This study evaluated the postoperative analgesic effect of ultrasound-guided single-point thoracic paravertebral nerve block (TPVB) combined with dexmedetomidine (DEX) in patients undergoing video-assisted thoracoscopic lobectomy. n = 20 each). G group: patients received routine general anesthesia; PR group: patients received 0.5% ropivacaine; and PRD group: patients received 0.5% ropivacaine with 1 μg/kg DEX. TPVB was performed in the T5 space before surgery, and then, general anesthesia induction and video-assisted thoracoscopic lobectomy were performed. Analgesics were administered through the patient-controlled analgesia (PCA) device intravenously. The background infusion of each PCA device was set to administer 0.02 μg/kg/h sufentanil, with a lockout time of 15 min, and a total allowable volume is 100 ml.  Sixty adult patients of the American Society of Anesthesiologists (ASA) I–III were randomly assigned into three groups (p < 0.05). Also, the duration of first time of usage of the patient-controlled analgesia (PCA) was longer. The heart rate (HR) and mean arterial pressure (MAP) during operation were lower in the PRD group as compared with the other two groups in most of the time. However, hypotension and arrhythmia occurred in three groups with no statistically significant difference.  Compared to PR and G groups, the total sufentanil consumption after operation, the times of analgesic pump pressing, the pain score, and the incidence of postoperative nausea or vomiting in the PRD group were significantly reduced ( A small volume of TPVB with ropivacaine and DEX by single injection produced longer analgesia in patients undergoing video-assisted thoracoscopic lobectomy, reduced postoperative opioids consumption, and the incidence of side effects. Video-assisted thoracoscopic surgery (VATS) is mainly used for the treatment of the lung, mediastinum, and pleural lesions. The main advantage is to avoid the injury of thoracotomy. Compared with thoracotomy, the operation time is shorter, the postoperative morbidity is lower, and the time for returning to normal activities is earlier . HoweverThoracic paravertebral block (TPVB) as part of a multimodal analgesia strategy after thoracotomy  and breaα-2 receptor agonists, due to their own sympathetic properties, provide stable hemodynamics during surgery and reduce the amount of narcotic analgesics. When DEX is used as an anesthetic for epidural anesthesia [Dexmedetomidine (DEX) is a novel alpha-2 adrenergic receptor agonist with dose-dependent sedative, anxiolytic, and analgesic effects and has an advantage of minimum respiratory depression compared with alternative drugs . It is westhesia , subaracesthesia , 13, PVBesthesia , 15, andesthesia , the timTherefore, this study was designed to evaluate whether TPVB combined with ropivacaine and DEX could improve the analgesic effect of patients undergoing VATS, thereby reducing postoperative opioid drugs consumption.ChiCTR-IOR-17013034. After obtaining approval and written informed consent from the institutional ethics committee, the study enrolled 60 ASA I–III patients  who were scheduled to undergo elective thoracoscopic lobectomy. Patients undergoing elective general anesthesia and video-assisted thoracoscopic unilateral lobectomy had no history of cardiopulmonary disease, no sinus bradycardia, no functional lesions of the sinoatrial node, no serious arrhythmia, and no thoracic deformity. Surgery time was more than 2 hours. Exclusion criteria: minimental state examination scores below 23 points; the history of dementia, psychosis, or other central nervous system disease or drug dependence and poor compliance; and those who did not complete video-assisted thoracoscopic lobectomy under general anesthesia or have to convert to thoracotomy.This study was approved by the Affiliated Suzhou Science and Technology Town Hospital of Nanjing Medical University' Ethics Committee, and written informed consent was obtained from all patients. The trial is registered at the Chinese Clinical Trial Registry, number μg/kg DEX for TPVB. Paravertebral blockade was performed under ultrasound guidance. A linear ultrasound transducer (HITACHI Arietta 60) was placed intercostally to identify the thoracic paravertebral space (TPVS), and a 20-gauge needle was inserted into the plane of the transducer. When the needle tip reached TPVS, 10 mL 0.5% ropivacaine with 1 μg/kg DEX in T5 paravertebral space was injected.All patients did not use preoperative medication, and in the preoperative preparation room, the patients were explained in detail the numerical rating scale (NRS) scoring rules . Patients were randomized into three groups of 20 individuals each using a computer-generated random number table (random number table method) with sealed envelope technology for assignment concealment (distribution hidden). In the operating room, ultrasound-guided right internal jugular vein catheterization for infusion and left radial artery catheterization for monitoring the changes of pressure have been performed. Conventional general anesthesia induction included sufentanil 5 ug/kg + etomidate 2 mg/kg + cisatracurium 2 mg/kg intravenous slow bolus injection; anesthesia maintenance: propofol 2.5 ug/ml, sevoflurane 0.8 MAC, remifentanil 3 ng/ml, bispectral index monitoring, and the depth of anesthesia is maintained between 40 and 60. Patients were classified into three groups as follows: G group: patients received routine general anesthesia; PR group: patients received 0.5% ropivacaine for TPVB; PRD group: patients received 0.5% ropivacaine with 1 μg/kg/h sufentanil, with a lockout time of 15 min, and a total allowable volume is 100 ml. The anesthetist conducted postoperative monitoring, pain assessment, and management was blinded to the patient groups. The flowchart of the study protocol is shown in The gender, age, ASA grade, height, weight, body mass index (BMI), heart rate (HR), mean artery pressure (MAP) baseline, duration of surgery (h), anesthesia (h), the time to first analgesic request since paravertebral injection (TFR1), first use of patient-controlled analgesia (PCA1) intravenously, total sufentanil dosage, and the pressing times of analgesic pumps were all recorded in three groups. The intraoperative MAP and HR were measured after starting TPVB bolus injection. Hypotension defined as a 30% decrease or less than 80 mmHg in systolic blood pressure from baseline was treated with ephedrine 5 mg intravenously and further boluses as required. Bradycardia defined as heart rate <55 beats per minute was treated with 0.6 mg intravenously. Analgesics were administered through the PCA device . The background infusion of each PCA device was set to administer 0.02 Ramsay sedation scores of patients were recorded at 30 minutes, 1 hour, 2 hours, 4 hour, 6 hours, 8 hours, 12 hours, and 24 hours after anesthetic recovery in three groups . The intraoperative MAP and HR were recorded at 10 minutes, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, and 100 min after starting TPVB bolus injection. Postoperative pain ratings (NRS) during rest and movement were recorded every two hours within 24 hours .All values are mean ± SEM. Two-way ANOVA for repeated measures was used when appropriate . The Student–Newman–Keuls multiple comparison post hoc test was used to differentiate within the groups. A probability value less than 0.05 was considered to indicate a significant difference between the groups, while a value greater than 0.05 was considered to indicate no significant difference between the groups.p > 0.05)  . All patp < 0.05; p > 0.05). There was no significant difference of postoperative Ramsay sedation scores among all groups before 12 h; however, the score values in the G group were significantly lower than that in the PR or PRD group  consumptions were reduced significantly in the PRD group compared to other two groups (p < 0.05) and G group (p < 0.05) (G group (p < 0.05).The NRS pain scores of ipsilateral arm were, respectively, indicated in  < 0.05) . There wp < 0.05). In the PR group and G group, the intraoperative HR tended to stabilize and increased slowly until 40 minutes, but the HR of the PRD group was lower than other G groups at 80 and 100 minutes. In the PR group and G group, the intraoperative MAP decreased significantly around 20 minutes, while MAP of the PRD group decreased to (77.1 ± 1.7) mmHg around 40 minutes, and it gradually returned to the baseline level around 100 minutes (p < 0.05). However, there was no significant difference in the consumption of sufentanil dose or the incidence of hypotension and the occurrence of bradycardia during the surgery.Hemodynamic parameters were monitored during surgery. HR and MAP trends are shown in Figures TPVB has been used wildly to reduce postoperative opioid consumption and provide effective pain control. The injection site and volume are quite different in different studies, and there are single and multiple injections with volumes of 5, 7, 10, 15, 20, or 0.3 ml/kg . A cadavDEX acts as a potential adjuvant for both axons and peripheral nerve blocks , 18, andIn this study, the requirement of vasopressors for maintenance of stable hemodynamic parameters did not reveal any significant difference during the induction period among groups. However, the HR and MAP in the PRD group were lower than those in the other two groups during surgery. It has been reported that the stable hemodynamics may possibly be explained on the basis of lower volume of local anesthetics used and a suitable selection of the dose of adjuvant . The sedμg/kg DEX) at site of T5 space before surgery also achieved effective postoperative analgesia. It may have beneficial clinical effects for reducing the side effects of drugs, puncture injury, and discomfort to patients and enhancing recovery after surgery.More recently, Hong et al. reported that DEX as an adjunct in TPVB provided effective pain relief and significantly reduced opioid requirement in VATS . Our resThere are certain limitations to our study. First, it was a retrospective study with a small sample size and the findings therefore have to be confirmed with a larger randomized controlled trial. Second, the small sample size and recruitment of relatively healthy patients (ASA I and II) limited the possibility of drawing a definitive conclusion. Greater numbers of patients and patients with multiple comorbidities  need to be included in future studies to verify these findings. Third, since a single dose of TPVB can provide sufficient pain relief, inserting a catheter into the paravertebral space for continuous infusion of local anesthetics may provide protracted postsurgery analgesia. Furthermore, different doses and drugs used in TPVB may cause bias of the results. More studies have to be conducted to clarify this question.μg/kg DEX combined with 0.5% ropivacaine by single injection at T5 space before surgery for patients undergoing thoracoscopic lobectomy prolonged the overall analgesia time after surgery and reduced the dose and side effects of opioids.In summary, a small volume of TPVB with 1


