SARS-Cov-2 infects not only adults, but also children, including pediatric surgery patients with acute abdomen. Here, we report a pediatric surgery case with incarcerated inguinal hernia and suspected COVID-19.i.e., full personal protective equipment (PPE) for COVID-19. Intraoperatively, we found a small intestine loop trapped in the scrotum and stuck in the inguinal canal. Postoperatively, the baby was continued to be managed as a patient with COVID-19 while waiting for the real-time reverse transcription polymerase chain reaction (RT-PCR) results.A 11-month-old male was brought to our emergency department with the main complaint of recurrent yellowish-green vomiting that was experienced from one day before admission. High fever and shortness of breath were also reported. This patient was also suffering from moderate dehydration. Neither history of contact with a confirmed case of COVID-19 nor traveling from any local transmission area were found. However, a SARS-CoV-2 rapid antibody test revealed a positive result. A lump in the left scrotum that persisted during admission was found. Fluid resuscitation and nasogastric tube placement for decompression was performed. Manual reduction was attempted but failed to reduce the lump. Accordingly, we decided to perform an emergency high ligation using tertiary protection regulations, Manual reduction is standard treatment for incarcerated inguinal hernia in children. The successful rate of manual reduction is about 70%, therefore, if the manual reduction fails, an emergency surgery is mandatory.During the COVID-19 pandemic, all medical procedures require clarity of the patient's status including whether infected with COVID-19. Along with proper precautions, great care must be taken during surgery to minimize the risk of cross infection to health workers.During the COVID-19 pandemic, surgeons should always be aware of the possibility of cross-transmission from the patient, since children are also susceptible to SARS-CoV-2 infection. When and wherever possible, surgeons should perform the procedure in the quickest and most effective manner to shorten exposure time with patient and anesthetic aerosols as well as using appropriate PPE. •Manual reduction is standard treatment for incarcerated inguinal hernia in children.•Children are also susceptible to SARS-CoV-2 infection.•Surgeons should perform the procedure in the most effective manner during pandemic. Until January 19, 2021 this global pandemic has infected more than 96 million people in over 220 countries/territories and caused 2,063,299 deaths [However, at the same time, pediatric surgery services have become severely affected . For pat2An 11-month-old male was brought to our emergency department with the main complaint of recurrent yellowish-green vomiting that was experienced from one day before admission. The patient's abdomen was bloated, while the last defecation was the day before coming to the hospital. High fever and shortness of breath were also reported. On physical examination, his general condition was lethargic, and body weight was 7 kg. Vital signs revealed pulse of 140 times per minute, respiratory rate 40 times per minute, body temperature of 39.5 °C, and capillary refill time <2 seconds. In the head examination, a sunken fontanel and eyes were found, indicating moderate dehydration. His abdominal wall was distended, and auscultation revealed an increase in bowel sounds, without any abdominal tenderness. A lump in the left scrotum that persisted during admission was found . The lumi.e., with full PPE for COVID-19. Intraoperatively, we found a small intestine loop trapped in the scrotum and stuck in the inguinal canal. Neither necrosis nor perforation were found in the intestinal loop. The intestine was returned into the peritoneal cavity followed by a high ligation. Postoperatively, the baby was continued to be managed as a patient with COVID-19 while waiting for the real-time reverse transcription polymerase chain reaction (RT-PCR) results.Accordingly, we decided to perform an emergency high ligation using tertiary protection regulations, 3Manual reduction is standard treatment for incarcerated inguinal hernia in children. Moreover, this technique is very useful to decrease the need for any emergency procedure during the COVID-19 pandemic . Howeveri.e., with full PPE for COVID-19, while waiting for the RT-PCR results. The final RT-PCR results of the patient were negative for two consecutive swab tests. In spite of this, there is a possibility of false negative results of RT-PCR in patients with COVID-19 [During the COVID-19 pandemic, all medical procedures require clarity of the patient's status including whether infected with COVID-19. Along with proper precautions, great care must be taken during surgery to minimize the risk of cross infection to health workers. This includes PPE and control of aerosols or body fluids produced during operations that might be contaminated [COVID-19 .High fever in our patient could be caused by ileus complications due to fluid sequestration to the third cavity which results in dehydration and may be accompanied by bacterial translocation . It mighThe use of operating rooms specifically for patients with COVID-19 which are separated from non-COVID-19 patients is also one way to reduce the risk of nosocomial infections . In our The surgical procedures for open high ligation in children before and during the COVID-19 pandemic are the same. However, considering the possibility of the surgeons' and other surgical team members’ exposure time to anesthetic aerosols and body fluids that might be contaminated with the SARS-Cov-2, it is recommended to perform the operation in the quickest and most efficient manner . FurtherNotably, while the diagnosis of COVID-19 is according to RT-PCR, there are several obstacles of detection of SARS-CoV-2 in developing countries, such as the PCR reagents supply, the qualified laboratory staffs and the laboratories with appropriate biocontainment levels accessibility ,21. Ther4During the COVID-19 pandemic, surgeons should always be aware of the risk of cross-transmission from the patient, since children are also susceptible to SARS-CoV-2 infection. When and wherever possible surgeons should perform the procedure in the quickest and most effective manner to shorten exposure time with the patient and anesthetic aerosols as well as using appropriate PPE.10.13039/501100009509Indonesian Ministry of Research and Technology/National Agency for Research and Innovation.This study was funded by The informed consent form was declared that patient data or samples will be used for educational or research purposes. Our institutional review board also do not provide an ethical approval in the form of case report.Written informed consent was obtained from the parents’ patient 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.Gunadi, Munawir Makkadafi and Amsyar Praja conceived the study and approved the final draft. Gunadi and Aditya Rifqi Fauzi drafted the manuscript. Kemala Athollah and Marcellus critically revised the manuscript for important intellectual content. All authors read and approved the final draft. All authors facilitated all project-related tasks.The manuscript is a case report, not considered a formal research involving participants.Gunadi.Not commissioned, externally peer-reviewed.No potential conflict of interest relevant to this article was reported.


To the Editor—The rapidly spreading pandemic of coronavirus disease 2019 (COVID-19) poses a huge challenge to global public health. Healthcare systems in many countries are facing severe shortages of medical supplies.34 Three ophthalmologists in Wuhan Central Hospital were infected with SARS-CoV-2 and died from severe COVID-19.6 At the ophthalmic center of Guangxi Province, we have continued to provide medical services for outpatients who need timely services during the COVID-19 pandemic. However, in response to the lack of personal protective equipment (PPE)  during the epidemic peak in China,7 we have adopted an easy measure to perform slit-lamp examinations. After thorough disinfection, we installed a home-made shield at the slit-lamp to separate patients and ophthalmologists. As shown in Figure Healthcare workers in ophthalmology departments are susceptible to cross infection because routine ophthalmic examinations, such as like slit-lamp microscopy, are usually performed in a setting of close doctor–patient contact.The protective shield could prevent contamination of the slit-lamp by respiratory droplets. Although the shortage of medical supplies is currently being alleviated in China, we are encouraging the continuation of this easy protective measure to curb cross transmission in ophthalmology and to protect the ophthalmologists on the frontline of the pandemic.


