The gels of cellulose and its derivatives have a broad and deep application in pharmaceutics; however, limited attention has been paid to the influences of other additives on the gelation processes and their functional performances. In this study, a new type of electrospun core–shell nanohybrid was fabricated using modified, coaxial electrospinning which contained composites of hydroxypropyl methyl cellulose (HPMC) and acetaminophen (AAP) in the core sections and composites of PVP and sucralose in the shell sections. A series of characterizations demonstrated that the core–shell hybrids had linear morphology with clear core–shell nanostructures, and AAP and sucralose distributed in the core and shell section in an amorphous state separately due to favorable secondary interactions such as hydrogen bonding. Compared with the electrospun HPMC–AAP nanocomposites from single-fluid electrospinning of the core fluid, the core–shell nanohybrids were able to promote the water absorbance and HMPC gelation formation processes, which, in turn, ensured a faster release of AAP for potential orodispersible drug delivery applications. The mechanisms of the drug released from these nanofibers were demonstrated to be a combination of erosion and diffusion mechanisms. The presented protocols pave a way to adjust the properties of electrospun, cellulose-based, fibrous gels for better functional applications. Cellulose, as one of the most abundant natural resources, has been exploited in the field of drug delivery for many years ,3,4,5,6.However, cellulose’s functional applications have not yet been fully expanded ,24,25,26Electrospinning, as an electrohydrodynamic technique, is broadly exploited to treat cellulose and its derivatives for biomedical and other applications ,37,38. TIn this study, with a common cellulose derivative, i.e., hydroxypropyl methyl cellulose (HPMC) as the filament-forming matrix and also the drug carrier, its homogeneous electrospun nanocomposite (containing APP from a single-fluid blending process) and its heterogeneous core–shell nanohybrids  were fabricated and characterized in parallel. A well-known poorly water-soluble drug, acetaminophen (AAP), was explored as the model’s active ingredient ,63. The In an electrospinning system, the spinneret is the most vital component ,65,66. Tfs) is adjusted to 0 mL/h, the process is degraded to a single-fluid blending process of the core fluid, and the final products are, correspondingly, homogeneous nanocomposites. In contrast, when fs is larger than 0 mL/h, the final products are core–shell nanohybrids  and their different electrospun products are included in Both HPMC and PVP are water-soluble, polymeric excipients broadly utilized in the pharmaceutical industry ,72. HoweShown in A punch pin with an inner hole of 10 mm, as shown in the upper-right inset of The core–shell nanohybrids F2 exhibited different behaviors to nanocomposites F1 in the following aspects: (1) the circle was slightly enlarged, but the pink color was light, which was attributed to the diffusion of basic fuchsin dissolved from the PVP–sucralose shell sections; (2) the colors at different regions were different—some showed a slightly pink color and some showed a deep purplish color, which was closely related to the core–shell nanostructures; (3) the deep purplish color section self-assembled into a strange shape, which was attributed to the movement and aggregation of the core HPMC molecules during the gelation processes and further indicated that the HMPC molecules had a strong capability of holding the basic fuchsin.The in vitro drug release profiles of the electrospun nanocomposites F1 and the core–shell nanohybrids F2 are included in t and Q0 represent the drug released into the dissolution media from its carriers at time point (t), k and n are two constants and P represents accumulative drug release percentage. The drug release mechanisms can be judged by the value of n. It is common knowledge that an n value smaller than 0.45 indicates a diffusion mechanism, a value larger than 0.90 indicates an erosion mechanism and a value between 0.45 and 0.90 represents a complex mechanism involving both diffusion and erosion.HPMC is frequently utilized both in the pharmaceutical industry and in scientific research as a film coating agent, thickening agent, drug release modifier, drug stabilizer, table binder and suspending ingredient in some liquid dosage forms for oral administration ,78. It iThe regressed equation for the electrospun nanofibers is included in For the electrospun core–shell nanohybrids F2, the equation is:Thus, it is clear that AAP from the electrospun nanocomposites F1 and the core–shell nanohybrids are all a combination of erosion and diffusion mechanisms because of an n value between 0.45 and 0.90.Although the drug release behaviors from the two types of electrospun nanoproduct involved both diffusion and erosion mechanisms, it is obvious that the core–shell nanohybrids F2 were more closed to the erosion mechanism due to a value of 0.68 compared to 0.48 for the nanocomposites F1. A schematic showing the different behaviors of the monolithic nanocomposites F1 and core–shell nanohybrids F2 is given in With HPMC as a key filament-forming polymeric matrix, both single-fluid blending electrospinning and modified, coaxial electrospinning were implemented to preparing HPMC–AAP monolithic nanocomposites F1 and core (HPMC–AAP)–shell  heterogeneous nanohybrids F2. Both F1 and F2 had linear morphologies without any discerned beads of spindles on them, as demonstrated by the SEM images. TEM images verified that F1 were homogeneous nanocomposites, and F2 were core–shell nanohybrids containing two layers of composites. XRD results suggested that AAP presented in F1 and F2 in an amorphous state. This was attributed to the favorable interactions between APP and HPMC, which were demonstrated by ATR-FTIR measurements. Water contact angle experiments and tongue-mimicking tests clearly demonstrated the differences between F1 and F2 in their hydrophilicity and gelation processes. In vitro dissolution tests demonstrated that the core–shell hybrids F2 were able to offer a faster release of the loaded AAP. The drug release was demonstrated to be controlled by a complex combination mechanism involving both diffusion and erosion. The present study shows a new way of adjusting the properties of electrospun, cellulose-based nanofibers for better drug delivery applications.Mn = 428,000 g/mol, methoxy content 28.0–30.0%, hydroxypropoxy content 7.5–12.0%) was purchased from Shandong Fine Chemical Co., Ltd. . Sucralose, polyvinylpyrrolidone  dichloromethane (DCM), anhydrous ethanol and basic fuchsin were bought from Shanghai Chemical Regents Co., Ltd. . All other chemicals were analytical reagents. Water was doubly distilled before usage.Acetaminophen AAP  was obtained from Hua-Shi Big Pharmacy . Hydroxypropyl methyl cellulose  was fixed at 2.0 mL/h. The sheath fluid flow rate (fs) was 0.0 mL/h and 0.5 mL/h for generating F1 and F2, respectively. The ambient conditions were a temperature of 21 ± 4 °C and a relatively humidity of 52 ± 5%.Two types of electrospun nanofiber were fabricated. One was the monolithic, fibrous nanocomposite (denoted as F1) from single-fluid electrospinning of the core liquid; the other was the core–shell fibrous nanohybrid (denoted as F2) from modified, coaxial electrospinning. The applied voltages were adjusted to ensure continuous spinning and that, meanwhile, no droplets were dropped during the working processes; the values were between 11 and 14 kV. The core fluid flow rate . The cross-sections of fibers were prepared by manually breaking the fibrous strip after immersion in liquid nitrogen for about 20 min. The inner structures were evaluated using transmission electron microscope  under an applied electron voltage of 300 kV.All the raw materials  and their electrospun products F1 and F2 experienced X-ray diffraction  tests. Attenuated total reflectance Fourier-transform infrared  was exploited to investigate the compatibility between the polymeric carriers and active ingredients.Water contact angle measurements and a homemade experiment on wet paper were conducted to evaluate the fast disintegrating properties of the electrospun nanocomposites F1 and core–shell nanohybrids F2, and the gelation processes were recorded using a camera .max = 243 nm. All the measurements were repeated 6 times.In vitro dissolution tests were carried out using paddle methods according to the Chinese Pharmacopoeia (Ed. 2020). An RCZ-8A dissolution apparatus  was used. Samples equivalent to 20 mg AAP were placed into the dissolution vessels in which 600 mL physiological saline (PS) was kept at a temperature of 37 ± 1 °C. The paddle rotation rate was 50 rpm. A UV–vis spectrophotometer  was used to measure the AAP concentration at a λ


Glomus tumour typically occurs in subcutaneous tissue but rarely in the viscera! organs. Most glomus tumours are benign but few atypical glomus tumours have been reported. Herein, we report a case of a 44-year-old male who presented with hematuria. Transurethral resection of bladder tumour was done. Microscopic examination showed nests and sheets of tumor around the blood vessels. Spindle cells resembling smooth muscle were also observed. An increase in mitosis was observed. These tumour cells show diffuse and strong cytoplasmic positivity for smooth muscle actin and negative for Pancytokeratin, Desmin, Synaptophysin, Chromogranin, S100, and Cluster of Differentiation 34. Ki-67 index was approximately 5%. To our knowledge, this is the first report of Glomangiomyoma of uncertain malignant potential in the urinary bladder which is considered as an unusual variant of atypical glomus tumor. This case emphasizes the importance of broad differential diagnosis which has to be considered in the urinary bladder mass. The patient didn't report any other urinary symptoms. He didn't take any medications and was non alcoholic as well as non smoker. There was no relevant past history and family history. No abnormal findings were noted on physical examination. On laboratory examination, his hematological and biochemical parameters were within normal limit. Routine urine examination reveals red blood cells in urine.The sonographic test showed a round to oval hypoechoeic mass of 1.6x1.3x0.7cm in the urinary bladder. The clinical diagnosis of Urothelial neoplasm was made. Then, the patient was planned for cystourethroscopic evaluation and biopsy. On cystourethroscopy, 1.5cm polypoidal mass was observed in the right lateral wall of urinary bladder. The mass has smooth surface and was firmly attached to the urinary bladder wall. TURBT was done and the mass was sent for histopathological examination.On gross pathological examination, the biopsy consists of four pieces of grey white soft tissue size ranging from 0.2 to 0.5cm and weighing 5gm. Microscopic examination revealed diffuse sheets and nests of tumor clustered around thin, dilated and branching blood vessels . The celSubsequently, immunohistochemical examination was performed .Capillary endothelial cells are positive and tumor cells are negativeTumor cells were positive for smooth muscle actin (SMA) . Tumor cThe patient was discharged on 4th post-operative day. Regular follow-up for four years revealed no signs of recurrence.5 Glomus tumour are considered as a