Paraneoplastic neurological syndromes (PNSs) are broad-spectrum disorders that can affect any part of the nervous system varying in core symptoms. Onconeural antibodies, including Hu, Yo, Ri, anti-CV2, amphiphysin, Ma2, and Tr are well-characterized and commonly used for the diagnosis of definite PNS. Generally, anti-CV2 antibodies have usually been associated with cerebellar ataxia, chorea, peripheral and autonomic neuropathies, myelopathy, optic neuritis, and retinitis. However, Parkinsonism has not been reported as the core symptom in patients with anti-CV2 antibodies.We report a patient with anti-CV2 antibody manifested as Parkinsonism and autonomic dysfunction, which may lead to the diagnosis of multiple system atrophy with predominant Parkinsonism (MSA-P). A lumbar puncture examination was undergone to find a positive anti-CV2 antibody in cerebrospinal fluid. PET-CT showed no tumor. Immunotherapy was adopted and the symptoms were relieved for 5 months. However, with no evidence of tumor, he died after 8 months.Our findings indicate that PNS with anti-CV2 antibody can be shown as MSA-P mimic. Considering that MSA is a neurodegenerative disease with a poor prognosis, screening for other treatable or controllable factors like PNS presented in this case is necessary when encountering a rapid progressive MSA-mimic patient.The online version contains supplementary material available at 10.1186/s12883-021-02448-6. PNS represents the remote effects of tumors on the nervous system, which is not associated with invasion or compression of the tumor but by activation of the immune system. Antibodies produced by the body against the tumor antigen may damage the nervous system. These antibodies are divided into 1) antibodies against neuronal nuclear and cytoplasmic antigens and 2) antibodies against cell surface and synaptic proteins. As a member of the former first identified as a biomarker of PNS in 2001 , 4. AntiA 70-year-old man presented to hospital with a 6-month history of slow movements with leaning forward and postural instability. Erectile dysfunction also occurred for more than half a year, before the motor symptoms. A month ago, the symptoms worsened. Meanwhile, he suffered from severe dizziness while standing, constipation and urinary incontinence in this month.. He was a heavy smoker previously without subjective loss of smell, seizures, or changes in body weight. He had no diabetes, family history of genetic diseases or exposure to toxic substances.His supine blood pressure (BP) was 106/76 mmHg with heart rate (HR) 82 beats/ min, while it could not be measured accurately when standing. He showed hypophonia and reduced facial expression. The muscle strength score in extremities was 5/5. He also showed bradykinesia, bilateral reduced arm swing and right upper limbs and both lower limbs rigidity without tremor. The deep tendon reflexes were normal and plantar reflex was flexor bilaterally.The values of thyroid function test, tumor markers were within the normal range. Results of serum anti-HIV antibody and hepatitis C antibody were negative. Urinary ultrasound showed 130 mL of post-void residual urine. Midbrain sonography and thymus computed tomography (CT) were normal. Multi-detector row CT scan of the lungs showed nodules with no thickening of bronchial wall, enlargement of hilar and mediastinal lymph nodes or atelectasis. Brain magnetic resonance imaging (MRI) showed no brain stem or cerebellar atrophy , protein was 460 mg / L and total leukocyte count was 4.00 × 10 for PNS , we diagIntravenous immunoglobulin was given at 0.4 g/kg/day. After 5 days of immunotherapy, his dizziness was significantly improved and he could walk independently. His supine BP was 130/83 mmHg with HR 79 beats/min. When standing, his BP was 123/75 mmHg with HR 82 beats/min at 1 min and 115/66 mmHg with HR 85 beats/min at 3 min. The BP was higher than that on admission with the absence of orthostatic hypotension. He received active hydration and medication every day since admission. However, the BP did not improve until the use of immunoglobulin, suggesting that it was the immunotherapy that worked. Moreover, there was no post-void residual urine in reexamination. As the symptoms relieved, intravenous immunoglobulin was stopped after 10 days. In consideration of the age and side effects, immunotherapy was refused by his relatives in the follow-up therapy. Considering that neurological symptoms may appear before the discovery of tumor in some PNS patients, we a followed him up dynamically.Five months later, his symptoms worsened characterized by frequent falls, inability to walk independently, dysphagia and unintelligible speech. His limbs were too tense to be bent. Likewise, no positive results were found in tumor screening. The BP was 140/89 mmHg, and levodopa-benserazide 50 mg /12.5 mg were given orally three times a day for months. However, it did not work. Unfortunately, he died after 8 months. We also organized the patient’s clinical manifestations and treatment options as a timeline  hoarse voice , autonomic nerve damage , sleep disturbance , and loss of taste . Basal ganglia changes were found in all the 4 patients (100%) whose brain MRI results were described, which could account for Parkinsonism. One case was combined with the thalamus and brainstem changes (25%). Two cases of MRS examination showed a reduction of NAA peak located in bilateral basal ganglia  and bilateral lateral ventricles . Lumbar puncture results were shown in 4 cases, 2 patients had elevated CSF protein levels (50%), with a maximum CSF protein of 629 mg/L. Three patients underwent EEG examination, and 2 showed abnormal results (66.7%) with slow waves in the temporal region. Four cases described the sites of antibody in detection, 2 in both blood and cerebrospinal fluid (50%), and 2 merely in blood (50%). Four cases gave the information about complicated tumor, treatment and prognosis. Two patients suffered from SCLC (50%), and radiotherapy  and chemotherapy  were adopted to perform clinical improvements. One patient diagnosed with breast cancer (25%) got improvements in life quality after surgery. No tumor was found in one patient (25%) who received hormone treatment: dexamethasone 10 mg for 3 days and 15 mg for 30 days by intravenous drip, then 6 mg orally once a day with 0.75 mg reduced every 7 days. During the following up, the patient suffered from originally uncontrollable tremors and new onset of seizure. In all, patients achieved cancer treatment when tumors were found are more related to better outcomes than patients accessed to immunotherapy with no tumor.Among the 7 patients (excluding this case), the ratio of male: female was approximately equal (42.9%). The mean age of onset was 60.3 (range 45-72 years old) during the 4 patients whose ages were recorded. Clinical manifestations were described in detail in 4 patients, with concomitant symptoms including: dysarthria  PNS patients with anti-CV-2 antibody have been reported to be associated with Parkinsonism  Considering the absence of evidence of tumors and economic reasons, the patient did not recheck PET-CT and the antibody after immunotherapy and during his follow-up. 2) Autopsy was not performed after death, so the exact cause of death could not be determined, nor could it be determined whether the patients had antigen-antibody complex deposition and pathological changes in basal ganglia and autonomic nervous system in the brain tissue, so as to explore the mechanism of anti-CV-2 antibody in cerebrospinal fluid causing Parkinsonism and autonomic nervous dysfunction. 3) Due to limited conditions, the titer of anti-CV-2 antibody in cerebrospinal fluid and the specific binding ratio of dopamine were unknown. This study reminds physicians of the differential diagnosis of MSA-P, especially PNS when encountering patients with Parkinsonism and rapidly progressing autonomic failure.Additional file 1: Table 1. Clinical characteristics of anti-CV-2 related PNS presenting with Parkinsonism.Additional file 2: Table 2. Summary of diseases manifested as Parkinsonism and dysautonomia.


Nigella sativa L.) and basil (Ocimum basilicum L.) seeds using conventional petroleum-based solvent and green solvent 2-methyltetrahydrofuran (MeTHF) was performed. MeTHF extraction allowed obtaining the highest oil yield in black cumin (34%). Regarding fatty acids composition, linoleic acid (61%) and α-linolenic (78%) were relevant in black cumin and basil green and conventionally extracted oils, respectively. Besides, MeTHF allowed obtaining higher tocopherols and total phenolics contents in black cumin (400 mg/kg of oil and 12 mg EGA/g oil) and basil (317 mg/kg oil and 5 mg EGA/g oil) compared to hexane-extracted ones. The content of major phenolic compounds in the two seed oils, trans-hydroxycinnamic acid, rosmarinic acid, and thymol was enhanced by MeTHF extraction. Furthermore, MeTHF-extracted oils possess stronger antioxidant activities  and high and similar anti-inflammatory capacity to hexane-extracted oils. In conclusion, the results revealed that MeTHF is efficient to replace hazardous solvents to extract oil from black cumin and basil seeds rich in compounds relevant to the human diet, including essential polyunsaturated fatty acids (n-6 and n-3), tocopherols, and phenolic compounds with improved biological activities.A comparative study of bioactive lipids extraction from black cumin ( In recent years, the demand for edible oils is steadily growing due to their importance for the nutritional security of the global population. Functional lipids are responsible for the health benefits of oils. Polyunsaturated fatty acids (PUFA) belong to two main classes, omega-3 and omega-6 which are essential bioactive compounds with special dietary and functional properties. PUFA are precursors that are metabolized to varied lipid mediators such as eicosanoids and docosanoids with known biological activity. Omega-3 fatty acids have anti-inflammatory activity and are associated with the reduction of the risk for chronic diseases . Moreove2-methyltetrahydrofuran (MeTHF) is one of the green solvents with a high potential to replace n-hexane. This bio-based solvent is cyclic ether, generated from carbohydrates derived from lignocellulosic biomass, which represents the most abundant biomass resources on earth [The most commonly applied methods for the recovery of oil from seeds are conventional methods that involve the use of high volumes of organic and toxic solvents. Hexane and chloroform/methanol mixture are the most employed ones. Due to their high volatility, organic solvents pose a serious problem for human and environmental health . Hexane on earth . During on earth .Recently, good results in terms of lipids yield extracted using MeTHF were reported ,11. Publn-Hexane was applied to obtain oils that were used for comparison. The soxhlet extraction, which is recognized as standard analytical practice for the extraction of oils from solid samples was used. The oil yield, fatty acid composition, bioactive compounds profiling, including tocopherols and phenolics, antioxidant and anti-inflammatory activities were analyzed.Nigella sativa L.) seeds were obtained from Menzel Temim (Northeast of Tunisia) in July 2018, while basil (Ocimum basilicum L.) seeds were purchased from Kairouan (Center of Tunisia) in September 2018. Seeds ground powder was used for oil extraction.Black cumin  of oils were analyzed by means of a colorimeter . The oil sample was placed in a glass cell against the light source to get color values. The L* represents the lightness (0–100 represents darkness to lightness), a* value represents red/green coordinate (+ to −), and b* value indicates yellowness to blueness (+ to −).Fatty acids composition investigation was carried out by means of gas chromatography–flame ionization detection (GC-FID) after derivatization to fatty acid methyl esters (FAMEs). The FAMEs analysis was carried out by GC using HP 6890 gas chromatograph  equipped with RT-2560 capillary column . The carrier gas was Nitrogen. The initial oven temperature was held at 170 °C for 2 min, increased at a rate of 3 °C/min ramp to 240 °C, and finally held there for 15 min. The injector and detector temperatures were 225 °C. Fatty acids identification of black cumin and basil oils was achieved by comparing their retention times with those of certified FAME mix. The results were expressed as the percentage of each fatty acid in the total.v/v). The extracts were combined and then evaporated to dryness. The residue was dissolved in a methanol/isopropanol/hexane mixture  and analyzed for tocopherol content using An Agilent Technologies 1100 series liquid chromatography (RP–HPLC) coupled with a UV-Vis multiwavelength detector. Column 250 × 4.6-mm, 5-μm Eclipse XDB-C18 was used for tocopherols separation. The samples were eluted using solvents combination where solvent A was (2% acetic acid in water), B (methanol), C (acetonitrile), and D (isopropanol). The following parameters were used: 95% A/5% B/2 min; 60% A/10% B/30% C/8 min; 25% B/75% C/22 min, and this percentage was held 10 min; 40% C/60% D/10 min; and this proportion was maintained 15 min; 25% B/75% C/2 min, and lastly, 95% A/5% B/3 min. Flow rate = 1 mL/min. The sample injection volume = 20 µL. Compounds were identified by comparing their retention time with those of standards.Tocopherols content was determined according to Tasioula-Margari and Okogeri  procedurThe polyphenols content of oil samples was assessed using Folin–Ciocalteu reagent method. Firstly, methanol was used to extract total polyphenols from oil samples . Oil . Zorbax Eclipse XDB C18 column (4.6 × 100 mm and 3.5 μm particle size) was used. The temperature of the column = 25 °C. The scanning range for the diode array detector was set at 200–400 nm. Mobile phase flow rate = 0.4 mL/min. A combination of solvent A (methanol) and solvent B (milli-Q water with 0.1% formic acid) was used as the mobile phase. The following gradient elution was used: 0–5 min, 10–20% A; 5–10 min, 20–30% A; 10–15 min, 30–50% A; 15–20 min, 50–70% A; 20–25 min, 70–90% A; 25–30 min, 90–50% A; 30–35 min, return to initial conditions. Sample injection volume = 2 μL. Compounds in oils were identified in accordance with their retention time with those of standards at 254 nm. The quantity of each compound was determined as microgram per gram of oil (μg/g oil).Three different antioxidant models  were employed to evaluate the antioxidant activity of methanol extracts obtained from black cumin and basil oils.50 (μg/mL), the antiradical dose required to cause a 50% inhibition. For that, each methanolic extract of seed oils, was mixed with a methanolic solution of DPPH [DPPH assay. The potential of methanolic extracts of oils to reduce the free DPPH radical  was expressed as IC of DPPH . The mixTotal antioxidant capacity. The total antioxidant capacity of methanolic extracts was evaluated through the assay of the green phosphate/Mo5+complex . The abs50 values (µg/mL).