the combination therapy of vancomycin and tobramycin is a justifiable approach. Bacterial keratitis can be better managed by understanding the local etiology and antibacterial drug susceptibility patterns.

Fibroblast Activation Protein-Targeted PET/CT With Al18F-NODA-FAPI-04 for In Vivo Imaging of Tendon Healing in Rat Achilles Tendon Injury Models.

BACKGROUND: Fibroblast activation protein (FAP) has shown high expression in inflammatory responses and fibrosis. HYPOTHESIS: We speculated that FAP could serve as a diagnostic and monitoring target in the tendon healing process. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 72 Sprague-Dawley rats were randomly divided into a tendon crush group and a half-partial tendon laceration group. Four rats in each group were injected with radiotracers weekly for 4 weeks after surgery, with aluminum fluoride-labeled 1,4,7-triazacyclononane-N,N',N″-triacetic acid-conjugated FAP inhibitor (Al18F-NODA-FAPI-04) administered on the first day of each week and 18F-fludeoxyglucose (18F-FDG) on the next day. Small animal positron emission tomography (PET) imaging was performed, and tendon tissue was collected for pathology and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis each week after surgery. RESULTS: One week after surgery, both radiotracers showed signal concentration at the lesion site, which was the highest radioactive uptake observed during 4 weeks postoperatively, consistent with the severity of the lesion. Consistent trends were observed for inflammatory cytokines during qRT-PCR analysis. Additionally, Al18F-NODA-FAPI-04 PET exhibited a more precise lesion pattern, attributed to its high specificity for naive fibroblasts when referring to histological findings. Over time, the uptake of both radiotracers at the injury site gradually decreased, with 18F-FDG experiencing a more rapid decrease than Al18F-NODA-FAPI-04. In the fourth week after surgery, the maximum standardized uptake values of Al18F-NODA-FAPI-04 in the injured lesion almost reverted to the baseline levels, indicating a substantial decrease in naive fibroblasts and inflammatory cells and a reduction in inflammation and fibrosis, especially compared with the first week. Corresponding trends were also revealed in pathological and qRT-PCR results. CONCLUSION: Our findings suggest that inflammation is a prominent feature during the early stage of tendon injury. Al18F-NODA-FAPI-04 PET allows accurate localization and provides detailed morphological imaging, enabling continuous monitoring of the healing progress and assessment of injury severity.

Effects of drought and salt stress on seed germination and seedling growth of Elymus nutans.

Drought and soil salinization are global environmental issues, and Elymus nutans play an important role in vegetation restoration in arid and saline environments due to their excellent stress resistance. In the process of vegetation restoration, the stage from germination to seedling growth of forage is crucial. This experiment studied the effects of PEG-6000 simulated drought stress and NaCl simulated salinization stress on the germination of E. nutans seeds, and explored the growth of forage seedlings from sowing to 28 days under drought and salinization stress conditions. The results showed that under the same environmental water potential, there were significant differences in responses of seed germination, seedling growth, organic carbon, total nitrogen and total phosphorus of above-ground and underground parts of E. nutans to drought stress and salinization stress. Using the membership function method to comprehensively evaluate the seed germination and seedling indicators of E. nutans, it was found that under the same environmental water potential, E. nutans was more severely affected by drought stress during both the seed germination and seedling growth stages. E. nutans showed better salt tolerance than drought resistance.

[18F]AlF-NOTA-ADH-1: A new PET molecular radiotracer for imaging of N-cadherin-positive tumors.

