Global navigation satellite system (GNSS) refractometry enables automated and continuous in situ snow water equivalent (SWE) observations. Such accurate and reliable in situ data are needed for calibration and validation of remote sensing data and could enhance snow hydrological monitoring and modeling. In contrast to previous studies which relied on post-processing with the highly sophisticated Bernese GNSS processing software, the feasibility of in situ SWE determination in post-processing and (near) real time using the open-source GNSS processing software RTKLIB and GNSS refractometry based on the biased coordinate Up component is investigated here. Available GNSS observations from a fixed, high-end GNSS refractometry snow monitoring setup in the Swiss Alps are reprocessed for the season 2016/17 to investigate the applicability of RTKLIB in post-processing. A fixed, low-cost setup provides continuous SWE estimates in near real time at a low cost for the complete 2021/22 season. Additionally, a mobile, (near) real-time and low-cost setup was designed and evaluated in March 2020. The fixed and mobile multi-frequency GNSS setups demonstrate the feasibility of (near) real-time SWE estimation using GNSS refractometry. Compared to state-of-the-art manual SWE observations, a mean relative bias below 5% is achieved for (near) real-time and post-processed SWE estimation using RTKLIB. The estimation of the spatial distribution of the water equivalent of the snow cover (SWE) in mountain catchment areas is presently the most important unsolved challenge in snow hydrology . In situGlobal navigation satellite system (GNSS) refractometry permits accurate and continuous in situ SWE monitoring ,8. A GNSIn contrast to manual ,10 pointThe temporal and spatial resolution is comparable with automatic SWE observations by buried snow pillows, scales, ground radar, cosmic ray, and acoustic snow gauges ,18,19,20d, being equivalent to the SWE above a submerged/buried GNSS antenna. Refs. ) e) eδLw asA and a satellite j, impacted by the atmospheric, relativistic, and multipath delays, as well as the satellite and receiver clock biases Thereby, h or the Up (height) component of the baseline were strongly correlated with the SWE [Instead of introducing and estimating the additional SWE parameter ( the SWE .h) and the receiver clock δLz. The path delay The The by 99.9% . The infThe station clock error and thus the constant part of The GNSS refractometry method based on the bias in the coordinate Up component  was applTo test the performance of the open-source standard GNSS processing software RTKLIB, the available seasonal dataset of the fixed, high-end GNSS refractometry snow monitoring setup from Davos Weissfluhjoch  was postTo evaluate the feasibility of near real-time SWE monitoring using GNSS refractometry, the fixed, low-cost GNSS snow monitoring system at Davos Laret operated autonomously in RTK GNSS positioning mode. RTKLIB was also used in real-time applications, as it was implemented in the Emlid Reach (M2) RTK receivers whereby the mobile ReachView application serves, among others, as a web interface for RTKLIB . The GNSmeasured , the δSWThe mobile, low-cost GNSS refractometry snow monitoring setup was also configured in RTK GNSS positioning mode to investigate the feasibility for (near) real-time SWE estimation directly in the field. The base station coordinates were determined and fixed over a 10 min observation interval in the field. For the absolute positioning of the measurement location, a virtual reference station (VRS) of the Swiss Positioning Service (swipos) was used to achieve an accuracy in the centimeter range.Measurements of the mobile setup were triggered manually and the baseline in East, North, Up components was estimated over a 15 min observation interval. The estimated total baseline was directly displayed in the Emlid ReachView mobile application when connected to the rover receiver. Since the sub-snow GNSS antenna was mounted straight below the base antenna, the total baseline corresponded to the Up (height) component when the probe was held vertically.The difference between the baseline, estimated with RTK GNSS refractometry to the actual baseline, measured physically on the avalanche probe, corresponds to the bulk SWE above the sub-snow GNSS antenna (Equation ).