β-carotene bleaching test. The antioxidant activity of methanolic extracts of seed oils was determined using the β-carotene/linoleic acid bleaching test . An aliqFirstly, the cytotoxicity of oil samples on macrophage-like cell line RAW 264. 7 was assessed using the resazurin reduction test as described by O’Brien et al. . Fluoresv/v) to avoid solvent toxicity. For the determination of nitric oxide, RAW 264.7 cells were seeded in 24-well plates at a density of 2 × 105 cells/ well and were allowed to attach for 24 h/ 37 °C. Cells were then treated with various concentrations of oils and stimulated with 100 μg/mL lipopolysaccharide (LPS). Nitrite production in supernatants was measured using the Griess method. A reference curve of nitrite was prepared on a 96-well plate. The nitric oxide produced was determined by measuring the absorbance at 540 nm (Varioskan Ascent plate reader), and by comparison with the standard calibration curve.Then, the anti-inflammatory potential of oil samples was evaluated by producing nitric oxide formed in RAW 264.7 cells. Oils were dissolved in DMSO to obtain stock solutions, which were further diluted to obtain final concentrations where solvent in the culture medium was maintained at 0.1% (p < 0.05) was performed to determine significant differences among means of three independent experiments.All the experiments were repeated in triplicate and the data were expressed as means ± standard deviations. 1-way ANOVA, Post-hoc homogeneous groups Tukey HSD test  which is better than obtained by hexane . Howeveri et al. , TunisiaOverall, our results demonstrate that MeTHF may become a suitable substitute for hexane in terms of oil extraction yield. Additionally, MeTHF is derived from a renewable source and is accepted as safe. Its use in pharmaceutical chemical processes has been agreed .The CIELAB color parameters of the oils  from black cumin and basil seeds are shown in The conventional extracted from black cumin and basil oils showed high L* and b* values. Our results are in accordance with those of Cheikh-Rouhou et al.  and GhalThe fatty acid profile of the oil seeds obtained with conventional and green extraction from black cumin and basil is summarized in Carum carvi, anise, fennel, and rapeseed seeds [As shown in ed seeds ,10,11.On the other hand, our results are similar to those found by Cheikh-Rouhou et al.  and SellOn the other hand, a low PUFA/SFA ratio in food (below 0.45) is a risk factor for augmented blood cholesterol levels. In this study, PUFA/SFA ratios in green extracted oils were high; about 3 and 6 for black cumin and basil oils, respectively. Moreover, these ratios are similar to those obtained in oils extracted conventionally . Thus, tApart from triacylglycerols as major components in black cumin and basil oils, there are also some minor components like tocopherols and polyphenols that could be recognized as micronutrients. Consumption of different isomeric forms of tocopherols like α-, β-, γ- and δ- contribute substantially towards the health improvement. Tocopherols have main immunomodulatory functions and can delay the pathogenesis of a variety of degenerative illnesses. They are effective as lipid antioxidants. The activity is linked mostly to their capacity to scavenge peroxyl radicals and so break free chain reactions .O. basilicum, γ-tocopherol was identified as being the one quantified at high concentration in seed oil [Lamiaceae family [Analysis of the tocopherols in seed oils extracted using conventional solvent hexane  showed tseed oil ,26. Moree family .Opuntia ficus–Indica L. seeds with similar tocopherols contents [As can be seen in contents .In brief, MeTHF proposed in this study, in addition to constitutes an ecological friendly alternative, permit obtaining the greatest tocopherols content in black cumin and basil seed oils. The study proved that the use of green solvents guarantees little chemical toxicity and elevated bioactive compounds extraction effectiveness.Phenolic compounds have a great effect on the sensory and nutritional quality of oils and may protect against lipid oxidation through quenching of radical reactions. Oils containing phenols may have the potential for health-improving particularly in the prevention of heart illnesses .The total phenolic content (TPC) of the two seed oils was analyzed. As given in As reported in It is interesting to identify antioxidant compounds in the black cumin and basil seeds oils since they may contribute to the quality and health benefits of the oils. Analysis of phenolic compounds in seed oils extracted using conventional and green solvents allowed the identification of 15 phenolic compounds. The compounds include 9 phenolic acids, 4 flavonoids, and two oxygenated monoterpenes .trans-hydroxycinnamic acid (5 µ/g oil), quercetin (6 µg/g oil), and isorhamnetin (7 µg/g) were also identified with important levels. Thymoquinone, a typical monoterpene of black cumin was identified at a low concentration (0.1 µg/mL). This monoterpene ketone was already identified in Tunisian black cumin essential oil [Black cumin oil extracted using hexane was characterized by a high amount of sinapic acid 16 µg/g oil). In addition, tial oil . However µg/g oiltial oil .Green extracted oils showed a clear difference compared to conventional extracted ones . Interestrans-hydroxycinnamic acid and thymoquinone in black cumin which contents are enhanced by 6 and 4 times compared to conventionally extracted oil. In the same line, thymol, rosmarinic acid, and circimaritin amounts increased by about 4, 105, and 6 times in green extracted basil oil compared to hexane-extracted oil. These results may be related to the relative solubility of phenolic metabolites in both solvents. In accordance with our results, Nutrizio et al. [p-hydroxycinnamic acid and demonstrated that 2-MeTHF can be an excellent candidate to extract p-coumaric acid. Besides, Wang et al. [Scutellaria baicalensis Georgi and reported that MeTHF was found to be efficient to recover flavones from the plant material after ionic liquids extraction with more than 97% baicalein being recovered.Except for sinapic acid which was not detected in black cumin green extracted oil, the results indicate that MeTHF provides oils with similar or higher contents of individual compounds compared to conventional extracted oils. Indeed, MeTHF was found to favor the extraction of o et al.  assessedo et al.  and carvo et al.  from plao et al.  tested ag et al.  exploredThus, agro solvent MeTHF demonstrated the potential to be a better alternative to petroleum solvent n-hexane applied in industry, in terms of phenolic profile.50 = 1.7 mg/mL), while basil oil showed the highest total antioxidant activity (16.6 mg GAE/g of oil). Moreover, the two oils exhibited a significant and similar ability to inhibit the bleaching of β-carotene (IC50 about 3 mg/mL).In this study, the antioxidant activities of black cumin and basil seed oils obtained by conventional and green solvents were investigated. Generally, the two seed oils extracted by the conventional extraction method by means of hexane possess important antioxidant activity . Indeed,Interestingly, black cumin and basil oils extracted with MeTHF showed significantly higher antioxidant activity than the ones extracted conventionally . In factLikewise, it is clear that the higher antioxidant power of green extracted oils compared to hexane-extracted ones could be explained by the different phenolic compositions. In our study, MeTHF extracted oils were specifically rich in phenolic acids. These compounds have been reported as strong antioxidant agents and effective intracellular ROS scavengers. The effectiveness of phenolic compounds as antioxidants depends on numerous factors, such as the number of hydroxyl groups bonded to the aromatic ring, the site of bonding, and the mutual position of hydroxyls in the aromatic ring . In our Overall, our results suggest that MeTHF could be an excellent substitute for hexane to recover black cumin and basil oils with enhanced antioxidant activity. Beyond the health benefits related to polyphenols intake from foods, the significance of producing oils with prominent phenolic compound contents and antioxidant activity is associated also with the fact that oils rich in PUFA are very vulnerable to oxidative degradation. Thus, the enhanced bioactivity in green extracted black cumin and basil oil might facilitate decreasing lipid peroxidation at ambient and elevated temperatures and could be so used as functional oils for dietary supplements.Firstly, the cytotoxicity effect of conventional and green extracted oils on RAW 264.7 cells was investigated using the resazurin test. After 24 h of incubation with a wide range of concentrations, no significant cytotoxic effects of the four oils to RAW 264.7 cells were detected at up to 150 μg/mL concentrations . At 200 A well-known inflammatory marker, specifically NO, was chosen in order to assess the anti-inflammatory activity of black cumin and basil oils. In macrophages and invading immune cells, the elevated quantity of NO generated by inducible nitric oxide synthase (iNOS) following LPS and/or inflammatory cytokines plays a central function in inflammation.50 values were 22 and 25 µg/mL, in hexane and MeTHF-extracted oils, respectively. In the case of basil, the highest activity was achieved at 150 µg/mL, in fact. NO production decreased by 60 and 64% in hexane and green extracted basil oil, respectively.Cells challenged with LPS trigger an important NO accumulation (16 μM), however, the incubation of murine macrophages with a graded concentration of hexane and MeTHF-extracted oils induced inhibition of NO production in a concentration-dependent manner as it is depicted in Nigella sativa at low level (inhibited NO production by 95.0% at very low concentration (4.1 μg/mL) [Independently to the species, the results showed similarity of anti-inflammatory activity in hexane and green extracted oils which suggest that the green solvent is as able as hexane to extract anti-inflammatory compounds from the two plant seeds. Our data are in accordance with previously published work showing that some MeTHF extracted oils exhibited important anti-inflammatory activity ,42. The 1 μg/mL) , was eff1 μg/mL) . However1 μg/mL) .trans-hydroxycinnamic and rosmarinic acids could probably participate to their anti-inflammatory activity since they can inhibit the NO generation and iNOS expression in LPS-stimulated Raw 264.7 [Furthermore, other minor compounds mainly phenolics could be involved in the bioactivity of black cumin and basil oils. For instance, the abundance of phenolic acids in the seed oils including aw 264.7 .The results show that the biobased solvent MeTHF is a promising green alternative to hexane to recover enhanced functional oils from black cumin and basil seeds. The black cumin oil yield extracted using bio-based solvent was increased compared to conventional extraction. Green extracted oils were rich in PUFA mainly linoleic (61%) and α-linolenic (56%) acids. For both seeds, the oils extracted with MeTHF gave the highest tocopherols and total polyphenols contents as well as the most pronounced in vitro antioxidant capacity. Green extracted oils possess potent anti-inflammatory activity also. The content of individual phenolic compounds including phenolic acids, flavonoids, and monoterpenes was enhanced after green-extraction. Taken together, our results show that using a substitute solvent to extract oil from black cumin and basil seeds would be beneficial to obtain oils with enhanced bioactive compounds content and functional properties which can lead to additional innovations and their integration in cosmetic and beneficial formulations. Biobased-solvent MeTHF has the advantages to be eco-friendly, safe with biodegradability, and easily recycled power. Being economical, it shows a mainly competitive edge when it comes to the economical equation of extraction procedure compared to other green technologies. Due to the initial high costs of purchasing and setting up the equipment, the efficiency of other technologies such as supercritical carbon dioxide extraction is hindered. Finally, further study must be undertaken using a cellular model to verify the protection ability of green extracted oils to protect cells against oxidative cell damage. Moreover, storage effects on oil quality needs to be conducted to explore oxidative stability of the MeTHF-extracted oils.