The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mixed-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar severe acute respiratory syndrome (SARS) Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins showed major differences in both shape and size, indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding site’s conformational changes during the simulation time indicated its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicated that the virus’ mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design. In early December 2019, the first atypical pneumonia outbreak associated with the novel coronavirus of zoonotic origin (SARS-CoV-2) appeared in Wuhan City, Hubei Province, China ,4,5. In The genome of coronaviruses typically contains a positive-sense, single-stranded RNA but it differs in size ranging between ≈26 and ≈32 kb. It also includes a variable number of open reading frames (ORFs), from 6 to 11. The first ORF is the largest, encoding nearly 70% of the entire genome and 16 non-structural proteins (nsps) . Of the On the basis of the three sequenced genomes of SARS-CoV-2 , Wu et al. performed a detailed genome annotation. The results were further compared to related coronaviruses—1008 human SARS-CoV, 338 bat SARS-like CoV, and 3131 human MERS-CoV, indicating that the three strains of SARS-CoV-2 have almost identical genomes with 14 ORFs, encoding 27 proteins including 15 non-structural proteins (nsp1–10 and nsp12–16), 4 structural proteins , and 8 accessory proteins . The only identified difference in the genome consisting of ≈29.8 kb nucleotides consisted of five nucleotides. The genome annotation revealed that SARS-CoV-2 is fairly similar to SARS-CoV at the amino acid level, however, there are some differences in the occurrence of accessory proteins, such as the fact that the 8a accessory protein, present in SARS-CoV, is absent in SARS-CoV-2, as well as the fact that the lengths of 8b and 3b proteins do not match. The phylogenetic analysis of SARS-CoV-2 showed it to be most closely related to SARS-like bat viruses, but no strain of SARS-like bat virus was found to cover all equivalent proteins of SARS-CoV-2 alanyl-L-valyl-N~1--4-(benzyloxy)-4-oxo-1-{[(3R)-2-oxopyrrolidin-3-yl]methyl}but-2-enyl)-L-leucinamide (N3 or PRD_002214) . The samIn this study, we investigated how only 12 different residues, located mostly on the protein’s surface, may affect the behaviour of the active site pocket of the SARS-CoV-2 Mpro protein. To this end, we performed classical molecular dynamics simulations (cMD) of both SARS and SARS-CoV-2 Mpros, as well as mixed-solvents molecular dynamics simulations (MixMD) combined with small molecules’ tracking approach to analyse the conformational changes in the binding site. The experiment setup and methodology workflow is presented in N3 and SARS-CoV-2 Mpro, respectively. We also used two structures of the SARS-CoV main protease: one, referred to as SARS-CoV MproN3 (PDB ID: 2amq), co-crystallised with the same N3 inhibitor, and the other without an inhibitor (PDB ID: 1q2w), which we refer to as SARS-CoV Mpro. The SARS-CoV-2 Mpro and SARS-CoV Mpro structures differ by only 12 amino acids located mostly on the proteins’ surface   as a com surface . Both en surface B. It shoA total of 2 µs classical molecular dynamics (cMD) simulations of both SARS-CoV-2 and SARS-CoV Mpros with different starting points were run to examine the plasticity of their binding cavities. As different starting points we used (i) SARS-CoV-2 Mpro apo structure; (ii) SARS-CoV-2 Mpro with an N3 ligand, which was removed before starting the simulation; (iii) SARS-CoV Mpro apo structure; and (iv) SARS-CoV Mpro with the same N3 ligand, which was also removed before starting the simulation. A total of 10 replicas of 50 ns classical molecular dynamics simulations were performed for each protein. To improve conformation sampling, the starting geometry for each system was kept but the initial vectors were randomly assigned. A combination of the cMD approach with water molecules used as molecular probes is assumed to provide a highly detailed picture of the protein’s interior dynamics . The smap < 0.05). Both proteins reduced their MAV upon inhibitor binding by approximately 20%, but the maximal volume of SARS-CoV was over 50% larger than those of SARS-CoV-2 , benzene (BNZ), dimethylsulfoxide (DMSO), methanol (MEO), phenol (PHN), and urea (URE). Cosolvents were used as specific molecular probes, representing different chemical properties and functional groups that would complement the different regions of the binding site and the protein itself. Using small molecules tracking approach, we analysed the flow through the Mpros structures and identified the regions in which those molecules were being trapped and/or caged, located within the protein itself  and probably even distant to active site mutations modify Mpro binding properties. Secondly, the C44-P52 loop regulates access to the active site and can contribute to the discrimination of potential inhibitors. Therefore, additional mutations in the above-mentioned regions, which could appear during further SARS-CoV-2 evolution, can significantly change the affinity between Mpro and its ligands. To verify potential threat of further mutability of the Mpro protein, we performed (i) correlated mutation analysis (CMA) on multiple sequence alignment, (ii) the analysis of the contribution of already identified differences between the SARS-CoV and SARS-CoV-2 Mpros to protein stability, and (iii) prediction of further possible mutations caused by the most probable mutations, the substitution of single nucleotides in the mRNA sequence of Mpro.Indeed, the analysis performed with Comulator software  showed tIn the interest of examining the energetical effect of the 12 amino acid replacement in the SARS-CoV-2 Mpro structure, we calculated their energetic contributions to the protein’s stability using FoldX . As expeTo investigate further possible mutations of SARS-CoV-2 Mpro, single nucleotide substitutions were introduced to the SARS-CoV-2 main protease gene. If a substitution of a single nucleotide caused translation to a different amino acid compared to the corresponding residue in the wild-type structure, an appropriate mutation was proposed with FoldX calculations. The most energetically favourable potential mutations were chosen on the basis of a −1.5 kcal/mol threshold . Most ofAdditionally, the potential mutability of the binding cavity was investigated. Residues belonging to the binding cavity were found within 7 Å from the N3 inhibitor. Then, we calculated the differences in the Gibbs free energy of protein folding with respect to the wild-type protein  and presThe analysis of water molecules’ distribution and trajectories can be used for the analysis of proteins’ structural features and biochemical properties. It also provides additional support to the drug design and investigation of protein interior ,37,38. AAs reported in the previous research, the overall plasticity of Mpro is required for proper enzyme functioning ,40. In tThe comparison of MD simulations of both main proteases initiated from different starting conformations (with and without N3 inhibitor) suggests that besides plasticity of the whole protein, there can be large differences between the accessibility to the binding cavity and/or the accommodation of the shape of the cavity in response to the inhibitor that can be bound. There are also differences in the outer pockets’ maximal accessible volumes between the two structures of SARS-CoV main proteases; the apo SARS-CoV Mpro structure used as a starting point of MD simulations has shown the largest MAV of all the analysed systems. These results suggest that the SARS-CoV main proteases’ binding cavity is highly flexible and changes both in volume and shape, significantly altering the ligand binding. This finding indicates a serious obstacle for a classical virtual screening approach and drug design in general. Numerous novel compounds that are considered as potential inhibitors of SARS-CoV have not reached the stage of clinical trials. The lack of success might be related to the above-mentioned plasticity of the binding cavity. Some of these compounds have been used for docking and virtual screening research aimed not only at SARS-CoV ,45 but aThe continuous effort of Diamond Light Source group  performiThe analysis of the water hot-spots shows the catalytic water hot-spot dominated water distribution inside the binding cavity. The remaining water hot-spots corresponded to a much lower water density level and were on the borders of the binding cavity, which suggests a rather hydrophobic or neutral interior of the binding cavity. The MixMD simulations performed with various cosolvents further confirmed these observations. The largest number and the densest hot-spots were located within the binding cavity and the region essential for Mpro dimerisation , betweenThe difficulties in targeting the active site of the Mpros are also explained by evolutionary study and potential mutability analysis. As already pointed out, the C44-P52 loop is likely to regulate the access to the active site by enabling entrance of favourable small molecules and blocking the entry of unfavourable ones. The second important loop, F185-T201, which starts in the vicinity of the binding site and links I and II domains with the III domain contributes significantly to Mpro dimerisation .The initial analysis of the effect of the 12 amino acid replacements in SARS-CoV Mpro on the SARS-CoV-2 Mpro structure stability was expected to provide neutral or stabilising contribution to protein folding. Indeed, all replacements were found to stabilise the protein’s folding  or have an almost neutral character . The analysis of the potential risk of further Mpro structure evolution within the binding cavity suggests that mutations of residues that contribute to ligand binding or access to the active site are energetically favourable, and are likely to occur. Some of the residues that are prone to mutate would provide the inactive enzyme  and therefore could be considered as a blind alley in enzyme evolution, but others  could significantly modify the inhibitors binding mode of Mpro. The locations of residues on the regulatory loop, which are prone to mutate puts in question the efforts to design inhibitors of the MPro active site as a viable long-term strategy. However, our results also indicate residues that are energetically unfavourable to mutate , which could provide an anchor for successful drug design that can outlast coronavirus Mpro variability in future. Alternatively, we would suggest targeting the region between II and III domains, which contributes to the dimer formation.+ ions were added to the SARS-CoV-2 and the SARS-CoV, respectively. Water molecules were placed using the combination of 3D-RISM [−1 × Å−2) and a final minimisation with no constraints of conjugate gradient energy minimization. Next, gradual heating was performed from 0 K to 300 K over 20 ps using a Langevin thermostat with a collision frequency of 1.0 ps−1 in periodic boundary conditions with constant volume. Equilibration stage was run using the periodic boundary conditions with constant pressure for 1 ns with 1 fs step using Langevin dynamics with a frequency collision of 1 ps−1 to maintain temperature. Production stage was run for 50 ns with a 2 fs time step using Langevin dynamics with a collision frequency of 1 ps−1 to maintain constant temperature. Long-range electrostatic interactions were modelled using the particle mesh Ewald method with a non-bonded cut-off of 10 Å and the SHAKE algorithm. The coordinates were saved at an interval of 1 ps. The number of added water molecules is shown in The H++ server  was used 3D-RISM  and the  3D-RISM . The AMB 3D-RISM  was used 3D-RISM  was usedBecause our analysis was focused on the binding site that is surrounded by short loops only, to keep a reasonable combination of the number and length of simulations, the single simulation length was set to 50 ns. As has been shown elsewhere ,52, longNormal mode analysis for each system was conducted using cpptraj from AmberTools 18. Only heavy atoms of the protein were included for analysis.Six different cosolvents: acetonitrile (ACN), benzene (BNZ), dimethylsulfoxide (DMSO), methanol (MEO), phenol (PHN), and urea (URE) were selected to perform the mixed-solvent MD simulations. The chemical structures of cosolvent molecules were downloaded from the ChemSpider database , and a d+ ions to the SARS-CoV-2 Mpro and the SARS-CoV Mpro, respectively. It was assumed that the percentage concentration of the cosolvent should not exceed 5% , or should be about 1% in the case of BNZ and PHN phenol , defined as the r pocket . PocketsTo analyse the significance of the changes of the maximal accessible volume between systems, we used generalized linear models with a Poisson distribution based on AIC comparisons and model fit. Wald tests were used to test the significance of the variables. All tests were performed in Statistica (StatSoft 2019).Object  or just the Scope without visiting the Object ; here, they were considered as potential binding sites. For clarity, the size of each sphere representing a particular hot-spot was changed to reflect its occupation level. The selection of the most significant hot-spots consisted of indicating points showing the highest density in particular regions. From the set of points in the space, small groups of hot-spots were determined. Groups were further defined by distance (radius) from each other. Any point found within a distance shorter than the determined radius (3 Å) from any other point being part of a given group was counted toward the group. For each so designated group of points, one showing the highest density was chosen as representing the place.AQUA-DUCT  was usedMN985262.1). Blastx [YP_009725295.1) amino acid sequence, identical with the previously downloaded SARS-CoV-2 Mpro, was received.SARS-CoV-2 Mpro was downloaded from the PDB as a complex with an N3 inhibitor (PDB ID: 6lu7). Tblastn  was run . Blastx  calculatFoldX software  was usedSARS-CoV Mpro was downloaded from the PDB (PDB ID: 1q2w). Blast  was run In this paper, we reported on molecular dynamics simulations of the main protease (Mpro), whose crystal structures have been released. We compared the Mpro for SARS-CoV-2 with a highly similar SARS-CoV protein. In spite of a high level of sequence similarity between these two homologous proteins, their active sites showed major differences in both shape and size, indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, a detailed analysis of the binding pocket’s conformational changes during simulation time indicated its flexibility and plasticity, which dashes hope for rapid and reliable drug design. Moreover, our findings show the presence of a flexible C44-P52 loop regulating the access to the binding site pocket. A successful inhibitor may need to have an ability to relocate the loop from the entrance to bind to the catalytic pocket. However, mutations leading to changes in the amino acid sequence of the C44-P52 loop, although not affecting the folding of the protein, may result in the putative inhibitors’ inability to access the binding pocket and provide a probable development of drug resistance. To avoid this situation in which the future evolution of the Mpros can undermine all our efforts, we should focus on key functional residues or those whose further mutation will destabilise the protein . Alternatively, we would suggest targeting the region between II and III domains, which contributes to the dimer formation. Our results provide the basis for drug design efforts aimed at this important protein target as part of the multifaceted global effort to eradicate COVID-19. In view of the presented challenges to finding a potent drug targeting Mpro, in our opinion, the most successful strategy would be to screen a large database of compounds with diverse structures involving or designing inhibitors de novo using a fragment-based approach. Both of these strategies, unfortunately, take much longer than a currently preferred approach based on repurposing existing FDA-approved compounds and hence should be pursued as a long-term plan of preparedness for future outbreaks of COVID epidemics involving this and other strains of the virus.