(5)SWESWE estimation results are presented for the post-processed  fixed, hThe post-processed SWE derived from the fixed, high-end GNSS snow monitoring setup at Davos Weissfluhjoch using the open-source software RTKLIB is shown in The estimated SWE is directly compared to reference data from close-by snow scale (blue) and manual observations (black) where a maximum SWE of approximately 800 mm w.e. is observed. The GNSS-derived SWE follows closely the trend observed by the reference sensors, with a root mean square error (RMSE) of 34.0 mm w.e. to the snow scale and 45.8 mm w.e. to the manual data. The mean relative bias (MRB) is below 10% compared to manual data and around 10% compared to the snow scale observations. Snow depth observations are illustrated for comparison and show strong settling of the snow after the accumulation of new snow, whereby the SWE is constant due to the increase in density. The GNSS-refractometry-derived SWE seems to detect snowmelt events, where the snow depth and the reference SWE decrease.Differences to the snow scale and manual observations were below 70 mm w.e. for the dry snow period until April 2016 b. AfterwThe post-processed SWE estimates are compared to the reference observations in The resulting SWE time series demonstrates the applicability of RTKLIB for SWE estimation in post-processing. It is thereby important that the ambiguities are resolved (fixed). Since RTKLIB does not offer the option to choose the GNSS solution type , only fixed GNSS solutions need to be selected for further SWE estimation. The SWE time series derived by near real-time GNSS refractometry processing from the fixed, low-cost setup are illustrated in Only fixed RTK solutions are selected and illustrated. GNSS data from the base is missing from 25 November to 16 December due to a power outage which led to the loss of the RTK GNSS mode and missing real-time baseline data after an automatic reboot. The RTK GNSS mode was manually re-established on December, 16, providing real-time data again. The near real-time GNSS SWE observations show a high level of agreement to the manual observations over the whole season, with differences below 50 mm w.e, an RMSE of 21 mm w.e, an MRB below 5%, and a cross correlation coefficient of 0.99 b,d. SettNoticeable is the big difference (approximately 200 mm w.e.) to the snow scale at the end of March and beginning of April which were caused by the over-measurement of the snow scale. Furthermore, a big difference (approximately 50 mm w.e.) to the snow scale and manual observation as well as the increased standard deviation of the GNSS-derived SWE is observable at the very end of December 2021. Heavy rainfall was observed that day, possibly influencing the RTK GNSS performance by increasing the path delay significantly. The constant part of the increased path delay may have been taken over by the receiver clock parameter and mitigated during double difference processing. Additionally, higher multipath due to the strong reflectivity of rain on snow and grounds could cause the strong deviations.Results of the mobile, low-cost, and (near) real-time GNSS refractometry snow monitoring setup  at DavosThe present study investigated the applicability of the open-source GNSS post-processing software RTKLIB as well as the (near) real-time feasibility of the GNSS refractometry method using low-cost GNSS equipment. As the SWE induced GNSS path delay is highly collinear with the receiver clock and the height (Up) coordinate, the biased baseline Up component was successfully used for SWE estimation.The seasonal SWE could be accurately determined over 15 min observation periods in post-processing using the GNSS refractometry method based on the biased GNSS baseline up component. In contrast to previous studies ,24, the Moreover, the SWE was successfully estimated in (near) real time directly in the field with the developed mobile, low-cost GNSS snow monitoring setup. Fixed GNSS solutions are crucial so that the SWE can be estimated with a mean relative bias of 2.5% compared to manual observations. This is contradictory to a previous study which concluded that “resolving the GPS float ambiguities is shown to have no significant impact on the SWE estimation” . There, The present results show the possibility of SWE monitoring based on 1 Hz RTK GNSS refractometry Up component estimation. In this case, subsequent outlier screening and data filtering is significant for achieving accurate and reliable SWE time series. A highly resolved (10 min) SWE monitoring is very advantageous for new casting applications such as the temporal detection of precipitation or run-off events .The SWE estimated with GNSS refractometry is currently only representative for a small spatial scale due to the point-wise measurement principle, estimating the SWE over an area of several square meters  could be utilized instead of the local GNSS reference for a distributed SWE monitoring in whole mountain catchment areas. Synergies can be used, and only the buried GNSS station needs to be installed in the snow monitoring site, reducing material, costs, and maintenance and allowing the installation in areas prone to avalanche danger. The feasibility of using long GNSS baselines will be investigated in a future study. The application of RTK GNSS refractometry in shallow snowpacks should be analyzed in more detail due to the limitation in the RTK GNSS positioning accuracy.