Background: The cell adhesion molecule (CAM) N-cadherin has become an important target for tumor therapy. The N-cadherin antagonist, ADH-1, exerts significant antitumor activity against N-cadherin-expressing cancers. Methods: In this study, [18F]AlF-NOTA-ADH-1 was radiosynthesized. An in vitro cell binding test was performed, and the biodistribution and micro-PET imaging of the probe targeting N-cadherin were also studied in vivo. Results: Radiolabeling of ADH-1 with [18F]AlF achieved a yield of up to 30% (not decay-corrected) with a radiochemical purity of >97%. The cell uptake study showed that Cy3-ADH-1 binds to SW480 cells but weakly binds to BXPC3 cells in the same concentration range. The biodistribution results demonstrated that [18F]AlF-NOTA-ADH-1 had a good tumor/muscle ratio (8.70±2.68) in patient-derived xenograft (PDX) tumor xenografts but a lower tumor/muscle ratio (1.91±0.69) in SW480 tumor xenografts and lowest tumor/muscle ratio (0.96±0.32) in BXPC3 tumor xenografts at 1 h post-injection (p.i.) These findings were in accordance with the immunohistochemistry results. The micro PET imaging results revealed good [18F]AlF-NOTA-ADH-1 tumor uptake in pancreatic cancer PDX xenografts with strong positive N-calcium expression, while lower tumor uptake in SW480 xenografts with positive expression of N-cadherin, and significantly lower tumor uptake in BXPC3 xenografts with low expression of N-cadherin, which was consistent with the biodistribution and immunohistochemistry results. The N-cadherin-specific binding of [18F]AlF-NOTA-ADH-1 was further verified by a blocking experiment involving coinjection of a non radiolabeled ADH-1 peptide, resulting in a significant reduction in tumor uptake in PDX xenografts and SW480 tumor. Conclusion: [18F]AlF-NOTA-ADH-1 was successfully radiosynthesized, and Cy3-ADH-1 showed favorable N-cadherin-specific targeting ability by in vitro data. The biodistribution and microPET imaging of the probe further showed that [18F]AlF-NOTA-ADH-1 could discern different expressions of N-cadherin in tumors. Collectively, the findings demonstrated the potential of [18F]AlF-NOTA-ADH-1 as a PET imaging probe for non-invasive evaluation of the N-cadherin expression in tumors.

The value of PET/CT for the diagnosis and evaluation of immunotherapy in occult primary renal cell carcinoma: a case report.

Skin and soft tissue diffusion metastasis (also known as occult cancer) is rare in renal cell carcinoma (RCC). Here, we report an extremely rare case of a 67-year-old male patient with occult primary RCC who developed metastases to the gums, skin, and diffuse soft tissue. The primary renal lesion was missed by computed tomography (CT), ultrasound, and 18F-fluorodeoxyglucose positron emission tomography (PET)/CT, and the diagnosis was confirmed by biopsy of gums and subcutaneous nodules. Subsequent enhanced CT revealed a lesion in the left kidney. The patient had progression-free survival of 16 months after treatment with axitinib and pembrolizumab. Pseudoprogression and tumor heterogeneity pose major challenges in the evaluation of immunotherapy. PET/CT is indispensable especially for cases with multiple metastases, widespread distribution of lesions, and major heterogeneity. In this case, the total lesion glycolysis was calculated by PET/CT and was used to evaluate systemic tumor load before and after immunotherapy, which was calculated as the product of the metabolic tumor volume and the mean standardized uptake value of the target lesion, which increased the accuracy of assessing diffuse lesions. Total lesion glycolysis can be used as a new method to quantitatively evaluate the efficacy of immunotherapy.

Development of 99mTc-Hynic-Adh-1 Molecular Probe Specifically Targeting N-Cadherin and Its Preliminary Experimental Study in Monitoring Drug Resistance of Non-Small-Cell Lung Cancer.

BACKGROUND: N-cadherin is considered a characteristic protein of EMT and has been found to be closely related to tumor resistance. In this study, a novel molecular imaging probe, 99mTc-HYNIC-ADH-1, was developed, and its diagnostic value in monitoring drug resistance in NSCLC was preliminarily investigated. METHODS: ADH-1 was labeled indirectly with 99mTc. Radiochemical purity and stability, partition coefficients and pharmacokinetics were evaluated. Additionally, the fluorescent probe of ADH-1 was synthesized to study tumor uptake in cells level and in vivo. Biodistribution analysis and small animal SPECT/CT were performed in PC9GR and PC9 tumor-bearing mice. RESULTS: 99mTc-HYNIC-ADH-1 was highly stable (radiochemical purity ≥ 98% in PBS and serum after 24 h). A cell binding study and fluorescence imaging showed that the uptake was significantly higher in PC9GR cells (gefitinib-resistant) than in PC9 cells (nonresistant) (p < 0.05). Biodistribution analysis showed rapid blood clearance and significant uptake in the kidney and resistant tumor. Small animal SPECT/CT studies showed that uptake in PC9GR tumors (T/NT = 7.73 ± 0.54) was significantly higher than that in PC9 tumors (T/NT = 3.66 ± 0.78) at 1 h (p = 0.002). CONCLUSIONS: The 99mTc-HYNIC-ADH-1 molecular probe has a short synthesis time, high labeling rate, high radiochemical purity and good stability, does not require purification, is characterized by rapid blood clearance and is mainly excreted through the urinary system. 99mTc-HYNIC-ADH-1 is considered a promising probe for monitoring drug resistance in NSCLC.