The turn of the decade will be remembered for the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has spread throughout the globe. While the fight to contain the virus continues, the impact of the pandemic on healthcare systems around the world is becoming apparent. Of particular concern to urologists are data from early stages of the pandemic showing SARS-CoV-2 infection rates of 3% in postoperative patients undergoing urological procedures in the Republic of Ireland between 16 March 2020 and 1 May 2020 [et al. when managing pneumoperitoneum in patients with or suspected of SARS-CoV-2 infection [2 circulating in the pneumoperitoneum may generate aerosols that contain SARS-CoV-2 virus [consider laparoscopy only in selected individual cases where clinical benefit to the patient substantially exceeds the risk of potential viral transmission in that particular situation. Where nonoperative management is possible (such as for early appendicitis and acute cholecystitis) this should be implemented” [When compared with open surgery, there are concerns that laparoscopic surgery confers a higher risk from an infection control perspective, of particular importance during the COVID-19 pandemic. This concern arises from the increased aerosolisation risk associated with desufflation of the pneumoperitoneum and exposure to surgical plumes, particularly with the use of ultrasonic scalpels and electrical surgical devices . Desufflnfection ,6. To da-2 virus . This isemented” .et al. who compared the AirSeal® System valve-less trocar systems with standard Versaport™ Plus V2 Trocar system in patients undergoing robotic-assisted radical prostatectomy[Applying this advice to urological procedures being undertaken during this pandemic is not straightforward. tatectomy. This brtatectomy. AdditioWe are still interrogating the safest ways to approach surgery in the COVID-19-era but there are certain aspects of the growing literature that have become clear. There is no evidence to confirm direct infection of members of a theatre team with the SARS-CoV-2 virus, or other viruses, from laparoscopic or open approach. Nevertheless, the ability of the SARS-CoV-2 virus to survive in certain environments has also been established. It remains viable in aerosols for 3 h with a half-life of 1.1–1.2 h . It can et al. documenting the limitations of the current reverse transcriptase (RT-PCR) method [et al. also asserts the limits of detection for the RT-PCR prevents the detection of positive patients with a low viral load, either in the initial phase of infection or following symptom relief [et al. in clinical specimens other than respiratory samples, including feces from 29% of patients, and in blood from a small number of patients. Importantly, for urologists, the virus has not been isolated in urine to date [There are also questions when it comes to the fidelity of testing, with Lippi ) method  . The RT) method . Lippi em relief . As such to date . This is to date . DespiteWhile concern regarding the COVID-19 pandemic remains ever present, it is important to understand the current gaps within the literature. An example of this ambiguity is the proposed utility in using Bacillus Calmette Guérin (BCG) vaccine to improve SARS-CoV-2 morbidity and mortality. Recent studies have shown countries without a recent or current universal BCG vaccination policy observed a slightly higher SARS-CoV-2 related morbidity and mortality than those countries that did . This reWe are learning more about COVID-19 daily, and as we look toward resumption of elective operating in Australia, it is important to consider which surgical procedures remain safest for both the patient and the surgical team alike. The absence of SARS-CoV-2 in urine provides urologists with some degree of reassurance when performing endoscopic work. However, when weighing the risk and benefits of an open, laparoscopic or robotic procedures, it remains a difficult decision. The theoretical risk of increased transmission posed by laparoscopic surgery needs to be considered. Nevertheless, the benefits of a minimally invasive surgical approach are long established, and these theoretical risks remain just that – theoretical. The increase in length of stay, in a high risk hospital environment after open surgery should also not be understated to a patient’s overall risk of infection . We do k


Left unchecked, pre-diabetes progresses to diabetes and its complications that are important health burdens in the United States. There is evidence of geographic disparities in the condition with some areas having a significantly high risks of the condition and its risk factors. Identifying these disparities, their determinants, and changes in burden are useful for guiding control programs and stopping the progression of pre-diabetes to diabetes. Therefore, the objectives of this study were to investigate geographic disparities of pre-diabetes prevalence in Florida, identify predictors of the observed spatial patterns, as well as changes in disease burden between 2013 and 2016.The 2013 and 2016 Behavioral Risk Factor Surveillance System data were obtained from the Florida Department of Health. Counties with significant changes in the prevalence of the condition between 2013 and 2016 were identified using tests for equality of proportions adjusted for multiple comparisons using the Simes method. Flexible scan statistics were used to identify significant high prevalence geographic clusters. Multivariable regression models were used to identify determinants of county-level pre-diabetes prevalence.p ≤ 0.05) from 8.0% in 2013 to 10.5% in 2016 with 72% (48/67) of the counties reporting statistically significant increases. Significant local geographic hotspots were identified. High prevalence of pre-diabetes tended to occur in counties with high proportions of non-Hispanic black population, low median household income, and low proportion of the population without health insurance coverage.The state-wide age-adjusted prevalence of pre-diabetes increased significantly (Geographic disparities of pre-diabetes continues to exist in Florida with most counties reporting significant increases in prevalence between 2013 and 2016. These findings are critical for guiding health planning, resource allocation and intervention programs. People with pre-diabetes are considered to be at higher risk of developing diabetes and subsequent complications than those that do not have the condition. In 2015, an estimated 33.9% of adults had pre-diabetes in the United States, and the percentage of pre-diabetics among seniors aged 65 years and older was estimated to be 48.3% . LifestyLike many other chronic conditions, there are geographic disparities in the prevalence of pre-diabetes. Previous studies investigating these disparities have been limited in that they have been descriptive in nature and very few have used rigorous statistical/epidemiological spatial cluster investigation techniques to identify these disparities and disease hotspots at sub-state levels and yet findings from such investigations are critically important for guiding health planning and resource allocation. Moreover, many of them have focused on diabetes and not pre-diabetes. However, one of our previous studies, that used rigorous statistical/epidemiological cluster investigation techniques and data from the 2013 Florida Behavioral Risk Factor Surveillance System (BRFSS), detected multiple high-prevalence clusters of both pre-diabetes and diabetes across the state . MoreoveThis study was approved by the University of Tennessee, Knoxville Institutional Review Board (Number: UTK IRB-19-05440-XM).This study was performed in Florida, which has 67 counties. Based on the American Community Survey (ACS) 5-year estimates for 2012–2016, the state had a population of 19.9 million people . The mosThis retrospective study used secondary data and hence did not involve obtaining consent. A list of data sources used in the study is summarized in Diagnostic criteria for pre-diabetes is either a fasting plasma glucose (FPG) level between 100 mg/dL and 125 mg/dL, a two hour plasma glucose level between 140 mg/dL and 199 mg/dL during an oral glucose tolerance test (OGTT), or glycated hemoglobin (A1C) level between 5.7% and 6.4% . Pre-diaCounty-level socioeconomic, demographic, and commute data were obtained from the 2012-2016 ACS 5-year estimates . SocioecRural–urban classification data for each county were obtained from the 2013 National Center for Health Statistics (NCHS) classification scheme . This clThe number of primary care physicians per county was obtained from the 2016 Area Health Resource Files (AHRF) from the Health Resources and Services Administration (HRSA) . The 201All descriptive analyses were performed in SAS 9.4 . ShapiroA one-tailed test of equality of proportions was performed in Stata version 15  to identify significant changes in pre-diabetes prevalence between 2013 and 2016, using the Stata command prtesti. The Simes method was used to adjust for multiple comparisons. This method was also used to identify differences in county-level characteristics between the two time periods. County level median household incomes between 2013 and 2016 were considered significantly different if their 90% confidence intervals did not overlap.Tango’s flexible spatial scan statistic (FSSS) was implemented in FleXScan  to invesp-value was ≤0.05. Only secondary clusters with a prevalence ratio (PR) greater or equal to 1.2 were reported to avoid reporting clusters with very low risk.In this study, the maximum spatial scanning window size was set at 10 counties, specifying binomial probability model. Restricted log-likelihood ratio (LLR) and 999 Monte Carlo replications were used for statistical inference. The most likely clusters were ordered on the basis of the restricted log-likelihood ratios. The primary or most likely cluster was identified as the cluster with the largest value of the restricted log-likelihood ratio. The null hypothesis of complete spatial randomness was rejected when the simulated r ≥ 0.7) potential predictors of pre-diabetes prevalence was assessed for potential association with the outcome. The choice of the variable to retain was based on biological and statistical considerations.To avoid multicollinearity during investigation of predictors of pre-diabetes prevalence, Spearman’s rank correlation coefficient was used to identify highly correlated potential predictor variables. Only one of a pair of highly correlated  manipulations and cartographic displays were performed in ArcGIS . Age-adjusted pre-diabetes prevalence for 2013 and 2016 were displayed in choropleth maps. The critical intervals used in the choropleth maps of 2013 were determined using Jenk’s optimization classification scheme. For consistency and to facilitate comparisons, the intervals used to display the 2013 prevalence data