As the COVID-19 pandemic continues to threaten public health, the vaccination of children against the disease appears to be a key factor to control the pandemic. Our aim was to investigate the prevalence of parents who have vaccinated their children against the COVID-19 and the factors influencing this decision.We conducted a web-based cross-sectional study in Greece during the first week of September 2021. The study questionnaire was distributed through social media and a convenience sample was obtained. Only parents with children aged 12–17 years old could participate in the study. We collected socio-demographic data of parents and we measured their attitudes towards vaccination and COVID-19 pandemic.Study population included 656 parents. Regarding vaccination, 27.1% of parents had their children vaccinated against the COVID-19, while almost all children had a complete vaccination history (98.9%). The most important reasons for decline of COVID-19 vaccination were doubts about the safety and effectiveness of COVID-19 vaccines (45.3%) and fear of side effects (36.6%). Regarding the information about the COVID-19 vaccines, parents showed more trust in family doctors than in scientists and the government. We found that increased parents’ age, increased trust in COVID-19 vaccines, and positive attitude of parents towards vaccination had a positive effect on children’s vaccination.Understanding the factors influencing parents’ decision to vaccinate their children against the COVID-19 is crucial to increase the COVID-19 vaccination coverage rate. Implementation of public health policies is necessary to spread knowledge about COVID-19 vaccines and to regain vaccine confidence. The Coronavirus disease 2019 (COVID-19) pandemic continues to cause several problems at the individual, social and economic level, although from the beginning of 2021 there are several vaccines with proven safety and efficacy . SuccessUnfortunately, in recent years there has been an increase in the general population’s negative attitude towards vaccination. Vaccine hesitancy refers to the reluctance of individuals to accept the vaccination offer. According to the World Health Organization, vaccine hesitancy is one of the top ten threats to public health . RegardiUnfortunately, COVID-19 cases in children continue to increase due mainly to the highly transmissible delta variant ,14. It iWe conducted a web-based cross-sectional study in Greece during the first week of September 2021. From 15 July 2021 until the time of our study, the Greek government offered a free COVID-19 vaccine to all children aged 15–17 years old throughout the country. The vaccine was also offered to children aged 12–14 years old from 30 July 2021. The vaccination was voluntary and was carried out after the agreement of the parents or guardians. Data collection was carried out online using Google forms to create an anonymous version of the study questionnaire. The questionnaire was distributed through social media and a convenience sample was obtained. Only parents with children aged 12–17 years old could participate in the study. Information on the purpose and design of the study was provided at the beginning of the on-line questionnaire, and parents provided informed consent to participate anonymously in the study. Given that the percentage of children who are vaccinated against the COVID-19 is unknown, we considered a prevalence of 50% to estimate the largest sample size. Thus, considering the precision level as ±5% and the confidence level as 95%, a sample size of 385 parents was obtained. We subsequently decided to substantially increase the sample size to minimize random error. The Ethics Committee of Department of Nursing, National and Kapodistrian University of Athens approved the study protocol .We collected socio-demographic data of parents and we measured their attitudes towards vaccination and COVID-19 pandemic. Regarding socio-demographic data, we collected the following: gender, parents’ age, marital status, educational level, self-perceived financial status, self-perceived health status, parents’ chronic disease, parents’ previous COVID-19 diagnosis, family/friends with previous COVID-19 diagnosis, and living with elderly people or vulnerable groups during the COVID-19 pandemic. We used the five-point Likert scale from 0 (“very poor”), to 4 (“very good”) to measure financial status and self-perceived health status.Regarding vaccination, we measured children’s and parents’ COVID-19 vaccination uptake, parents’ seasonal influenza vaccination in 2020 and children’s complete vaccination history. In all cases, we used “yes/no” answers. Also, we recorded the reasons why parents refuse to vaccinate their children.Additionally, we recorded attitudes of parents towards vaccination and COVID-19 pandemic by measuring the following variables: self-perceived severity of COVID-19, self-perceived knowledge regarding COVID-19 and COVID-19 vaccines, concerns about the side effects of COVID-19 vaccination, trust in COVID-19 vaccines, the role of vaccination in ensuring public health, and trust in the government, scientists, and family doctors regarding the information about the