hamartomatous proliferation of normal glomus cells which are involved in thermal regulation and was first described by Masson in 1924.6 Based on histopathology examination, Glomus tumour are classified into typical and atypical.2 Typical Glomus tumour are subclassified into solid Glomus tumour, glomangioma and glomangiomyoma depending on the relative proportion of round glomus cells, vascular smooth muscle cells and spindle cells.6 Solid Glomus tumours are composed of solid sheets of glomus cells interrupted by vessels of varying size whereas glomangiomas are less well circumscribed and are composed of hemangioma like vascular structure with small clusters of glomus cells in their wall.2 Likewise glomangiomyoma resemble to those of solid Glomus tumour and glomangioma however there is gradual transition of round to oval glomus cells to elongated mature smooth muscle cells.7 On the other side, Folpe and colleagues based on study of 52 cases suggested the criteria and subclassified atypical Glomus tumour into malignant Glomus tumour (glomangiosarcoma), glomus tumour of uncertain malignant potential (GLUMP), symplastic Glomus tumour and glomangiomatosis.8 They establish the criteria based on the fact that metastatic lesions were seen in 38% of tumour fulfilling the criteria of malignant Glomus tumour while metastasis was not seen in others.8Genitourinary tract Glomus tumour are extremely rare and the majority of cases are reported in cervix, vagina, kidney and renal pelvis.6 After extensive search of database, we found only three reported cases of Glomus tumour in the urinary bladder of which one is benign,2 one is malignant9 and one is Atypical GLUMP10 however to the best of our knowledge glomangiomyoma of uncertain malignant potential has not been reported previously.Glomus tumour in urinary bladder is exceedingly rare as glomus bodies are typically absent in visceral organ and exact pathogenesis in not well understood.8 In our case, the tumour is of visceral origin which describes the deep location and has high mitotic activity. The diagnosis has been confirmed with the aid of immunohistochemistry. It is necessary to differentiate Glomus tumour from other tumours such as urothelial carcinoma, hemangiopericytoma, epithelioid leiomyoma, carcinoid tumour and paraganglioma. The nested areas in the lesion suggest urothelial carcinoma however histomorphological features as well as negative IHC stains for AE1/AE3 exclude urothelial carcinoma. The presence of hemangiopericytic (staghorn) vascular pattern in the present case suggest the diagnosis of hemangiopericytoma but strong immunopositivity of SMA in the tumour cells and absence of CD 34 immonostaining in tumour cells in conjunction with the histomorphological features ruled out hemangiopericytoma. Furthermore, in our case the tumour cells were negative for desmin, excluding and epithelioid leiomyoma. The reported case here in was unlikely to be a carcinoid tumour and paraganglioma, since tumour cells lack both neuroendocrine marker (chromogranin and SYP) as well as S100.We diagnosed this case as Glomangiomyoma of uncertain malignant potential based on histomorphology and the criteria proposed by Folpe, et al.Conclusively, we can say that this is an exceptional case presented as a urinary bladder mass which resembles urothelial neoplasm clinically. Histopathological finding requires confirmation with IHC, which include positivity for SMA to establish the definite diagnosis and help to differentiate it from other mimickers.


To determine biological variation of the aggrecanase-generated aggrecan ARGS neoepitope in serum (sARGS) and synovial fluid (sfARGS) within and between patients with osteoarthritis (OA) or anterior cruciate ligament (ACL) injury.Matched samples of serum and synovial fluid were available, as parts of clinical trials, from i) 16 subjects with early-stage OA on 8 occasions over 1 year, and ii) 120 subjects with acute ACL injury with samples available from at least 2 of 6 visits over 5 years. We used an in-house immunoassay to quantify ARGS and one-way ANOVA for statistical analyses.Variability in ARGS was higher in synovial fluid than in serum in both patient groups. Subjects with OA had the lowest variability both within and between patients and showed no variation over time in the degree of variability or in the cross-sectional mean, neither in serum nor in synovial fluid. After ACL injury, the concentration and the variability of ARGS was highest immediately after injury, with a subsequent decline both in concentration and in variability with time. In both patient groups there was a positive correlation between sfARGS and sARGS both within and between individuals (correlation coefficients between 0.16 and 0.20).The biological variation of ARGS is lower in serum than in synovial fluid, and lower in OA than after ACL injury. Serum ARGS is a measure of the total release of ARGS aggrecan from the whole body and a poor reflection of the release of ARGS aggrecan within the affected joint. The fieFor aggrecanase-generated ARGS several studies have reported on differences between patient groups , tempora22.1Subjects were selected from two previous study cohorts.2.1.1We analyzed samples available from the ARTZAL-study comprising 52 patients with knee pain and arthroscopically verified deep cartilage fissures and villous-like flakes in the symptomatic knee, considered to represent early-stage OA, randomly assigned to receive intraarticular injections of 2.5 ml of either hyaluronan or vehicle, weekly for 5 weeks, and followed-up for one year . No diff2.1.24) from baseline to 2 or 5 years . I. I17]. IThe biological variation of ARGS in these subjects with ACL injury was highest at baseline and in synovial fluid where the between-subjects CV was 98%, 3 times higher than at follow up visits 4 months to 5 years after injury (CV = 31%), and 4 times higher than in early-stage OA , whereas the variation within subjects at 42% was more than 3 times higher in synovial fluid compared to the 13% in serum .1 value of 0.278 for synovial fluid, which indicates that approximately 70% of the total variation of sfARGS in synovial fluid could be attributed to within-subject variation  and within subjects (r = 0.16) . However3.4The relationship between ARGS in synovial fluid and in serum, calculated as the ratio sfARGS to sARGS, changed with time after injury from an initial ratio of 91 acutely after injury to a ratio of approximately 10 in the following visits A. In conThe point estimates of the within-subject correlation measured at each visit spanned over a wider range and fluctuated more after ACL injury than in early OA B.4In two distinctly different patient groups – ACL injury and early-stage knee OA – both of which potentially could be treated by DMOADs involving inhibition of aggrecanases, we here report the biological variation of the aggrecanase generated ARGS neoepitope in joint fluid from the affected knee, as well as in serum. In addition to the reported measures of variability being important for planning, powering, and interpretation of studies, three main observations can be made. 1) The variation of ARGS is larger after injury than in early-stage OA. 2) The variation of ARGS is larger in synovial fluid than in serum. 3) Although ARGS in synovial fluid and serum are correlated, the individual variation is substantial, and within a patient a measurement in one fluid cannot substitute for measurement in the other.Injurious compression of cartilage gives an immediate increased expression of the aggrecanase ADAMTS-5 without affecting the expression of aggrecan , suggestIn the ACL injury cohort, we chose to look at the variability in the acute phase after injury separately from the follow up period from 4 months to 5 years. The primary reason for this was that the baseline sampling of synovial fluid and serum was not done at the same time point, which complicates interpretation in differences in variability and correlation between synovial fluid and serum ARGS. The heterogeneity in baseline sampling (ranging from 0 to 44 days after injury), in combination with a known rapid change in both expression of aggrecanases after injury , and ARGThis is to our knowledge the first study that reports on variability of ARGS aggrecan in both serum and synovial fluid. The previous few studies have looked at short term variability in serum only. One study investigated the sensitivity of sARGS to exercise and found minute increases in the hours following moderate exercise, very low day-to-day variation in the individual, but a substantial variation between individuals . Two pubWe have previously noted that ARGS concentrations in serum and synovial fluid are correlated in subjects with ACL injury ,25. In tBiological variation may be regarded as the random variation around a homeostatic set point . The varThis study has some limitations. When studying biological variation in a biomarker, a contributing factor is the analytical source of variation. The assay used to quantify ARGS involves a pre-treatment step that increases the sensitivity of the assay, but also adds handling that may increase the analytical variation. To minimize the effect of analytical variation we analyzed all sequentially taken samples in duplicate on the same analytical run on the same plate. Since only between 6 and 8 subjects were run on each plate, and the between-plate variation is higher than the within-plate variation, the influence of analytical variation is greater on the between-individual variation than on the within-subject variation. We cannot rule out that this may have overall increased the absolute values for the ARGS variability between subjects, but differences in the variability between groups or between fluids remain unaffected. Collection of synovial fluid through knee puncture is more complex and difficult than collection of blood samples, and the resulting volume is often small. This has limited the numbers of subjects with matched samples of synovial fluid and serum in both cohorts. The lack of matched samples at baseline in the ACL injury cohort is a further limitation in this study.5The biological variation of ARGS is larger in synovial fluid than in serum, and larger after injury than in early-stage OA. Although ARGS concentrations in synovial fluid and serum are correlated, the individual variation is substantial, and a measurement within a patient in one fluid cannot substitute for measurement in the other.Staffan Larsson acquired and had access to all data, drafted the manuscript and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors were involved in study conception and design of the study, in the analysis and interpretation of data, and in the critical revision and approval of the final manuscript.10.13039/501100004359Swedish Research Council (VR), Governmental funding of clinical research within the National Health Services (ALF), the 10.13039/501100007949Swedish Rheumatism Association, Greta and Johan Kocks Foundations, and from Galapagos NV in Mechelen, Belgium. Funding sources had no role in the design, collection, and interpretation of the data or the decision to submit for publication.This study received funding from the Staffan Larsson has no conflict of interest. André Struglics has no conflict of interest. Stefan Lohmander has no conflict of interest in relation to the subject of this report.