Influence of relative positions of the heat carrier and lateral canal opening on gutta-percha obturation of lateral canals in a three-dimensional-printed model.

Background/purpose: Effective filling of the lateral canals is of great significance in successful root canal treatment, but it is generally being challenging. This study aimed to evaluate the influence of relative positions of the heat carrier and lateral canal opening on gutta-percha obturation of lateral canals in a three-dimensional (3D)-printed model. Materials and methods: Thermal conductivity and real-time temperature transmission of gutta-percha were investigated using laser flash and thermal infrared analyses. 3D-printed root canal models with lateral canals at 1, 3, and 5 mm from the apex were fabricated, and different relative positions of the heat carrier were tested. The obturation process was recorded on video, and the obturation depth of the lateral canals was observed using X-ray micro-computed tomography. Results: Gutta-percha showed low thermal conductivity of 1.07 W/(m·K), and heating increased the temperature of gutta-percha above 60 °C only within 1 mm beyond the heat carrier tip. For lateral canals at 1 and 3 mm from the apex, gutta-percha penetrated further with deeper penetration of the heat carrier (P < 0.05). For 5-mm lateral canals, the heat carrier was always at apical level and the gutta-percha obturation depth was more at 2 mm apically than at 3 or 4 mm (P < 0.05). Conclusion: Gutta-percha is a poor thermal conductor. The position of the heat carrier in relation to the lateral canal opening affects obturation depth. Only when the heat carrier reaches or passes the lateral canal opening can gutta-percha penetrate a lateral canal.

Preliminary Study on Human Adipose Stem Cells Promoting Skin Wound Healing through Notch Signaling Pathway.

BACKGROUND: Mesenchymal stem cells (MSCs) have been documented as possible candidates for wound healing treatment because their use could reinforce the regenerative capacity of many tissues. Human adipose stem cells (hADSCs) have the advantages of easy access, large quantity and easy operation. They can be fully applied in the treatment of skin wounds. OBJECTIVE: In this study, we aim to explore the roles and potential mechanisms of hADSCs in cutaneous wound healing. METHODS: hADSCs were obtained from human subcutaneous fat. Adipocytes and osteocytes differentiated from hADSCs were determined by staining with Oil Red O and alkaline phosphatase (ALP), respectively. We assessed the effects of hADSCs and hADSC conditional medium (CM) on wound healing in an injury model of mice. Then, we investigated the biological effects of hADSCs on human keratinocytes HaCAT cells in vitro. RESULTS: The results showed that hADSCs could be successfully differentiated into osteogenic and lipogenic cells. hADSCs and hADSCs-CM significantly promote skin wound healing in vivo. hADSCs significantly promoted HaCAT cell proliferation and migration by activating the Notch signaling pathway and activated the AKT signaling pathway by Rps6kb1 kinase in HaCAT cells. In addition, we found that hADSCs-mediated activation of Rps6kb1/AKT signaling was dependent on the Notch signaling pathway. CONCLUSION: We demonstrated that hADSCs can promote skin cell-HaCAT cell proliferation and migration via the Notch pathway, suggesting that hADSCs may provide an alternative therapeutic approach for the treatment of skin injury.

Gamma knife radiotherapy in a neurofibromatosis type 1 Chinese pedigrees with NF1 gene frameshift mutation: A case report.