To evaluate the clinical presentation of choanal atresia (CA) in tertiary centers across Canada.Multi-centre case series involving six tertiary care pediatric hospitals across Canada. Retrospective chart review of patients born between 1980 and 2010 diagnosed with CA at a participating center.The health charts of 215 patients  with CA were reviewed and included in this study. The mean age of patients at time of CA presentation was 0.4 months (range 0.1 to 7.2 months) for bilateral CA and 37.8 months (range 0.1 to 164.1 months) for unilateral cases. The most common presenting symptoms for bilateral CA in decreasing order were respiratory distress (96.4%), feeding difficulties (68.2%), and rhinorrhea (65.5%), and for unilateral cases in decreasing order were rhinorrhea (92.0%), feeding difficulties (24.7%), and respiratory distress (18.0%). For the majority of patients (73.2%), the obstruction comprised mixed bony and membranous tissue, with only 10.5% presenting with a purely membranous obstruction. Familial history of CA was confirmed in only 3.3% of cases. One half of patients with CA presented with one or more associated anomalies and 30.6% had a syndrome.The present investigation is the first national multi-institutional study evaluating the clinical presentation of CA over three decades. The present cohort of CA patients presented with a breadth of co-morbidities with highly variable presentations, with bilateral cases being more severely affected than unilateral cases. Further investigation into hereditary linkages to CA development is warranted. Choanal atresia (CA) is a congenital condition resulting in obstruction of the posterior nasal passage(s), known as the choana, that may be bony, membranous or mixed bony-membranous. The incidence of this uncommon entity is approximately one in 5000 to 8000 live births , 2. The To date, no agreement regarding best practices for management of CA exist. Ongoing controversy regarding best practices for clinical management of CA lies, in part, in the rarity of the disorder and consequently the limited level of evidence available in published studies on this subject matter. The literature often reports case series of single surgeon or single institution experiences, involving small sample sizes –9, with Pediatric otolaryngologists practicing in tertiary care centers across Canada were invited, via phone or email, to participate in the study. Of the nine centers who were contacted, six centers  agreed to participate in the chart review. Representatives of each participating center formed the Canadian Choanal Atresia working group (in acknowledgment). Health charts of patients born between 1980 and 2010 diagnosed with CA who underwent treatment at participating centers were included in this study. Patients born before 1980 or after 2010 or patients who did not receive a definitive diagnosis of CA were excluded from the study. A standardized checklist was completed for each patient presenting with CA who met study inclusion/exclusion criteria. The following variables were collected from patient chart review: incidence of CA in Canada, presenting signs and symptoms of CA, requirement of intubation, reports of CT scans, use of suction catheter to aid in diagnosis, characteristics of CA, associated syndromes and co-morbidities, family history, and, a comparison of CA characteristics for unilateral versus bilateral cases. Ethical approval for this study was obtained from the Health Research Ethics Boards at each participating center.Descriptive analyses including frequencies, means and standard deviations of study outcomes were undertaken. In addition to descriptive statistics, results of chi-square tests/Fisher’s exact tests, and independent samples t-tests were reported where appropriate. The incidence rate of CA in Canada was estimated based on the following calculation: total number of births in provinces in Canada that were well represented by participating centers between 1980 and 2010 , 17, divn = 26), Toronto (n = 83), Edmonton (n = 17), Vancouver (n = 60), Montreal (n = 11), Halifax (n = 18)] met the study inclusion criteria and were included in this national study. Health charts of 215 patients were reviewed. One-hundred and twenty-seven patients (59.6%) were female and 88 (40.9%) were male. The mean age of patients at time of CA presentation was 0.4 months (range 0.1 to 7.2 months) for bilateral CA and 37.8 months (range 0.1 to 164.1 months) for unilateral cases. Patients were born pre-term on average [mean (SD) for gestational age was 35.0 (9.0) weeks].Two-hundred and fifteen patients across the six participating centers  presented with one or more associated anomaly. Sixty patients (30.6%) were identified with a syndrome; of the 93 patients with bilateral CA, 37 (45.1%) presented with a syndrome, and, of the 122 patients who had unilateral CA, 23 (20.2%) presented with a syndrome. Thirty-five patients  had confirmed CHARGE syndrome. Table Table For the CHARGE subgroup, the prevalence of CHARGE-related anomalies ranged from 5.7 to 40.0%, with a high prevalence of coloboma, heart defects, ear anomalies and sensorineural hearing loss (see Table n = 7), Pierre Robin sequence (n = 2), Treacher-Collins Syndrome (n = 2), fetal alcohol syndrome (n = 2), Apert’s syndrome (n = 1), Autism (n = 1), Cerebral palsy (n = 1), Clefting syndrome (n = 1), Cornelia de Lange (n = 1), Crouzon’s (n = 1), Di George syndrome (n = 1), Duane-radial ray syndrome (n = 1), Goldenhar syndrome (n = 1), Kabuki syndrome (n = 1), Moebius syndrome (n = 1), Pallister-Hall syndrome (n = 1), Pfeiffer syndrome (n = 1), Riley-day syndrome (n = 1), Ring chromosome 18 (n = 1), Turner Syndrome (n = 1) and undetermined genetic syndrome (n = 1).Of available syndrome information in patient charts, thirty patients (50.0%) had been diagnosed with other syndromes, and six of these patients were diagnosed with a syndrome in addition to CHARGE. Syndromes included Down syndrome (Only thirteen patients (8.5%) had a confirmed abnormal karyotype. Varied karyotype abnormalities were found including: 45, X0; 45, XX; 46, XX, (9p+); 47, XY; Partial trisomy 15; Trisomy 18; Trisomy 21; Trisomy 22; Ring chromosome 18; Robertsonian translocation between 14 & 22, 14 & 15; and, SALL4 gene mutation.Table 2(1) = 18.8, p < .001] and were also significantly more likely to present with a syndrome  than individuals with unilateral CA. Respiratory distress was a predominant presenting symptom in bilateral CA cases (96.4%) and occurred in only 18.0% of unilateral cases . Rhinorrhea was a predominant symptom in unilateral cases and, also in bilateral cases but statistically significantly less .Table The present investigation explored the clinical presentation of CA over a 30-year period (1980 to 2010). To address some of the limitations of previous studies that were mostly single surgeon or single institution case series, resulting in small sample sizes from which to infer conclusions, this national study took a multi-institutional approach. This is the first national multi-institutional study reporting on a large sample size of 215 patients with CA, providing stronger empirical support to help elucidate some of the outstanding controversies related to the clinical manifestation of this condition. This study will contribute to the body of knowledge on clinical presentation of CA, particularly relative to CA characteristics and associations, diagnosis, and familial factors.Based on the synthesis of cases of patients with CA reviewed in this present investigation, the incidence rate for CA in Canada was estimated to be between one in 30,000 to 40,000. As such, the present investigation found the incidence of CA to be much lower than incidence rates of approximately one in 5000 to 8000 live births commonly reported in the literature , 2.Signs and symptoms of CA are well known in the literature, however, the specific rates of symptoms are not well reported. In the present study, presenting symptoms included rhinorrhea in 82.6% of cases and respiratory distress in 55.8% of cases, of which 67.7% required intubation. In addition, a significant number (43.7%) also presented with feeding difficulties. Patients who presented with bilateral CA had statistically significantly higher rates of symptoms than unilateral CA. Most patients with bilateral CA presented with respiratory distress (96.4%) and many presented with rhinorrhea (65.5%) and feeding difficulties (68.2%).Persistent symptoms of rhinorrhea, respiratory distress, and/or nasal obstruction usually prompt further evaluation of the nasal and oral cavity. Inability to pass a suction catheter through the nasal passages usually prompts a more thorough evaluation of the anatomy of the nasopharynx, with the use of nasal endoscopy and, possible CT scan  to confiAs is reported in most case series, a predominance of females over males , and unilateral over bilateral  cases were observed in the present study. Furthermore, our findings confirm previous investigations indicating one half of patients with CA present with an anomaly, and that the most common syndrome associated with CA is CHARGE , 18. PatBased on CT scan review of 47 scans from the literature and 16 from the authors’ clinical experience with CA, Brown et al. 1996) found that 29% of cases were pure bony, and 71% were mixed bony-membranous, with no cases of pure membranous being reported in this small case series 96 found .In the present study, the study cohort presented with a variety of anomalies affecting varied bodily systems including urinary, skeletal, pulmonary, gastro-intestinal, airway abnormalities, palatal anomalies and CHARGE-associations including coloboma, heart defects, retarded growth and development, genital hypoplasia, and ear anomalies. Although the majority of cases of CHARGE-associations belonged to the CHARGE-syndrome subgroup, with the exception of genital hypoplasia, all other CHARGE associations  were found in some patients who belonged to the non-CHARGE syndrome subgroup as well. This finding may indicate unconfirmed cases of CHARGE-syndrome or a natural variability in the clinical profile of CA. Given the varied anomalies present in CA and its association with CHARGE, a CHARGE workup may be warranted with a diagnosis of bilateral CA.The hereditary aspects of CA have not been clearly delineated to date and are likely multifactorial . In the This study was limited by its retrospective nature and missing data. Furthermore, three tertiary care centres in Canada did not participate in the present investigation, limiting the generalizability of study findings.This retrospective multi-center investigation reported on over 30-years experience of patients with CA across tertiary care centers in Canada. In line with previous literature, our study cohort presented with a breadth of co-morbidities and variability in presentation, with over one half presenting with one or more anomalies. Observed findings indicated limited hereditary linkages. Health practitioners involved in the care of patients with CA should investigate the presence of multiple anomalies. Future research investigating hereditary linkages and CA development is warranted.


