Transverse testicular ectopia (TTE) is a rare anomaly characterized by the presence of both testicles in the same hemiscrotum or inguinal region. The most common clinical findings of TTE are unilateral nonpalpable testis in the scrotum and inguinal hernia on the side of palpable testis in the scrotum. It should be kept in mind that TTE may coexist with Persistent Mullerian Duct Syndrome (PMDS). Therefore, appropriate treatment should be performed considering PMDS.Type 2 transverse testicular ectopia was diagnosed in one patient who was operated with bilateral undescended testis. He was treated with transseptal orchiopexy and excision of mullerian structures.In case of TTE with PMDS, optimal surgical approach with orchiopexy and excision of Müllerian duct is necessary. Intraoperative aggressive dissection of vas deferens and testicular vessels should be avoided in TTE patients. They should be closely followed because of the increased risk of malignant transformation in the postoperative period.An investigation of transvers testicular ectopia should be performed in all nonpalpable undescended testis anomalies. It is usually found incidentally in patients operated for inguinal hernia or undescended testicles. Preoperative ultrasonography and laparoscopic evaluations are helpful in diagnosis, and the treatment of TTE. Standard treatment of TTE is mainly surgery, including inguinal hernia repair, transseptal orchiopexy, and the repair of congenital anomalies . In this2An eight-month-old male patient was admitted to our hospital, pediatric surgery clinic with the complaint of bilateral undescended testes. According to medical history; he was born at term, and there was no medical problem during the postnatal period. On the physical examination, an uncircumcised penis, left undescended testis in the inguinal canal, and unpalpable right testis were observed. The urethral meatus was in normal localization, and there was no finding related to the hernia in both inguinal canals. In ultrasonography, right testicle was detected in the inguinal canal, but left testicle could not be detected. Hence, the patient underwent laparoscopy for diagnostic and therapeutic purposes:The operation was started by entering with a 3-mm port from the umbilicus. In the diagnostic laparoscopy performed with 3-mm optics, the left testis was observed in the inguinal canal, and the right testis was close to the left inguinal canal opening. The right testis was ectopically located on the left side with the left testis. The dissection of the adherent hernia sac was decided during this operation in this patient with TTE A. Howeve3TTE may be seen alone or accompanied by inguinal hernia, hypospadias, scrotal anomalies, true hermaphroditism, and PMDS. The ectopic testis may be located in the abdomen, at the level of the internal inguinal ring or in the same hemiscrotum . The incTTE may occur with different clinical conditions. Both testicles have a common processus vaginalis sac, and inguinal hernia is the most common situation where the ectopic testis should be present. On the side of the ectopic testis, incarcerated inguinal hernia is an expected condition. Transverse testicular ectopia is the first pathology to be considered in the presence of unilateral non-palpable testis and contralateral reducible or irreducible hernia . In the Ultrasonography, MRI, and laparoscopy may be used for diagnosis in addition to physical examination .The basic treatment principles of TTE are the preservation of fertility, the repair of congenital anomalies, and hernia, orchiopexy, follow-up due to increased malignancy risk. Surgery may be performed with inguinal approach, laparoscopy, laparoscopy assisted inguinal aproach, or laparotomy. Laparoscopic exploration will improve the appearance of the anatomy .The surgical techniques used in the treatment of TTE are transseptal orchiopexy and transperitoneal orchiopexy. In the transseptal orchiopexy method, the ectopic testis is moved from the window created in the scrotal septum to the opposite side. In the transperitoneal orchiopexy method, the ectopic testis is placed to the extraperitoneal area by crossing the root of the penis and fixed into the other side of the hemiscrotum . HoweverThe primary surgical approach in type 2 TTE cases with PMDS is similar to type 1 TTE. Besides, the excision of Mullerian structures is needed in type 2 TTE cases. The main purpose of the surgery is not to damage the vas deferens, which is usually located lateral to the uterus and testicular vessels. Microsurgical techniques can be performed to separate fused structures. But, in spite of these techniques, if the fusion is at an advanced level and the injury to the vas deferens is unavoidable, salpingectomy should be performed at the proximal level, the pedicle of the myometrium should be left intact, and the mesentery of the fallopian structure attached to the upper pole of the testis should be left without separation from the testicle due to the injury risk of the epididymis . Patient4In cases with the undescended testis, it should be considered that the underlying pathology may be TTE. On physical examination, the inguinal canal on the side of the descended testis into the scrotum should be palpated for the presence of the ectopic testis up to the internal inguinal ring, if there is a suspicion for TTE.The case report had no sponsors.This case report is exempt from ethical approval by our institutionWritten informed consent was obtained from the patient’s relatives for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.This is not a ‘first in humans’ report, so it is not in need of registration.mevlitkorkmaz@gmail.com.Mevlit Korkmaz, MD, Assoc. Prof2 Email: Address: Çamlık Mah. Selçuklu Cad. No:22 Pendik İstanbul Türkiye.OrcID:https://orcid.org/0000-0001-5494-5868.Not commissioned, externally peer-reviewed.Tural Abdullayev: Writing - original draft, Writing - review & editing, Data curation, Conceptualization. Mevlit Korkmaz: Writing - original draft, Writing - review & editing, Data curation.No potential conflicts of interest.


We report a novel strategy, combining surface molecularly imprinting and self-assembly directed electro-polymerization with high densely imprinting PO molecules in the PATP/AuNPs film. Firstly, AuNPs were in situ electrodeposited at the electrode surface, and then assembled with electropolmerizable functional monomer p-aminothiophenol (ATP). Subsequently, PO molecules were assembled onto the ATP monolayer-modified AuNPs, forming a basis of surface molecular imprinting. After that, replenished PO molecules were embedded in the PATP/AuNPs film by PO and the ATP molecular self-assembly directed electro-polymerization in the polymerization precursor mixture. The resulting imprinted PATP/AuNPs/SPCE possesses high sensitivity, affinity, and selectivity toward PO, with a low detection limit of 1 × 10−9 M. The proposed sensor was successfully applied for the determination of PO in fruit and vegetables, giving satisfactory recoveries. The strategy reported herein can be further expected to fabricate various molecular imprinted sensors for the determination of other pesticide residuals.This paper presents the fabrication of a molecularly imprinted, polymer-based disposable electrochemical sensor for paraoxon (PO) determination. The sensor was based on a screen-printed carbon electrode (SPCE) modified with a surface molecularly imprinted poly ( Highly neurotoxic organophosphates, such as paraoxon (PO), are widely used as persistent pesticides and nerve agents, and are responsible for a number of poisonings. Organophosphate can disrupt the cholinesterase enzyme that catalyzes the breakdown of acetylcholine, a neurotransmitter needed for proper nervous system function, which often causes convulsions, coma, respiratory paralysis, and death ,2. In viAs an alternative approach—molecularly imprinted polymers (MIPs) that exhibit high selectivity and affinity to the predetermined specific species—are now experiencing fast research growth, and have been applied as the sensor recognition element for organophosphates determination ,12,13. TAddressing the limitations of conventional molecular imprinting techniques, several research groups have begun to explore alternative approaches for developing new imprinting methodologies. An effective approach is to control templates to locate at the surface of imprinted materials, typically exampled by surface imprinting, which is carried out by immobilizing template molecules at the surface of suitable substrates, forming thin imprinted films ,20. SurfHerein, we fabricated a novel disposable electrochemical sensor based on surface molecularly imprinted PATP/AuNPs composite film for sensitive and selective analysis of PO. Specially, we demonstrated a self-assembly directing strategy for the highly dense imprinting of the template molecules in the electropolymerized PATP/AuNPs film at the disposable screen printed carbon electrode (SPCE) surface see . For thep-nitrophenol, isocarbophos, and parathion were purchased from Dr. Ehrenstorfer GmbH . An individual stock solution of pesticide was prepared by dissolving each compound in ethanol with a concentration of 0.1 M of both solutions and stored in amber bottles at 4 °C. p-aminothiophenol was obtained from Alfa Aesar . HAuCl4·4H2O (Au% > 47.8%) was purchased from Sinophacm Chemical Reagent Co., Ltd . The other reagents used were commercially available as analytical reagent grade or better.Paraoxon, monocrotophos, Disposable SPCEs were purchased from Dropsens, Inc. , which consisted of an Ag/AgCl reference electrode, a carbon working electrode (3 mm in diameter), and a carbon auxiliary electrode. A sensor connector  allowed for the connecting of the SPCE to a Bio-analytical System  CV-50w electrochemical workstation. The scanning electron microscopy (SEM) images were obtained by using a JEOL-6700F SEM instrument . UV spectra were recorded using a UV-2501 spectrophotometer .−1 until a steady current–voltage curve was obtained. The electrode was further washed three times with distilled water and then dried under nitrogen gas. To electrodeposit AuNPs on the SPCE surface, the electrode was applied at a potential of −0.2 V for 80 s in 0.2 g·L−1 HAuCl4 solution. Optimization of the deposition parameters can be seen in 2SO4/H2O2, 7:3) for about 300 s, rinsed again with doubly distilled water, and finally dried under nitrogen gas. A SPCE was first applied at a potential of +1.2 V in acetate buffer (pH 5.0) for 300 s, and then cycle scanned from +0.3 V to +1.1 V at a rate of 50 mV·sATP modification was carried out by immersing the pretreated AuNPs/SPCE into a 10 mM solution of ATP in ethanol. After reacting at RT for 24 h, the AuNPs/SPCE was taken out and then rinsed three times with ethanol and distilled water, respectively. Afterwards, the AuNPs/SPCE was immersed into a PO solution (1 mM) for 6 h, and then was taken out, rinsed with ethanol, and subsequently dried under nitrogen flow at RT.