RATIONALE: Neurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disorder. NF1 is a multisystemic disease and its pathogenesis involves mutations in the NF1 gene on chromosome 17q11.2 causing RAS overactivation to stimulate abnormal cell proliferation. In this article, a Chinese family with neurofibromatosis type 1 was reported and the relationship between the phenotype and gene mutation was analyzed. PATIENT CONCERNS: The patient was a 9-year-old-male child diagnosed with right eye exophthalmos combined with right eye glioma, optic edema, and peripheral visual field defect. There were multiple cafe-au-lait spots in the whole body of the child. His mother had multiple cafe-au-lait spots, and the eye examination showed no abnormalities. DIAGNOSIS: The proband was diagnosed with NF1 and a heterozygous frameshift mutation (c. 6641delG p. Arg2214Asnfs*30) in the NF1 gene was identified, and his mother also carried the same pathogenic mutation. INTERVENTIONS: To protect the vision of the right eye, he was treated with gamma knife radiotherapy. OUTCOMES: After therapy, his fundus optic disc edema was decreased and the best corrected visual acuity of the right eye was increased. LESSONS: Gene detection is helpful to diagnose the disease and guide the treatment. Gamma knife radiotherapy can preserve better neurological function.

Regulation of Semaphorin3A in the process of cutaneous wound healing.

Semaphorin 3A (Sema3A) has been recognized as a crucial regulator of morphogenesis and homeostasis over a wide range of organ systems. However, its function in cutaneous wound healing is poorly understood. In our study, we demonstrated that Sema3A adenovirus plasmids transfection limited keratinocyte proliferation and decreased migrative capacity as assessed by in vitro wound healing assay. Sema3A transduction inhibited TGF-ß1-mediated keratinocyte migration and EMT process. Besides, we applied mice with K14-Cre-mediated deletion of Sema3A and found that Sema3A depletion postponed wound closure with decreased re-epithelialization and matrix growth. Contrary to the results obtained with full-length Sema3A plasmids transfection, increased keratinocyte migration with recombinant Sema3A proteins resulted in quicker closure of the wounding area after a scratch. Further, exogenously applied recombinant Sema3A worked with EGF to maintain the activation of EGFR by interacting with NRP1 and thereby regulated the internalization of the EGFR-NRP1 complex. Taken together, these results indicated a paradoxical role of autonomous and non-autonomous Sema3A expression during wound healing. Combined administration of recombinant EGF and Sema3A proteins could accelerate the process of wound repair, thus providing promising treatment prospects in the future.

Compound heterozygous missense mutations in a Chinese mucopolysaccharidosis type VI patient: a case report.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is a rare autosomal recessive inherited disease caused by mutations in the arylsulfatase B (ARSB) gene. MPS VI is a multisystemic disease resulting from a deficiency in arylsulfatase B causing an accumulation of glycosaminoglycans in the tissues and organs of the body. In this report, we present the case of a 16-year-old Chinese male who presented with vision loss caused by corneal opacity. MPS VI was confirmed by genetic diagnosis. CASE PRESENTATION: A 16-year-old Chinese male presented with a one-year history of binocular vision loss. The best-corrected visual acuity was 0.25 in the right eye and 0.5 in the left eye. Although slit-lamp examination revealed corneal opacification in both eyes, the ocular examinations of his parents were normal. At the same time, the patient presented with kyphotic deformity, short stature, joint and skeletal malformation, thick lips, long fingers, and coarse facial features. Genetic assessments revealed that ARSB was the causative gene. Compound heterozygous missense mutations were found in the ARSB gene, namely c.1325G > A (p. Thr442Met) (M1) and c.1197G > C (p. Phe399Leu) (M2). Genetic diagnosis confirmed that the patient had MPS VI. CONCLUSIONS: This paper reports a case of MPS VI confirmed by genetic diagnosis. MPS VI is a multisystem metabolic disease, with corneal opacity as a concomitant ocular symptom. As it is difficult for ophthalmologists to definitively diagnose MPS VI, genetic testing is useful for disease confirmation.

[Animal Manure Fertilization Promotes Antibiotic Resistance Gene Dissemination Among Manure, Soil, and Vegetables].