N-isopropylacrylamide)/polyvinyl alcohol nanoparticles were prepared. Core-shell polymeric nanoparticles, bacterial cellulose/chitosan membranes, and biocomposites were independently loaded with silver sulfadiazine, a well-known sulfonamide antibacterial agent used in the therapy of mild-to-moderate infections for sensitive organisms. The chemistry, structure, morphology, and size distribution were investigated by Fourier transformed infrared spectroscopy (FTIR-ATR), RAMAN spectroscopy, Scanning electron (SEM) and Transmission electron microscopy (TEM), and Dynamic light scattering (DLS). In vitro release behaviors of silver sulfadiazine from polymeric nanoparticles and biocomposites were investigated. The biological investigations revealed good biocompatibility of both the nanoparticles and the biocomposites in terms of human dermal fibroblasts viability and proliferation potential. Finally, the drug-loaded polymeric biomaterials showed promising characteristics, proving their high potential as an alternative support to develop a biocompatible and antibacterial wound dressing.Dressing biomaterials play a key role in wound management keeping a moisture medium and protecting against external factors. Natural and synthetic materials could be used as dressings where chitosan and bacterial cellulose is one of the most important solutions. These biopolymers have been used for wound dressing based on their non-toxic, biodegradable, and biocompatible features. In this study, biocomposites based on bacterial cellulose and chitosan membranes tailored with antimicrobial loaded poly( New approaches try to overcome daily medical challenges, such as drug delivery systems, tissue engineering, or wound dressing. The wounds are generally regarded as a disruption of the skin epithelial cells continuity. The healing process represents a dynamic and complex interaction between cells, growth factors, blood, or extracellular matrix. In some clinical conditions, such as diabetes or extended cancers dermal wounds like ulcers, burns or even surgical cuts frequently do not heal and become a problem that demands dedicated clinical care. Without such an approach these wounds can lead to delayed healing, infections, and many other conditions that may lead to amputation. With this respect, wound dressings evolved in recent years to meet the requirements for modern tissue repair and regeneration. The repairing process requires several steps, such as coagulation, inflammation, proliferation, and maturation. To accomplish these steps and to avoid the microorganisms’ invasion into the wound, the use of an appropriate dressing is crucial  proved to be efficient solutions for the treatment of chronic wounds and burns , methyl oleate (MO), and potassium persulfate initiator were purchased from Sigma–Aldrich, 3050 Spruce Street, St. Louis, MO 63103, USA. Bacterial cellulose (BC) was provided by National Chemical-Pharmaceutical for Research and Development Institute, Bucharest, Romania. Silver sulfadiazine drug was purchased from ACROS Organics, New Jersey, USA.Low molecular weight chitosan  with a 75% percent of deacetylation, anhydrous calcium chloride, sodium hydroxide, polyvinyl alcohol (PVA) with 88% hydrolysis degree , 2.2.N-isopropylacrylamide)/polyvinyl alcohol/methyl oleate (PNIPAM/PVA/MO) nanoparticles is based on a radical polymerization mechanism of the corresponding monomer and polyvinyl alcohol (PVA) and methyl oleate (MO). Briefly, aqueous solutions of polyvinyl alcohol (0.5 and 5 wt. % concentration) were prepared. In the meantime, aqueous solutions of N-isopropylacrylamide monomer (NIPAM) with 0.5 and 5 wt. % concentration was prepared, and the potassium persulfate initiator (KPS) was dissolved accordingly. Solutions of NIPAM/PVA 5 wt. % and NIPAM/PVA 0.5 wt. % were prepared by stirring for 2–3 h. After obtaining NIPAM/PVA 0.5 and 5% solutions, the mixtures were divided into four parts and methyl oleate (MO) was added in ratios 0.125, 0.25, 0.5, and 1 v/v within the mixture  solutions were subjected to vigorous stirring for 12 h to prepare the nanoparticles suspension. The NIPAM/PVA/MO nanoparticles suspension was heated at 60 °C for 4–5 h for NIPAM grafting and final stabilization of nanoparticles suspension. The final systems were obtained as core-shell nanocapsules with a methyl oleate core, PVA interfacial stabilizer, and PNIPAM as a polymeric shell. The layered core-shell structuration is beneficial to get nanocarriers with surface entrapped drugs for higher release efficiency , native and treated chitosan, and BC/chitosan membranes were recorded on a Bruker Vertex 70 FT-IR spectrophotometer with attenuated total reflectance (ATR) accessory with 32 scans and 4 cm2.6.2.The size and morphology of the nanoparticles together with membrane morphological characteristics were investigated by Scanning Electron Microscopy (SEM) using a Quanta Inspect F50, with a field emission gun (FEG) having 1.2 nm resolution and an Energy Dispersive X-ray Spectrometer (EDXS) having 133 eV resolution at MnKα. Morphology, geometrical evaluation (size and shape) of nanostructural characteristics of the core-shell constructs were investigated by high-resolution transmission electron microscopy (HR-TEM) and selected area electron diffraction (SAED) using a TECNAI F30 G2 S-TWIN microscope operated at 300 kV with Energy Dispersive X-ray Analysis (EDAX) facility.2.6.3.θ = 10–80. An X-ray beam characteristic of Cu Kα radiation was used (λ = 1.5418 Å).X-ray diffraction (XRD) spectra were registered on a Panalytical X’PERT MPD X-ray Diffractometer, in the range, 22.6.4.tW and 0W denote the weight of the wet hydrogel at a predetermined time and the weight of the dry sample, respectively. The equilibrium swelling degrees (ESD) were measured until the weight of the swollen hydrogels was constant. At least three swelling measurements were performed for each sample and the mean values were reported. Furthermore, gel fraction experiments were performed by leaching tests.The swelling behavior of the biocomposites was evaluated in saline solution at 37 °C. The weight changes of the samples were recorded at regular time intervals during swelling. The swelling degree of the hydrogels was determined according to the following equation:fW denotes the weight of the dried sample after water extraction.The gel fraction (GF) of the samples was determined according to the following equation   using a Zetasizer Malvern DLS device. Data were collected for PNIPAM nanoparticles obtained from 0.5 and 5 wt. % polymer concentration.2.6.8.In vitro release behaviors of silver sulfadiazine from PNIPAM/PVA/MO nanoparticles and biocomposites were evaluated in time. Briefly, the dried loaded PNIPAM/PVA/MO nanoparticles and subsequently membrane biocomposites were entrapped in a cellulose membrane, immersed in 50 mL of PBS , and incubated in a precision water bath  at 400 rpm and 37 °C. Aliquots (5 mL) containing a mixture of PBS and released drug were collected at defined time points and the release medium was refreshed with the addition of an equal amount of fresh PBS after each withdrawal to maintain the total volume of the sample constant. The silver sulfadiazine release profiles from nanoparticles and biocomposites were evaluated by UV-VIS spectroscopy.The silver sulfadiazine encapsulation within both nanoparticles and BC/chitosan membranes followed two pathways: in the case of nanoparticles, the drug was dissolved into Phosphate buffer saline (potassium phosphate/sodium hydroxide) (PBS) solution (pH 7.45). The drug solution concentration was: 0.1 mg/mL for nanoparticles from 5% polymer concentration and 0.05 mg/mL for nanoparticles from 0.5% polymer concentration. The dried nanoparticles were added to the drug solution (0.1 g of nanoparticles/mL of drug solution). The resulted nanoparticle suspension was stirred for 24 h. Finally, the nanoparticles were separated by filtration. In the second case, the dried bacterial cellulose/chitosan membrane was immersed into drug solution and the encapsulation took place by swelling. For both cases, the encapsulation evaluation was done by UV–VIS analysis of the drug that remained in the solution. 2.7.® CRL-2091) were used as in vitro cell culture model to screen the cytotoxicity potential of the developed nanoparticles and to evaluate the biocompatibility of the proposed biomaterials. In this view, CCD-1070Sk human dermal fibroblasts were grown in Minimum Essential Medium (MEM), supplemented with 1% (v/v) penicillin/streptomycin  and 10% (v/v) fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2, at 37 °C. The culture medium was refreshed every two days and cells were split 1:3 weekly, by trypsin/EDTA treatment.Considering the final application of the novel biocomposites in the field of wound healing, human dermal fibroblasts /polyvinyl alcohol nanoparticles was investigated after 24 and 48 h of treatment with pristine PNIPAM/PVA/MO. In this view, 104 cells/cm2 were seeded in well plates and allowed to adhere to the substrate for 24 h. The next day, the culture media were exchanged with the following PNIPAM/PVA/MO dilutions prepared in complete culture medium: 20, 15, 10, 7.5, and 5 mg/ml. These treatments were applied 24 h post-seeding and maintained for 24 and 48 h.The cytotoxic potential of the poly. An untreated control was prepared following the same procedure and used as a reference.2.8.2.This test was employed as an additional viability investigation to evaluate the ratio between the living and the dead cells. In this view, after 24 and 48 h of treatment, cell culture media were harvested and the CCD-1070Sk monolayers were washed with a serum-free culture medium. A mixed solution of calceinAM and ethidium bromide was prepared according to the manufacturer’s recommendations . All the samples, including the untreated reference, were incubated for 30 min at room temperature and darkness with this staining solution. In the end, the monolayers were investigated using an Olympus IX73 inverted fluorescence microscope and CellSense imaging software for image capturing.2.8.3.In Vitro Toxicology Assay Kit  according to the manufacturer’s instructions. After 20 min of incubation in darkness at room temperature, the absorbance of the samples was determined at 490 nm using FlexStation III (Molecular Devices) microplate reader.The cytotoxic potential of the PNIPAM/PVA/MO on CCD-1070Sk human dermal fibroblasts was investigated by spectrophotometric evaluation of Lactate Dehydrogenase (LDH) activity in the culture media after 24 and 48 h of treatment. Briefly, 500 µl of culture media were harvested at the above-mentioned time points and mixed with the components of the 2.9.6 cells/sample.A preliminary biocompatibility investigation of the biocomposites was conducted by performing the MTT assay. In this view, human dermal fibroblast cells from CCD-1070Sk cell line were seeded in direct contact with the biomaterials at an initial density of 102.9.1.After 24 h and 5 days of culture, cell viability was investigated. In this view, cell culture media were harvested and the monolayers from the membranes’ surfaces were washed with a serum-free culture medium. The samples were then incubated for 6 h at 37 °C in 1 mg/ml MTT solution. The formazan crystals produced by the metabolically active cells were solubilized in DMSO and the absorbance of the resulting solutions was determined at 550 nm by using FlexStation III multimode microplate reader (Molecular Devices).2.10.in vitro experiments were performed in triplicate (n = 3) and results were analyzed using one-way ANOVA and Bonferroni test. All the results were presented as mean value ± standard deviation (SD) using GraphPad Prism Software 6.0 .All the 3.3.1.3.1.1.