−1. Optimization of the scan parameters on the PATP film formation can be seen in The pretreated PO–ATP–AuNPs/SPCE was immersed in the HAc–NaAc (pH 5.0) aqueous electrolyte solution containing 5 mM ATP and 0.1 mM PO. The electropolymerization was performed by seven cyclic scanning from −0.20 V to +0.60 V at a scan rate of 50 mV·sThe modified electrodes were allowed to pre-absorb PO by incubating in the acetate buffer solution containing varying PO concentrations for 10 min. After removing the physically adsorbed substances on the electrode surface by doubly distilled water rising, 20 µL of acetate buffer solution was placed onto the working electrode. The amount of bound probe was quantified by DPV measurements from −0.50 V to −1.00 V. The corresponding maximum current was used for concentration determination. All the measurements were performed at RT.Apple and cabbage samples were purchased from a local market at Wuhan, China. The real samples were prepared by peeling and chopping the fruit and vegetables into small pieces. Twenty-five grams of the homogenized sample was mixed with 50 mL of acetonitrile and subsequently blended for 3 min. The extract was then filtered through a filter paper into an evaporation flask with 5–7 g of sodium chloride. The mixture was blended for 1 min and let stand for 30 min to let the phases separate. After phase separation, 10 mL of the upper acetonitrile layer was drained and filtered through a 0.2 μm pore size membrane into an evaporation flask, and then evaporated to dryness, following which ethanol (5 mL) was added. Spiking of the samples was achieved by adding different levels of PO stock solution into the samples, and then homogenizing the mixtures.In this work, we aim to enhance the sensitivity and selectivity of PO analysis by modifying the electrode surface with electron-rich amino groups, as well as π-donor aromatic ring groups that would selectively concentrate PO at the electrode surface using hydrogen bonds and π-donor-acceptor interactions. ATP is an appropriate molecular anchor for anchoring template molecules on Au surfaces. Thus, ATP was chosen to act as the polymerizable functional monomer for preparing the PO imprinted polymer films. Furthermore, AuNPs were introduced onto the sensor surface, serving as a high conductive nanostructured imprinted substrate that has a higher surface area, for further increasing the amount of effective imprinted sites . The pre4 at a constant potential of −0.20 V for 80 s. Then the AuNPs-modified SPCE was immersed into the ATP solution for 24 h. The ATP molecules could absorb onto the AuNPs surface based on the Au–S bonds and the exposed an array of the amino groups towards the solution, forming an ATP SAM [In the first procedure, AuNPs were easily formed in situ on the surface of SPCE by electrochemical deposition of HAuCl ATP SAM   was determined using the criterion S/N = 3 was 1 × 10−9 M. For comparison, curve b depicts the calibration curve observed with the nonimprinted PATP/AuNPs/SPCE. The LOD for analyzing PO by the nonimprinted PATP/AuNPs/SPCE is 1.7 × 10−7 M. The results show that the LOD for PO by the imprinted PATP/AuNPs/SPCE is about two orders of magnitude lower than that with the nonimprinted control.The DPV responses that correspond to the analysis of PO by the imprinted PATP/AuNPs/SPCE are shown in Kα) of PO to the imprinted sensing surface on the basis of Equation (1) [Kα is given by Equation (2), where α is defined as the amount of imprinted sites in the system, and θ is defined as the amount of imprinted sites that are occupied by PO, which can be derived from the coulometric analysis of the first wave of the reduction of PO at any concentration. Equation (2) can also be rearranged to form Equation (3). The charge associated with the bound PO is proportional to the amount of occupied, imprinted sites. Curve a in Kα with the imprinted PATP/AuNPs corresponded to (2.5 × 104 M−1), that is, ca. 7-fold higher than that with the nonimprinted control  . Kα is gkPO/kPT) of the imprinted PATP/AuNPs/SPCE equals 8 (see We studied the selectivity of the imprinted PATP/AuNPs/SPCE and compared it to the nonimprinted control. Parathion (PT) was used to verify the response selectivity of the imprinted PATP/AuNPs/SPCE towards PO. The response selectivity factor , respectively. The competitive selectivity of the imprinted PATP/AuNPs/SPCE electrode to PO molecules was evaluated by calculating the peak current ratio (Ip/Ia), where Ip and Ia were the anodic peak current of 10 µM PO at −0.77 V in the presence and absence of interfering substances. The results showed a 50-fold excess of PT, MC, IC, and p-NP over PO hardly causes the significant change of peak current of PO, in which the peak current ratio only slightly varied from 0.93 to 1.06 , isocarbophos (IC), and 1.06 see , which iThe reproducibility of the developed imprinted sensor was determined by comparing the DPV responses of ten sensors prepared in parallel under the same conditions towards 10 µM PO in acetate buffer solutions. A relative standard deviation (RSD) of 1.53% was obtained, indicating a good reproducibility in the sensor fabrication. When not in use, the sensor can be put in a plastic casket filled with nitrogen and stored at 4 °C. No obvious deterioration in the response of the sensor towards PO was determined in the first 10-day storage. After a month in storage, the electrode showing a ca. 10% decrease in the PO signal, and a 16% decrease after three months in storage. The obtained results indicate that the developed sensor possesses excellent stability.n = 6). The presented results demonstrate that the proposed method can be a promising approach for PO determination in real samples. The proposed sensor was compared with other published methods [To investigate the feasibility of the imprinted PATP/AuNPs/SPCE for practical applications, apple and cabbage samples were used for the quantitative analysis by the developed sensor. None of these real samples had electrochemical responses when analyzed by using the developed imprinted sensor. It is assumed that there is no PO in the samples, or the concentration of PO is too low to be detected. Thus, the recovery experiments were performed by adding known concentrations of PO into the samples. The samples were then spiked with different levels of PO.  methods ,36,37,38A novel molecularly imprinting strategy, combining surface molecularly imprinting and self-assembly directed electro-polymerization for the sensitive electrochemical sensing of PO was developed. The proposed method relies on the π–donor–acceptor and hydrogen bond interactions between PO, ATP, and ATP–AuNPs. The tailor-made cavities formed in the imprinted film showed good selectivity and affinity toward PO. The selectivity, reproducibility, and stability of the MIP sensor are satisfactory. The good sensitivity, affinity, and selectivity of the sensor is attributed to the following reasons: on the one hand, the employment of the AuNPs inner layer as a high-conductive, nanostructured, imprinted substrate enlarges the electro-active surface area; on the other hand, the surface imprinting of the template and the functional, monomer, self-assembly directed electro-polymerization increases the quantity of the template molecule in the unit area, and creates the imprinted materials with a high ratio of effective imprinted sites. Therefore, this novel, facile strategy reported herein can be further expected to fabricate various molecular imprinted polymers for detecting the pesticide residuals and other environmentally deleterious chemicals.


Turnera diffusa (damiana) for their inhibitory activities against recombinant human monoamine oxidases (MAO-A and MAO-B) in vitro identified acacetin 7-methyl ether as a potent selective inhibitor of MAO-B (IC50 = 198 nM). Acacetin 7-methyl ether  is a naturally occurring flavone that is present in many plants and vegetables. Acacetin 7-methyl ether was four-fold less potent as an inhibitor of MAO-B when compared to acacetin (IC50 = 50 nM). However, acacetin 7-methyl ether was >500-fold selective against MAO-B over MAO-A as compared to only two-fold selectivity shown by acacetin. Even though the IC50 for inhibition of MAO-B by acacetin 7-methyl ether was ~four-fold higher than that of the standard drug deprenyl , acacetin 7-methyl ether’s selectivity for MAO-B over MAO-A inhibition was greater than that of deprenyl (>500- vs. 450-fold). The binding of acacetin 7-methyl ether to MAO-B was reversible and time-independent, as revealed by enzyme-inhibitor complex equilibrium dialysis assays. The investigation on the enzyme inhibition-kinetics analysis with varying concentrations of acacetin 7-methyl ether and the substrate (kynuramine) suggested a competitive mechanism of inhibition of MAO-B by acacetin 7-methyl ether with Ki value of 45 nM. The docking scores and binding-free energies of acacetin 7-methyl ether to the X-ray crystal structures of MAO-A and MAO-B confirmed the selectivity of binding of this molecule to MAO-B over MAO-A. In addition, molecular dynamics results also revealed that acacetin 7-methyl ether formed a stable and strong complex with MAO-B. The selective inhibition of MAO-B suggests further investigations on acacetin 7-methyl as a potential new drug lead for the treatment of neurodegenerative disorders, including Parkinson’s disease.The investigation of the constituents that were isolated from  Calea urticifolia [O-, from Agatache rugosa was also reported as a selective potent MAO-B reversible inhibitor [The monoamine oxidases  are FAD (flavin adenine dinucleotide)-dependent enzymes that are responsible for the metabolism of neurotransmitters, such as dopamine, serotonin, adrenaline, and noradrenaline, and for the oxidative deamination of exogenous arylalkyl amines . Due to icifolia . Similarnhibitor .Turnera diffusa Willd. ex Schult (known as damiana), which is a native plant to America and Africa, is traditionally used for the treatment of various diseases, including sexual impotence, neurasthenia, diabetes mellitus, urine retention, malaria, diarrhea, and peptic ulcer [Turnera diffusa [ic ulcer . The plaic ulcer . Phytoch diffusa ,15. Seve diffusa . Based oO-β-d-(6-O-pcoumaroyl) glucoside, echinaticin, tetraphyllin B, tricin-7-glucoside, diffusavone, turneradiffusin, rhamnosylorientin, rhamnosylvitexin, and turneradin were isolated from T. diffusa [A series of flavonoids and flavonoid glycosides, namely acacetin, acacetin 7-methyl ether, vetulin , apigeni diffusa .50 = 0.198 μM) as compared to MAO-A (IC50 = >100 μM) (T. diffusa only showed moderate inhibition of MAO-A and MAO-B (IC50 in the range of 13–61 μM), with no significant selectivity towards MAO-B or MAO-A  . The conor MAO-A . The stu50 value (100 and 150 nM), one around the IC50 value (200 nM), and two above the IC50 value (300 nM and 500 nM) for the inhibition kinetics experiments. The assays were run at varying concentrations of the substrate and fixed concentrations of the inhibitor. The Ki  values were computed with double reciprocal Lineweaver–Burk plots. The binding of acacetin 7-methyl ether to human MAO-B affected the Km value  without much effects on the Vmax (maximum enzyme activity), indicating that the inhibition of MAO-B by acacetin 7-methyl ether was competitive  with the MAO-B enzyme for 20 min and the resulting enzyme-inhibitor complex preparation was dialyzed overnight in phosphate buffer. The activity of the enzyme was analyzed before and after the dialysis . The incThe time dependence of binding of acacetin 7-methyl ether to MAO-B was analyzed. The recombinant enzyme was incubated for different times with the test compounds, namely, deprenyl, (0.100 μM), acacetin 7-methyl ether (0.500 μM), and acacetin (0.100 μM) . The conhMAO-A and hMAO-B. Three-dimensional (3D) protein-ligand interactions