COVID-19 vaccines. The above variables were measured on a scale from 0 to 10 with higher values indicating greater agreement.We used numbers (percentages) to present categorical variables and mean (standard deviation) to present continuous variables. The normality of the distribution of the continuous variables was assessed with the Kolmogorov–Smirnov test and normal Q-Q plots. The dependent variable was COVID-19 vaccine uptake in children. When parents vaccinated their children, the dependent variable was defined as 1. Socio-demographic variables and parents’ attitudes towards vaccination and COVID-19 pandemic were the independent variables. The relationship between the independent variables and the COVID-19 vaccine uptake in children was assessed with univariate and multivariate logistic regression analysis. Independent variables with p-values <0.20 in univariate logistic regression analysis were included in multivariate logistic regression analysis to eliminate confounding. We calculated unadjusted and adjusted odds ratios (OR), 95% confidence intervals (CI), and p-values. In multivariate logistic regression model, we applied the backward stepwise model. All tests of statistical significance were two-tailed. Statistical analysis was performed with the Statistical Package for Social Sciences software .Study population included 656 parents. Parents’ attitudes towards vaccination and COVID-19 pandemic are shown in Multivariate regression analysis identified that increased parents’ age , increased trust in COVID-19 vaccines  and positive attitude of parents towards vaccination  had a positive effect on children’s vaccination.To our knowledge, this is the first study that estimates COVID-19 vaccine uptake among children and investigates the factors that lead parents to vaccinate their children against the COVID-19. In a sample of parents in Greece, we found that 27.1% of them had already vaccinated their children aged 12–17 years old. This proportion seems low, but the absence of similar studies does not allow us to make direct comparisons with other countries and draw safer conclusions. Furthermore, our study was conducted just one month after the Greek government approved COVID-19 vaccines for children. As evidence from studies increases, it is quite likely that more parents will be persuaded to vaccinate their children against the COVID-19. Early results of randomized controlled trials on the safety and efficacy of COVID-19 vaccines in children are encouraging ,17, but Consistent with prior studies in adults –24, the An interesting finding of our study is the fact that most parents had been vaccinated against the COVID-19 (82.9%) and more than half had been vaccinated against influenza (54.3%). This finding is extremely encouraging, as it indicates a positive attitude of parents towards vaccination at a time when the COVID-19 vaccination rate among adults is low ,28–30. MOur multivariate regression model revealed that parents who had more trust in COVID-19 vaccines were more likely to vaccinate their children against the disease. Several studies have already shown that trust in COVID-19 vaccines is related with parents’ willingness to vaccinate their children against the COVID-19 –38. VaccOur findings should be interpreted in the context of several limitations. Given the convenience sample, the on-line data collection, and the unknown response rate, the extent to which our findings can be generalized to other parents is unknown. For instance, parents with limited internet access were less likely to participate in our study. There may have been an information bias in our study since vaccine uptake was self-reported and some parents may have falsely reported that they vaccinated their children. However, the fact that the questionnaire was completed anonymously may have reduced this bias. Moreover, we have explored several factors that may influence parents’ decision to vaccinate their children, but clearly there are other factors that can be studied such as psychological factors, mass media variables, impact of fake news, etc. Finally, our sample included mainly mothers with a high level of education. Thus, further studies with representative samples are critically needed to draw safer conclusions.We provide an early assessment of children’s COVID-19 vaccination status in Greece. Understanding the factors influencing parents’ decision to vaccinate their children against the COVID-19 is crucial to increase the COVID-19 vaccination coverage rate. Future research is needed in this area, as the factors influencing the parents’ decision are miscellaneous. In addition, evidence from studies regarding COVID-19 vaccines is constantly increasing which may influence parents’ attitudes. Mandatory vaccination does not seem to be the most appropriate approach as it generates strong reactions and disagreements. For this reason, the establishment of mutual trust between parents and the scientific community is an essential prerequisite for making a rational decision regarding COVID-19 vaccination in children. Implementation of public health policies is necessary to spread knowledge about COVID-19 vaccines and to regain vaccine confidence.