Increasing antibiotic resistant genes (ARG) are being detected in animal manure-amended soil, causing public concerns. However, the effects of animal manure fertilization on the ARG of vegetable endophytes remain unknown. In this study, high-throughput quantitative PCR (HT-qPCR) was used to explore the effects of poultry manure fertilization on the bacterial community and resistome in the soil, root endophytes, and leaf endophytes from Cichorium endivia L.. The results indicated that poultry manure fertilization not only enhanced the number of ARG in soil and root endophytes, but also increased the abundance of ARG in the soil, root endophytes, and leaf endophytes. The relationship analysis demonstrated that the ARG profile in the soil and vegetable endophytes was significantly correlated with the bacterial community, and the ARG abundance was highly correlated with Proteobacteria, Acidobacteria, Actinobacteria, and Cyanobacteria. In addition, the overlapping ARG between the Cichorium endivia L. endophyte and soil indicated the internal spread route of the ARG from the manure to Cichorium endivia L through the soil. In conclusion, poultry manure fertilization increased the diversity and abundance of the ARG in vegetables via the manure-soil-vegetable pathway.

Metastatic microcalcification in the lungs and stomach due to hyperparathyroidism: A case report.

We present a case of a 34-year-old man who was admitted to our hospital with a six-month history of nausea and vomiting that had worsened over the past two weeks. Diagnosis of primary hyperparathyroidism (PHPT) due to a single parathyroid adenoma was confirmed by laboratory results and imaging but following pharmacological therapy the patient developed a dry cough. Single-photon emission tomography/computed tomography (SPECT/CT) with technetium-99m-diphosphonate (99mTc-MDP) was used to assist in the diagnosis and results showed diffuse metastatic microcalcification in the lungs and stomach. The patient underwent right parathyroidectomy and 99mTc-MDP SPECT/CT scan six months later showed a significantly reduced radioactive distribution in the lungs and none in the stomach. Diffuse metastatic microcalcification in the lungs and stomach due to PHTP is rare and can easily be misdiagnosed.

Preclinical evaluation of [68Ga]Ga-MALAT-1-antisense oligonucleotides for specific PET imaging of MALAT-1 expressing tumours.

OBJECTIVE: The present study was to explore the feasibility of developing positron molecular probes for the metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), to evaluate the distribution and pharmacokinetics, and to explore whether the probe can be used for the imaging of malignant tumours with high MALAT-1 expression in vivo. METHODS: [68Ga]Ga labelling of MALAT-1 antisense oligonucleotides ([68Ga]Ga-MALAT-1-ASO) was synthesized by the conjugation of MALAT-1-NOTA-ASO and [68Ga] Ga3+. The radiochemical purity was shown by radio-HPLC. Pharmacokinetic studies and cellular uptake studies were performed. The biodistribution and metabolism of [68Ga] Ga-MALAT-1-ASO in normal ICR and MHCC-LM3 xenograft-bearing nude mice were studied in vitro and in vivo. RESULTS: [68Ga]Ga-MALAT-1-ASO was obtained in 98% radiochemical yield from a 10-min synthesis with 100 ± 50 MBq/nmol specific activity and >99% radiochemical purity. The Log D was -2.53 ± 0.19. The tracer displayed excellent stability in vitro. 68Ga-MALAT-1 ASO showed satisfactory binding ability to MHCC-LM3 cells; the biodistribution of [68Ga]Ga-MALAT-1-ASO in MHCC-LM3 tumour-bearing mice demonstrated specific uptake of the radiotracer (3.04 ± 0.11%ID/g). Micro-PET images of the MHCC-LM3 cell xenograft mouse model provided further evidence to support the hypothesis that [68Ga]Ga-MALAT-1-ASO can target tumours in vivo. CONCLUSIONS: We conclude that [68Ga]Ga labelling of MALAT-1 ASO is a convenient approach. The high accumulation of [68Ga]Ga-MALAT-1-ASO for tumours expressing MALAT-1 suggests that this radio compound may be used as a potential positron molecular probe. Molecular structure optimization studies need to be more in-depth to further reduce its background uptake and enhance tumour targeting.

CT5, a subtle in vitro DNA assembling method based on the combination of FnCas12a and T5 exonuclease.

OBJECTIVE: To develop a new DNA assembly method based on FnCas12a and T5 exonuclease. RESULTS: We developed a method named as FnCas12a and T5 exonuclease (CT5) cloning system. FnCas12a performs site-directed cleavage to the target DNA fragments, and T5 exonuclease generates 20-30 nt single-stranded region at each end of the DNA fragments for homologous recombination-mediated DNA assembly. CT5 was applied to multi-fragment assembly and DNA cloning of large vectors (> 10 kb). The efficiencies were approximately 91.4% and 97%, respectively. In addition, CT5 cloning is also utilized for the "walking" of DNA elements, which enables subtle modification of the relative distances of DNA elements in plasmids. CONCLUSIONS: The CT5 method was a precise and exquisite DNA operating system and provided an ideal platform for the study of gene functions, genetic engineering and synthetic biology.