of acacetin . The experimental data also supported this  to ted this . The docted this C. We alsted this . A previhMAO-B. Therefore, we carried out a 10 ns MD simulation of the complex of acacetin 7-methyl ether with MAO-B using Desmond software [Docking alone cannot provide full insight into the binding mode and dynamics of acacetin 7-methyl ether within software . The MD Strong π-π interactions were observed between Tyr326 and Ring B and Ring C of acacetin 7-methyl ether. In addition, the protein-ligand interaction diagram B indicatTurnera diffusa. Acacetin 7-methyl ether has also been isolated from a few other plants and showed antidiabetic, antiproliferative, and antioxidative activities [Acacetin 7-methyl ether  was identivities ,22,23. Mtivities . Howeverl-deprenyl), rasagiline, and safinamide. Selegiline and rasagiline are irreversible inhibitors of MAO and they may be associated with significant side effects [50 198 nM vs. 50 nM, respectively), but it dramatically improves the selectivity (>500) for MAO-B. This suggests a significant scope for further optimization of the 5, 7-dihydroxy-4′-methoxyflavone pharmacophore to achieve high potency and selectivity against the human MAO-B. 5, 7-Dihydroxy-4′-methoxyflavones represent a new class of selective reversible MAO-B inhibitors with potential therapeutic application for treatment of neurodegenerative disorders, including Parkinson’s disease in combination with standard drugs [O-, a derivative of acacetin that was isolated from Agastache rugosa plant leaves [The selective MAO-B inhibitors are used alone as well as in combination with carbidopa-levodopa for the treatment of motor symptoms of Parkinson’s disease . Reducti effects ,27. Both effects  and acacrd drugs . A recent leaves . The revt leaves .50 = 0.115 μM) than with acacetin 7-methyl ether (IC50 = >100 μM). To shed light on the interaction profile, binding mechanism, and dynamic properties of acacetin 7-methyl ether with MAO-B, we carried out a 10 ns MD simulation of their protein-ligand complex. The structural and dynamical properties of acacetin 7-methyl ether were analyzed using RMSD plots of the ligand and of the Cα atoms of the protein . Kynuramine, clorgyline, deprenyl, acacetin, and DMSO were purchased from Millipore Sigma . Safinamide was purchased from TCI Chemicals, Portland, OR, USA.O-β-d-(6-O-pcoumaroyl) glucoside (>97%), echinaticin (>96%), tetraphyllin B (>98%), tricin-7-glucoside (>98%), diffusavone (>97%), turneradiffusin (>96%), rhamnosylorientin (>97%), rhamnosylvitexin (>95%), turneradin (>95%), and acacetin (>96%) were isolated from T. diffusa at the National Center for Natural Products Research (NCNPR), University of Mississippi, University, MS, USA. Their identities and purities were confirmed by chromatographic  and spectroscopic - and two-dimensional (2D)-NMR, HR-ESI-MS) methods, as well as by comparison with the published spectroscopic data [Acacetin 7-methyl ether (purity >98%), vetulin (>97%), apigenin-7-pic data ,15.T. diffusa on recombinant human MAO-A and MAO-B, the kynuramine oxidative deamination assay was performed in 384-well plates, as previously reported, with minor modifications [50 values. Kynuramine concentrations for MAO-A and MAO-B were 80 μM and 50 μM, respectively. These concentrations of kynuramine were twice the apparent KM value for substrate binding [50 values for MAO-A and MAO-B inhibition were calculated from the concentration dependent inhibition curves using XLfit, a Microsoft Excel-based plug-in which performs Regression, curve fitting and statistical analysis .To investigate the effect of the isolated constituents from ications ,31. A si binding ,31. The 50 values for the inhibition of MAO-A and MAO-B by acacetin 7-methyl ether, the enzyme assay was performed at a fixed concentration of the substrate kynuramine for MAO-A (80 μM) and for MAO-B (50 mM) and with varying concentrations of inhibitor/test compounds for MAO-A and MAO-B (0.01 μM to 100 μM). The dose-response curves were generated using Microsoft® Excel and the IC50 values were analyzed using XLfit software.To determine the ICFor the determination of the binding affinity of the inhibitor (Ki) with MAO-A and MAO-B, the enzyme assays were carried out at different concentrations of kynuramine substrate (1.90 μM to 500 μM) and varying concentrations of the inhibitors/compound. Compounds acacetin and acacetin 7-methyl ether were tested at 0.030–0.100 μM and 0.100–0.500 μM, respectively, for MAO-B to determine the Km and Vmax values of the enzymes in the presence of the inhibitor. The controls without inhibitor were also simultaneously run. The results were analyzed by standard double reciprocal Line–Weaver Burk plots for computing Km and Vmax values, which were further analyzed to determine Ki values [The inhibitors bind with the target enzyme through the formation of an enzyme-inhibitor complex. The formation of the enzyme-inhibitor complex may be accelerated in the presence of the high concentration of the inhibitor. The reversibility of binding of MAO inhibitory compound acacetin 7-methyl ether was determined by the formation of the complex by incubating the enzyme with a high concentration of the inhibitor, followed by extensive dialysis of the enzyme-inhibitor complex and the recovery of catalytic activity of the enzymes. The MAO-B (0.2 mg/mL protein) enzyme was incubated with each inhibitor: acacetin (0.5 μM), acacetin 7-methyl ether , deprenyl (0.5 μM), and safinamide (0.2 μM) in a total volume of 1 mL, 100 mM potassium phosphate buffer (pH 7.4). After 20 min incubation at 37 °C, the reaction was stopped by chilling in an ice bath. All of the samples were dialyzed against potassium phosphate buffer  at 4 °C for 14–18 h (three buffer changes). Control enzyme (without inhibitor) was also run through the same procedure and the activity of the enzyme was determined before and after the dialysis [To analyze whether the binding of the inhibitor with MAO-B was time-dependent, the enzyme was pre-incubated for different time periods (0–15 min) with the inhibitors at a concentration that produces approximately 70–80% inhibition. The inhibitor concentrations used to test time-dependent inhibition were acacetin (0.1 μM), acacetin 7-methyl ether (0.5 μM), and deprenyl (0.1 μM) with MAO-B (12.5 μg/mL). The controls without inhibitors were also simultaneously run. The activities of the enzymes were determined, as described above, and the percentage of enzyme activity remaining was plotted against the pre-incubation time to determine the time-dependence of inhibition.The X-ray crystal structures of MAO-A (PDB accession number: 2Z5X ) and MAO3. The solvated system was energy-minimized with the DESMOND minimization algorithm for 2000 iterations when considering a convergence threshold of 1.0 kcal/mol/Å. The constructed system was simulated with the relaxation protocol in Desmond [Monoamine oxidase B consists of a two-domain molecular architecture. Each identical monomer consists of 520 amino acids. In this study, we used one monomer (chain A) for the docking and MD simulations. It is reported  that the Desmond  and at a Desmond . The simTurnera diffusa (damiana) identified three O-methyl flavones, namely, acacetin, acacetin 7-methyl ether, and vetulin as selective inhibitors of human MAO-B. Acacetin 7-methyl ether was a highly potent and selective MAO-B inhibitor with >500-fold selectivity towards MAO-B when compared to MAO-A. Further studies suggested that acacetin 7-methyl ether is a reversible competitive inhibitor of MAO-B. Computational docking analysis confirmed the selective binding of acacetin 7-methyl ether to human MAO-B as compared to MAO-A. Acacetin 7-methyl ether may have a potential therapeutic application for the treatment of neurodegenerative disorders, including Parkinson’s disease.Biological screening of constituents of


Interpreting troponin concentrations in this context is challenging because the kinetics of troponin release after physical exercise are not well understood. Most previous research has used assays that are not able to quantify troponin within the reference range, or has involved endurance athletes and evaluated a single time point rather than serial sampling.3The latest generation of high-sensitivity cardiac troponin assays are able to quantify very low concentrations of troponin in the majority of people. Indeed, international guidelines now recommend the use of low concentrations of troponin to risk-stratify patients with suspected acute coronary syndrome.STAT high-sensitive troponin I assay (Abbott Laboratories). This assay has a limit of detection of 1.2 ng/L and an upper reference limit (99th percentile) of 34 ng/L in men and 16 ng/L in women. Change in troponin concentration within each study visit was evaluated by a 1-way repeated-measures ANOVA and paired t tests. We compared troponin concentrations between visits by a 2-way repeated-measures ANOVA.In this study, we evaluated the effect of intensity and duration of physical exercise on troponin concentration in 10 physically active healthy volunteers . The study was approved by the Research Ethics Committee and was conducted with written informed consent. Eligibility was assessed using the Physical Activity Readiness Questionnaire, the American Heart Association Pre-Participation Screening Questionnaire, and a 12-lead electrocardiogram. All participants underwent an initial fitness test using a maximal graded exercise test on a cycle ergometer to exhaustion to quantify their lactate threshold. They subsequently attended 3 study visits, at least 7 days apart, during which they underwent exercise on a cycle ergometer. The first visit involved low-intensity cycling (50%–60% of the participant’s lactate threshold) for 60 minutes. During the second visit, participants cycled at high intensity (80%–90% lactate threshold) for 60 minutes, and during the third study visit, participants cycled at moderate intensity (60%–70% lactate threshold) for 4 hours. Troponin concentrations were measured at the start of exercise and then hourly for up to 6 hours during each study visit, followed by measurements at 1, 2, and 7 days thereafter using the ARCHITECTP<0.001 for both), but was unchanged after low-intensity exercise (P=0.055). Troponin concentrations were significantly higher after the shorter duration of high-intensity exercise  compared with the longer duration of moderate-intensity exercise . After moderate- and high-intensity exercise, troponin concentration returned to baseline within 48 hours . The median heart rate and peak power output were 112 bpm  and 113 W (88–135), respectively, during low-intensity exercise, 151 bpm (135–162) and 139 W (127–159) during moderate-intensity exercise, and 155 bpm (139–165) and 178 W (161–205) during high-intensity exercise.Study participants had a median troponin concentration of 1.8 ng/L  at baseline. Troponin was elevated after moderate- and high-intensity exercise , a Senior Clinical Research Fellowship (FS/16/14/32023), and a Research Excellence Award (RE/18/5/34216).Drs Lee, Chapman, Anand, and Shah have received honoraria from Abbott Diagnostics. Dr Mills reports research grants awarded to the University of Edinburgh from Abbott Diagnostics and Siemens Diagnostics outside the submitted work, and honoraria from Abbott Diagnostics, Siemens Diagnostics, Roche Diagnostics and Singulex. The other authors report no conflicts.