A possible link between influenza immunization and susceptibility to the complications of COVID-19 infection has been previously suggested owing to a boost in the immunity against SARS-CoV-2.This study aimed to investigate whether individuals with COVID-19 could have benefited from vaccination against influenza. We hypothesized that the immunity resulting from the previous influenza vaccination would boost part of the immunity against SARS-CoV-2.We performed a population-based cohort study including all patients with COVID-19 with registered entries in the primary health care (PHC) electronic records during the first wave of the COVID-19 pandemic  in Catalonia, Spain. We compared individuals who took an influenza vaccine before being infected with COVID-19, with those who had not taken one. Data were obtained from Information System for Research in Primary Care, capturing PHC information of 5.8 million people from Catalonia. The main outcomes assessed during follow-up were a diagnosis of pneumonia, hospital admission, and mortality.We included 309,039 individuals with COVID-19 and compared them on the basis of their influenza immunization status, with 114,181 (36.9%) having been vaccinated at least once and 194,858 (63.1%) having never been vaccinated. In total, 21,721 (19%) vaccinated individuals and 11,000 (5.7%) unvaccinated individuals had at least one of their outcomes assessed. Those vaccinated against influenza at any time , recently , or recurrently  before being infected with COVID-19 had a higher risk of presenting at least one of the outcomes than did unvaccinated individuals. When we excluded people living in long-term care facilities, the results were similar.We could not establish a protective role of the immunity conferred by the influenza vaccine on the outcomes of COVID-19 infection, as the risk of COVID-19 complications was higher in vaccinated than in unvaccinated individuals. Our results correspond to the first wave of the COVID-19 pandemic, where more complications and mortalities due to COVID-19 had occurred. Despite that, our study adds more evidence for the analysis of a possible link between the quality of immunity and COVID-19 outcomes, particularly in the PHC setting. COVID-19 is caused by SARS-CoV-2, a novel coronavirus that emerged in China in 2019, which became the primary agent of a new pandemic that rapidly spread worldwide .Although new variants of SARS-CoV-2 have emerged since December 2020, the coronavirus’ genome is composed of RNA and depends on the RNA polymerase to generate its proteins, with a mechanism of error correction that results in a lower mutation rate than the influenza virus . This loCOVID-19, similar to influenza A and B, is caused by RNA virus and produce similar symptoms. The influenza virus needs the hemagglutinin and neuraminidase surface proteins to infect cells, whereas SARS-CoV-2 needs the S protein . PreviouA study conducted in Australia assessed the cellular and humoral immune responses during and after disease occurrence in a patient with a mild COVID-19 infection. They found that the immune response in different cell types is associated with clinical recovery. These results are coincident with similar findings among patients with influenza reported by the same authors ,16.Other studies observed differences in the susceptibility to COVID-19 in children of different ages with a lower infection rate than that in adults and older individuals . AlthougConsequently, a link between the quality of the immunity and recovery from COVID-19 may exist. Thus, we hypothesized that the immunity resulting from the previous influenza vaccination would boost part of the immunity against SARS-CoV-2, and we aimed to investigate whether individuals with COVID-19 could have benefited from vaccination against influenza.We performed a population-based cohort study including all adults with COVID-19 in Catalonia, Spain, who were registered as confirmed cases (through the polymerase chain reaction [PCR]) or as probable cases -10 codes registered that are compatible with COVID-19) in the primary health care (PHC) system. All individuals with COVID-19 were diagnosed from the pandemic’s onset (March 2020) to June 30, 2020. Participants were compared on the basis of their influenza vaccination status between those having received the influenza vaccine before having COVID-19   2016/679).We included 309,039 individuals with COVID-19 during the first wave of the pandemic in accordance with their influenza immunization status ; 114,181Of those who received the influenza vaccine, 66,611 (58.3%) had been recently vaccinated (2019-2020) and 75,311 (66%) had been systematically vaccinated against influenza at least during 3 different years .Of the participants with COVID-19, 11,000 (5.7%) unvaccinated and 21,721 (19%) vaccinated participants presented at least one of the following events: hospital admission, pneumonia, or death. For those who received the influenza vaccine at any time before having COVID-19, the risks of hospitalization  and death  were higher than those among unvaccinated participants. For the recently vaccinated participants, the risk was higher for hospitalization , pneumonia , and death . For people with