Endometrial stromal tumours are uncommon tumours of the uterus. They mainly occur in perimenopausal women. Tumours with typical clinicopathological features do not usually pose diagnostic problems. However, rare clinicopathological features can occur, and clinicians without significant experience may have difficulty diagnosing these tumours and managing these patients.Herein, we report a case of endometrial stromal sarcoma that occurred in a 25‐year‐old woman. The pathological features, immunophenotype, treatment and prognosis were discussed.The tumour revealed morphological heterogeneity, and there were similar proliferative‐type endometrial stromal cells, an extensive amount of mature adipose tissue, and prominent rhabdomyoblastic and smooth muscle cells. Histopathological and immunohistochemical studies confirmed low‐grade endometrial stromal sarcoma with smooth muscle, adipocytic and rhabdomyoblastic differentiation (approximately 60% were differentiated tissues). The final treatment of the tumour was total abdominal hysterectomy with bilateral salpingo‐oophorectomy. There was no evidence of recurrence for 109 months postoperatively.We found that low‐grade endometrial stromal tumours with extensive adipocytic and prominent rhabdomyoblastic differentiation are misdiagnosed because they are infrequent. They must be differentiated from rhabdomyosarcoma with accurate identification of adipocytes, and long‐term follow‐up is needed. Morphologic features of the low‐grade endometrial stromal sarcoma on haematoxylin–eosin stain. The tumour revealed morphological heterogeneity, there were similar proliferative‐type endometrial stromal cells, an extensive amount of mature adipose tissue, smooth muscle and prominent rhabdomyoblastic cells.  Nine months after delivery, the patient's mass was more pronounced in her lower abdomen. Ultrasound showed a mass on the left rear wall of the uterus measuring 10.4 × 9.1 cm, which was diagnosed as a uterine leiomyoma. The pelvic examination revealed a normal vagina, unremarkable cervix and a uterus that was consistent with a pregnancy at 16 weeks of gestation. Laboratory data, including blood cell count, chemistry and tumour markers, were within normal limits. Past medical history and family history were insignificant. Preoperatively, the patient requested fertility‐sparing surgery to treat the mass. The tumour was removed laparoscopically.Gross examination of the tissue block revealed a 12.0 × 9.0 × 3.5 cm tumour displaying grey‐to‐yellow cords and block tissue with a soft to firm texture. The cut surface of the tumour showed a focal haemorrhage.On a low‐power microscopic examination, the edge of the tumour was had obviously irregular margins and infiltrated into the surrounding myometrium Figure . It was Immunohistochemical staining was performed. The tumour cells were diffusely (approximately 90%) and strongly positive for cluster of differentiation 10 . The decision was made to perform a total abdominal hysterectomy with bilateral salpingo‐oophorectomy 1 month after the initial surgery. Residual tumour and intravascular tumour thrombus were not found in the pathological examination. Considering the above situation, the patient chose not to receive adjuvant treatment.Postoperative follow‐up was continued, and there was no evidence of recurrence in the 109 months to date.3LG‐ESS is a rare tumour accounting for <1% of uterine malignancies.There are no reliable preoperative imaging modalities that can distinguish LG‐ESS from a uterine mesenchymal tumour.Postoperatively, LG‐ESS with typical pathological features is usually characterised by the proliferation of small uniform cells. The mitotic index is low, usually <5 mitotic figures per 10 high‐power fields. Immunohistochemical staining indicates positivity for CD10, ER and PR. However, in rare instances, LG‐ESSs show unusual morphologic features. There are various differentiation types, including smooth muscle differentiation, fibromyxoid change and sex cord‐like differentiation. Endometrioid‐type gland appearance and clear cell change are uncommon.The presence of mature fat is much more commonly seen in smooth muscle tumours of the uterus. To our knowledge, our case of endometrial stromal sarcoma with adipocytic differentiation is the second reported case.The main diagnostic difficulty was distinguishing between rhabdomyosarcoma and endometrial stromal sarcoma with extensive rhabdomyoblastic differentiation, especially spindle cell rhabdomyosarcoma. The spindle cell variant of rhabdomyosarcoma is uncommon and most often encountered in the paratesticular region of children. Patients have a good prognosis. Only isolated cases in adulthood have been described.Another important differential diagnosis was highly cellular leiomyoma. The cells in highly cellular leiomyoma tend to be slightly larger and have an eosinophilic cytoplasm. There is the typical bundle area migration. In addition, highly cellular leiomyomas usually contain thick‐walled vessels. There can be irregular extension between tumour tissue and adjacent myometrium, but there is no invasive growth or necrosis. h‐caldesmon staining was positive, and CD10 staining was negative. In this case, we observed that some tumour cells resembled endometrial stromal cells and other tumour cells resembled smooth muscle cells. Abundant thin‐walled arteries were found in the tumour background, and there was an obvious infiltrative margin. Immunohistochemical staining showed positivity of tumour cells for CD10, SMA  and h‐caldesmon . These features supported the diagnosis of LG‐ESS with smooth muscle differentiation.Endometrial stromal nodules are also commonly confused with LG‐ESS. Microscopically, the histologic features of ESNs and LG‐ESSs are very similar. However, ESNs are benign tumours and have a well‐demarcated border but focal finger‐like projections or immediately adjacent nests of tumour cells (measuring <3 mm in greatest extent from the main mass and <3 in number). In the tumour, infiltrative margins were obvious. Therefore, LG‐ESS was diagnosed.The standard treatment for LG‐ESS is hysterectomy and bilateral salpingo‐oophorectomy. Lymphadenectomy or lymph node biopsy remains debatable.LG‐ESS is an indolent tumour with a favourable prognosis.4In summary, we presented an extremely rare case of LG‐ESS, which had complex morphological features with extensive multiple tissue differentiation, especially extensive adipocytic and prominent rhabdomyoblastic differentiation. Such an unusual variant of ESS not only causes diagnostic challenges but also has not been reported in terms of its prognosis. Based on long‐term follow‐up, we described the diagnosis, treatment and prognosis of this case for the first time. However, further large‐scale studies with long‐term follow‐up are required to confirm our findings.The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.