The Israeli organ donor law was established in 2008. In the ensuing 10 years there have been some improvements in deceased donation and living donor rates and a reduction in the unethical practice of transplant tourism. There is, however, controversy regarding increased access to transplant for those who have been living donors, who are family members of deceased donors, or who have registered their intent to donate. The issue of routine retrieval versus obtaining consent for organ donation has also been raised. This commentary will address these issue, and propose some steps for improvement of the current Israeli organ donation system. Israel’s organ transplant and brain-death law was passed in Israel in 2008 and fully implemented in 2010. It was developed as a response to the three major challenges to organ procurement and transplantation in Israel: 1) confusion regarding determination of death, 2) organ trafficking and unethical/illegal transplant tourism, and 3) the critical dearth of transplantable organs.A major objective of the law, was an attempt to strengthen brain death determination to satisfy both medical and religious needs. This required voluntary training by the critical care physicians responsible for the determination of death by neurologic criteria. To date, the uptake by these physicians has been less than adequate. Unfortunately, brain death as a medical criterion for death is not uniformly accepted within the Israeli population, particularly amongst the ultra-Orthodox community.A second objective was to stem the tide of transplant tourism as both an illegal and unethical means of recipients’ seeking solid organ transplantation. This has proven to have been successful, as demonstrated by the marked decrease in transplant tourism over the last 10 years [Perhaps the most controversial part of the law was a priority structure, favoring access to transplantation based on a point system. The highest priority was given to those whose first-degree relatives were deceased organ donors, or those who themselves had been a previous living donor. The next level of priority was given to those who choose to register as a donor. The last priority level is for individuals with first-degree family members who have registered as donors. This is in the context of an opt-in system whereby donor families must still provide consent in order to proceed with the organ donation, regardless of registration status.In a recent IJHPR article , Berzon Although the point system only comes into effect after medical need is taken into account, Berzon argues that medical need alone should be the highest priority, and that access to transplantation must not be based on a predetermined hierarchy . There iIt should be noted however, that in all transplant allocation policies around the world, societal factors are often employed ahead of medical need. One example illustrating this point is that uniformly, allocation systems for kidney transplants give higher priority to pediatric recipients versus their adult counterparts. In addition, patients with liver failure secondary to alcoholic cirrhosis are often obligated to wait a 6-month sobriety period before acceptance onto a wait list for liver transplantation . There aIn the general population, the likelihood of requiring a life-saving organ transplant is fivefold greater than the chance of being a deceased organ donor. This imbalance between supply and demand means that transplantation is highly dependent upon an adequate supply of both deceased and living organ donors. To date, no jurisdiction has been able to meet the need of an abundant supply of transplantable organs. Thus, different countries have had varying approaches to trying to address the imbalance. In the Israeli system, registration as a donor increases a person’s priority if he/she were ever to need a future transplant. In the absence of an adequate supply of organ donors (those willing to donate), the likelihood of receiving a transplant diminishes.An analogy to organ donor registration is childhood vaccination policy. A child who is vaccinated may achieve some future health advantage. However, unless vaccination occurs within the vast majority of the population, preventable communicable diseases will still occur, and even a vaccinated child may still be at risk if immunity doesn’t develop. Conversely, parents who choose not to vaccinate their children may still be afforded protection so long as there is enough herd immunity (vaccination rates close to 100%). Similarly, if registration for organ donation ever approached levels nearing 100%, then the priority point system would become irrelevant, and organ Israeli donation rates could be amongst the highest in the world.When the Israeli organ donor law first came into existence in 2008 and implemented in 2010, there had been a lot of public and media attention. However, over time there has been less awareness and donor registration rates have been flat. The success of countries such as Spain is not based on an opt-out, or routine retrieval policy, but rather on a very well-resourced, well-organized organ donation community within that country . Organ dWithin Israel’s ultra-orthodox community, there are many who do not accept brain death as a definition of death. This issue became very apparent with the establishment of the new brain death determination laws . HoweverA shift in policy from obtaining family consent to routine retrieval as posited by Berzon  is unlikhttps://www.organdonor.gov/index.html).The United States does not practice routine referral and like Israel, requires consent from next-of-kin. The US has a donation rate of ~ 28–30 donors per million, which is better than the majority of jurisdictions with routine referral policies. Approximately 50% of its population are registered as organ donors, and in some states this number exceeds 85% . Not only will this increase the organ donor supply, but it may help in part circumvent the issues surrounding neurological death determination in some religious communities.In Ontario Canada with a population of 13.5 million, DCD accounts for approximately 33% of all deceased organ donors and has resulted in an additional 2000 transplants since its implementation in 2006. During this same period, donation after brain death criteria also increased . Since 2Israel has made tremendous improvements in the 10-year history of the 2008 organ donation laws. Some of these changes were bold and not without controversy. The organ donation system in Israel continues to face significant challenges. There is room for improvement without the need to abandon the current structure. Jurisdictions that have seen significant improvements in organ donation rates have had major shifts culture and compelling resource allocation.


We present a case of an oropharyngeal cerebrospinal fluid (CSF) fistula in a patient that presented with headache, rhinorrhea, and pneumocephalus years after an anterior cervical discectomy and fusion. Imaging suggested a defect in the fovea ethmoidalis, but endoscopic surgery revealed the defect in the oropharynx. A second procedure was performed to remove the spinal hardware and repair the leak. This case is not only unique in the literature but also highlights the importance of maintaining a broad differential diagnosis to include rare complications and shows that despite dramatic improvements in imaging, locating CSF leaks still presents a challenge. Cerebrospinal fluid (CSF) leaks represent a pathological connection and communication between the subarachnoid space with another nonnative cavity. CSF leakage can result from innumerable processes: iatrogenic, trauma, tumors, or spontaneous disruption of a barrier. Patients often present when the CSF leaks are large enough to result in pneumocephalus or meningitis, a serious and life-threatening condition that can result in permanent neurologic deficits.Identification of the site of the leak can pose a challenge. Multiple imaging modalities including high-resolution computed tomography (HRCT), CT cisternography/myelography, and magnetic resonance imaging can be and have been used. All have been shown to be useful, but the sensitivity and specificity varies greatly depending on size, location, etiology, and status of leak (active versus inactive) –4.Anterior cervical discectomy and fusion (ACDF) procedures are very common and relatively safe. Although rare, patients undergoing ACDF surgeries can develop intraoperative CSF leaks through accidental dural breach during the procedure (0.2% to 1.8% of cases) with the vast majority of these being noted and repaired at the time of surgery , 6. DeveThe purpose of this study is to discuss the workup and management of a patient that presented five years after ACDF with chronic dysphagia, acute headache, pneumocephalus, and signs of meningitis that was subsequently found to have a CSF-oropharyngeal fistula. Imaging suggested that the site of the CSF communication was the left fovea ethmoidalis. The correct anatomic location of the fistula, however, was not identified until a negative endoscopic exploration of the skull base led to identifying the oropharyngeal defect.After a thorough literature review, we identified 3 case reports of post-ACFD fistula presenting as a neck mass and dysphagia –9 and onA 64-year-old male presented to the emergency room with a 2-week history of a severe, persistent headache. The patient's pertinent past medical and surgical history included obesity and an anterior cervical discectomy and fusion five years prior. A CT scan of the head followed by an MRI of the brain and spine revealed extensive pneumocephalus and concerns for meningitis. The MRI of the spine showed the ACDF hardware but did not reveal surrounding defects. The neurosurgery team was consulted, and the patient was admitted.CT cisternogram/myelogram and high-resolution CT sinus were obtained. The cisternogram/myelogram was negative for leak at the skull base and cervical spine. CT imaging showed an air-fluid level within a left posterior ethmoid air cell with an apparent 2 mm adjacent osseous dehiscence along the fovea ethmoidalis, suspicious for the source of the CSF leak in this patient . The radThe otolaryngology/rhinology team was consulted due to the radiographic findings on the sinus CT. On further history and exam, the patient reported intermittent clear rhinorrhea and occasional salty tasting drainage. He denied any significant history of rhinosinusitis. Physical exam demonstrated an obese man who was uncomfortable. Holding the neck in flexion demonstrated clear fluid from the left nostril. The nasal endoscopy was normal. Based on clinical presentation and imaging, there was concern for a left skull defect.The patient was taken to the operating room for identification and repair of the CSF leak. The neurosurgery team placed a lumbar drain and dilute fluorescein dye injected intrathecally. On nasal endoscopy, fluorescein dye was noted to be pooling in the nasopharynx throughout the case. A total sphenoethmoidectomy was performed, and the area of the potential defect was located and confirmed with intraoperative surgical navigation. Inspissated mucus was found at the area of mucosal thickening on CT  without Subsequent endoscopic evaluation of the nasopharynx and oropharynx revealed significant pooling of fluorescein, but both openings to the eustachian tubes were normal. Close inspection of the mid-oropharynx, however, revealed a bulge in the posterior oropharynx, and a small, pinpoint fistula with actively leaking fluorescein-dyed CSF was found .The CT scan of the neck region was reviewed to evaluate the cervical hardware , and theThis case illustrates two points. The first is that CSF leak localization can be challenging , 14. TheAs observed in this case, localizing a CSF leak can be difficult. Multiple imaging modalities are used to localize defects. High-resolution CT scan has the benefit of wide available and noninvasive to the patient. It is useful for evaluating the sinonasal and bony anatomy –3. A sysMRI is another common option. CSF is bright on T2-weighted imaging and can be seen passing through a defect. While also noninvasive, it is more expensive than CT scanning. The sensitivity and specificity ranges from 56–94% and 57–100% [CT cisternography/myelography combines intrathecal contrast injection followed by CT scanning to identify a fistula. Studies report relatively lower accuracy compared to other options . This loOther options include MRI cisternography and single proton emission CT. Some studies suggest improved sensitivity and specificity with these modalities, but they are not widely available and are further associated with higher cost. Additionally, MR cisternography is invasive and requires intrathecal injection , 4. UsinDespite the use of multiple imaging modalities preoperatively in our patient, only operative exploration with the use of intrathecal fluorescein positively located the fistula. Intrathecal fluorescein is suggested to be safe at diluted doses with the benefit of localizing defects , 16. IntThe best localization technique would be one that is widely available, relatively low in cost, noninvasive, and highly accurate. This case highlights the fact that such a technique does not currently exist. Also, a thorough literature search reveals that most published reports on CSF leak identification are retrospective series with relatively small patient populations. Additional research is needed to determine the best approach to CSF leak localization.After a thorough literature review, we identified 3 case reports of post-ACFD fistula presenting as a neck mass and dysphagia –9 and onCSF leak after ACDF is an uncommon complication in the postoperative period, and it is a rare fistula to develop years after the procedure.Despite using several imaging modalities to identify the CSF leak location, none of these identified the site of fistula in our patient. This case highlights the need for research to identify the best approach for defect localization that is accurate, cost effective, and minimally invasive.