were also used in the choropleth maps for 2016 data.Significant spatial clusters of high pre-diabetes prevalence were also displayed using ArcGIS. In addition, statistically significant changes in county-level pre-diabetes prevalence estimates between 2013 and 2016 were mapped at the county level using ArcGIS. Significant predictors of pre-diabetes were also displayed in choropleth maps, as were significant changes in these characteristics between 2013 and 2016.A total of 36,955 respondents participated in the 2016 Florida BRFSS Survey of whom 584 had missing age data and were excluded from analysis, leaving 36,371 for analysis. Respondents ranged in age from 18 to 99 years, with a median of 60 and an interquartile range of 45 to 71 years. Among respondents who reported race/ethnicity, the most commonly represented group was non-Hispanic white (57.9%), followed by Hispanic (23.4%), non-Hispanic black (14.1%), and Asian (2.7%). Those who identified as American Indian or Alaska Native represented 0.3% of the population while 1.6% identified as “other” race or ethnicity.The state-wide age-adjusted pre-diabetes prevalence in 2013 was 8.0%. This increased to 10.5% in 2016, but varied from 4.5% (DeSoto County) to 20.2%  , 2A–2B. p < 0.0001) between 2013 and 2016. Significant changes between 2013 and 2016 were identified for all but six counties   –3B. Of tp = 0.001). In contrast, in 2013, some of these southern Florida counties were among those with the lowest prevalence of pre-diabetes in the state. Each of the five counties within the cluster exhibited an increase in pre-diabetes prevalence between 2013 and 2016. Three secondary clusters with prevalence ratios ≥1.2 were identified in 2016, spanning from north-central Florida to the more rural eastern and central panhandle.Six significant high-prevalence pre-diabetes spatial clusters were identified in 2013, and four were identified in 2016 –4B. In 2Summary statistics of the investigated potential predictor variables are shown in Overall, 14.1% of the state population was non-Hispanic black, but this also varied across counties. The highest proportions of non-Hispanic black residents tended to be in counties in the central to eastern panhandle along the state’s northern border with Georgia, as well as in counties with urban centers such as the Miami area, Jacksonville, and Tampa.There was also geographic variation in the unemployment rate, with relatively high levels of unemployment in rural areas such as the inland-south central region as well as the northeastern and north-central panhandle. Higher proportions of the population with limited access to healthy foods also tended to be located in the inland south-central portion of the state, as well as along the central Atlantic coast. Counties with relatively high proportions of the population reporting physical inactivity tended to be located in the rural eastern panhandle and the central panhandle counties just west of the Tallahassee region, as well as in the inland south-central region. Central Florida counties surrounding the Orlando area tended to have high proportions of the population with arthritis, as did several of the rural counties in south-central Florida, while low prevalence proportions of arthritis were observed in Miami-Dade County, the Tallahassee area, and the Jacksonville area.p = 0.0113) and proportion of the population without health insurance coverage (p = 0.0007), but a significant positive association with proportion of non-Hispanic black population (p = 0.0215).Results of the univariable associations of county-level characteristics with age-adjusted county-level pre-diabetes prevalence are displayed in Relative changes in significant county-level predictor variables between 2013 and 2016 are displayed in  Significant changes in the proportion of non-Hispanic black population between 2013 and 2016 occurred in 36 (53.7%) counties, with many of these counties exhibiting a relative change of less than 5%. The proportion of non-Hispanic black population increased in most of the counties surrounding Orlando, but otherwise there were no clear geographic patterns or obvious overlap with changes in pre-diabetes prevalence.This study investigated county-level geographic disparities of pre-diabetes prevalence in Florida, identified predictors of the observed disparities as well as changes in disease burden between 2013 and 2016. The findings are useful for guiding the allocation of resources for the continued implementation of targeted prevention and control programs.The results indicated that geographic disparities in pre-diabetes prevalence continue to exist in Florida. While previous studies have investigated the geographic distribution of this condition, few have used rigorous spatial statistical/epidemiological techniques to investigate and identify these disparities. To best inform needs-based health planning, there is need for continuous monitoring and rigorous assessments to characterize the distribution of pre-diabetes and identify predictors of high-risk areas so as to target intervention programs. The existence of spatial disparities of pre-diabetes prevalence observed in this study is consistent with findings from the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study, which reported that the odds of pre-diabetes among adults ≥45 years old living in the “stroke belt” were higher than for those living outside this region .Spatial clustering of county-level pre-diabetes prevalence has previously been described. However, no rigorous epidemiological approaches have been consistently used in these investigations. The current study mitigates this problem by using Tango’s flexible spatial scan statistic (FSSS) which is a rigorous approach that is robust and does not have the problem of multiple comparisons that other methods such as Moran’s Local Indicators of Spatial Association (LISA) do have. Moreover, application of this approach can be scaled to other states to provide useful information to guide evidence-based health planning. Additionally, Tango’s FSSS identifies clusters without the need for pre-specification of the suspected cluster location or size and thus eliminates pre-selection bias. An additional advantage of using Tango’s FSSS is the flexibly shaped scanning window, which has high power for the detection of clusters that are not circular in shape .It is worth pointing out that other studies have used FSSS to identify spatial clusters of other health outcomes such as measles , cancer Pre-diabetes awareness reportedly varies with factors such as age, level of educational attainment, health insurance coverage, and food security . Data frThe significant positive associations observed between county pre-diabetes prevalence and the proportion of the population that was non-Hispanic black suggests that the geographic disparities in the prevalence of the conditions in Florida are at least partly attributable to racial disparities. Significantly higher odds of pre-diabetes have been reported among black participants in the REGARDS study in comparison to the white participants, a finding which changed only minimally after adjusting for region .Median household income was a significant predictor of county pre-diabetes prevalence. Economic stability is a key social determinant of health . It is kThere was an increase in the statewide prevalence of pre-diabetes as well as among the majority of counties between 2013 and 2016. However, since prevalence estimates were based upon self-reports, it is difficult to determine the extent to which the observed increases are attributable to true increases within the population rather than changes in diagnostic and reporting practices. However, the overlap in the spatial patterns of improved health insurance coverage and increased pre-diabetes prevalence may suggest that these increases might, in part, be due to increased awareness and reporting of the condition. Improved diagnosis and reporting may also account for some of the observed increases in pre-diabetes prevalence.Temporal changes were also observed in some county-level characteristics that may have contributed to the changes in pre-diabetes prevalence during the study period. The findings suggest that areas with indicators of economic stagnation were more likely to have increases in pre-diabetes prevalence during the study period. For example, the rural counties in the panhandle and south-central Florida tended not to have significant changes in median household income. The observed temporal changes warrant ongoing monitoring of the conditions within Florida. Additionally, county characteristics exhibited changes that varied from county to county across the state, further emphasizing the importance of evidence-based planning that recognizes local differences in population characteristics, disease burden and health needs.This study has used rigorous spatial epidemiological tools to investigate pre-diabetes disparities and predictors in Florida. Continued use of such approaches is crucial for guiding evidence-based health planning and service provision. Understanding the local and regional changes in health conditions is important for guiding the targeting of control efforts to improve help outcomes for all.Due to the nature of BRFSS data, pre-diabetes status of respondents was based upon self-reports and therefore, the exact diagnostic criteria used were not available. The use of differing definitions of the condition or methodological changes may impact prevalence estimates at the population level , and mayThis study confirmed the persistence of geographic disparities in the prevalence of diagnosed pre-diabetes at the county level in Florida. It demonstrated the usefulness of Tango’s flexible scan statistic for identifying high prevalence clusters of the condition. The study also showed an overall, state-wide increase in pre-diabetes prevalence in Florida. Counties with stagnant median income levels tended to have temporal increases in pre-diabetes prevalence, highlighting the importance of place-based factors in chronic disease risk management. Continued monitoring of pre-diabetes distribution is warranted, with careful attention to factors such as healthcare access and patient awareness of the condition, which may affect reporting. The identification of high-prevalence clusters of pre-diabetes with flexible scan statistics is useful for identifying populations at greatest risk, and informing the allocation of resources within the state. Ongoing monitoring and epidemiologic analyses are important for identifying trends at county and state levels and for identifying factors associated with pre-diabetes that may represent potential targets for health planning and interventions.10.7717/peerj.10443/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.10443/supp-2Supplemental Information 2Click here for additional data file.