HPMC-L illustrated a more stable gel structure than the HPMC-H solution. Nanoemulsions showed lower moduli values, firmness, and work of shear than conventional emulsions due to the influence of high-pressure homogenization. A combination of lecithin and HPMC improved the physical and lipid oxidative stability of the emulsions, presenting a lower creaming index and thiobarbituric acid reactive substances (TBARS). In conclusion, HPMC-L at 2% w/w could be a suitable type and concentration combined with lecithin to formulate a saturated fat replacer that could mimic butter technological performance during food manufacturing operations.The combination of two emulsifiers, lecithin and hydroxypropyl methylcellulose (HPMC), into emulsions is an interesting strategy to design fat replacers in food matrices. The objective of this study was to investigate the effect of HPMC type and concentration on the formation, stability, and microstructure of conventional emulsions and nanoemulsions. Two different types of HPMC with low and high content of methyl and hydroxypropyl groups (HPMC-L and HPMC-H) were evaluated. The results showed that the molecular structure and concentration of HPMC play a major role in the viscoelastic behaviour, the gelation temperature, and the strength of gel formed. The firmness and work of shear of HPMC solutions increased significantly ( The consumption of saturated fatty acids has adverse health effects, promoting cardiovascular disease (CVD) ,2. One iIn recent years, structured emulsions have been developed to be used as effective fat replacers in different food products by providing the desirable texture and organoleptic properties. Structured emulsions are oil-in-water systems where oil droplets are encapsulated in the layers of hydrated gelators . These sThe combination of the oil component and the emulsifier plays an important role in the formation of the nanoemulsion. Several studies reported on the effect of oil type, with different fatty acid chain lengths, and the effect of fatty acid profile emulsion stability and bioaccessibility of lipophilic bioactive ,19,20,21Hydroxypropyl methylcellulose (HPMC) is a hydrocolloid with surface activity, mechanical, and thermo-reversible gelation properties ,27. TherTwo different types of commercial hydroxypropyl methylcellulose (HPMC) were used ; one with low methoxy and hydroxypropyl content (HPMC-L) and another one with higher content of methoxyl and hydroxypropyl (HPMC-H) . Butter Trichloroacetic acid, thiobarbituric acid and 1,1,3,3-tetraethoxypropane (TEP) , and hydrochloric acid  were used. High purity water was used for the preparation and dilution of reagents.w/w of HPMC-L and 2, 3, 4, and 5% w/w of HPMC-H) were prepared following the method described by Liu, et al. [Concentrations of HPMC solutions were selected after preliminary work was carried out to achieve HPMC solutions that exhibit viscoelastic moduli values within the same range. HPMC solutions  in water (85% w/w) at 200 rpm for 30 min at ambient temperature to ensure complete dispersion. Then, the oil was added (10% w/w) to the aqueous phase during continuous stirring. Secondly, the emulsions were homogenised with a high-speed homogeniser  at 10,000 rpm for 5 min to produce conventional emulsions (CE) or for 10 min to produce nanoemulsion (NE). The obtained CE and NE were further processed into lecithin and HPMC-L stabilised CE and NE. After assessing HPMC thermogelation properties  to conventional emulsions . The mixtures were stirred using a magnetic stirrer  at 200 rpm for 3 h at ambient temperature to ensure complete dispersion, then left at 4 °C for at least 24 h in order to allow the HPMC to be completely hydrated before the measurements [HPMC-L was then added at three different concentrations  to NE . The NE was processed through a high-pressure homogeniser (HPH)  at 400 bars for 1 cycle . It was w/w were prepared to measure critical micelle concentration (CMC). CMC was determined at the intersection points of the interfacial tension values versus the HPMC concentration (logarithm) plot [The interfacial tension between HPMC solutions and extra virgin olive oil was determined using a pendant drop analyser  at 20 °C. An axisymmetric drop (20 µL) of HPMC dispersion was delivered and allowed to stand at the tip of the needle inside a quartz container of extra virgin olive oil (9 mL) for 15 min to achieve emulsifier adsorption at oil–water interface ,42. Threhm) plot ,44. All The rheological measurements were performed using a rheometer  equipped with a Peltier temperature control device. A serrated parallel plate geometry was used; the diameter of the lower stationary plate (PPTD 200/56/1) and upper plate (PP50/P2) was 50 mm; the gap between the plates was 1 mm. The samples were allowed to rest in the measurement position for 5 min for sample relaxation and temperature equilibration before measurements. Amplitude sweep and frequency sweep measurements were performed at 20 °C for HPMC solutions and emulsions, and at 30 °C for butter. In bakery products, shortening is usually incorporated into the mixing process at 25 °C, to achieve a more plastic and spreadable behaviour . All theThe strain sweeps were carried out at a strain amplitude range of 0.001 to 1000% and at a constant frequency of 1 Hz in order to determine the linear viscoelasticity region (LVR) of HPMC solutions and emulsions. The LVR was identified where G’ and G” were not influenced by applied strain.Frequency sweeps were conducted from 0.1 to 100 Hz at a constant strain amplitude of 1.0% for HPMC solutions and emulsions, and 0.01% for butter, in order to determine G’ and G” values.Temperature ramp viscoelasticity was determined according to the method of Sanz, Laguna, and Salvador  and Yoo A TA-XT2 texture analyser equipped with the Texture Exponent software  was used to determine the firmness and spreadability of the butter, HPMC solutions, and emulsions. Spreadability was determined according to the method of Stefan and Kocevski , ChetanaThe particle size and polydispersity index of emulsions were determined by a dynamic light scattering (DLS) instrument  following the method of Guerra-Rosas, et al.  and SharHt is the total height of the emulsion (mm) and Hc is the height of cream layer (mm). Measurements were performed in triplicate.The creaming index (%) was evaluated based on the method reported by Arancibia, Navarro-Lisboa, Zúñiga, and Matiacevich  with somw/w). Samples were heated in a water bath at 95 °C for 10 min, then the samples were allowed to cool down to room temperature for 10 min, followed by centrifugation  at 10,000 g for 15 min. The absorbance of the supernatant was measured at 532 nm using a UV spectrophotometer . The concentrations of TBARS values were determined by using a standard curve prepared using a 1,1,3,3-tetraethoxypropane (TEP) standard curve (coefficient correlation (R2) = 0.9994). TEP standards between 0.01 to 0.20 µg/mL were prepared with trichloroacetic acid 7.5%. Measurements were conducted in triplicates for each emulsion batch.TBARS were determined according to the method of Qiu, et al.  and Sharw/w) gel samples and the emulsions, which were lyophilized in a vacuum freeze dryer  for three days and stored in a desiccator for 24 h. All the samples were cut into small slices and fixed on a brass holder. Then, the surface of the samples was coated with gold-palladium and observed in the microscope  at an accelerating voltage of 20 kV and a magnification of 500×.SEM observations were performed according to the method described by Ding, Zhang, and Li  and Perop < 0.05). A two-way ANOVA was conducted to evaluate the influence of two independent factors: the type of emulsion  and storage time  on the creaming index and TBARS results.One-way analysis of variance (ANOVA) was performed using SPSS version 25.0 . Multiple pairwise comparisons using Tukey’s HSD test were used to compare the mean values  and methoxyl (21.4%) substitution levels in HPMC-L in comparison with HPMC-H .Higher concentrations of both HPMCs gave place to an increase in G’ and G” moduli values and lower frequency dependence compared with lower concentrations of the polymers. These results could be due to the formation of a more structured solution as the concentration of the polymer increased. Other authors also found an increase in moduli values for the more concentrated HPMC solutions ,30. WhenWhen hydroxyl groups in methylcellulose (MC) are substituted by hydrophobic groups, some hydrogen bonds within MC are prevented and the polymer derivate interacts more with the surrounding water . MoreoveOn cooling, a two-stage process was observed B,D: firsp < 0.05) on the firmness and spreadability of HPMC solutions. It was found that firmness and work of shear of HPMC solutions increased significantly (p < 0.05) with increasing concentration; high values of work of shear suggest a higher force is needed to induce shearing deformation, which indicates low spreadability. An increase in HPMC concentrations could lead to an increase in cellulose hydrophobicity and the formation of a transient three-dimensional network [w/w, the HPMC-H solution presented significantly (p < 0.05) higher values of firmness and work of shear than the HPMC-L solution. These results could be explained by the higher mechanical strength of the HPMC-H solution, at room temperature, as explained in The firmness and spreadability properties of a colloid define how it will behave during certain unit operations ; thus, these parameters will give us an indication of the technological functionalities of the HPMC solutions. The firmness and spreadability of HPMC solutions were analysed and compared with butter in order to select the suitable type and concentration of HPMC that could be used to formulate emulsions that could be used as a saturated fat replacer. As shown in  network ,59. Regaw/w as shown in Scanning electron microscopy (SEM) was used to further understand the microstructure of HPMC gels at 4% The molecular structure of HPMC and its concentration in solution were determinant factors for the behaviour of the polymer in solution during heating, specifically in the mechanical strength of the structures and the onset of the gelation temperature. HPMC-L had a higher surface activity than HPMC-H  and its The dynamic spectra of CE and NE stabilised with lecithin and HPMC in comparison to butter are shown in Emulsions with higher concentrations of HPMC (CE-4) showed higher G’ and G” moduli values and lower frequency dependence compared