Epithelioid hemangioendothelioma (EHE) is a rare tumor that is vascular in origin, arising from the endothelial or preendothelial cells. It can occur at different sites of the body, commonly in the liver, lungs, and bone. Pulmonary EHE is usually symptomatic, sometimes presenting with a cough, dyspnea, or chest pain. Rarely, it presents with pleural effusion. Here, we are presenting a case of pleural epithelioid hemangioendothelioma presenting as a case of spontaneous bilateral hemothorax. Epithelioid hemangioendothelioma (EHE) is a rare vascular tumor of endothelial or preendothelial cell origin, and it represents less than 1% of all vascular tumors. EHE affects various sites of the body, but it typically affects the lungs and liver [A 49-year-old male, a smoker of 15 packs/year, previously healthy, presented with a ten-day history of a sudden cough, associated with shortness of breath and left-sided chest pain. The patient also gave a history of loss of appetite and weight loss of about 15 kilograms for the past three months. On admission, his blood test revealed a drop in hemoglobin from 14.3 g/dL to 8.6 g/dL. The chest X-ray on admission showed bilateral lung opacity , and pleA CT scan of the chest with IV contrast was done, and it showed massive bilateral hemothorax, more on the left side causing lung collapse, but no evidence of contrast extravasations Figures –5. ImmedHistopathological examination of the pleural biopsies revealed extensive infiltration of the pleura by atypical epithelioid cells arranged in clusters, in strands, or lying individually Figures  and 8. TThe case was discussed in a multidisciplinary meeting, and the consensus is to start him on chemotherapy.Epithelioid hemangioendothelioma (EHE) is a rare vascular tumor of endothelial cell origin, with an estimated prevalence of less than one in 1 million . It was Histologically, the tumor is composed of small nests or short cords of rounded to slightly spindled endothelial cells that blend. A typical finding seen in immunohistochemistry is Weibel-Palade bodies seen in the cytoplasm of the endothelial cells . In EHE,Fifty to seventy-six percent (50%-76%) of patients with pulmonary EHE are asymptomatic and are incidentally diagnosed by abnormal chest radiography . EHE mayPleural epithelioid hemangioendothelioma carries a poor prognosis with a mean survival of 10–12 months. Due to the rarity of this condition, there is still no standardized treatment.Epithelioid hemangioendothelioma is a rare vascular tumor with poor prognosis; a bilateral spontaneous pleural hemothorax may well be the single presentation. Treatment of such condition is not adequate which may require further investigation.


Semantic dementia (SD) is a neurodegenerative disorder characterised by progressive language problems falling within the clinicopathological spectrum of frontotemporal lobar degeneration (FTLD). The development of disease-modifying agents may be facilitated by the relative clinical and pathological homogeneity of SD, but we need robust monitoring biomarkers to measure their efficacy. In different FTLD subtypes, neurofilament light chain (NfL) is a promising marker, therefore we investigated the utility of cerebrospinal fluid (CSF) NfL in SD.This large retrospective multicentre study compared cross-sectional CSF NfL levels of 162 patients with SD with 65 controls. CSF NfL levels of patients were correlated with clinical parameters , neuropsychological test scores and regional grey matter atrophy .sr=−0.32, p=0.002) and with smaller grey matter volume of the parahippocampal gyri . However, cross-sectional CSF NfL levels were not associated with progression of grey matter atrophy and did not predict survival.CSF NfL levels were significantly higher in patients with SD  than in controls (577 (446–766), p<0.001). Higher CSF NfL levels were moderately associated with naming impairment as measured by the Boston Naming Test (CSF NfL is a promising biomarker in the diagnostic process of SD, although it has limited cross-sectional monitoring or prognostic abilities. Semantic dementia (SD) is a sporadic neurodegenerative disorder characterised by loss of semantic knowledge, impaired naming and word comprehension, with preserved speech production.A promising biomarker in frontotemporal dementia (FTD) is neurofilament light chain (NfL), a major component of the neuronal cytoskeleton involved in axonal and dendritic growth, signalling and transport.In a large series of patients with SD from 14 different centres, we investigated our hypothesis that CSF NfL levels are elevated compared with controls and correlate with disease severity, atrophy and clinical progression in SD.1-42 and high phospho-tau and/or total-tau,et al. in those cases diagnosed before 2011 or Gorno-Tempini et al. from 2011 onwards).In total, 168 patients with SD with one CSF collection from 14 different centres in Europe and the USA . The remaining 21 patients with CSF NfL concentrations fulfilled the clinical diagnostic criteria of SD, but had no neuroimaging available (n=18) or had bilateral atrophy (n=3). TDP-pathology was confirmed in all seven deceased patients with brain autopsy.1-42 levels and had either normal neurological examinations, neuropsychological testing scores and Clinical Dementia Rating (CDR) scores of 0 (n=44) or they were cognitively healthy family members without a mutation or spouses from patients with genetic FTD or a different neurodegenerative dementia (n=21).To compare NfL levels between controls and patients with SD, 65 sex-matched and age-matched healthy controls from our previous studies were included.Disease duration was defined as time between first symptoms noted by a caregiver (onset) and CSF collection. Survival was defined as time between CSF collection and death.The local ethics committees approved the study and all subjects or their legal representatives provided written informed consent.Most subjects (n=147) underwent global cognitive screening and/or neuropsychological assessment (NPA) at the local study site  according to the manufacturer’s instructions. Measurements were performed in three different laboratories: the Amsterdam University Medical Center , Bristol Myers Squib, Wallingford, Connecticut, USA (19 patients with SD and 44 controls) and the Washington University School of Medicine (8 patients with SD). All laboratories used the same ELISA, but the latter two added a dilution step . Thus, considering the optimal linearity of the assay used,In 87 patients, structural T1-weighted (T1w) 3T MR-images within 6 months of CSF collection were available for neuroimaging analysis , and in 32 patients a follow-up scan ≥6 months was available . Brain images were acquired locally and scans from a scanner that only contributed one or two scans were excluded, resulting in datasets from nine different scanners; detailed information on numbers and reasons for exclusion are presented in et al.The T1w images were processed according to procedures described by Bron Since SD is an asymmetric disorder, GM ROIs of the dominant brain side  were used for analysis; secondary analyses were undertaken on bilateral GM ROIs. All ROIs were corrected for head size by normalising to intracranial volume (ICV) and reported as percentage of ICV. The degree of asymmetry was assessed by calculating the ratio of temporal lobar GM volume from the dominant side to that of the non-dominant side, yielding a smaller ratio when more asymmetry was present. For follow-up images, progression of atrophy was assessed by change in volume (mL) per year uncorrected for ICV: (volume follow-up – volume baseline)/interval between scans (years).SPSS Statistics 21.0 for Windows  was used to analyse the data. Statistical significance was set at p<0.05 and Bonferroni correction for multiple testing was used when appropriate. Continuous data were compared between two groups by Mann-Whitney U or t-tests where appropriate; categorical variables were compared by χ² tests. CSF NfL levels were normalised by log transformation and compared between patients with SD with controls using analysis of covariance, correcting for sex, age at CSF collection and laboratory of NfL measurement. Diagnostic performance was assessed by receiver operating characteristic analysis, with optimal cut-off levels at the highest Youden index.sr) was used to correlate non-transformed NfL levels with age at CSF collection, disease duration at CSF collection, cognitive screening scales , neuropsychological tests and GM ROIs. Next, multivariate linear regression analysis (β) was also used to assess the association between (1) non-transformed NfL levels (independent variable) and neuropsychological tests (dependent variable), correcting for age, sex and laboratory and (2) non-transformed NfL levels and GM ROIs, correcting for age, sex, laboratory and scanner. The Bonferroni method was used for correction for multiple comparisons. NfL as predictor for survival (after CSF collection) in patients was analysed using the Log Rank test and Kaplan-Meier curves comparing NfL tertiles, and a Cox regression with correction for age, sex and laboratory, both on tertiles and NfL as continuous variable.Spearman’s correlation coefficient , trended towards an association with annual change of MMSE , and there was a trend for association with GM volume of the medial and inferior temporal gyri . Cross-sectional NfL levels did not correlate with progression of atrophy in the selected ROIs in the small series of patients with SD with follow-up scans (n=32) . Additionally, the bilateral medial and inferior temporal gyri, temporal pole and hippocampus were negatively associated with NfL; however, after correction for covariates, the association did not withstand Bonferroni correction.CSF NfL levels were negatively associated with GM volume of the parahippocampal gyrus on the dominant atrophic side (s (n=32) . When thCox regression analysis with cross-sectional CSF NfL as a continuous variable showed a trend for association between NfL levels and survival ). However, CSF NfL tertiles did not associate with survival after CSF collection . In contrast to other FTLD subtypes, NfL did not associate with progression of GM atrophy or survival.The elevated CSF NfL levels in patients with SD compared with controls is in line with those found in previous smaller SD series.While earlier studies found an association of NfL with CDR in different FTD types,The significant association between higher CSF NfL levels and smaller parahippocampal GM volume in our study is in line with studies showing positive correlations with temporal cortical regions in FTD.We showed a trend for a significant association between cross-sectional NfL levels and future survival in SD. This seems to be in contrast with the evidence of an association between high NfL levels and shorter survival in FTD, progressive supranuclear palsy, Alzheimer’s disease (AD) and amyotrophic lateral sclerosis.A major strength of this study was the large international series of CSF samples and clinical data from patients with this rare disease, which comprises 10%–15% of FTD cases.Since SD is one of the most homogeneous subtypes of FTD, it is a promising target for developing novel treatments. Our results are also important for future trials, as they suggest a limited role for cross-sectional CSF NfL as a monitoring or progression marker in SD.The multicentre approach also caused some limitations, especially considering the measurement of NfL levels at three different centres. Unfortunately, no controls are provided in the ELISA kit, and due to the current lack of a quality control programme, the assays of the different laboratories could not be compared directly. We accounted for differences between laboratories by normalisation and laboratory-correction, but in future multicentre studies, harmonisation of results could be improved by remeasuring a number of control and patient samples across the different laboratories.Other possible limitations include the different neuropsychological test batteries per centre and this might have introduced variability and loss of sensitivity; however, we transformed different versions of the same test to analyse them together. Furthermore, the variation in scanners and parameters of the T1-weighted imaging was reduced by including 3T data only, excluding data from scanners on which less than three datasets were acquired and correcting for scanner in the analysis. Additionally, we did not study different primary progressive aphasia subtypes and did not include serum—which is likely to replace CSF measurements in the near future. The lack of longitudinal NfL data did not allow us to draw conclusions about NfL and clinical or imaging markers over time; it is conceivable that a plateau phase or even a decrease of NfL may occur over time.In conclusion, our results show that cross-sectional elevated CSF NfL may be a useful biomarker for the neurodegenerative process in SD, which could lead to the use of CSF NfL as a diagnostic biomarker. However, the use of CSF NfL to monitor disease progression in SD remains debatable, since we only found a moderate association with language deterioration and atrophy and no relation of CSF NfL with survival (in a relatively short follow-up time). This is in contrast with previous reports on NfL in other neurodegenerative diseases