Meckel's diverticulum with polypoid hyperplasia of ectopic gastric mucosa diagnosed by double-balloon enteroscopy and single-photon emission computed tomography/computed tomography.

Meckel's diverticulum (MD) is a relatively common true congenital diverticulum on the ileum. Bleeding caused by polypoid hyperplasia of ectopic gastric mucosa in MD is rare. A 14-year-old Chinese boy presented with intermittent melena and haematochezia for 1 month. Laboratory data showed normocytic anaemia. Gastroscopic findings were normal. The patient underwent exploratory laparotomy without bowel preparation on day 2 because of sudden haematochezia and decreased haemoglobin. Intraoperative colonoscopy revealed inflammatory changes in the terminal ileal mucosa with diffuse haemorrhage. Melena with decreased haemoglobin recurred 20 days after the first operation. Computed tomography (CT) and angiography revealed a tubular lesion that was localised in the right lower abdominal quadrant. Single-photon emission computed tomography/computed tomography (SPECT/CT) fusion imaging with 99m-technetium pertechnetate confirmed moderately increased uptake in the distal ileum. Retrograde double-balloon enteroscopy (DBE) showed a diverticulum with prominent mucosal polypoid hyperplasia at an insertion depth of 100 cm from the anastomotic stoma. Diverticulectomy and end-to-end anastomosis were performed, and MD was confirmed by a histopathological examination. The patient's postoperative recovery was uneventful during the 2-month follow-up. MD with polypoid hyperplasia of ectopic gastric mucosa is rare. Complementary use of DBE and SPECT/CT can accurately diagnose MD by providing anatomical and functional information.

Persistent secondary hyperparathyroidism caused by parathyromatosis and supernumerary parathyroid glands in a patient on haemodialysis.

BACKGROUND: Secondary hyperparathyroidism (SHPT) is a common high-risk factor for mortality in end-stage renal disease, and parathyromatosis and supernumerary parathyroid glands are very rare causes of persistent SHPT. Preoperative diagnosis and removal of all hyperplastic parathyroid glands are challenging. We report a rare case of persistent SHPT due to parathyromatosis and supernumerary parathyroid glands and successful management by multiple imaging modalities. CASE PRESENTATION: A 53-year-old Chinese woman on haemodialysis experienced discomfort due to itching and bone pain due to persistent SHPT after parathyroidectomy. The supernumerary parathyroid glands and parathyromatosis were detected by multiple imaging modalities, including 99mTc-sestamibi (99mTc-MIBI) scans, ultrasonography and four-dimensional computed tomography (4D-CT) and then excised; pathological confirmation was performed. During follow-up, her serum calcium and parathyroid hormone levels were stable in the appropriate ranges, and no complications arose. CONCLUSIONS: Because of persistent SHPH after parathyroidectomy in patients with haemodialysis, multiple imaging modalities, including 99mTc-MIBI scans, 4D-CT and ultrasonography, are helpful for detecting supernumerary parathyroid glands and parathyromatoses. Accurate preoperative localization of this rare lesion is important for management, enabling the removal of all affected parathyroid tissues.

A single digestion, single-stranded oligonucleotide mediated PCR-independent site-directed mutagenesis method.

A PCR-independent in vitro site-directed mutagenesis method was established. Cas12a from Francisella novicida (FnCas12a) linearizes the plasmid with single digestion. T5 exonuclease removes the target nucleotide. A short single- or double-stranded mutagenic oligonucleotide introduces the mutation. This rapid and simple mutagenesis method is referred to as FnCas12a and T5 exonuclease mediated site-directed mutagenesis system (CT5-SDM). The platform is also suitable for the mutagenesis of plasmids larger than 10 kb. KEY POINTS: Site-directed mutagenesis mediated by single-stranded DNA. Removing target site with T5 exonuclease. Highly efficient cleavage of target DNA with FnCas12a.