to CE-2, CE-0, NE-0, and NE-2 . These rAs discussed in During the cooling cycle, from 90 °C–50 °C B, the mop > 0.05), suggesting that when NE-2 is used in mixing or dough sheeting operations, it will respond to the forces in a similar way to butter. The concentration of HPMC had a significant effect (p < 0.05) on the firmness and spreadability of emulsions (p > 0.05) penetration and shear forces to induce a deformation in the samples, thus CE-4 was firmer and more difficult to spread than CE-2. These results agree with the viscoelastic behaviour of the samples, where it was also observed that higher HPMC concentration gave place to more structured samples  lower values of firmness and shear (higher spreadability) than CE. Although this trend was not observed in the analysis of the viscoelastic behaviour of the samples (When comparing CE-2 and NE-2 with the same HPMC concentration (2%), NE presented significantly (SEM images of emulsions are shown in p < 0.05) between the emulsion type and storage time when the CI of the emulsions was evaluated (p < 0.05) higher CI value than CE-2, CE-4, and NE-2. These results could be attributed to the effect of HPMC in the mechanical properties of the emulsions, as higher concentrations of HPMC gave place to more structured and firmer viscoelastic gels  in CI values for CE-0 and NE-0. Oil droplet aggregation including flocculation and coalescence take place over time, leading to the formation of a creaming layer in emulsions [p < 0.05) than CE-0, which could be due to the smaller droplets size (MDD) and narrower size distribution values (PDI) of the NE  between the type of emulsions and storage time at any of the two storage temperatures tested. The mean plots are shown in p < 0.05) TBARS values than CE-0 and NE-0  at two temperatures (4 °C and 20 °C). There were no significant interactions . TBARS values increased when storage time increased from day 7 to day 14 at 4 °C and from day 1 to day 7 at 20 °C. There are many factors that could affect the oxidation reaction of emulsions such as the chemical structure of lipids, oxygen concentration, temperature, antioxidants, droplet characteristics, and light [The changes in the TBARS values of emulsions through storage time B were sind light ,71,72. TThis study showed that the molecular structure of HPMC and its concentration in solution were determinant factors for the behaviour of the polymer in solution during heating, specifically in the mechanical strength of the structures and the onset of the gelation temperature. A higher concentration of hydrophobic groups gave place to HPMC solutions with higher mechanical strength. However, lower molecular weight HPMC formed thermoreversible gels that presented higher stability during cooling and higher final strength. Moreover, this study revealed that the combination of emulsifiers (lecithin and HPMC) had a positive effect on the rheological and texture properties and on the physical and chemical stability of conventional emulsions and nanoemulsions. The results demonstrated that a combination of lecithin and HPMC improved the physical and lipid oxidative stability of the emulsions during storage, due to (i) the formation of a thicker protective layer of lecithin and HPMC adsorbed in the interface of the oil droplets and (ii) the formation of a stronger network in the continuous phase. Nanoemulsions showed higher spreadability than conventional emulsions due to the disruptive forces of high-pressure homogenization on the emulsion structure. A nanoemulsion formulated with 5% lecithin and 2% HPMC could be used as a suitable saturated fat replacer on bakery products, as it showed a similar firmness and spreadability to butter, indicating a similar deformation response in processing operations. These results will be the basis to formulate saturated fat replacers with specific technological functionalities to achieve the right performance in food manufacturing operations such as mixing, dough sheeting, baking, and storage.


G-quadruplexes (G4s) are well known non-canonical DNA secondary structures that can form in human cells. Most of the tools available to investigate G4-biology rely on small molecule ligands that stabilise these structures. However, the development of probes that disrupt G4s is equally important to study their biology. In this study, we investigated the disruption of G4s using Locked Nucleic Acids (LNA) as invader probes. We demonstrated that strategic positioning of LNA-modifications within short oligonucleotides (10 nts.) can significantly accelerate the rate of G4-disruption. Single-molecule experiments revealed that short LNA-probes can promote disruption of G4s with mechanical stability sufficient to stall polymerases. We corroborated this using a single-step extension assay, revealing that short LNA-probes can relieve replication dependent polymerase-stalling at G4 sites. We further demonstrated the potential of such LNA-based probes to study G4-biology in cells. By using a dual-luciferase assay, we found that short LNA probes can enhance the expression of c-KIT to levels similar to those observed when the c-KIT promoter is mutated to prevent the formation of the c-KIT1 G4. Collectively, our data suggest a potential use of rationally designed LNA-modified oligonucleotides as an accessible chemical-biology tool for disrupting individual G4s and interrogating their biological functions in cells. G-quadruplexes (G4s) are non-canonical secondary structures of DNA that can form from guanine-rich sequences in human cells . Over thCurrently molecules to disrupt G4s are limited, with only few molecular structures described to date . Recent in vitro is assessed using single stranded G-rich sequences, in the genome G4s are always exposed to their reverse-complementary strand (i.e. the C-rich strand) that competes with the G4 for duplex formation. These two structures (i.e. duplex and G4) are therefore likely to exist in a dynamic equilibrium  and investigated their ability to affect the kinetics of G4-disruption. We explored the effect of LNA-placement by designing sequences that differ in the positioning of the modifications along the probe, keeping the total number of LNA modifications under five. We observed that the LNAs placed opposite to guanines within a G-tetrad accelerated the rate of G4 disruption, whilst the very same sequences with the LNA targeting the loops of the G4 resulted either in non-significant changes in disruption kinetics or, counterintuitively, a deceleration of the G4-disruption. Strikingly, our investigations also revealed that an LNA-modified probe as short as 10 nucleotides retains the same G4-disrupting ability when compared to fully complementary sequences containing the same LNA modifications, suggesting that LNAs actively trigger the G4-disruption process and that short probes can be used to accelerate G4-disruption under physiological conditions. Single-molecule mechanical disruption experiments confirmed the enhanced G4-disruption by short LNAs, demonstrating the potential of such probes to deplete G4s sufficiently stable to stall polymerase. This led us to hypothesise that LNA-probes could facilitate the progression of polymerases through stable G4 sites across the genome during replication and transcription. We demonstrated this concept in a single-extension polymerase-stalling assay, revealing that treatment with LNA-probes can measurably enhance polymerase progression through G4-templates in a fashion that supports an LNA-mediated G4-disruption model. Remarkably, the probes with the greatest c-KIT1 G4-disrupting ability also caused an increase in c-KIT gene-expression, as measured by a dual luciferase assay. Altogether, our data demonstrates that short LNA probes can be easily designed to significantly disrupt G4-folding both in biochemical assays and in cells, suggesting a potential for rationally designed LNA probes as an accessible platform for systematic investigation of the biological function of individual G4s in the human genome.t) = 0. Experiments were performed at 25°C. The samples were excited at 488 nm with an 8 nm excitation slit and emission was measured at 518 nm with an 8 nm emission slit at regular time points using a BMG Labtech Clariostar Plus instrument. Data points were taken every 30s (for hTelo) or every 120s (for c-KIT1). All experiments were performed in three independent replicates. Data were normalised to define the signal at t = 0 as 0% and the saturation point of the curves as 100%. For each condition, each replicate dataset was fit to a non-linear model using a single exponential decay function to obtain apparent half-lives (s). Data analysis and visualisation were performed using GraphPad Prism 9.0.1. Error bars represent standard error of mean (SEM). Statistical analysis was performed using an unpaired, two-tailed Student's t test and P values less than 0.05 were considered statistically significant.Oligonucleotides used in this assay were purchased from Integrated DNA Technology (IDT) and stored as water solutions at − 20°C  and stored at −20°C. T4 DNA ligase and Exonucleases I and III were obtained from New England Biolab . Terminal transferase and Phi 29 polymerase were respectively obtained from Thermo Fischer  and Mclab . Digoxigenin dUTP (Dig-dUTP) was purchased from Roche (Swiss). Streptavidin and digoxigenin (Dig) antibody-coated polystyrene beads were purchased from Spherotech .TAA CCC TAA CCC TAA CCC TAA CCC TAA CAA TCC TG) that contain a 27-nt sequence (underlined) complementary to the hTelo DNA sequence was first slowly annealed (95–25°C in 2.5 h) with a splint . The hybridized DNA was circularized by T4 DNA ligase at 16°C for 16 h  to carry out RCA (Rolling Circular Amplification) by Phi 29 polymerase in presence of 10 mM dNTP at 30 °C for 5 min. After RCA, the product was labeled with digoxigenin (Dig) at the 3′ end through the terminal transferase with 0.2 mM Dig-dUTP at 37 °C for 1.5 h. The biotin- and digoxigenin-labelled, multi-GQ containing ssDNA construct was stored at −20°C and used within one week for mechanical disruption experiments.2 laser . The top and the bottom channels in the microfluidic chamber were used to inject streptavidin-coated polystyrene beads that had been attached with multi-GQ containing ssDNA and the anti-Digoxigenin-coated polystyrene beads, respectively. The top and bottom sections of the middle channel were connected with the top and bottom channels by micropipettes , respectively. Buffers with different chemical compositions were flowed