recurrent vaccination, the risk of the 3 outcomes was also higher that among unvaccinated participants  and compared those who had received the influenza vaccine with those who were never vaccinated. Those who received the vaccine any time before having COVID-19 were at a higher risk of complications than those who were unvaccinated. We obtained similar results for those who were recently vaccinated (2019-2020 campaign) and for those who were systematically vaccinated (at least 3 years), and the same comparisons were carried out after excluding individuals living in LTCFs. We also obtained similar results on matching vaccinated and unvaccinated individuals. Thus, we did not find a possible link between receiving the influenza vaccine and presenting better clinical outcomes after a COVID-19 infection.Some researchers have studied this possible association. Massoudi and Mohit  conducteCandelli et al  assessedA study conducted in Brazil  includedIn a systematic review  includinThere are reports from some countries with high influenza vaccination rates and high incidences of COVID-19 and mortality ,33. For We need to consider that our results correspond to the first wave of the COVID-19 pandemic, when there were more negative outcomes and mortalities due to COVID-19 than in the subsequent waves in our setting; thus, this higher statistical power allowed us to detect differences. Furthermore, in subsequent waves, more confounders might have been present, such as COVID-19 vaccination or effects of the different SARS-CoV-2 variants, making it more difficult to manage their potential effect in the analysis of the outcomes of the infection.We also need to bear in mind that the target population for the influenza vaccine in our country are people older than 60 years, individuals with chronic comorbidities or immunodeficiency, and health care workers among others , some ofAmong other limitations of our study is the reliability of the COVID-19 diagnoses; we included individuals without a confirmed result, as during the first wave of the pandemic in our setting, PCR tests were not always performed. This limitation has been described in other studies including those conducted at the beginning of the pandemic when diagnostic tests for COVID-19 were not widely available and clinical algorithms were used to assess COVID-19 diagnoses . We compIn conclusion, we were not able to establish a protective role of the immunity conferred by the influenza vaccine on the outcomes of COVID-19 infection. Nonetheless, our study adds more evidence to the analysis of the possible link between the quality of the conferred immunity and outcomes of COVID-19 infection, and it has some strengths, such as the large cohort size, its representativeness with respect to the general population, and the completeness of its sociodemographic data. We have already highlighted that our cohort comprises individuals who received care from the PHC system; hence, we have estimated the risk of complications for a different population from the hospitalized individuals who are usually assessed in multiple studies.


The aim of this study was to compare the reduced-port robotic approach and the conventional four-port approach in terms of postoperative pain and short-term surgical outcomes.Reduced-port laparoscopic gastrectomy can potentially reduce postoperative pain and improve recovery time. However, the inherent difficulty caused by the narrow manipulation angle makes this operation difficult, especially during lymph node dissection. The intrinsic advantage of the da VinciAll patients who underwent radical gastrectomy with D2 lymph node dissection using the da Vinci Xi robotic system, including reduced-port or conventional four-port approach, were analyzed retrospectively. The primary outcome was postoperative pain assessed using the numerical rating scale (NRS). The secondary outcomes were the number of harvested lymph nodes, operation time, length of hospital stay, and postoperative 30-day complications.p = 0.047, POD 3: 4 vs. 3, p = 0.178]. After propensity score matching, there were no significant differences in the median number of harvested lymph nodes, operation time, and length of hospital stay between the groups. The postoperative 30-day complications were more frequent in the conventional four-port group, but there was no significant difference compared with the reduced-port group after propensity score matching.Forty-eight patients were enrolled in the study, 10 cases in the reduced-port and 38 in the conventional four-port group. Postoperative NRS revealed no significant difference between the reduced-port and conventional four-port groups [postoperative day (POD) 1: 4.5 vs. 3, Reduced-port robotic gastrectomy with D2 lymph node dissection might be comparable to the conventional four-port robotic operation in terms of postoperative pain, surgical quality, and short-term outcomes. However, further studies are required to confirm our results and clarify the advantages of the robotic reduced-port approach. Minimally invasive surgery is widely performed as one of the treatment modalities for gastric cancer –3. CurreWith advances in medical equipment and surgical techniques, reduced-port or single-incision