Supplemental Digital Content is available in the text. ATP) channels are thought to couple cellular metabolism with membrane excitability, calcium entry, and endothelial mediator release. We hypothesized that endothelial KATP channels have a broad role protecting against high blood pressure and atherosclerosis. Endothelial-specific Kir6.1 KO mice (eKO) and eKO mice on an apolipoprotein E KO background were generated (A-eKO) to investigate the role of KATP channels in the endothelium. Basal blood pressure was not elevated in eKO mice. However, when challenged with a high-salt diet and the eNOS inhibitor L-NAME, eKO mice became more hypertensive than their littermate controls. In aorta, NO release at least partly contributes to the endothelium-dependent vasorelaxation induced by pinacidil. In A-eKO mice atherosclerotic plaque density was significantly greater than in their littermate controls when challenged with a high-fat diet, particularly in the aortic arch region. Levels of endothelial dysfunction markers were higher in eKO compared with WT mice; however, these were not significant for A-eKO mice compared with their littermate controls. Furthermore, decreased vascular reactivity was observed in the mesenteric arteries of A-eKO mice, but not in aorta when on a high-fat diet. Our data support a role for endothelial Kir6.1-containing KATP channels in the endothelial protection against environmental stressors: the maintenance of blood pressure homeostasis in response to high salt and endothelial integrity when challenged with a high-fat diet.In the endothelium, ATP-sensitive potassium (K ATP) channels are inwardly rectifying K+ channels activated by declining ATP or increasing ADP levels, thus regulating a range of biological activities by linking cellular metabolism with membrane excitability.1 KATP channels are ubiquitously expressed in the body and are particularly prominent in the cardiovascular system including heart and smooth muscles where they are involved in a range of physiological mechanisms such as the regulation of vascular tone and blood flow.5 At the molecular level, KATP channels are composed of 4 pore-forming Kir6.x subunits (Kir6.1 or Kir6.2) and 4 regulatory sulphonylurea receptor  subunits to form a functional hetero-octameric complex.6 Tissue-specific combinations of a particular SUR with a specific Kir6.x subunit generate channels with distinct pharmacological and electrophysiological properties.ATP-sensitive potassium channels  are also hypertensive, but they did not fully recapitulate the global KO phenotype suggesting the involvement of other cellular populations of KATP channels.2 It is known that K+ currents can influence endothelial function by hyperpolarizing the cell, promoting Ca2+ entry and thus leading to the release of vasoactive mediators.11 KATP channels have also been shown to be expressed in vascular endothelial cells (ECs) and Kir6.1 (and possibly Kir6.2) is thought to be the underlying pore-forming subunit for this channel.15 Using cre-loxP technology, we conditionally deleted the Kir6.1 subunit in vascular ECs and showed that KATP currents were abolished in ECs.5 Furthermore, Ca2+ entry into ECs in response to KATP channel activation was also eliminated and the response of the coronary circulation to hypoxia was compromised.5The importance of KATP is expressed in endothelium and that it plays an important functional role in response to stress. Furthermore, there is increasing interest in endothelial cell calcium signaling and metabolism and how they might regulate key processes such as angiogenesis.17 However, the broader role that the KATP channel may play in the circulation outside of the coronary vasculature remains to be investigated.5 Thus, in this study, we aimed to address the role of Kir6.1-containing KATP channels in the endothelium of conduit and resistance vascular beds, and to investigate how these channels affect vascular function at rest and in response to pathological challenges.It is clear from our work and that of others that KThe data that support the findings of this study are available from the corresponding author upon reasonable request.All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the British Home Office regulations (covered by project licence PE9055EAD) and by the US National Institutes of Health .Data Supplement.Details are given in the 2 Briefly, mice were anesthetized with isoflurane, 5% for initial induction and 1% to 1.5% for maintenance during surgery. The left carotid artery was isolated and the probe catheter inserted to a depth of ≈1 cm and secured with sutures. The probe body was placed subcutaneously on the left side of the abdomen. Two weeks post-surgery, continuous recordings were commenced using the Acquisition module of the Dataquest software  at a sampling rate of 2 kHz for 24 to 48 hours and analyzed using Ponemah P3 plus analysis software . Mice were further interrogated with a high-salt diet  and L-NAME in the drinking water (5 mg/10 mL) for 4 weeks. Twenty-four hour recordings were taken at 0, 7, 14, 21, and 28 days. At the end of the study, mice were sacrificed and the hearts harvested and weighed.Blood Pressure (BP) was measured using implanted radio-telemetry probes PAC-10  as described previously.Data Supplement for details on genotyping, organ bath and wire myography experiments, tail cuff BP measurements, analysis of atherosclerotic plaque formation, ELISA, and data analysis.See 2 To examine the possible contribution of endothelial Kir6.1 in BP regulation, we used continuous telemetric monitoring in conscious 8- to 12-week-old wildtype and eKO mice. Wildtype and eKO mice showed typical circadian variation in basal heart rate, systolic and diastolic blood pressure (SBP and DBP) with higher heart rate and BP at night than during the day when mice are active . The basal prediet heart rate was lower in eKO mice  compared with wildtype  mice, significantly so during the day (P<0.05). There was no difference in prediet SBP and DBP between wildtype  and eKO  mice suggesting KATP channels in the endothelium do not contribute significantly to basal BP regulation in mice . Analysis of the heart weight; body weight ratio did not reveal cardiac hypertrophy at this time point in either wildtype or eKO mice  and a high salt (HS) chow (8% NaCI) over a period of 4 weeks. Both groups became hypertensive within the first week on the diet with a decreased heart rate and increased SBP . Pulse pressure was also increased in both groups. All parameters reached a relative plateau at 14 days. Significantly, the effect on heart rate and BP was greater in mice lacking endothelial Kir6.1 . We also used a noninvasive tail cuff method to measure BP when mice were on a HS only diet (without L-NAME). Basal BP levels were similar in wildtype and eKO mice (P>0.05). After 7 days on the HS diet, both genotypes became hypertensive. In wildtype mice, SBP and DBP returned to baseline levels by day 21; however, in eKO mice, BP was significantly  higher than baseline and wildtype mice at 28 days .The absence of Kir6.1 in VSM leads to hypertensive mice, but the hypertension is not as pronounced as in mice with global deletion of Kir6.1.P<0.0001). The application of the NO synthase inhibitor L-NAME (300 μmol/L) to endothelium-intact vessels also significantly shifted pinacidil-induced relaxation to the right . In comparison, in eKO mice, removal of the endothelium had a much smaller but still significant effect on pinacidil-induced relaxation . The application of L-NAME also significantly shifted the pinacidil-induced relaxation to the right . We also tested the effect of the KATP inhibitor glibenclamide (10 μmol/L) with a KATP-independent, NO-dependent vessel relaxant, acetylcholine  and eKO mice (P<0.0001). The dose-response curves to pinacidil  and acetylcholine  between eKO and wildtype mice were not significantly different in endothelium-intact rings.To investigate the effect of deleting Kir6.1 in the endothelium of conduit vessels, we compared the relaxation of endothelium intact (+E) and denuded (−E) aortic rings from 10- to 12-week-old wildtype and eKO mice with pinacidil (0.1–100 μmol/L). In wildtype mice, endothelial denudation shifted the pinacidil dose-response curve to the right . The application of L-NAME to wildtype endothelium-intact rings had no effect on the pinacidil dose-response curve . This is likely due to the known actions of endothelium-derived hyperpolarization of VSM cells either through direct coupling or the release of mediators particularly in high resistance vessels to compensate for the lack of NO.23 In comparison, for eKO mice, removal of the endothelium had no effect in mesenteric rings  and in endothelium-intact rings L-NAME had no effect on the pinacidil-induced relaxation . Additionally, acetylcholine-induced relaxation was not affected by glibenclamide in either wildtype or eKO mice . As in the aortic rings, there was no significant difference for the pinacidil-induced  and acetylcholine-induced  relaxations of endothelium-intact mesenteric rings in wildtype and eKO mice. These data support the presence of a Kir6.1-containing endothelial KATP channel in mesenteric arteries, the actions of which are not sensitive to NO inhibition.To investigate the effect of endothelial Kir6.1 deletion in resistance vessels, we compared the relaxation of endothelium-intact and endothelium-denuded mesenteric artery rings from 10- to 12-week-old wildtype and eKO mice to pinacidil. Denudation of wildtype mesenteric rings shifted the pinacidil dose-response curve to the right  diet for a prolonged period of time. Groups of WT and eKO mice were placed on either a sham , HS (8% NaCI), or HF (42% fat) diet when they were 6- to 8-week-old for 4 (HS) and 26 (HF) weeks. Plasma E-selectin was used as a biomarker of endothelial dysfunction. Plasma E-selectin levels were similar in mice on the sham diet . However, in mice on a HS diet, plasma E-selectin levels were significantly higher in eKO compared with wildtype mice at 4 weeks indicating possible endothelial dysfunction in eKO mice . In contrast, in mice on a HF diet (26 weeks), although the mean plasma E-selectin levels were higher than mice on a sham diet, there was no significant difference between wildtype and eKO mice . We then compared the relaxation of the aorta from these mice to endothelium-dependent stimulation with acetylcholine; however, we did not see any difference between wildtype and eKO mice on all 3 diets . In addition, there was no difference in acetylcholine-induced relaxations in the mesenteric arteries of these mice on a sham or a HF diet . However, there was a clear rightward shift of the acetylcholine dose-response curve in eKO mice compared with wildtype mice fed a HS diet, . These data suggest endothelial dysfunction in eKO mice fed a HS diet occurring predominantly in resistance vessels but not with a HF challenge.Endothelial dysfunction is often observed in disease states such as hypertension and obesity, we therefore investigated the role of Kir6.1-containing K), or HF 2% fat di), or HF 2% fat di), or HF 2% fat di), or HF 2% fat di), or HF 2% fat di), or