into the two middle channels by a Harvard Apparatus PHD 2000 pump  with a flow rate maintained at 0.5 μl/min. The chamber was fixed in a sample bracket in a homemade laser optical tweezer instrument (ΔL) and construction of ΔL-force curves, see Supporting Information, t-test on the mean of at least 5 independent measurements. Statistical significance is under P values lower than 0.05.A multi-channel microfluidic chamber was used for single-molecule experiments . The chastrument . To starP values lower than 0.05 were considered statistically significant. Error bars in graphs represent standard error of mean (SEM).Polymerase extension assay was performed using Phi29 DNA polymerase  purchaseTm. The triplicates were then used to generate mean curves and standard errors of means.UV-melting experiments were performed as previously described . In a ty2.Cells were grown in DMEM Glutamax media  supplemented with 10% fetal bovine serum at 37°C, 5% CO4 cells/well in a 96-well plate (Corning® 96-well Clear Flat Bottom-3598). The transfection was performed with 330 ng of pc-KIT plasmid (Addgene plasmid number 118983) and 25 μM of LNA probe in a final volume of 5 μl by using FuGENE® HD Transfection Reagent (Promega-E2311). The level of expression of the dual luciferase reporter genes was measured 24 h post-transfection. The luminescence signals were acquired using a BMG CLARIOSstar Plus luminometer, using the dual luciferase reporter assay reagents (Promega-E1980) and following the manufacturer instructions. Luminescence data were acquired from three biological replicates each performed in three technical replicates.HEK293T cells were plated one day before transfection, at a cell density of 4 × 10N = 3 for each condition) was tested using Shapiro-Wilk test  and Normal QQ-plots. The statistical significance of the results was tested using one-way ANOVA first to assess overall significant differences, and successively using the Dunett's test . P values lower than 0.05 were considered statistically significant. Error bars on graphs represent standard error of mean (SEM).The dataset containing the biological replicates for each condition was normalised to impose that the highest value in each the dataset was considered 100%. The normalised datasets . As expected, all the LNA probes caused a significant, but comparable, increase in the melting temperature of the duplex formed  compared to the duplex obtained using a non-LNA modified DNA probe. This is in line with previous literature reports, which confirms that the positioning of the LNA probes along the sequence has a limited effect on the thermodynamic stability of the duplex formed as a result of G4-disruption . Since K+ is known to have a strong effect on G4-stability (+ concentrations to have sufficiently slow G4-disruption rates (∼30–60 min for c-KIT1) for accurate kinetics measurements. To achieve this, we performed our assay using 100 nM of labelled c-KIT1 annealed in presence of 5 times molar excess of DNA probe KIT_DNA1 in a Tris–HCl buffer (pH 7.4) containing 1 mM KCl. We then followed the disruption of the G4 by monitoring FAM emission over time, which yielded a half-life of c-KIT1 G4 of 1753 ± 70 s , corresponding to a ∼3-fold acceleration of the G4-disruption triggered by KIT_LNA3 invasion compared to what was measured with the DNA probe (KIT_DNA1). Conversely, when LNA modifications were placed in the loops (KIT_LNA1), the measured G4 half-life  was comparable to the DNA probe KIT_DNA1. Consistent with this trend, targeting the 3′ G-tetrad of c-KIT1 G4 with KIT_LNA2, also resulted in a significant reduction of the half-life . To confirm that targeting of Gs within the G-tetrads was essential to achieve accelerated G4-disruption, we next tested KIT_LNA4 in which the modifications are scrambled throughout the sequence without exclusively targeting the G-tetrads. Surprisingly, KIT_LNA4 yielded a significant increase of the G4 half-life , which corresponds to a ∼2.5-fold deceleration of the G4-disruption process compared to that with unmodified DNA (KIT_DNA1)  Figure . This obA clear trend that emerges from this initial screening is that LNA modifications, particularly when placed complementary to the guanines involved in G-tetrad formation, can significantly accelerate the rate of c-KIT1 G4-disruption. Collectively, this data revealed a strong position-dependent effect of LNA modification in oligonucleotide probes on the half-life of c-KIT1 G4, which could serve as guiding-principles for rational design of effective G4-disrupting probes.KIT-LNAs, we questioned if a similar trend existed among other G4s. To test this, we used the human telomeric G4 sequence (hTelo), which adopts a mixed type topology , corresponding to a ∼1.5-fold acceleration of G4-disruption compared to unmodified DNA probe hTelo_DNA1 (1967 ± 65 s)  relative to the hTelo_DNA1 control  Figure . In contl Figure . These rin vitro and in vivo models , corresponding to a ∼2-fold acceleration compared to the longer unmodified KIT_DNA1 (1753 ± 70 s)  is significantly more efficient in accelerating c-KIT1 G4-disruption than its full-length analogue KIT_LNA2 (1087 ± 24 s), suggesting the potential for an active role of the LNA modifications in disrupting this G4-structure. Conversely, with a truncated probe lacking LNA modifications KIT_DNA_short, we failed to observe any G4-disruption, confirming that LNA modifications are essential for G4-disruption. Similarly, for the hTelo system, we found that the short LNA probe hTelo_LNA_short under similar conditions (100 nM G4 in a Tris–HCl buffer at pH 7.4 in 100 mM K+) showed efficient disruption of the hTelo G4, yielding a lifetime of 1655 ± 105 s, which is comparable (P = 0.337) to that observed with the longer LNA-modified analogue hTelo_LNA1 (1539 ± 18 s), while remarkably faster than the iso-sequential DNA control hTelo_DNA_short   than hTelo_LNA_short, supporting that the position of LNAs in the probe is critical to achieve efficient G4-disruption. These results demonstrate that shorter LNA-modified probes are highly efficient to reduce the disruption half-life of G4s, displaying comparable or better efficiency than their full-length analogues. This key observation suggests a model in which the LNA modifications invade the G4s and promote its disruption independently from the length of the hosting DNA sequence.To target c-KIT1, we designed ) Figure . It is w) Figure . We notislow one , which m14 ± 18s , which iKIT_LNA_short and KIT_DNA_short to disrupt c-KIT1 G4s under higher K+ conditions. Briefly, we mixed (25 nM of Cy5-labelled) c-KIT1 G4 in Tris.HCl buffer at pH 7.4 containing 100 mM KCl with a 10-fold excess of the probe (either LNA or DNA), which was then followed by PAGE to separate products based on their molecular weights. The formation of a higher molecular weight species (indicative of LNA-DNA heteroduplex formation) was observed only when the c-KIT1 G4 was treated with KIT_LNA_short  in the presence of KIT_LNA_short  or hTelo_LNA_short (21 folded G4s) decreased the number of folded G4s in the construct (55 G4s in buffer). This confirms what was measured by FRET assays for hTelo, demonstrating that hTelo_LNA_short is a more efficient G4-disruptor compared to its DNA analogue , suct Figure  allows ue Figure .F1/2) at which 50% of all telomeric G4s present in the construct are mechanically disrupted under different conditions  significantly increased the average mechanical stability of the telomeric G4s compared to the untreated sample (F1/2 = 25.4 pN) or the hTelo_DNA_short sample (F1/2 = 26.8 pN). This suggests that in the presence of hTelo_LNA_short, although formation of G4s was reduced, the surviving G4s had conformations that render increased mechanical stabilities compared to those with or without hTelo_DNA_short  or do not favour G4 formation (100 mM Li+). The polymerase is expected to stall at the c-KIT1 G4 under K+ conditions, which leads to truncated extension products resolved in a urea-denaturing gel. Under conditions that do not promote G4-formation (i.e. Li+), more efficient formation of the full-length product should be observed. The ratio of the full-length product versus the stalled product can be quantified as the extent of the polymerase progression under different experimental conditions , we observed a ∼5-fold increase (P =0.04) in polymerase extension compared to the untreated template , we failed to observe any significant (P = 0.593) rescue of polymerase progression. These results suggest that short LNA probes can be used to abrogate polymerase-stalling at G4-sites by disrupting G4s under physiologically relevant conditions. These results also indicate that the acceleration in G4-disruption and the depletion of mechanically stable G4s triggered by LNA probes are sufficient to perturb G4-prevalence and stability in a way that can affect biochemical process, such as polymerase elongation. Altogether this further validates the potential for LNA-based probes to be used in cells to investigate G4-biology.In contrast, when the G4-containing template was treated with DNA probe in vitro results, we next questioned if the G4-disrupting properties of these LNA-probes could be used to perturb G4-folding in cells. To investigate this, we employed a previously described dual-luciferase reporter system . This plasmid was transfected into HEK293T cells in the presence or the absence of the KIT LNA/DNA probes for 24h, followed by the luminescence-based measurement of gene-expressions. Since the promoter that controls Firefly expression does not contain the c-KIT1 G4, we would expect that our probes would only affect the Renilla expression levels, which is controlled by the c-KIT promoter.Inspired by the m Figure  52). Br. Brin viKIT_LNA2 and its shorter version, KIT_LNA_short, led to a statistically significant increase in gene expression of the Renilla gene when compared to the untreated sample  24 h after transfection, whereas KIT_LNA_short led to an increase in gene expression of ∼1.5-fold (P = 0.03) compared to untreated sample. In contrast, KIT_LNA4 and the control DNA probe KIT_DNA_short, which are not expected to efficiently disrupt the G4, failed to provide any significant change in gene-expression relative to the untreated plasmid.Treatment of the transfected cells with e Figure . SpecifiKIT_LNA2. This further supports a model in which the increase in gene-expression measured in our experiments is a consequence of LNA-mediated disruption of the c-KIT1 G4. Our results suggest that LNA-probes can elicit G4-disruption