laparoscopic gastrectomy has been attempted to reduce the operative trauma and achieve quicker recovery –11. HoweIn this study, we analyzed the safety and benefits of robotic reduced-port and conventional four-port gastrectomy by using the intrinsic advantages of the robotic system to eliminate surgical difficulty.Data of all patients with gastric cancer who underwent robotic radical gastrectomy from October 2016 to December 2020 at our hospital were retrospectively analyzed. The surgical cases were classified into two groups: the reduced-port approach with an umbilical single-port access device and conventional four-port approach. Patients with metastatic lesions found during the operation, intraoperative conversion to open surgery, or conversion to palliative surgery were excluded. The selection for the reduced-port approach or conventional port approach is mainly patient-directed after fully explaining the risk and benefits of the two different kinds of techniques. All patients’ demographic data and perioperative and postoperative outcomes were collected for analysis. The primary outcome was the patient’s pain score assessed using the numerical rating scale . The secondary outcomes were the number of retrieved lymph nodes, operation time, length of hospital stay, and postoperative 30-day complications. Operation time was defined as the time from the start of the first wound incision to final wound closure, including robotic docking and console time. The severity of postoperative 30-day complications was assessed using the Clavien–Dindo classification. All gastrectomy procedures in these two groups were performed with standard radical resection with D2 lymph node dissection, followed by Billroth II or Roux-en-Y anastomosis reconstructions. All operations in the robotic reduced-port and conventional four-port groups were performed by two surgeons, who also played the roles of console surgeons and assistants. This study was approved by the relevant institutional review board and all informed consent was provided to each patient.® / da Vinci® Xi™ Surgical System . The patient was placed in the reverse Trendelenburg position with bilateral leg spilt, and the assistant stood between the legs. The robotic Xi system was set up on the left side of the patient, leaving more space for the anesthesiologist. In the robotic reduced-port group, only two port wounds were created on the patient’s abdomen: a 3–4-cm vertical transumbilical incision and an 8-mm trocar wound located over the patient’s right lateral abdomen  with “MatchIt.”All demographic and clinicopathological characteristics were summarized using descriptive analysis, and continuous data were compared using the two-tailed Student A total of 48 patients were enrolled in the study. After propensity score matching, 10 and 20 patients were included in the reduced-port and conventional four-port groups, respectively. Patient demographics and clinical outcomes before and after matching are shown in Table p = 0.047). The NRS scores on postoperative days 2 and 3 between the two groups were similar .The NRS scores on postoperative days 1, 2, and 3 between the two groups revealed no significant difference. In the reduce-port group, the NRS on postoperative day 1 was slightly higher than that in the conventional four-port group . At least 28 lymph nodes were retrieved from both groups. The median operation times of both groups were similar , and blood loss was also comparable. The median length of hospital stay in the two groups was 16 vs. 14 days (p = 0.659), and the longest hospital stay was 23 days.The total number of retrieved lymph nodes was higher in the reduced-port group than in the conventional four-port group, but the difference was not significant  (Table 0 vs. 19 No mortality was noted within 90 days after surgery. There was no significant difference in complications in the 30 days after surgery between the two groups after propensity score matching. Table In this study, we reported that robotic reduced-port gastrectomy is not inferior to conventional four-port gastrectomy in terms of short-term surgical outcomes and surgical quality. Meanwhile, we demonstrated an almost equal number of harvested lymph nodes between the two procedures. The reduced-port robotic gastrectomy used in our research might be applied to early gastric cancers and some selected advanced gastric cancers.There were no significant differences in operation time between the robotic two-port group and conventional four-port group; however, compared with previous studies, the operation time in our study was longer , 15. TheThe concept of reduced-port or single-incision surgery in the minimally invasive surgery field confers to the reduction of the number of port wounds or wound length to potentially improve postoperative pain, accelerate postoperative recovery, shorten length of hospital stay, and/or increase cosmetic satisfaction , 16, 17.The oncological outcome of gastric cancer surgery can be assessed by the number of retrieved lymph nodes –25. RetrTechnically, robotic surgery can provide 3D images, endowrist articulation, and tremor filtering, which can increase the number of retrieved lymph nodes without causing excessive