Parkinson's disease is a progressive degenerative nervous system disease. Recent studies have shown that secondary changes in the GABA system play directly affect the pathogenesis of PD. There is still much debate about GABA concentrations because currently, GABA concentrations in the brain tissue are obtained indirectly by measuring its concentration in the blood and cerebrospinal fluid. These results are unreliable. Magnetic resonance spectroscopy (MRS) is the only noninvasive method for evaluating the concentration of metabolites in living brain tissue and has been widely applied in research and clinical practice. In addition, combining MEGA-PRESS technology with LCModel software for quantitative GABA measurements is largely recognized. At present, the PD monkeys model in primates has been increasingly proficient. Primates are more similar to humans in terms of brain structure and function than other animals. However, 3.0 T MRS studies involving the PD monkey model to measure metabolites in living subjects with PD are still rare. The study was performed at 3.0 T MRI with control monkeys and PD monkeys that were injected methyl-phenyl-tetrahydropyridine (MPTP) in one side of common carotid artery before and 3 months after successful model establishment to measure GABA concentrations in the bilateral striatum. Behavioral observations were performed for all animals, and the behavioral score was recorded. After 3 months, the GABA concentration in the bilateral striatum was measured in both groups by high-performance liquid chromatography (HPLC). The data obtained from magnetic resonance spectroscopy (MRS) were compared with the actual measured GABA concentrations in tissues isolated from the corresponding regions, and their correlations with the behavior score were analyzed. The research objectives are to investigate the changes of γ-aminobutyric acid (GABA) concentration in the bilateral striatum of monkeys with Parkinson's disease (PD) and the value of quantitatively measuring its concentration by noninvasive 3.0 T spectroscopy.(1) The MRS results showed that the GABA concentration in the injured side of the striatum of the PD monkeys was higher than in the contralateral side, but the difference was not statistically significant (P = 0.154). Compared with that the blank control group, the GABA concentration in the striatum of the PD monkeys increased, but there was no difference between the groups . (2) The GABA concentration that determined from the isolated specimens by HPLC in the injured side of the striatum of the PD monkeys was significantly higher than that in the contralateral side (P < 0.01). Compared with the blank control group, the PD monkeys had higher GABA concentrations in both sides of the striatum, and there was a significant difference in the lesion side (P = 0.004), while there was a non-significant difference in the contralateral side (P = 0.475). (3) The mean GABA concentration in the injured striatum of PD monkeys determined by MRS was not significantly correlated with the behavioral score . The mean GABA concentration in the injured striatum determined from the isolated specimens was positively correlated with the behavioral score in the same period .The GABA concentration in the injured striatum of PD monkeys is increased and positively correlated with behavioral changes. Validity of noninvasive 3.0 T MRS to detect PD neurotransmitter changes is limited. Parkinson's disease is a progressive degenerative nervous system disease and is often accompanied by symptoms such as resting tremor, muscle stiffness, bradykinesia, and posture balance disorder. The pathology is mainly the degeneration of nigrostriatal dopaminergic neurons, which leads to the loss of dopamine production by neuronal projection of the substantia nigra to the dorsal striatum, thereby reducing the normal inhibition of GABA ergic neuron activity . ConventGABA is a amino acid neurotransmitter located in the central nervous system and is mainly generated by the decarboxylation of glutamate (Glu) by glutamic acid decarboxylase (GAD). It binds to GABA receptor and induces a strong biological effect to inhibit neuronal excitability, causing a series of neurodegenerative diseases such as PD. Recent studies have shown that secondary changes in the GABA system play an important and direct role in the pathogenesis of PD , and theDue to the low content of GABA, it is difficult to measure the GABA content via conventional MRS sequences; this measurement has special requirements in terms of spectral editing technology, equipment and field strength. Henry et al.  optimizeThe purpose of this study was to compare the GABA concentration in the striatum measured by the 3.0 T MRI MEGA-PRESS spectral editing sequence combined with LCModel software and HPLC with the cynomolgus monkey as an experimental animal, to investigate the changes in GABA concentration in Parkinson's striatum and to evaluate the value of 3.0 T noninvasive spectroscopy for quantitative measurement of GABA concentrations.One week after the model was successfully established, limb muscle stiffness began to appear on the side contralateral to the MPTP injury of the PD monkeys (6/6). In addition, typical PD symptoms, such as limb postural tremor and spontaneous body rotation were sometimes found on the MPTP lesion side. The lowest score was 6 and the highest score was 12.5; the scores remained constant. The limbs of the MPTP injury side did not show symptoms such as muscle stiffness and tremor  showed stable symptoms and were given intramuscular APO. Ten minutes after the injection, a rapid onset of symptoms began to appear on the side of the the MPTP injury and the symptoms accelerated with stimulation, reaching 8 laps/min. This value remained stable for 8 to 12 weeks (Table (1) As seen from the MRS results in Table statistically significant P = 0.154). Compared with that in the blank control group, the GABA concentration in the striatum of the PD monkeys increased, but the increase was not statistically significant . (2) The GABA concentration that determined from the isolated specimens by HPLC in the injured side of the striatum of the PD monkeys was significantly higher than in the contralateral side (P < 0.01). Compared with the blank control group, the PD monkeys had higher GABA concentrations in both sides of the striatum; the difference was significant in the lesion side (P = 0.004) but not the contralateral side (P = 0.475) (Table . CompareMPTP is oxidized to produce l-methyl-4-phenylpyridinium (MPP+ ), which has neurotoxic effects and destroys dopamine production channels, resulting in PD . It is bN-acetylaspartate (NAA), creatine (Cr), choline (Cho), lactate (Lac), myo-inositol (MI), glutamate (GIu), GABA, and glutamine (Gln). The main studies related to PD found changes in GABA, Cho, NAA, and Cr. As early as 1950, GABA was first discovered in mammalian brain extracts and the GABA brain content was extremely low, approximately 5–10 mmol/L [The metabolites detectable by noninvasive MRS include 0 mmol/L . In our In a study measuring GABA concentrations in the PD striatum, Chassain et al.  used MPTAt present, there are two main methods for accurately measuring GABA concentrations in vivo using MRS technology: (1) Using different spectral editing methods, such as two-dimensional J resolved spectroscopy, the chemical shift correlation spectrum, the magnetization transfer method, the multiple-quantum filter (MQF), and the J-modulation differential technique, to separate GABA at δ 3.0 ppm from the background of macromolecules and to determine the total creatine/high-energy phosphate (Cr/PCr) value; (2) Analysis of GABA and other metabolites using a special software package (LCModel and MRUI are commonly used in research currently) to provide the concentration .However, this study used the 3.0 T MRI MEGA-PRESS spectral editing sequence combined with LCModel software to measure the GABA concentration in the bilateral striatum of PD monkeys. Compared with the healthy side and the control group, the lesion side had higher striatum GABA concentrations, but the difference was not statistically significant. This finding is inconsistent with previous cell culture, animal experiment and autopsy results and may be related to the following: (1) GABA is present in 80% of the striatum, but its concentration is extremely low, and its resonance peak is easily hidden by the peaks of other metabolites. In addition, the sample size of this study is too small, resulting in no difference in the obtained data; (2) the 3.0 T MRI MEGA-PRESS spectrum editing sequence combined with LCModel software has certain limitations in measuring GABA concentrations in vivo. The problem of an unclear peak separation and a low signal-to-noise ratio has not been improved, and the quality of the spectrum still needs to increase. Some other factors may contributed this results, such as small head movements during the scan procedure, relation to akinetic-rigid and tremor dominant expressions.At present, there are four main ways to improve the quality of the spectrum: (1) increase the magnetic field strength ; (2) devIn this study, the GABA concentration in the striatum was measured by HPLC 3 months after the PD model was established in monkeys. It was found that the GABA concentration in the striatum of the PD monkey was increased and the elevated GABA concentration was positively correlated with the PD monkey behavioral score. However, for the measurement provided by the 3.0 T MEGA-PRESS technique, the GABA concentration on the affected side was not correlated with the behavioral score. It is not difficult to conclude that the GABA concentration obtained by the 3.0 T noninvasive spectroscopy technique has limitations and it has not been confirmed that there is a correlation between the GABA concentration measured by the 3.0 T MEGA-PRESS technique and the behavioral score in PD monkeys. Previously, the authors used this technique to investigate the relationship between the mean striatum GABA concentration, the clinical course of disease, and the unified Parkinson's disease rating scale (UPDRS) score in 45 patients with PD and found no significant correlation between them . MoreoveIn conclusion, this study used 3.0 T MRS to measure the concentration of GABA in the striatum of living animals