in a cellular context, indicating a potential link between half-life of G4-disruption and its implication on gene-expression that could be further leveraged to study G4-biology.A previously reported study using the same pc-KIT plasmid  showed tiso-sequential LNA probes vary greatly in their in vitro potency depending on their locations. We questioned if a similar positional-effect could be exploited to improve LNA-modified G4-disrupting probes.Current methods to investigate G4-biology mostly rely on either small-molecule or antibody-based approaches ,54, whicOur disruption assays with two topologically different G4s (c-KIT1 and hTelo) demonstrated that LNA-modified probes can not only disrupt but also accelerate the disruption rate of these model G4 systems. Crucially, our data revealed that the position of the LNA modification is key for ‘fine-tuning’ the G4-disrupting abilities. When LNA-modifications were placed complementary to the G-tetrad, we observed significant reductions in the half-life of G4-disruption. Strikingly, when the same number of LNA modifications were distributed in the loops or randomly dispersed, there was a significant increase in the half-life of G4-disruption, suggesting a slow-down of the G4 disruption kinetics. These observations were similar for both G4 systems studied here, suggesting that the design of LNA probes to target and disrupt specific G4s is a generalisable strategy.iso-sequential short DNA probes showed no such capability.To further assess the role of LNA-modifications to promote G4-disruption, we investigated shorter LNA probes of 10 nts in length. These probes target half lengths of the c-KIT1 and hTelo G4-forming sequences. We found they were still able to efficiently induce G4-disruption. By using a FRET-based assay, we demonstrated that the short LNA-modified sequences were able to efficiently disrupt and accelerate the disruption kinetics of G4s with a comparable or higher efficiency than the full-length probes, which further indicates an active role of the LNA-modifications in promoting G4-disruption. Application of such short LNA probes in a single-molecule optical tweezer platform revealed that these LNA probes were able to deplete hTelo G4s. This key observation suggested that LNA probes can abrogate stable G4s, which stall polymerase in replication or transcription bubbles. To test a potential application of such LNA probes to disrupt G4s in biochemical processes, we have employed a single step-extension assay to evaluate whether short LNA-probes could relieve polymerase stalling at G4-sites. Consistent with the single-molecule experiments, this assay demonstrated that short LNA probes were able to disrupt the G4 and increase polymerase extension of templates containing G4s, whilst the equivalent in vitro was also found to be correlated with elevated c-KIT1 expression in cells (We then tested these probes in a dual luciferase reporter assay to investigate their ability to perturb transcription in cells. By using a plasmid system that contains the promoter region of the oncogene c-KIT, we revealed that short LNA probes targeting c-KIT1 G4 were able to enhance gene-expression in cells. Strikingly, treatment with the very same sequences lacking LNA modifications did not display any change in gene-expression levels. These results are in agreement with the observed increase in gene-expression measured when formation of c-KIT1 G4 was prevented by sequence mutations in the pc-KIT plasmid . Similarin cells . The incAll the data reported in this paper are available in the manuscript or supplementary information. Raw gel and microscopy images can be requested to the corresponding author.gkac569_Supplemental_FileClick here for additional data file.


In this case report, we present a unique cause of coccygodynia due to an intradural lumbar spinal tumour in a patient with multiple filum terminale paragangliomas. We highlight the symptomatology, the clinical course, and the radiological findings. Our review of the literature proved our case to be the first report of an intradural cauda equina tumour presenting with coccygodynia in English literature. Based on the outcome and clinical response to treatment we make a bold hypothesis on the possible anatomical mechanism of his coccygodynia. Paragangliomas are neoplasms originating from the autonomic nervous system, found generally in adrenal and extra-adrenal locations. Extra-adrenal paragangliomas are rare and occur most commonly in the carotid bodies and the jugular glomus. Primary spinal paragangliomas are extremely rare, most frequently involving the cauda equina and the filum terminale . The mosA 47-year-old male patient was referred to our Neurosurgical Department due to a two-year-long history of coccygodynia not responding to pain management. The patient was assessed multiple times by General Practitioners and Orthopaedic surgeons during all that period and was finally referred for a pelvis MRI. Due to an abnormal finding, he underwent a complementary lumbar spine MRI and then was referred to our department.The patient was complaining of pain in his tail bone area, sometimes aggravated in the morning and he described no particular activities or positions to alleviate or provoke the pain. Especially, he did not seem to be affected by prolonged sitting or pressure in the area. Clinical and neurological examinations were unremarkable whereas palpation and manipulation of the coccyx did not reveal any tenderness. The patient had no past medical history and did not report any recent injury.MRI of his lumbar spine revealed a large intradural, homogeneously enhancing tumour at the level of the L3 vertebra, as well as a smaller one with similar radiological characteristics at the level of S2 Figure . A charaAfter careful evaluation of the MRI findings, we made a decision to operate on the large one and based on the histology and clinical response to decide on further treatment. The patient underwent an L3 laminectomy and successful total excision of the intradural lesion through a standard durotomy. Intraoperatively, the tumour was well encapsulated, vascular and firmly attached to the filum terminale. A large vein was noted running adhered to its surface, few nerve roots were loosely adhered but easily dissected off the tumour and prominent bleeding was noted if the capsule was violated. The tumour was carefully dissected off surrounding nerve roots, the filum terminale was coagulated and then cut proximally and then distally achieving complete removal of the tumour. Postoperatively, the patient had an excellent recovery and complete resolution of his coccygodynia was reported.Histological examination of the specimen revealed an encapsulated tumour with an alveolar pattern consisting of chief cells with granular, eosinophilic cytoplasm and spheroidal nuclei arranged in clusters separated by fine fibrovascular septae with dilated vessels (Zellbaten pattern). No nuclear atypia or necrosis was noted. Immunochemistry labeling was chromogranin (+), synaptophysin (+), S100 (+), GFAP (-), and keratin CK18.8 (+) confirming the diagnosis of a filum terminale paraganglioma.Interestingly, the patient was under close follow-up and reported relapse of his coccygodynia approximately 18 months postoperatively. A new MRI was performed which revealed total removal of the tumour at L3 with no local recurrence but increase in size of the smaller lesion at S2 Figure . The patFilum terminale paragangliomas are rare benign tumours that arise from the ectopic sympathetic neurons and become misplaced on the cauda equina rootlets or on the filum terminale ,4. ParagOn the other hand, coccygodynia is a well-known clinical entity with variable aetiology most commonly attributed to major or repetitive minor trauma of the coccyx. Non-traumatic causes of coccygodynia include hypermobility or hypomobility of the sacrococcygeal joint, infections, neoplasms, degenerative joint disease, anatomical variants of coccygeal morphology, or even somatization disorder and other psychological disorders. The hallmark of coccyx pain in local causes is the typical pain that worsens with prolonged sitting and even with defecation and sexual intercourse. Physical examination usually reveals tenderness over the coccyx and pain on manipulation ,7.However, atypical coccygodynia could represent a referred or radicular pain and should prompt further evaluation of the patient . In termTo our knowledge so far, there was no correlation between an intradural lumbar spine tumour presenting with coccygodynia. Therefore, we conducted a PubMed search using the terms coccygodynia/coccydynia/coccalgia, tailbone pain/coccyx pain/coccygeal pain, AND intradural tumour/spinal tumour or using specific tumour terms. According to our search, there were only two French articles in 1967 and 1968 reporting an ependymoma and a giant tumour of the cauda equina presenting with coccygodynia ,12. Our In this context, we attempted to shed light on the possible correlation of such a tumour causing coccyx area pain. Based on the complete resolution of pain after removal of the large L3 tumour, we hypothesized that most probably this was a referred pain alleviated by removal of the lesion and transection of the filum terminale. We assumed that the tumour was causing mechanical strain on the filum terminale, which terminates attached to the first segment of the coccyx, potentially causing referred pain.However, in our case, the patient experienced a relapse of his pain and underwent an MRI that revealed an increase in the size of the second smaller intradural S2 tumour and no recurrence at the original site of surgery. Obviously, this means that our original assumption was wrong and most probably the aetiology of pain should be radicular in nature and caused by compression of the lower sacral nerve roots whose dermatomes correspond to the area patients may describe as the tail bone area.Wall et al. described the cross-sectional anatomy of the cauda equina in 15 fresh human cadavers and discovered a highly consistent pattern . The lowCoccygodynia is a well-known entity with characteristic pain patterns and clinical signs. However, as highlighted by our case, first-line physicians should be aware of atypical coccygeal pain and suspect a referred or radicular pain mechanism so that they further evaluate patients and conduct a more thorough paraclinical investigation. Our case highlights such a paradigm and also demonstrates a unique clinical presentation of an intradural lumbar tumour.