HF 2% fat di−/− mice to generate A-eKO mice and their littermate controls (A-WT). ApoE−/− mice are known to develop fatty deposits in the aorta in response to high-fat feeding. In atherogenesis, endothelial dysfunction contributes to the formation of atherosclerotic plaques on the arterial wall. A-eKO and A-WT mice were put on a HF diet when at 5 to 6 weeks of age for 12 weeks. We found that there was a significant increase in atherosclerotic plaque formation in aortas of A-eKO mice compared with A-WT mice , with the highest plaque burden found in the aortic arch region . Surprisingly, although there were elevated levels of plasma E-selectin and P-selectin compared with mice on a normal background, these were not significantly different between A-WT and A-eKO mice . Both A-WT and A-eKO mice exhibit similar body weight development over the 12 weeks on an HF diet . In addition, the acetylcholine dose-response curve was shifted to the right in A-eKO mice compared with A-WT mice in the mesenteric arteries  but not in aortas . Taken together, these data suggest that endothelial Kir6.1-containing KATP channels may have a protective role in the vasculature against development of atherosclerosis.To investigate the effects of HF consumption in eKO mice we crossed these mice with ApoEATP channel in vivo and in vitro under normal and pathological conditions in novel mouse strains that lack the pore-forming Kir6.1 subunit in vascular endothelium. The endothelial cells lining the luminal surface of the entire vasculature play an important role in regulating the vascular tone of the surrounding VSM cells.24 They do so by secreting an array of vasodilators and vasoconstrictors, such as NO, prostacyclin, and endothelin and also by direct propagation of endothelial cell hyperpolarization via myoendothelial gap junctions.25 Endothelial dysfunction caused by an imbalance of these vasodilators and vasoconstrictors is present in many pathological conditions, for example, it could promote the development of hypertension and is regarded as an early marker for predisposition to atherosclerosis.27In the present study, we have investigated the role of the endothelial K2 Given the ability of the endothelial KATP channel to modulate cytosolic Ca2+5 and thus potentially the release of vasoactive mediators, it raises the question of the contribution of the endothelial KATP channel to BP regulation. From our in vivo telemetry data, we found that there was no change in basal BP parameters in eKO mice compared with wildtype littermate controls. This is perhaps not surprising as the dose-response curve to the KATP opener pinacidil was not significantly different in eKO mice in both aortas and mesenteric arteries compared with WT mice. Likewise, responses to the endothelium-dependent vasodilator acetylcholine in eKO mice were unaffected under basal conditions in both aorta and mesenteric arteries. Finally, there was no evidence of cardiac hypertrophy in eKO mice compared with littermate controls. One significant change was a slower heart rate in the eKO mice, which might be indicative of a compensatory change via baroreflex to normalize BP.In our previous work, the smooth muscle–specific Kir6.1 KO mouse was found to be moderately hypertensive but not as hypertensive as the mouse with global deletion of Kir6.1.ATP channels in vascular endothelium from pharmacological studies of pinacidil-induced relaxation in aortic and mesenteric artery rings. In both conduit and resistant vessels, responses to pinacidil were compromised when endothelium was removed, and this change was less or no longer occurred in eKO mice. Interestingly, the pinacidil-evoked vasorelaxation was higher in eKO mice compared with WT mice. It is unclear why this is but it could be because of an enhanced compensatory response from the smooth muscle. It is known that the relaxation evoked by acetylcholine in conduit vessels is endothelium-dependent and is mediated by nitro oxide synthesized endogenously by NO synthase (eNOS) from L-arginine.29 Organ bath studies on aorta suggest that the NO pathway might well be involved in KATP channel activity as addition of the eNOS inhibitor L-NAME had an attenuating effect on pinacidil-induced relaxations. Acetylcholine-induced relaxation was sensitive to the KATP blocker glibenclamide in both WT and eKO mice. Although speculative, this could be due to an enhanced blockade of VSM KATP channels by glibenclamide. Interestingly, mesenteric arteries were unaffected by either L-NAME or glibenclamide. This may simply reflect the fact that in these resistance arteries, other vasodilatory mechanisms involving EDHF and a direct propagation of endothelial cell hyperpolarization to smooth muscle play a more prominent role.30Complementary to our previous work and that of others, we provide further evidence for the widespread distribution of KATP channels may perform a compensatory role by offsetting some of the rise in BP. This could be through a direct release of vasoactive mediators or via endothelial KATP-mediated changes in endothelial cell membrane potential that is directly conducted to the smooth cells through myoendothelial gap junctions to produce vasorelaxation.23 However, the channel may more generally protect the endothelium against endothelial dysfunction such as would occur with the development of hypertension.32 In support, eKO mice also have elevated plasma E-selectin levels and diminished acetylcholine relaxation responses in mesenteric arteries. In WT mice, E-selectin levels are actually lower after a HS challenge compared with basal levels suggesting that KATP channels may protect against endothelial dysfunction by reducing E-selectin levels rather than preventing an increase. Elevated plasma E-selectin levels may also be indicative of an inflamed endothelium, and it is possible that endothelial KATP channels modulate the response to HS-mediated inflammation.33 Furthermore, the heart rate drops as BP rises in these mice, probably related to activation of the baroreceptor reflex without time to reset to a new set-point. Interestingly, heart rate was lower in eKO mice even under basal conditions before challenge, significantly so during the day. While as previously discussed this may result from changes in the baroreceptor reflex, cardiac ECs also express and release a range of auto- and paracrine mediators such as NO, PGI2, and endothelin which control important cardiac functions namely contractility and rhythm.34 It may be that KATP channels are also expressed in the endocardium as well as vascular ECs and contribute to the modification of cardiac function.Although we did not see a basal BP phenotype in eKO mice, we found that when the 2 groups of mice were made hypertensive by challenging them with a HS diet both with and without L-NAME administration, BP was significantly higher in eKO mice. Furthermore, mice lacking Kir6.1 in the endothelium had a faster initial increase in BP after introduction of the diet which was sustained over the 4-week period of the diet suggesting that under hypertensive stress endothelial K35 Endothelial dysfunction is often observed in obese animals and a reduction in endothelial NO bioavailability is often the leading mechanism.38 Endothelial dysfunction is closely associated with the development of atherosclerosis, with obesity and insulin resistance being the major risk factors.39 In mice fed on a HF diet over a course of 26 weeks, we observed no difference in the acetylcholine-induced relaxations in both aortas and mesenteric arteries in eKO mice compared with littermate controls. Although the E-selectin levels were higher than in mice on a sham diet, there was no difference between WT and eKO mice. In addition, there was no evidence of atherosclerotic plaque formation in both WT and eKO mice. This is likely due to the relatively high natural resistance of mice to atherosclerosis.40 Therefore, to unmask the potential role of endothelial KATP in response to a HF diet, we crossed the eKO mice onto the Apolipoprotein E (ApoE) KO background to generate A-eKO and A-WT mice. ApoE−/− mice have long been used as an animal model to investigate the progression of atherosclerosis and they develop atherosclerotic lesions resembling fatty streaks.41 Obvious plaque formation was seen in both control and A-eKO mice at 12 weeks on HF with the percentage area of atherosclerotic plaques significantly higher in the A-eKO mice, with most of the plaques formed around the aortic arch region. This is not surprising, given that in human vasculature, atherosclerotic lesions are often found at regions with altered wall shear stress such as bends, bifurcations and T-junctions.42 While plasma E-selectin and P-selectin levels were a lot higher in mice on the ApoE−/− background, this was not different between A-eKO mice and A-WT mice. However, there was an attenuated acetylcholine relaxation in mesenteric arteries of the A-eKO mice, which suggests some endothelial dysfunction.We also challenged the mice with a HF diet and this intervention can lead to increased body weight, hyperglycemia, hyperinsulinemia, cardiac dysfunction, and hypertension.ATP channels have been described in monocytes though the subunit composition is not clear.45 Thus, it is possible that inflammatory responses might be modulated. However, it is clear from the ex-vivo studies that there is intrinsic endothelial dysfunction. Some of the pharmacological agents have limitations; pinacidil has been reported to lead to vasorelaxation in a KATP independent manner and glibenclamide to block CFTR.47 However, our prior work indicates the predominant effect is via KATP channels and we use a concentration of glibenclamide (10 µmol/L) that leads to little inhibition of CFTR.47There are some limitations of our study. The tie2 cre recombinase driver line is widely used for endothelial deletion. However, it should be borne in mind that it also deletes in hemopoietic cells and KATP channel in both mouse aortas and mesenteric arteries. They seem to have minimal contribution under basal physiological conditions; however, upon pathological challenge, they (1) contribute to the regulation of BP when challenged with a hypertensive stimulus and (2) play a protective role in preventing excessive atherosclerotic plaque formation.In conclusion, the findings in our study support the existence of an endothelial Kir6.1-containing KATP channels expressed in the endothelium protect against the development of hypertension and atherosclerosis in response to increased dietary salt and fat intake. This reveals new roles for these channels in ameliorating cardiovascular pathology.We have shown that K−/− mice. Y. Li, Q. Aziz and A. Tinker conceived the study. Y. Li, Q. Aziz, N. Anderson, and L. Ojake performed the experiments and analyzed the data. Y. Li, Q. Aziz, and A. Tinker prepared the manuscript.We are grateful to Dr Amie Moyes for technical assistance and Professor Adrian Hobbs for kindly providing the Tie2Cre+ and ApoEThis research was supported by the British Heart Foundation (RG/15/15/31742) and was facilitated by the NIHR Cardiovascular Biomedical Research Centre at Barts.None.