and to compare it with the concentration of GABA in isolated striatum to determine the sensitivity and feasibility of using noninvasive MRS to measure neurotransmitter concentrations in early stages. We found that the elevated levels of GABA in the striatum of PD patients were positively correlated with behavioral changes. Furthermore, the 3.0 T MRS technique can only measure a limited concentration of GABA in vivo, so it is necessary to further increase the magnetic field strength and optimize the editing techniques. The sample size of this study was small; the field strength was 3.0 T and there was no comparison with high field magnetic resonance; thus, a further increase in the sample size and further comparative analysis are required.Eight healthy male cynomolgus monkeys weighing 8–10 kg and aged 6–9 years were used. The experimental animals were provided by the Guangxi Xiongsen Primate Experimental Animal Breeding Development Co., Ltd., and the experimental design was were approved by the ethics committee of the First Affiliated Hospital of GuangXi Medical University, China . In accordance with the feeding requirements for medical experimental animals, feeding was carried out in a single cage, 12 h of circular illumination daily, and sufficient water and fresh fruit were ingested. Six monkeys were PD models (numbered M1 to M6) and 2 were blank control group.For the cynomolgus monkeys in the PD model group, ketamine (1 mg/kg) intramuscular anesthesia was applied, the exposed common carotid artery was separated, and the external carotid artery was temporarily clamped. MPTP (1.0–1.2 mg/kg) was slowly injected into the Left side the common carotid. There is no anterior communicating artery between the left and right anterior cerebral arteries, and MPTP injected in one carotid artery can not reach the contralateral hemisphere. For the control group, only a neck skin incision was made where the common carotid artery branched into the external carotid artery, and no drugs were injected. All cynomolgus monkeys were injected with ampicillin (0.5 g/monkey) after surgery for 7 days to prevent surgical wound infection.According to the monkey Parkinson's syndrome score scale created by Kurlan et al. , the movSix PD monkeys were given an intramuscular injection of apomorphine  receptor agonist) at 4, 8, and 12 weeks after the PD model was established. The number of rotations per minute were recorded within 3 h of injection, and the average number of laps was determined.Eight monkeys underwent MRS examination before the MPTP model was established and 3 months afterward. The monkeys were fasted for 4 h before the examination and were intramuscularly injected with ketamine + promethazine  to ensure the animals were quiet during the MRS scanning.Scanning equipment: Siemens 3.0 T magnetic resonance scanner and 12-channel head phased array coil  was used. The scans for all subjects were completed by the same radiologist. Settings were as follows: 3D prepared rapid acquisition gradient echo (MP-RAGE) sequence localization, scanning range from occipital macropore to the semioval center level, thickness 1 mm, TR 1700 ms, TE 2.9 ms, TI 900 ms, matrix 256 × 256, FOV 220 mm × 220 mm. For the MEGA-PRESS sequence, the region of interest (ROI) was located in the bilateral striatum . (4) Derivatization reagent stored at 0–4 °C. (2) Preparation HPLC analysis of the sample supernatant, and the supernatant was taken and eluted according to the chromatographic conditions. (3) Take 5 μL of standard or pretreated sample, adding 25 μL of OPA derivatizing agent for derivatization reaction. The calculation method used is the external standard method, based on the peak area. The formula is as follows: GABA concentration = (A sample/A standard) × standard concentration × standard sample amount × total sample volume/ sample amount × sample weight (unit: μmol/g).After completing the MRS scan test at 12 weeks, monkeys were humanely euthanized by decapitation method in accordance with USAISR IACUC Policy. Briefly, monkeys without analgesics/anesthetics were restrained and were decapitated using a guillotine by a trained person. This method allows to obtain intact brain tissue without chemical contamination. The brain anatomical map of the cynomolgus monkey was compared, and the brain tissue of the same part of the MRS examination was stored , and the frozen tube was immediately placed in liquid nitrogen and stored in a − 70 °C refrigerator for later use. The striatum GABA concentration was measured by HPLC: (1) Preparation of main reagents: (1) stock solution of amino acid standards  (1.0 mmol/L), stored at -200C. (2) 0.1 M sodium tetraborate (Ph = 9. 6) 100 mL. (3) Mobile phase A: 0.1 mol/L KH2O2 to eliminate endogenous peroxidase. After washed with 0.01 M PBS, the sections were incubated with blocking solution  at room temperature for 1 h. (3) The sections were incubated with anti-TH antibody overnight at 4 °C. The sections were incubated with secondary antibodies for 1 h at room temperature after washed with 0.01 M PBS. (4) Immunoreactivity was visualized by incubated in DAB and counterstained with hematoxylin. Control staining was performed by using PBS as the primary antibodies. (5) TH-positive cells in SNc were detected under a light microscope.(1) Brain was fixed in 4% paraformaldehyde. SN sentions (40 mm) were made by using freezing sliding microtome. The SN sections were washed with 0.01 M PBS. (2) The SN sections were blocked with 3% HStatistical calculations were performed with SPSS 17.0 software. Two independent sample t tests were used for comparisons between two groups and between two sides within the same group. The correlation between GABA concentration and behavioral score was determined by Pearson's correlation. The measurement data are expressed as the mean ± SD. P < 0.05 was considered statistically significant.


Society expects professionals to promote their businesses in an ethical manner, refraining from misleading or deceptive marketing due to the potential to harm members of the community. In Australia this expectation resides in the Australian registration board advertising guidelines or the Health Practitioner Regulation National Law. Registration board data indicate there are many health care professionals failing to meet these expectations. The aims of this research were to determine the frequency, type and nature of at-risk advertising by Australian chiropractors and physiotherapists and whether there is a correlation between professional association membership and advertising guideline compliance.A cross sectional audit examining practitioner advertising was performed on representative samples of Australian chiropractors and physiotherapists. Two auditors examined advertising by 380 physiotherapists and 359 chiropractors for material potentially in breach of the regulatory authorities’ advertising guidelines. The advertising appeared on practitioner websites and linked Facebook pages.Two-hundred and fifty-eight (72%) audited chiropractors and 231 (61%) audited physiotherapists had breaches of the Advertising Guidelines on their websites and linked Facebook pages. The frequency of breaches by chiropractors was higher. The type and nature of the breaches by chiropractors was potentially more harmful. Membership in a professional association influenced neither the frequency nor the severity of breaches with chiropractors.Advertising breaches were common in both samples even though regulators and professional associations provide practitioners with explicit information on how to comply with advertising guidelines. Breaches by chiropractors were more numerous and more serious due to their greater potential to lead consumers to make inappropriate and potentially harmful healthcare decisions.Stronger enforcement strategies may have a positive effect on compliance. Advertising by health professionals is an integral part of practise. Providing consumers with ethical accurate advertising assists with making informed health related decisions.Chiropractors and physiotherapists are amongst Australia’s 15 regulated health professions. All must abide by the same advertising guidelines. The guidelines stipulate what constitutes unacceptable advertising. Unacceptable advertising includes advertising that is false, misleading or deceptive or likely to deceive. Unacceptable advertising has the potential to cause harm. Recent Australian court cases have highlighted the potential harm to consumers when health care providers publish false advertising . AlthougThis research examined advertising by a representative sample of Australian chiropractors and physiotherapists. Practitioner websites and linked Facebook pages were audited. It reports on the frequency, type and nature of advertising at risk of being non-compliant with the advertising guidelines. In addition, this research determined whether there is a correlation between professional association membership and advertising guideline compliance.Since 2010 all registered Australian health care providers have had uniform nationwide legislation – The National Law (NL). The NL outlines the regulatory obligations for advertising a regulated health service. Obligations include: advertising must not be false, misleading or deceptive or likely to be misleading or deceptive and must not include testimonials; any claims made must be able to be substantiated; offers of gifts, discounts or other inducements must come with terms; advertising must neither create and unreasonable expectation of beneficial treatment nor directly or indirectly encourage the indiscriminate or unnecessary use of regulated health services .Under the NL, the definition of a regulated health service is very broad and is not restricted to direct clinical services. Uniform Advertising Guidelines (AG) are in place for each of the 15 regulated health professions governed by its own Board. A breach of advertising provisions of the NL is a criminal offence punishable by fine. Other enforcement approaches include the respective Board placing restrictions on an individual’s registration and their ability to practise. Legislative authorities in other countries including the United States of America, Canada, and the United Kingdom have also legalised advertising by health care professionals and have similar regulations governing professionals’ advertising.the astute and the gullible, the intelligent and the not so intelligent, the well-educated as well as the poorly educated, men and women of various ages pursuing a variety of vocations. … all persons exposed to the conduct should be considered although conduct which is only likely to mislead or deceive an extraordinarily stupid person would not fall within the ambit of the provisions .Before considering the regulatory authorities’ compliance and enforcement strategy, it is appropriate to consider one of the most challenging aspects of the AG, namely what is meant by misleading or deceptive. The Chiropractic Board of Australia (CBA) advises that misleading someone may include lying to them, leading them to a wrong conclusion, creating a false impression, leaving out important information, or making false or inaccurate claims . And theThe courts will consider whether a reasonably significant number of potential purchasers would be likely to be misled or deceived. Intent is not a necessary element for conduct to breach the misleading conduct provisions. Conduct may be regarded as misleading or deceptive even if the originator of the conduct did not intend to mislead or deceive members of the target audience. The test is objective. The factor is conduct taken at face value .The Australian Health Practitioner Regulation Agency (AHPRA) prosecutes breaches of the AG. The high volume of complaints received by AHPRA during 2016 triggered the establishment of a dedicated