There were no significant differences in final HB grade. All ENoG values of the VZV DNA-positive group were significantly higher than those of the control group . Conclusions: RHS patients with salivary VZV DNA have more severe clinical symptoms than the control group. There were no differences in prognosis and associated symptoms. A VZV DNA test using saliva samples of patients with RHS can evaluate the clinical symptoms and provide early confirmation of VZV infection, enabling timely treatment in a non-invasive way.Background: The relationship between varicella zoster virus (VZV) collected from saliva and Ramsay Hunt syndrome (RHS) remains unclear. Therefore, this study aimed to investigate whether VZV DNA in saliva alters the clinical symptoms and prognosis of RHS. Methods: To measure the severity of clinical symptoms of 100 RHS patients, the initial House–Brackmann (HB) grade and associated symptoms were evaluated. The final HB grade at the end of treatment was measured to determine the prognosis. Electroneurography (ENoG) was performed on four facial muscles, including the frontalis, oculi, nasalis, and oris. Results: Salivary VZV DNA was isolated from 72 patients with RHS. The VZV DNA-positive group was 34.023 times more likely to have severe initial HB grade than the control group (95% CI, 3.21–359.68;  Ramsay Hunt syndrome (RHS) is a neurological disease caused by the varicella zoster virus (VZV) and is the second most common cause of acute facial palsy. RHS shows clinical symptoms of vesicles in the ears or oropharynx, accompanied by facial palsy . In someThis retrospective study included 100 patients diagnosed with RHS at Kyunghee Medical Center, Seoul, Korea between January 2015 and October 2021. RHS patients were diagnosed based on clinical symptoms. Clinical symptoms of RHS include a vesicular rash on the ear (herpes zoster oticus) or in the oral mucosa accompanied by acute peripheral facial nerve paralysis. Patient medical records included information regarding sex, age, treatment period, underlying disease, and morbidity. Comorbid symptoms, such as dizziness, tinnitus, hyperacusis, and hearing distance, were also assessed. The House–Brackmann (HB) grading system was used to evaluate the degree of facial palsy . The HB The inclusion criteria were patients hospitalized within seven days of onset and who were not lost to follow-up until the end of treatment. Patients with incomplete medical records, whose cause of facial palsy was malignancy or central lesion, those who were lost to follow-up during treatment, or those who had previous herpes zoster infection were excluded.All patients who met the inclusion criteria were treated with oral corticosteroids , oral antiviral agents (famciclovir for 1 week), and protective eye care by administration of artificial eye drops or ointment, taping of the paralyzed eyelid during the night, and patient education [Saliva samples were collected from the hospitalized patients. The saliva samples were collected by patients themselves spitting 1~2 mL of saliva into the sample container. Samples were collected once on the day of hospitalization. After the samples were collected, these were immediately sent to the molecular genetic testing room and immediately stored in a refrigerator at 4 °C until examination. Viral NA Small Volume Kit (Roche) reagents were used for DNA extraction from saliva samples using the MagNA Pure 96 (Roche). The Pathogen Universal 200 protocol was used for viral DNA isolation from 200 μL of each saliva sample using 100 μL of eluate as per the purification protocol. A low denaturing temperature PCR (LDT-PCR) was performed using conventional PCR reagents and VZV PCR primers. The PCR mixture included 10 μL 2× master mix, 5 μL VZV primers, and 5 μL template. The PCR conditions were 94 °C for 15 min, 94 °C for 30 s, 65 °C for 1 min, 72 °C for 1 min, and 72 °C for 10 min. PCR results were analyzed using electrophoresis and an image analyzer, the Gel Doc XR System  [Venous blood samples of the patients were analyzed using enzyme-linked immunosorbent assay (ELISA). Anti-VZV IgM and IgG titers were measured in the blood samples. Antibody titers of anti-VZV IgM and IgG were interpreted as positive if their ratio was ≥1.10.Electroneurography (ENoG) was performed between 5 and 14 days after the onset of facial palsy to evaluate its prognosis using a constant current high-voltage stimulator (model DS7A) by a highly experienced examiner ,12. ENoGt-tests. Categorical variables, such as initial and final HB grade, sex, direction, diabetes mellitus (DM), hypertension (HTN), and comorbid symptoms, were compared using chi-squared or Fisher’s exact tests. Multiple regression analysis was performed to determine the effect of VZV DNA in saliva on the initial and final HB grade by controlling confounding variables, such as sex, age, direction, DM, and HTN, and presented as the odds ratio and 95% CI. All statistical analyses were performed using IBM SPSS statistical software. Statistical significance was set at p < 0.05.Continuous variables, such as ENoG values and age, were compared using A total of 100 patients with RHS were enrolled in this study. The demographical data of the study participants are presented in p = 0.014); the probability of severe initial HB grade in patients with RHS was 34.023 times higher than that of the negative group .p = 0.003; orbicularis oculi, p = 0.001; nasalis, p < 0.001; orbicularis oris, p = 0.037).In this study, we examined differences in clinical and comorbid symptoms as well as prognosis according to the presence or absence of VZV DNA in the saliva of patients with RHS. VZV DNA in saliva was detected in 72% of the patients with RHS, and VZV IgM was detected in 33% of the patients with RHS. The detection efficiency of VZV DNA in saliva was higher than that of VZV IgM in patients with RHS, which confirmed that PCR analysis of VZV DNA in saliva showed a higher diagnosis rate than serological tests in patients with RHS. In previous studies, the detection rates of VZV DNA in the saliva of patients with RHS and ZSH were reported to be between 23 and 63% and 7 and 59%, respectively ,14. The The initial HB grade according to the presence or absence of VZV DNA in saliva and the number of patients who showed severe clinical symptoms increased significantly in the VZV DNA-positive group. When comparing the ENoG values in the four facial muscles  according to the presence or absence of VZV DNA in the saliva of patients with RHS, the VZV DNA-positive group showed higher ENoG values in all four muscles than the control group. This means that the VZV DNA-positive group showed more severe axonal loss than the control group. Thus, detecting VZV DNA in the saliva of patients with RHS is associated with worse clinical symptoms. Moreover, there was no significant difference in the final HB grades categorized as complete recovery and satisfactory recovery, according to the presence or absence of VZV DNA in the saliva. Additionally, comorbid symptoms such as dizziness, tinnitus, otalgia, hyperacusis, and hearing disturbance did not vary significantly depending on the presence or absence of VZV DNA in the saliva. A few studies have shown the association between salivary VZV DNA and RHS. According to one study, the VZV DNA-positive group of patients with facial palsy had a higher final HB grade and no difference in the initial HB grade. Moreover, when compared to the VZV DNA-negative group, the VZV DNA-positive group showed more symptoms of dizziness and hearing disturbance, except otalgia . These fContinuous inflammation of the facial nerve in the temporal bone and VZV reactivation in the geniculate ganglia cause facial palsy in patients with RHS. The facial nerve is composed of motor, parasympathetic, and sensory fibers. The cell bodies of sensory fibers are in the geniculate ganglion. Some afferent fibers are supplied to the mucous membrane of the oropharynx, external auditory canal, and auricular skin . ReactivThis study has the advantage of presenting statistical validity by collecting data from a relatively large group of patients. In addition, the ENoG value was measured in the four facial nerves for a more accurate evaluation of clinical symptoms. Despite facial palsy being caused by VZV infection, the diagnosis of RHS is delayed because of the late appearance of vesicles, interfering with early antiviral treatment. Nevertheless, PCR analysis of VZV DNA in saliva can help overcome this problem through early diagnosis and clinical evaluation of RHS. A limitation of this study is that the difference in clinical symptoms according to the VZV DNA level could not be analyzed because the DNA levels in saliva could not be measured quantitatively using our instrument. Moreover, accurate VZV DNA analysis was uncertain, as there were differences in the time of expression of facial palsy and the time of collecting saliva samples for VZV DNA testing among patients.The initial HB grade was higher in the VZV DNA-positive group than that in the VZV DNA-negative group. Moreover, there were more patients with severe initial HB grades (V and VI) in the VZV DNA-positive group than in the VZV DNA-negative group. The ENoG values measured in the four facial muscles  were higher, indicating more severe clinical symptoms in the VZV DNA-positive group than the VZV DNA-negative group. However, there was no difference in the final HB grade, and hence no differences in prognosis and comorbidities between the two groups.Thus, a VZV DNA detection test using saliva samples of patients with RHS can evaluate the clinical symptoms and provide early confirmation of VZV infection, enabling timely treatment through non-invasive strategies.