Patients with metastatic small-intestinal neuroendocrine tumours (NET) have been shown to have a reduced quality of life compared to the general population and many have disabling symptoms during somatostatin analogue (SSA) treatment. The aim of this prospective study was to document the patient-reported symptoms, coping and quality of life during SSA treatment and to measure patients’ fat-soluble vitamin levels.Patients with metastatic small-intestinal NET on treatment with long-acting SSA were included. Data on patient characteristics, blood samples, questionnaires (EORTC-QLQ-C30 and GI.NET-21) and structured patient interviews were collected at inclusion and after 1 year.Eighty-eight patients were included, 77 (88%) attended 1 year follow-up. Approximately 50% of patients reported symptoms, the most common symptoms at baseline and after 1 year follow-up were diarrhoea, flatulence, fatigue, abdominal discomfort and sore injection lumps. Diarrhoea and fatigue were reported as their main complaint, 23% had > 5 daily episodes of diarrhoea and 59% reported fatigue. However, patients reported a high perceived quality of life, high daily activity, coped with their symptoms and managed their daily life well. Deficiency of vitamin D (27%) and A (13%) were observed.Patients with metastatic small-intestinal NET on SSA treatment reported a high frequency of symptoms. Minor improvements were seen after 1-year of follow-up, illustrating that many symptoms might be difficult to improve, or may not be recognised by the health service. Patients, however, generally reported a high quality of life. Care for NET patients on SSA treatment should include a regular systematic symptom registration and vitamin measurements. The incidence of neuroendocrine tumours (NET) is increasing , 2. NET In the present study we performed a systematic registration of symptoms, treatment of symptoms and overall well-being of NET patients using SSA at study inclusion and after 1 year. A detailed overview of these aspects could be helpful in designing approaches to improve the quality of life of this patient group. Vitamin measurements were also included as a these patients can develop deficiencies in fat-soluble vitamins .This was a 1-year prospective observational cohort study performed at the two largest NET centres in Norway. All NET patients in the uptake area are treated at these centres, thus the patients should represent a population-based cohort on SSA treatment. The aim was to evaluate and describe the symptoms, quality of life and fat-soluble vitamin levels in patients with metastatic small-intestinal neuroendocrine tumours. The data were collected during Jan 2014 to Aug 2017.Inclusion criteria were patients with non-resectable metastatic neuroendocrine tumours with a Ki-67 < 20% (WHO G1-G2) with either a known origin in the small-intestine or a highly probable origin in the small-intestine. Patients had to be on treatment with a long-acting SSA and have a life expectancy of more than 6 months. The study was approved by the regional Ethics Committee and informed consent was obtained from each patient.The following data were collected from medical records: surgery, disease location, Ki-67 tumour estimate, general clinical condition, medical treatment, co-morbidity, laboratory markers including Chromogranin A (CgA) in blood and 5-hydroxyindoleacetic acid (5-HIAA) in urine, age, gender, occupation and disease status based on last computed tomography (CT) evaluation (stable disease/progression). Blood samples were taken at baseline and at 1-year follow-up to detect possible deficiencies in fat-soluble vitamins .Patients were asked to respond to standardised and validated quality of life questionnaires, including one general questionnaire and one supplementary NET questionnaire, both developed by the European Organisation for Research and Treatment of Cancer . The EORTC QLQ.C30 includes 30 questions incorporated in five functional scales: physical, role, cognitive, emotional, and social; nine symptom scales: fatigue, pain, nausea, vomiting, dyspnea, appetite loss, sleep disturbance, constipation, diarrhea; and a global health and quality of life scale . The queGiven the descriptive purpose and the non-clinically relevant differences from baseline to 1-year follow-up, we decided to avoid unnecessary statistical testing. The results from this study are therefore only presented descriptively. Continuous data are summarised using descriptive statistics and categorical data are presented using absolute frequency and percentage. All available patients and data are included in the descriptive tables. The denominator for percentage calculations is the total number of patients with available data. For variables with missing values, the number of patients with missing values is presented.In total 88 patients fulfilled the inclusion criteria and provided data at baseline and 77 at 1-year follow-up. Patients who did not complete 1-year follow-up had either died (7 patients), did not want to participate in the follow-up (2 patients), had been transferred to another hospital (1 patient) or was not able to perform the questionnaires due to dementia (1 patient). Main baseline characteristics are shown in Table Octreotide LAR was used by 61% of patients while 39% used lanreotide. The median time since the start of SSA treatment was 3.1 years (range: 0.2–15.3 years). The injections were given at 3-to-4-week intervals for the vast majority of patients . The type of SSA treatment and injection intervals remained approximately the same throughout the year of the study (data not shown). Almost all patients reported that the main indication for treatment were to live longer (92%) and reduce symptoms (57%). A vast majority of the patients responded that they had no chance of a cure from the disease (90%).In the EORTC QLQ-C30 questionnaire, patients reported high mean scores, corresponding to good self-assessed quality of life at baseline for global health status and the five functional scales  Table . There wn = 34) versus patients on treatment with octreotide LAR (n = 54) is shown in Table In the nurse interviews, approximately half of the patients reported immediate and persistent symptoms from the injections both at baseline and at the 1-year follow up Table . More thAt baseline, levels of vitamin D were below the recommended value in 24 (27%) patients. For vitamin A, vitamin K and vitamin E, the number of patients with low values were 11 (13%), 6 (13%) and 2 (2%) respectively. At the 1-year follow-up, similar results were reported. Two-thirds of patients (67%) reported taking vitamin supplements at the baseline visit  or pancreatic enzyme supplements (30%) compared to the symptom burden the patient cohort had. There are limited data on the true benefit of these drugs on symptoms in NET patients and collecting such data prospectively would be useful. Our study indicates a need for new symptomatic treatment options and an evaluation of their usefulness. Telotristat indicated for diarrhoea in NET patients was not approved during the study period and might have been beneficial for some of these patients . A novelIt is difficult to find other studies for comparison of our results. In published quality of life studies on NET, the study populations are frequently heterogeneous, the methodology differs and the quality of the data handling and reporting is variable . PublishA substantial proportion of the patients in our study had vitamin deficiencies. Vitamin deficiency in NET patients has been shown in prior studies. Vitamin B3 deficiency was found in 45% of patients with serotonin producing tumours  and vitaAn interesting finding of the study was that though these patients report a high symptom burden, they also reported a high quality of life and coped well, arguing against a direct correlation between symptom burden and QOL. This speaks to the potential for human adaptation and resiliency even when living with an incurable disease. As many as 90% of the patients knew that they would eventually die from their disease, and 92% knew that the main indication of the SAA treatment was to live longer but with no chance for cure. This is quite in contrast with previous observations in cancer patients before the immunotherapy era. In a study of patients with metastatic lung or colorectal cancer given palliative chemotherapy, 69% of lung cancer patients and 81% of colorectal cancer patients did not report understanding that chemotherapy was not likely at all to cure their cancer .A limitation of our study is that we could not discriminate whether symptoms were post-operative, due to the SSA treatment or the NET disease itself. To better understand the pathophysiology and to offer specific treatments, such discrimination would probably be necessary. To address this issue, symptoms should be recorded prospectively before start of SSA treatment or any surgical procedures and then regularly thereafter. The aim of this study was not to compare the NET population to the general population. We already know that the NET population has a lower health related quality of life compared to the general Norwegian population . It woulOverall, it can be concluded that patients with metastatic small-intestinal NET have a high symptom burden during long-term treatment with SSA, but despite this maintain a high quality of life. To further improve NET patient care, it may be beneficial to regularly use short questionnaires for collection of symptoms during SSA treatment. It will be important to try to discriminate between symptoms related to prior surgical procedures, the ongoing SSA treatment or from the NET disease itself to optimise the care. Many of these patients may need differentiated management strategies and a closer structured follow-up to achieve optimal care. Future studies are needed to explore how these symptoms can be managed in a more effective way to further improve the care and ensure that quality of life is as good as possible in each patient.


Although hearing aids (HAs) are sometimes efficacious in abating tinnitus, the precise mechanism underlying their effect is unclear and predictors of symptom improvement have not been determined. Here, we examined the correlation between the amount of tinnitus improvement and pre-HA quantitative electroencephalography (qEEG) findings to investigate cortical predictors of improvement after wearing HAs. QEEG data of thirty-three patients with debilitating tinnitus were retrospectively correlated with the percentage improvements in tinnitus handicap inventory and the numerical rating scale scores of tinnitus. Activation of brain areas involved in the default mode network  were found to be a negative predictor of improvement in tinnitus-related distress after wearing HAs. In addition, higher pre-HA cortical power at the medial auditory processing system or higher functional connectivity of the lateral/medial auditory pathway to the DMN was found to serve as a positive prognostic indicator with regard to improvement of tinnitus-related distress. In addition, insufficient activity of the pre-treatment noise canceling system tended to be a negative predictor of tinnitus perception improvement after wearing HAs. The current study may serve as a milestone toward a pre-HAs prediction strategy for tinnitus improvements in subjects with hearing loss and severe tinnitus. Tinnitus is an auditory symptom characterized by the perception of sound or noise in the absence of an external sound source. The overall prevalence of tinnitus in the general population is 10–15%  with aboSince Saltzman and Ersner first reported effectiveness of HAs for controlling tinnitus about 70 years ago , many stSeveral hypotheses have been proposed to explain the possible mechanism of tinnitus improvement associated with the use of HAs . For exaPrevious researchers have suggested several mechanisms of tinnitus generation in subjects with reduced peripheral auditory input. That is, auditory deprivation may result in a compensation mechanism called “homeostatic plasticity,” a phenomenon affecting the neuronal activity of the auditory system at several levels along the ascending auditory pathway to compensate for the reduced peripheral input . At the Hearing aid have long been proposed as one of the most useful treatment options for tinnitus, and most previous studies have reported positive effects of HAs on tinnitus. However, the mechanism underlying tinnitus improvement associated with HA usage remains unclear. In particular, despite previously suggested theories of tinnitus improvement by HAs implying the possible involvement of cortical neuroplastic changes, the neural substrates associated with the improvement of tinnitus by HAs have not been investigated. Based on previous studies of ours showing that pre-treatment cortical activities in the auditory cortex, posterior cingulate cortex, and the parahippocampus are important predictors of tinnitus improvement after hearing restoration by cochlear implantation , and alsA total of 33 non-pulsatile subjective tinnitus subjects with hearing loss who had used HAs for more than 6 months were enrolled in this study. The mean age of the participants was 64.6 ± 14.6 years (range: 26–88 years), and 14 (42.4%) were male. During the initial visit, we obtained a structured medical history regarding the characteristics of tinnitus, such as the affected side, the psychoacoustic nature , matched tinnitus frequency and loudness, and the duration of tinnitus perception. Out of the total of 33 subjects, 14 (42.4%) complained of unilateral tinnitus, while the remaining 19 had bilateral tinnitus. More than half of the participants  reported pure-tone tinnitus, while 11 (33.3%) had narrow-band noise, and one (3.0%) had warble-tone tinnitus. The most frequently matched frequency of tinnitus was 8 kHz , and the others showed variable tinnitus frequencies . The mean