−1 specific for O–H stretching from hydroxyl bacterial cellulose groups and an intense peak at 2886 cm−1 attributed to symmetric stretching of C–H bond from methylene and methine groups. The maximum at 1645 cm−1 corresponds to hydroxyl group bending vibration including absorbed water molecules. The peaks at 1471, 1464, and 1342 cm−1 are attributed to methyl and methine bending vibration from the backbone. The peaks at 1280 and 1240 cm−1 were attributed to the stretching vibration of C–O bonds. The intense peak at 1103 cm−1 is attributed to C–O–C ether bonds from piranozic cycles backbone and between piranozic cycles  generated by sodium hydroxide neutralized the –NH3+ species needed for chitosan dissolution. The –NH3+ species neutralization supported by the high polymer concentration (7%) and deacetylation degree (75%) ensured the optimal conditions for the generation of the novel physically crosslinked network. Thus, there were developed structural and dimensional stable membranes in specific basic medium. The BC/chitosan membrane spectrum  crystalline structure. The new peaks at 2 theta of 26.7, 29.7, 39.4, 43.2, 47.5, and 48.3° completed the file of calcium oxide crystalline structure. Furthermore, the peak at 14.55° was split into two independent peaks probably due to a more organized crystalline structure of native BC with calcium contribution. XRD results revealed major interactions of BC with calcium cations leading to a stabilized BC network which can be considered as a pseudo-crosslinked structure with the association of microparticles into a unitary structure. The pseudo-crosslinking of BC microparticles into a unitary structure had a major role in the design of (BC)/chitosan membranes as interpenetrated network (IPN).The interaction of bacterial cellulose (BC) with calcium cations was further investigated by XRD. The diffractograms of bacterial cellulose and calcium-treated bacterial cellulose are shown in 3.3.The evaluation of the mechanical properties of physically crosslinked chitosan membrane is an interesting approach for a further understanding of the 3 D network. There are too many tensile assays in the literature on physically crosslinked chitosan . The physical crosslinking process could be an ideal approach for future similar systems based on chitosan to express the desired mechanical properties.The investigation of rheological behavior was performed on swollen samples at swelling equilibrium. The stress was optimized to maintain a linear viscoelastic domain with frequency dependence. 3.6.High-resolution SEM images  reveal t3.6.1.TEM images  revealed3.6.2.2+ and negatively charged alcoholate ions in BC  and core part which can also be chemically bound by the shell layer.It is known that persulfates are capable of initiating the crosslinking of polyvinyl alcohol chains by the radical mechanism  obtained from 0.5% polymer concentration were subjected to a drug release test showing different efficiencies. The membranes with a lower amount of drug-loaded nanoparticles expressed higher release efficiency with a release time of about 5 h, and higher control (The silver sulfadiazine release profile for membranes loaded with drug entrapped nanoparticles was carried out to improve the control and sustain a prolonged release. The nanoparticles with 0.125% methyl oleate (MO) content were chosen due to the high release efficiency. The higher drug control release profile was studied by varying the BC/chitosan ratio and the ratio of nanoparticles  control . A highe control . Membran control . Here, t3.9.The cell viability and proliferation potential results showed that the majority of the seeded cells displayed good viability both at 24 and 48 h post-seeding when treated with the pristine PNIPAM/PVA/MO . More, aThese results are supported by the fluorescence microscopy images obtained after Live/Dead double staining of the cells with calceinAM and ethidium bromide. As shown in Further, the cytotoxic potential of the PNIPAM/PVA/MO was investigated after performing the LDH assay. The obtained results indicated that the pristine PNIPAM/PVA/MO do not exhibit any cytotoxic effects on the CCD-1070Sk human fibroblast cell line as low levels of LDH activity were found after 24 h of treatment .3.10.CCD-1070Sk human dermal fibroblasts' cell viability and proliferation potential were investigated in direct contact with the developed BC/chitosan membranes decorated with silver sulfadiazine PNIPAM/PVA/MO. MTT assay was employed after 24 h and 5 days of culture. The spectrophotometric data represented in 4.in vitro screening revealed good biocompatibility on CCD-1070Sk human dermal fibroblasts in terms of 5 days cell viability. The biocomposite was designed with a layered structure to fulfill the main features of the modern wound dressing in terms of wound protection, moisture, and drug release. The two polymeric networks acting together represent a novel approach that can be more suitable for the next generation of wound dressing materials. Overall, the results presented in the current manuscript indicate that the novel biocomposites are promising candidates for modern dressings for chronic wounds management and could be further developed by in vivo validation on animal models.Here, bacterial cellulose/chitosan membranes tailored with silver sulfadiazine loaded PNIPAM/PVA/MO nanoparticles were prepared. Bacterial cellulose/chitosan membranes are formed as a result of the restructuring of the system of intermolecular interactions, which is accompanied by an increase in the density of the network. The increased network density provided a simultaneous decrease in the degree of swelling and an increase in the mechanical strength, as well as the elastic modulus of the bacterial cellulose/chitosan membranes. The composite membranes with a lower amount of drug-loaded polymeric nanoparticles had higher release efficiency and good control due to the good dispersion of the NPs within the BC/chitosan membrane. More, silver sulfadiazine was first released in the support membrane and finally in the medium. The pristine PNIPAM/PVA/MO core-shell polymeric nanoparticles displayed good biocompatibility on CCD-1070Sk human dermal fibroblasts as the treatment with various dilutions did not alter cell viability and proliferation potential as compared with untreated control. We treated CCD-1070Sk cells with 20, 15, 10, 7.5, and 5 mg/ml pristine PNIPAM/PVA/MO core-shell polymeric nanoparticles and after 48 h of exposure, we observed that the cells viability and proliferation potential was not altered by the treatment, suggesting that the new delivery systems display good biocompatibility on human dermal fibroblasts. After this confirmation, we embedded the core-shell polymeric nanoparticles into a biocomposite material composed of bacterial cellulose and chitosan for its prospective use as a wound dressing. Its basic


Vibrio vulnificus is an opportunistic human pathogen and autochthonous inhabitant of coastal marine environments, where the bacterium is under constant predation by heterotrophic protists or protozoans. As a result of this selection pressure, genetic variants with antipredation mechanisms are selected for and persist in the environment. Such natural variants may also be pathogenic to animal or human hosts, making it important to understand these defense mechanisms. To identify antipredator strategies, 13 V. vulnificus strains of different genotypes isolated from diverse environments were exposed to predation by the ciliated protozoan Tetrahymena pyriformis, and only strain ENV1 was resistant to predation. Further investigation of the cell-free supernatant showed that ENV1 acidifies the environment by the excretion of organic acids, which are toxic to T. pyriformis. As this predation resistance was dependent on the availability of iron, transcriptomes of V. vulnificus in iron-replete and iron-deplete conditions were compared. This analysis revealed that ENV1 ferments pyruvate and the resultant acetyl-CoA leads to acetate synthesis under aerobic conditions, a hallmark of overflow metabolism. The anaerobic respiration global regulator arcA was upregulated when iron was available. An ΔarcA deletion mutant of ENV1 accumulated less acetate and, importantly, was sensitive to grazing by T. pyriformis. Based on the transcriptome response and quantification of metabolites, we conclude that ENV1 has adapted to overflow metabolism and has lost a control switch that shifts metabolism from acetate excretion to acetate assimilation, enabling it to excrete acetate continuously. We show that overflow metabolism and the acetate switch contribute to prey-predator interactions.IMPORTANCE Bacteria in the environment, including Vibrio spp., interact with protozoan predators. To defend against predation, bacteria evolve antipredator mechanisms ranging from changing morphology, biofilm formation, and secretion of toxins or virulence factors. Some of these adaptations may result in strains that are pathogenic to humans. Therefore, it is important to study predator defense strategies of environmental bacteria. V. vulnificus thrives in coastal waters and infects humans. Very little is known about the defense mechanisms V. vulnificus expresses against predation. Here, we show that a V. vulnificus strain (ENV1) has rewired the central carbon metabolism, enabling the production of excess organic acid that is toxic to the protozoan predator T. pyriformis. This is a previously unknown mechanism of predation defense that protects against protozoan predators. Vibrio vulnificus is a Gram-negative, halophilic bacterium that thrives in warm marine and estuarine waters. Despite its environmental origin, the bacterium is associated with opportunistic infections that include gastrointestinal infections caused by ingestion of raw or undercooked seafood as well as wound infections caused by exposure of wounds or broken skin to estuarine water or seawater resulting in sepsis  distinguishes the environmental  E-genotype strains (vcgE) from the clinical genotype strains (vcgC)  , 4. Moreclusters  or five clusters  of straiclusters –9. Bactelusters –.Comamonas acidovorans , that can kill Tetrahymena thermophila , encoded by the rtxA1 gene, that causes plasmolysis of amoebae  (T. pyriformis