Advertising Compliance Team which works closely with the Legal Team and the Policy and Communications Team. To assist in their decision making, AHPRA utilise experts to evaluate advertising claims.AHPRA considers that education and engagement are effective tools as part of its strategy to achieve behaviour change and compliance with the regulations. AHPRA has developed advertising education tools accessible via the AHPRA website under the heading: Check, Correct, Comply. This section of the website includes numerous examples of non-compliant advertising common to all professions and examples specific to chiropractors. In addition, the National Boards consider that some words have a greater capacity to mislead or deceive when used in advertising and recommend that advertisers be cautious when using them. The “words to be wary [of]: cure, safe, effective, and can help/ improve/treat or effectively treats” . In addiIn response to a recognised need to reduce non-compliant advertising AHPRA announced a pilot audit of chiropractic and dentist advertising commencing in the 2019 registration period. This step marks a shift from reactive enforcement to proactive enforcement and is expected to improve compliance across the entire sector. The data generated by AHPRA will inform a review of the compliance strategy, identify profession-specific differences in compliance rates, inform future strategic directions and ensure sustainable change .In Australia there are 657,621 registered health practitioners across 15 professions. The 5167 registered chiropractors make up 0.8% of the total health practitioner registrant base, while the 28,885 registered physiotherapists make up 4.5% of the base .Advertising complaints are considered separately from conduct complaints. AHPRA Annual Reports covering the period 2013–2017 –15 demonA clear picture exists regarding conduct complaints against chiropractors and physiotherapists in Australia  and wherThe reactive nature of AHPRA’s approach has obvious limitations. Spittal, Bismark and Studdert  suggest There is no evidence chiropractic care benefits babies or can treat them for medical conditions and there is not enough evidence to suggest it [chiropractic] can achieve general wellness or treat various organic diseases and infections.it [CBA] was concerned about a number of practitioners who were falsely advertising that chiropractic care for spinal problems could also treat a range of other ailments .A less clear picture exists regarding advertising complaints. Regulatory authority reports confirm that Australian chiropractors and physiotherapists are the subject of significant numbers of advertising complaints –15. In 2Emphasising the need for concern, a 2018 high profile Australian case establishes that serious breaches do occur and that misleading advertising by health practitioners can harm members of the community . Overall, 55.0% (198) of the chiropractic dataset were members of a chiropractic professional association. Amongst these, 72.3% (142) had a breach, whereas 70.7% (114) of non-members had a breach. A chi-square test was performed using Microsoft Excel® to determine whether there was any difference in numbers of breaches between association members and non-members. The chi-square statistic is 0.036 and the These data demonstrate that within the samples audited, neither profession exhibited a high level of compliance with the advertising guidelines. As a group, chiropractors had more at-risk advertising and the nature of the at-risk advertising had a greater potential to cause harm. Membership of a professional chiropractic association did not appear to increase compliance by chiropractors. No physiotherapist had at-risk advertising classified as a major misleading breach. Physiotherapist breaches were confined to minor misleading breaches: displaying association membership as postnominals and using testimonials.Previous research has examined conduct breaches by these professions and claims made in chiropractic patient brochures and on chiropractic college web sites. Within Australia, research in 2018 indicated that both professions had conduct breaches with chiropractors having 6 times more complaints than physiotherapists . Ryan, TElsewhere, at the turn of the twenty-first century, patient brochures from the largest State, Provincial, and National Chiropractic Associations and Research Agencies in Canada and the USA were found to contain many unsubstantiated, potentially harmful claims , 29. MorAHPRA’s annual reports only provide the number of advertising complaints broken down by profession. The rate of advertising compliance and the types of at-risk advertising within the professions is unknown. This appears to be the first study to examine the extent and the nature of practitioner advertising breaches in a representative sample of Australian chiropractors and physiotherapists using advertising guidelines as a standard.The chiropractic findings are of major concern for two reasons, the first being public safety. Society expects and accepts that professionals advertise their services to assist consumers in making informed choices. To meet societal expectations and legal obligations, advertising must be socially responsible, truthful, appropriate and not misleading or deceptive. Advertising that fails to meet these expectations has the potential to harm. To assist practitioners in fulfilling their obligations, regulators formulated specific rules about advertising of health services to protect the rights of consumers however the data indicate that both professions and chiropractors in particular are not fulfilling their obligations.The second reason is the high percentage of chiropractors advertising in an unacceptable manner. This raises questions about the profession’s culture and understanding of its obligations under the social contract. It is beyond the scope of this paper to examine this; however, this topic has been the subject of papers by observers both within the profession and external to it over several decades –41. The In this electronic age most health care providers have a web presence and increasingly use social media in their practices in response to consumer demand –46. WithTraditionally regulatory authorities have been reactive to complaints; however, there is an argument to be made for increased public protection by the authorities becoming proactive. Recent experience by the College of Chiropractors of British Columbia (CCBC) demonstrates that auditing practice websites and linked Facebook pages is a simple, comprehensive and cost-effective way of identifying breaches and achieving compliance with its Efficacy Claims Policy [advertising policy]. The CCBC’s 1200 registrants had 1 months’ notice of an upcoming audit of webpages and social media. Within 2 weeks of commencement of the audit procedure, 97% of the practitioners notified of a potential breach had voluntarily complied with the CCBC’s directive . The CCBIt is conceivable a greater understanding of advertising practices by each profession will encourage practitioners to comply and provide regulators, educators, professional associations with an in-depth understanding of the number and nature of breaches. Perhaps the CBA’s and AHPRA’s enhanced Advertising compliance and enforcement strategy for the National Scheme , 48 willA key strength of this study is its comprehensiveness. Using representative samples, advertising by 739 practitioners was audited on 214 websites plus associated Facebook pages. Staff profiles, frequently asked question sections, video presentations, and educational materials were scrutinised. When scientific publications were referenced within the advertising they were sourced and reviewed for accuracy. The audit was conducted using a detailed template based on 33 categories of inappropriate advertising. The template was prepared using examples provided within registration board advertising guidelines.Confirmation bias is frequently a problem with this type of research. Both auditors are registered chiropractors with careers involving chiropractic guideline compliance matters and consumer law. This could have made them more sensitive to breaches by chiropractors and, due to their comparative lack of experience with physiotherapist compliance matters, less sensitive to compliance breaches by physiotherapists. While the auditing may have been strengthened by having a physiotherapist conduct the physiotherapy audit, both professions work in musculoskeletal medicine and have the same advertising guidelines. Both auditors are conversant with the musculoskeletal medicine literature and are confident that the physiotherapist audit data are accurate.Comparability of individual auditor’s findings could be a limitation. This was minimized because each auditor performed ½ the audits in each profession’s sample using a comprehensive breach template developed from the AGs and associated explanatory notes/examples. While there was no formal check of the reliability of the auditors’ findings, an informal check emerged when the data was being inputted into NVivo. Due to the number of group practices, there were about 10 instances in which both auditors had examined the same website for a different group member. In all instances the breach allocation was the same.Clustering of practitioners due to group practices may skew the results. Locating webpages for 380 physiotherapists required 72 practice websites versus 141 for 359 chiropractors which indicates that the number of physiotherapists per group practice is larger than the number of chiropractors per group practice. While a larger sample from each profession would reduce the possibility of skewed results due to group practice clustering, this limitation can largely be discounted because of the distinction between the physiotherapy results and chiropractic results as depicted in Figs. An additional limitation is underreporting of breaches amongst chiropractors. It was not uncommon to find multiple breaches within a single category on a practitioner’s website but only one example was recorded. Similarly, many breaches could be indexed into multiple categories however only the category with the greatest harm potential was used. This was in keeping with the aim of the research, namely to determine what percentage of each profession had websites containing AG breaches with examples included for explanatory purposes.Advertising by health care professionals is an accepted part of practice. It informs the public about the profession and the professional enabling the public to make better health care choices. Advertising by the 15 Australian registered health care professions is regulated by a registration board and AHPRA governed by specific advertising guidelines and the National Law. The main objective of this study was to examine advertising by chiropractors and physiotherapists to determine the frequency and nature of advertising guideline breaches. While both professions had advertising that breached the guidelines, breaches by chiropractors were more frequent and more serious.The study highlights areas for future research. Given that chiropractors are over-represented in both conduct and advertising breaches is there a nexus between the two? Are chiropractors who breach advertising guidelines more likely to generate conduct breaches? Are there alternate compliance measures that would be more effective for chiropractors? Is there something in the professional development of chiropractors and physiotherapists that makes them prone to breaching advertising guidelines? AHPRA, professional associations and educational bodies may find the data from this audit helpful in designing further research and developing interventions that raise compliance by chiropractors and physiotherapists and protect the public from harm.