duration of tinnitus perception was 8.4 ± 9.8 years (range: 3 months – 40 years). This retrospective study was approved by the Seoul National University Bundang Hospital Institutional Review Board (IRB) and the requirement for informed consent from the subjects was waived (IRB- B-1702/383-104) and the IRB waived the need for informed consent for this study. All methods were performed in accordance with the relevant guidelines and regulations.The perceived tinnitus handicap was measured using the tinnitus handicap inventory (THI), a self-reported tinnitus handicap measure consisting of 25 items evaluating the extent of tinnitus-related distress . We evalpost hoc stratification approach, to compare the resting-state oscillatory brain activities between the subjects who showed marked improvement (MI group) and slight improvement (SI group) after tinnitus treatment with HAs. The MI and SI groups were assigned according to percentage improvement of NRS tinnitus distress scores. All subjects in the MI group showed >44.4% improvement of NRS tinnitus distress intensity, while all subjects in the SI group showed improvement <37.5%.We used the median split method , a data-® equipment using the algorithm provided by each manufacturer’s software. The presence of tinnitus and its matched frequency were not considered during the fitting process, and additional functions for tinnitus management, such as sound generation implemented in HAs, were not applied in this study so as to evaluate pure contribution of HAs to the improvement of tinnitus. HAs were fitted according to the individual’s configuration of hearing loss as well as to participant’s comfort and preference. The participants were counseled to increase the usage time of HAs gradually, up to full-time usage. Follow-up and re-fitting were performed 2 – 3 times a month until the patients were satisfied with the amplification of sound and alleviation of discomfort from HAs. After using HAs for over 6 months with proper fitting and full-time usage, aided threshold levels at frequencies of 250, 500, 1000, 2000, 3000, and 4000 Hz were evaluated. The average follow-up duration was 5.3 ± 0.9 months . In addition, questionnaires on THI and NRS scores were obtained after 6 months of HA usage.At the initial visit, the hearing level of all participants was evaluated with pure tone audiometry (PTA) and speech audiometry, and all subjects were shown to have mild to severe hearing loss. The average hearing threshold   of parti1. The impedances at all electrodes were maintained below 5 kΩ. Data were obtained at a sampling rate of 1024 Hz and then filtered using a high-pass filter with a cutoff of 0.15 Hz and a low-pass filter with a cutoff of 200 Hz. After initial data acquisition, the raw data were processed with resampling to 128 Hz and then with band-pass filtering using a fast Fourier transform filter and application of a Hanning window at 2 – 44 Hz. The data were then imported into Eureka! Software  without assuming a predefined number of active sources. The LORETA-KEY software divides the Montreal Neurological Institute (MNI)-152 volume -KEY softwarestraints . Source straints . The ele2 volume , includi2 volume . Perform2 volume .Phase synchronization and the extent of coherence between the MI group and SI group corresponding to different regions of interest (ROIs) were calculated to evaluate the difference of functional connectivity between two groups using the LORETA-KEY connectivity toolbox. This toolbox defines measures of linear and non-linear dependence  between multivariate time series; the measures are expressed as the sums of lagged/instantaneous dependencies. For functional connectivity analysis, a total of 16 ROIs defined by the Brodmann area (BA), known to be related to tinnitus according to the literature, were selected as possible nodes: the bilateral primary auditory cortices  , the bilFor ROIs that showed a significant correlation with the percentage improvements in NRS tinnitus-related distress or perception, log-transformed electric current density was averaged. Each ROI was defined by a single voxel that was closest to the center of the area where a significant difference was found in the source localization analysis. Then, the correlation between the log-transformed electric current density in each ROI and the percentage improvements in NRS tinnitus-related distress or perception was evaluated by calculating the Pearson correlation coefficient.t statistic between MI and SI groups with a threshold of P < 0.05. Correction for multiple comparisons in SnPM using random permutations has been shown to yield similar results to those obtained from a statistical parametric mapping approach using a general linear model with multiple comparison corrections . All other descriptive statistical analyses were performed using SPSS software . In all analyses, P < 0.05 was taken to indicate statistical significance.To identify the resting-state cortical oscillatory activities related to tinnitus improvement, the LORETA-KEY images were analyzed by the statistical non-parametric mapping (SnPM) method for each contrast using LORETA-KEY’s built-in voxel-wise randomization tests (5000 permutations). In this way, we correlated pre-HAs source-localized activities among whole-brain areas and a connectivity of 16 ROIs with the percentage improvement in the THI score, NRS loudness, NRS distress, and NRS perception. Also, to test the reliability of our results, we have split the enrolled subjects into 2 groups according to the order of enrollment (an odd-numbered subject group and an even-numbered subject group) and performed the same analyses to see if the correlations are maintained in these two subgroups. Moreover, we employed a rections . For lagP < 0.01 for all frequencies)] ] . The aveP < 0.01). In addition, NRS tinnitus loudness , tinnitus distress , and tinnitus perception  improved significantly after 6 months of wearing HAs. To evaluate the relationship between the degree of tinnitus improvement and the functional gain with HAs, we performed simple correlation analysis between the functional gains and the tinnitus questionnaire scores. The percentage improvements in the THI scores were not significantly correlated with the average functional gain with use of HAs . The percentage changes of NRS loudness , distress , and perception  were also not significantly correlated with the functional gain in use of HAs.All questionnaire scores pertaining to tinnitus severity improved significantly after using HAs . The meaP < 0.01) . In subgroup analysis, we have confirmed negative correlations between the percentage improvements in NRS tinnitus-related distress and the right IPL, PHC, and PCC in each subgroup , right PHC (BA 27), and right posterior cingulate cortex  for the gamma frequency band ( < 0.01) . The corsubgroup , in accosubgroup . For theP = 0.08) (P = 0.10). In subgroup analysis, the tendency of negative correlation between the percentage improvements in NRS tinnitus-related perception and the bilateral sgACC for beta 3 frequency band was also confirmed in each subgroup  exhibited negative correlations of marginal significance with the percentage improvements in NRS tinnitus perception for the beta 3 frequency band ( = 0.08) . The logsubgroup , in accosubgroup . No signMeanwhile, the percentage improvement of NRS tinnitus loudness did not show significant correlations with pre-HA source-localized activities at any of the eight frequency bands examined.P < 0.01) , bilateral dACC (BA 24), and bilateral sgACC (BA 32) for the alpha 1 frequency band ( < 0.01) . There wP < 0.05). For the other four frequency bands , no significant differences were found between the MI and SI groups with regard to functional connectivity among the 16 ROIs.On correlation analysis between lagged linear functional connectivity of the 16 ROIs and questionnaire scores (THI and NRS scores), no significant correlations were found. However, on lagged linear functional connectivity analysis with regard to tinnitus-related distress, the MI group showed significantly increased functional connectivities in comparison to the SI group between the left A1 and left IPL for the delta frequency band , betweenIn the current study, we sought to evaluate the treatment effect of HAs in subjects with debilitating tinnitus and to gain a better understanding of the underlying neural predictors of a beneficial effect related to HAs. We observed significant tinnitus improvement 6 months after wearing HAs and found several positive- and negative pre-treatment cortical indicators that may be important for predicting the amount of improvement with regard to tinnitus-related distress and tinnitus perception during the daytime. In addition to previous observational studies showing improvement of tinnitus after wearing HAs in subjects with tinnitus, the current study is the first to demonstrate treatment outcome may be predictable based on the status of the brain before HAs are applied.As described above, the percentage improvements in NRS tinnitus-related distress showed statistically significant negative correlations with the pre-HAs source-localized activities at the right IPL, right PHC, and right PCC for the gamma frequency band . These tPrevious studies have suggested that tinnitus generators may become integrated in the DMN in subjects with tinnitus . In thisOn median split analysis with regard to tinnitus-related distress, the MI group showed significantly higher cortical powers in the bilateral DLPFC, bilateral dACC, and bilateral sgACC for the alpha 1 frequency band in comparison with the SI group . AnalogoThis is also consistent with a previous electrical stimulation study indicating activation of the DLPFC and the dACC in patients with limb and back pain undergoing burst stimulation of the spinal cord . As the Functional connectivity contrast analysis between the MI and SI groups revealed significantly increased functional connectivities between the left A1 and the left IPL in the MI group compared to those in the SI group for the delta frequency band , and betP = 0.08) with the pre-HA source-localized activities at the bilateral sgACC , which was suggested in previous functional MRI studies to form part of the core of tinnitus perception , 2012. OTo our knowledge, this is the first study to show the possibility of treatment outcome prediction with regard to the amount of tinnitus improvement using pre-HA cortical oscillatory features.However, several limitations of the present study should be noted. First, as we evaluated the treatment outcomes of HAs 6 months after the first fitting, the long-term effects of HAs with regard to improvement of tinnitus should be re-evaluated in a follow-up study. That is, future studies correlating pre-treatment rs-qEEG data with long-term outcomes should be conducted to determine the cortical predictors of the final tinnitus treatment outcome after wearing HAs. Second, information on the post-HA status of the included subjects was limited because only questionnaires, such as THI and NRS scales, were used for outcome evaluation of tinnitus improvement. In this regard, future follow-up studies exploring post-HA qEEG changes to determine objective cortical oscillatory activity changes after partial peripheral reafferentation by HAs, and to correlate changes in cortical oscillatory activities with changes in subjective tinnitus and tinnitus-related distress, should be performed. Third, we enrolled tinnitus patients with severe distress . That is, only a few subjects had mild distress in this retrospective cohort. To explore HA-related tinnitus improvement and relevant cortical activity changes, and to minimize the effects of improved distress, further studies are required to explore subjective symptom changes and objective oscillatory power changes in subjects with tinnitus, with minimal distress caused by their symptoms. Fourth, dividing the subjects into groups with- and without meaningful THI improvements, and comparing pre-HAs qEEGs to see the pre-treatment differences between good- and poor responders to HAs would give us further valuable information on the clinical setting. However, there were only 3 of 33 subjects who showed clinically an “improvement,” defined as a decrease of more than 20% in the THI score , and thuTaken together, we evaluated the effects of HAs in subjects with severe tinnitus, with reference to pre-treatment cortical oscillatory patterns measured by rs-qEEG. This study underscored activation of the DMN as a negative predictor of tinnitus-related distress improvements even after partial peripheral reafferentation by HAs. In addition, we found that higher pre-HA cortical oscillatory power at the medial auditory processing system or functional connection of the lateral/medial auditory pathway to the DMN may act as positive prognostic indicators with regard to improvement of tinnitus-related distress. Finally, we observed a tendency toward insufficient activity of the pre-treatment noise canceling system as a negative predictor of tinnitus perception improvement after wearing HAs. The present study may facilitate the development of a pre-HA prediction strategy for tinnitus improvements in subjects with hearing loss and severe tinnitus.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Seoul National University Bundang Hospital Institutional Review Board. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.J-JS led the analysis and interpretation of the result, and drafted the first manuscript. JH and J-JS conceived the investigation, revised the manuscript for important intellectual content. JH, DR, SV, Y-CC, J-WK, and J-JS contributed to all aspects of the investigation, including methodological design, data collection and analysis, interpretation of the results, and revision of the manuscript for important intellectual content. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.