predation (T. pyriformis cells was not significantly different from that of the control (T. pyriformis. The toxicity of ENV1 cell-free supernatants (CFS) was also tested on the saltwater ciliate, Uronema marinum. The addition of ENV1 CFS caused the same cell death observed in T. pyriformis after less than 5 min (data not shown).Thirteen strains of  sources , were aswn alone , and thecontrol) . In contredation  and the  control . This suT. pyriformis with the CFS of ENV1 grown for 24 h in 0.5× VNSS medium. After 10 min at room temperature, the ciliates stopped swimming and sank to the bottom of the microtiter plates. After 1 h, the ciliates were dead (100%) with their cytoplasm leaking out  filters did not alter CFS toxicity, suggesting that the secreted factor was likely smaller than 10 kDa. Collectively, the results indicate that the toxic factor is not an enzyme or a protein. The CFS pH was approximately 4, and therefore, the role of pH in toxicity was examined. Neutralization of the pH to 7 using sodium hydroxide rendered the CFS nontoxic to T. pyriformis  under acidic pH.The CFS analysis indicated that the toxic factor is not an enzyme or a protein (heat treatment), and thus, is likely a small molecule (ultrafiltration), and that its function is affected by protonation of functional groups (active in acidic pH). Free fatty acids are small organic compounds produced by several bacteria that have antimicrobial properties, which are dependent on the pH of the environment . Therefociliates . Furtherriformis . These rT. pyriformis only when ENV1 grew in the presence of iron  direct oxidation of pyruvate by pyruvate oxidase (PoxB) and (ii) decarboxylation of pyruvate to acetyl-CoA, followed by the conversion of acetyl-CoA to acetate by phosphotransacetylase (Pta) and acetate kinase (AckA). Decarboxylation of pyruvate to acetyl-CoA can occur both aerobically, by pyruvate dehydrogenase complex (PdhC), and anaerobically, by pyruvate-formate lyase (Pfl)  was downregulated and pfl genes (BJD94_01215 and BJD94_08715) were upregulated (ackA genes (BJD94_01780 and BJD94_2925), both of which were upregulated under iron-replete conditions. This indicates that conversion of acetyl-CoA to acetate follows the Pta-AckA pathway and suggests that ENV1 is adapted for acetate excretion through pyruvate fermentation. Moreover, the upregulation of genes associated with alcohol/acetaldehyde dehydrogenase  (BJD94_06220) suggested that acetate accumulates through conversion of acetyl-CoA to ethanol and acetaldehyde and contributes to replenishing NAD+ from NADH. In addition to decarboxylation of pyruvate, Pfl also catalyzes pyruvate to formate, which is a metabolic intermediate that also helps replenish the NAD+ pool. Furthermore, fdhA  that converts formate to carbon dioxide was also upregulated under the iron-replete conditions (Acetate excretion in se (Pfl) . V. vulnnditions .E. coli  and regenerates NAD+ by excreting partially oxidized intermediates, such as ethanol and formate , through pyruvate fermentation  initially excreted acetate but switched to assimilation after 3 h (The acetate switch is a phenomenon when cells switch from acetate excretion through overflow metabolism to assimilation through the Pta-AckA or the PoxB (if present) pathways. For example, a grazing-sensitive fter 3 h . In contfter 3 h . In suppfter 3 h . The diffter 3 h , suggestV. vulnificus strains, representing different genotypes and isolation sources, for resistance to predation by T. pyriformis. Only ENV1 showed resistance to predation by T. pyriformis  by adding NaOH rendered the CFS nontoxic, indicating that the active ingredient that confers toxicity is less likely to be a protein or enzyme but a small compound that is sensitive to pH, such as organic acids. High concentrations of acetate (up to 4 mM) were detected in the CFS of ENV1  and anaerobically by pyruvate-formate lyase (Pfl)  under aerobic conditions or by decarboxylation of pyruvate (under both aerobic and anaerobic conditions) to acetyl-CoA followed by generation of acetyl-phosphate and then acetate, catalyzed by phosphotransacetylase (Pta) and acetyl kinase (AckA). ENV1, like most se (Pfl) . ENV1 ensome two . Transcr acetate .E. coli, anaerobic respiration is regulated by ArcA that represses TCA cycle genes , the enzyme responsible for lactate synthesis, was not significantly upregulated, lactate and formate were detected at millimolar concentrations in the CFS of ENV1 . Furthermore, genes responsible for the synthesis of formate (pfl) and ethanol (adh) were induced  modulation of the cAMP and NAD+ pool required for the transcription and activation of acetyl-CoA synthase  resulted in hypoxic growth in V. cholerae that led to increased transcription of the virulence gene regulator toxT that in turn induces the production of the cholera toxin. Furthermore, arcA expression-linked repression of TCA cycle and increased acetate excretion are also associated with increased toxT transcription , and as a result, the excreted acetate and other organic acids acidify the environment. Therefore, acetate excretion under an acidified environment is a novel bacterial antipredator strategy that provides protection for V. vulnificus ENV1.In conclusion, we found that V. vulnificus strains were kindly provided by J.D. Oliver, UNC Charlotte, Charlotte, NC and Shin-Ichi Miyoshi of Okayama University, Japan. Bacterial strains (−1). The iron chelator, 2-2′ bipyridyl (BPD) (100 μM) , was added to the medium to induce iron limitation. T. pyriformis was routinely passaged in 15 mL peptone-yeast-glucose (PYG) medium  added to 1 L 0.1× M9 minimal medium  supplemented with 0.1 M sterile-filtered glucose in 25 cm2 tissue culture flasks with ventilated caps  and incubated statically at room temperature (RT) for 3 days. To remove the nutrient media and to acclimatize the ciliate to phagotrophic feeding, 500 μL of the T. pyriformis culture was added to 20 mL of 0.5× NSS medium   were prepared as described previously  (1:1). Videos and images were recorded under these conditions. Uronema marinum (Dujardin 1841) was isolated by Martina Erken in 2011 at the Sydney Institute of Marine Science (SIMS) harbor and kept as a nonaxenic culture.All  strains  were rou strains  as appro1 H3BO3)  supplemeeviously . The heaV. vulnificus, 106 cells mL−1 of overnight cultures in 0.5× VNSS   and the plates were incubated at RT with shaking at 60 rpm for 24 h. The cell density of each well was measured at an optical desntiy at 600 nm (OD600) . Planktonic fractions were collected for CFU mL−1 counts. T. pyriformis was enumerated by microscopy.To assess predation of planktonic .5× NSS)  were addV. vulnificus ENV1 were toxic to protozoa, overnight cultures of V. vulnificus ENV1 were adjusted to 106 cells mL−1 in 0.5× VNSS and incubated for 24 h. Cell-free supernatants (CFS) were collected by centrifugation at 3,100 × g for 5 min and filtered . Various treatments of the CFS, including heating (95°C for 2 h), freezing/thawing (–20°C for 24 h), ultrafiltration , proteinase K (200 μg mL−1) from Tritirachium album , proteinase (1 mg mL−1) from Streptomyces griseus (pronase E) , and pH adjustment with hydrochloric acid  and sodium hydroxide (NaOH 1 to 3 mM) were tested to assess what types of biomolecules may be responsible for toxicity. The CFS was added to 24-well microtiter plates  containing T. pyriformis (104 cell mL−1), and numbers of live T. pyriformis cells were determined by microscopy. Sterile medium controls (0.5× VNSS) were included for each treatment to ensure none of the treatments were toxic to T. pyriformis. Images of the whole field of view at different time points were taken using inverted phase-contrast microscopy. Cells that die due to cytotoxicity lose their shape, become more spherical, and leak cytoplasm through compromised cell membranes. These cells sink to the bottom of the well. Hence, only misshapen, disintegrating cells at the bottom of the well were counted as dead.To determine if factors secreted by For the estimation of short-chain fatty acids , CFS was collected as described previously. CFS was acidified with formic acid  and analyzed by gas chromatography flame ionization detector (GC-FID) using a DB-FFAP column (Agilent) with standards of each fatty acid prepared in water at concentrations between 1 and 200 ppm.V. vulnificus were adjusted to 106 cells mL−1 (OD600 = 0.001) in 0.5× VNSS (iron-replete) or 0.5× VNSS supplemented with a 100 μM concentration of the iron chelator, 2-2′ dipyridyl (iron-depleted) in 24-well microtiter plates . Plates were incubated for 10 h at RT with shaking at 60 rpm (early stationary phase), and the supernatant toxicity was determined. The samples were fixed in RNAprotect bacteria reagent (Qiagen). Total RNA was extracted by lysozyme digestion and the RNeasy plus minikit (Qiagen) following the manufacturer’s instructions. RNA concentration and purity were determined by spectrophotometer (NanoDrop ND-1000), and the integrity of the RNA was determined by agarose gel electrophoresis and using an Agilent Bioanalyzer 2100. The RNA was stored at −80°C until it was prepared for sequencing using the Illumina standard kit following the manufacturer’s protocol (Illumina). Samples were sequenced by paired-end sequencing on the Illumina Hi-Seq 2500 platform with read lengths of 100 bp.Overnight cultures of https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Illumina adaptors, short reads, and low-quality reads were removed using cutadapt (version 1.11)  was performed using sortmeRNA (version 2.0)  . In siliion 2.0) . mRNA re1 genome  using Bo1 genome . The num1 genome .P value was smaller than 0.05. The normalized transcripts were then log2 (N + 1) transformed prior to principal-component analysis and unweighted pair group method using average linkages (UPGMA) hierarchical clustering for the sample dendrogram on the heatmap.The raw count table of transcripts was then used as an input for the Deseq2 R package for differential expression analysis . BrieflyarcA start codon, a 750-bp region downstream of arcA, a gentamicin resistance cassette, and the linearized suicide vector pCVD442 (Addgene no. 11074). The assembled construct was transformed into E. coli BW20767 via heat shock transformation. The correct insertion of fragments in pCVD442 was confirmed by sequencing .A four-fragment construct was generated using NEBuilder HiFi DNA assembly master mix (New England Biolabs), consisting of a 750-bp region upstream of the V. vulnificus ENV1 using electroporation  and 10 mM citrate (C). Counter selection of the clones was achieved by spreading mid-log-phase cultures of the 1st recombination clones on counter selection agar containing sucrose . The deletion was confirmed by PCR and sequencing of the PCR product .The construct was introduced into poration . Electrod Maaløe  supplemet tests were used to compare means between experimental samples and controls. For experiments including multiple samples, one-way or 2-way analyses of variance (ANOVAs) were used for the analysis and Sidak’s or Dunnett’s multiple-comparison test provided post hoc comparison of means when appropriate.Statistical analyses were performed using GraphPad Prism version 7.03 for Windows . Data that did not follow Gaussian distribution, as determined by frequency distribution graphs, were natural log transformed. Two-tailed student’s PRJNA448800, BioSample accession number SAMN08866536, and SRA accession numbers SRR6942709, SRR6942712, SRR6942711, SRR6942710, SRR6942713, and SRR6942708.Data from this study have been deposited in the NCBI database under BioProject accession number


To determine factors that influence insurance approval for definitive proton therapy (PT) for prostate cancer.Between 2014 and 2018, 1592 insured patients with localized prostate cancer were evaluated and recommended to undergo definitive PT; 547 patients (34.4%) had commercial insurance, whereas 1045 patients (65.6%) had Medicare/Medicaid. Of those with Medicare, 164 patients (15.7%) had Medicare alone; 677 (64.8%) had supplemental plans; and 204 (19.5%) had secondary commercial insurance. Insurance that “covered” PT for prostate cancer implied that it was an indication designated in the coverage policy. “Not covered” means that the insurance policy did not list prostate cancer as an indication for PT. Of all 1592 patients, 1263 (79.3%) belonged to plans that covered PT per policy. However, approval for PT was still required via medical review for 619 patients (38.9%), comparative dosimetry for 56 patients (3.5%), peer-to-peer discussion for 234 patients (14.7%), and administrative law judge hearings for 3 patients (<0.1%). Multivariate analyses of factors affecting approval were conducted, including risk group (low/intermediate versus high), insurance type , whether PT was included as a covered benefit under the plan (covered versus not covered), and time period (2014-16 versus 2017 versus 2018).P < .001); insurance type ; and time, with 877/987 patients (88.9%) approved between 2014 and 2016, 255/312 patients (81.7%) approved during 2017, and 255/293 patients (87.0%) approved thereafter (P = .02). Clinical factors, including risk group, had no bearing on insurance approval (P = .44).On multivariate analysis, factors affecting PT approval for prostate treatment included coverage of PT per policy  in this study. More work is needed to help navigate appropriate access to care and to assist patients seeking definitive PT for prostate cancer treatment. Definitive proton therapy (PT) is an excellent treatment option for patients with localized prostate cancer. Moreover, PT produces cure rates comparable to both prostatectomy and intensity-modulated radiotherapy (IMRT) but with increasing evidence for reducing late morbidity –3. HowevIn addition, access to PT and its benefits is further affected by it being more costly than IMRT and other traditional modalities. This is, in part, due to the increased time and resources needed to effectuate advanced PT technology for a given patient –6. The iAlthough prostate cancer is more indolent than many other malignancies, such as breast and head and neck cancers, delays in treatment may cause anxiety . AdditioGiven that many PT centers treat patients with prostate cancer, and the few reports on PT approval for prostate cancer, this study aimed to identify factors affecting the likelihood of insurance approval for definitive prostate PT at the University of Florida Health Proton Therapy Institute (UFHPTI).Between 2014 and 2018, 1592 insured patients with localized prostate cancer were evaluated at UFHPTI and were deemed to be good candidates for PT. An additional 17 commercially insured patients were excluded from consideration in this series because their insurance company was not a major plan and provided minimal benefits. Patient selection was based on the extent of disease and medical co-morbidities, such that PT would likely reduce the risk of late complications, including radiation proctitis and radiation cystitis and possibly improve the probability of cure [Table 1.Patients provided consent to participate in an institutional review board–approved outcomes tracking protocol. Additionally, some patients were offered the opportunity to participate in clinical trials, such as a recently initiated Patient Centered Outcomes Research Institute pragmatic trial comparing PT with IMRT (NCT 03561220 ). PatienInsurance status included 547 patients (34.4%) with commercial insurance and 1045 patients (65.6%) with Medicare (including Medicare Advantage). Medicare patients were further stratified according to secondary non-Medicare plans: 164 patients (15.7%) had Medicare alone, 677 patients (64.8%) had supplemental plans, and 204 patients (19.5%) had secondary commercial insurance. The purpose for this breakdown resulted from the recognized differences in processes and approval strategies required to address each form of coverage: supplemental plans, such as AARP and ColonialPenn, are purchased by the patient to provide the 20% co-insurance that Medicare does not pay. Commercial secondary plans, such as BlueCross BlueShield, follow variable rules; some may have a deductible and/or out-of-pocket cost because of prior payments and some may not pay for certain procedures at all, based on plan benefit-coverage stipulations. Notably, many commercial secondary plans are acquired through previous employers after a person has retired, which is common given the average age of prostate cancer diagnoses. Traditionally, supplemental plans tend to cover and approve PT if covered and approved by Medicare, whereas commercial plans have their own independent coverage policies entirely.Overall, 1263 (79.3%) patients had insurance plans stating coverage for PT, meaning the prostate cancer was a listed diagnosis for which PT could be approved. Conversely, “not covered” meant that prostate cancer was not designated as a diagnosis for which PT could be considered. For those with insurance plans that covered PT, approval was not guaranteed; a lengthy process of medical review and decision appeals often ensued before a final decision of definitive prostate PT, regardless of the plans stated coverage. The process differed notably among insurers. For commercial payors, typically, if a request for approval was denied, there was a first-level appeal, a second-level appeal, followed by an external, independent review organization review. Comparative dosimetry, if requested, was obtained early in the process and was frequently followed by a peer-to-peer discussion. The latter may have been with a radiation oncologist or a physician without significant expertise in oncology. For Medicare plans, a final appeal may have been to an administrative law judge, which is a legal proceeding usually attended by the patient, a representative from the insurance company, and the patient's radiation oncologist or representative. Some patients with prostate cancer pursued all steps in the PA or appeals process, whereas others did not. The endpoint of this study was insurance approval for reimbursement for PT by insurance type, stated coverage of PT, clinical risk group, and time period.2 analysis assessed any univariate association between binary approval endpoints and risk group (low and intermediate versus high), insurance type , insurance coverage (covered versus not covered), and time period (2014-16 versus 2017 versus 2018). Time period was included as a variable because it was considered that, as the number of proton facilities increased, insurance policies might change to reflect cost-containing and resource-allocation measures. Thus, 2014-16 was considered the past and 2017 and 2018 were considered to be more recent. Multiple nominal logistic regressions were then used to assess the simultaneous significance of these factors to affect insurance approval.The software JMP Pro (version 14.0.0) was used for statistical analysis . Likelihood-ratio χTable 2. Patients whose insurance covered protons had few steps required in an effort to obtain approval . The number of appeals for the group of 1592 patients were as follows: no appeals, 1424 patients (89.4%); 1 appeal, 149 patients (9.3%); and 2 or more appeals, 19 patients (1.2%).The steps required in seeking approval for covered versus not-covered patients are depicted in Table 3. Approval was obtained for 877/987 patients (88.9%) treated between 2014 and 2016, 255/312 patients (81.7%) treated during 2017, and 255/293 patients (87.0%) treated during 2018.Overall, 1263 patients (79.3%) had insurance that covered PT. Of those 1263 covered patients, 1227 (97.1%) were ultimately approved; 939 of the 1227 patients (76.5%) whose insurance covered PT and who were approved had a Medicare/Medicaid HMO product. Of the 329 patients whose insurance did not cover PT, approval was obtained for 160 patients (48.6%). Insurance approval for PT by insurance type  and coverage (yes versus no) is shown in P = .35; insurance type, P < .001; coverage (yes versus no), P < .001; and time period, P = .01. However, multiple nominal logistic regressions then assessed which of those factors could significantly affect insurance approval when accounting for all other factors. Multiple logistic regressions found approval varied significantly by insurance type, P < .001; coverage, P < .001; and time period, P = .02. Risk group as a clinical correlate had no effect on approval (P = .44) (Table 4).Univariate analysis approval varied by risk group, After the approval process, treatment was eventually delivered as follows: 1166 patients (73.2%), UFHPTI protons; 21 patients (1.3%), UFHPTI photons; and 405 patients (25.4%), treatment elsewhere or no data.There are limited data pertaining to insurance approval for PT –9. Even We additionally found that, despite reports suggesting or showing PT benefits for prostate cancer , 18, PT Future studies on factors affecting PT access for prostate cancer should be conducted. It is likely that approval varies geographically depending on the health care system and the available medical resources of the country. Furthermore, the health insurance companies in the United States have a financial model that aims to contain costs . The appOne of the limitations of our study is the lack of detailed data related to the time required to secure final insurance approval for each patient. Another limitation is that we do not have data related to participation in a clinical trial and its effect on insurance approval. Finally, we are unsure what proportion of patients who sought approval for PT but did not receive it dropped out in the process because of frustration and fear of disease progression, rather than preference for another therapy.In conclusion, most patients with prostate cancer have insurance plans that approve PT for the treatment of localized prostate cancer. Factors that affect the likelihood of insurance approval include the type of insurance, whether the policy covers PT, and the time period.