bleeding compared with laparoscopic surgery , 18, 28.In our study, regardless of the reduced-port or conventional port approach, only three robotic arms and two robotic instruments were used, including the fenestrated bipolar and harmonic scalpel for dissection. The only differences in these two approaches are the instrument approaching site and manipulation angle. In addition, the hemoclip, Hem-o-lok, or endostapler device were all applied via the assistant port using a laparoscopic instrument applier. Owing to the endowrist instrument and tremor filtering properties of the robotic equipment, intracorporeal dissection and reconstruction procedures were almost the same between the two groups, despite the different manipulation angles. Therefore, if the there was no significant difference in the NRS scores between the two groups, the subsequent recovery, length of hospital stay, and 30-day complications will theoretically have no significant differences between the groups. However, we did not measure the cosmetic satisfaction of the patient in either group, although theoretically, the cosmetic satisfaction will be slightly higher in the reduced-port group .This study has several limitations. The number of patients in the reduced-port group was small. The main reason for this is the relatively higher cost of the robotic reduced-port procedure. Compared with the conventional port, additional US 1000 dollars were charged for the single-port access system advice. In addition, owing to the older age of the patients in our study, the possible cosmetic gain from the reduced-port procedure was somewhat neglected by the patients. We collected the data of both robotic total and distal gastrectomy cases because the purpose of the study was to compare robotic reduced-port to conventional robotic four-port surgery in terms of postoperative wound pain and short-term outcomes. The overall complications of the total gastrectomy might be relatively higher than that of the distal gastrectomy; however, the patient who received total gastrectomy operation between the two groups were similar in our study.Our study revealed that the reduced-port robotic distal or total gastrectomy with D2 lymphadenectomy might be comparable to the conventional robotic four-port approach for selected patients. There were no significant differences in terms of postoperative pain, surgical quality, or short-term postoperative outcomes. However, further comparative or large-scale randomized control studies are required to confirm our results and clarify the advantages of the robotic reduced-port approach.


Magnetic resonance susceptibility-weighted imaging (SWI) can identify small brain blood vessels that contain deoxygenated blood due to its induced magnetic field disturbance. We observed focal clusters of possible dilated small vessels on SWI in white matter in severe small vessel disease (SVD). We assessed their prevalence, associations with SVD lesions, and vascular reactivity in patients with sporadic SVD and in patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).Secondary cross-sectional analysis of a prospective multicenter observational study of patients with either sporadic SVD or CADASIL (INVESTIGATE-SVD) studied with 3 Tesla MRI including blood-oxygen-level-dependent MRI cerebrovascular reactivity (CVR). Two independent raters evaluated SWI sequences to identify “vessel-clusters” in white matter as focal low-signal dots/lines with small vessel appearance . We assessed per-patient and per-cluster associations with SVD lesion type and severity on structural MRI sequences. We also assessed CVR within and at 2-voxel concentric intervals around the vessel-clusters using contralateral volumes as a reference.t, p) within vessel-cluster volumes  and in the surrounding volume expansion shells up to 4 voxels  in vessel-clusters with complete cavities, but not in vessel-clusters without complete cavitation.Among the 77 patients enrolled, 76 had usable SWI sequences, 45 with sporadic SVD  and 31 with CADASIL . We identified 94 vessel-clusters in 36 of the 76 patients . In covariate-adjusted analysis, patients with vessel-clusters had more lacunes  , higher white matter hyperintensity (WMH) volume , and lower CVR in normal appearing white matter , compared with patients without vessel-clusters. Fifty-seven of the 94 vessel-clusters (61%) corresponded to noncavitated or partially cavitated WMH on fluid-attenuated inversion recovery, and 37 of 94 (39%) to complete cavities. CVR magnitude was lower than in the corresponding contralateral volumes (mean difference [SD], Vessel-clusters might correspond to maximally dilated vessels in white matter that are approaching complete tissue injury and cavitation. The pathophysiologic significance of this new feature warrants further longitudinal investigation. Recent work suggested that the decreased number and altered morphology of deep medullary veins may be related to WMH load.5-8 On inspection of high-quality MRI scans from patients with severe SVD, we recently observed small clusters of linear-like structures in deep WMH with different grades of cavitation that showed similar signal characteristics to the medullary veins on SWI (links.lww.com/WNL/C29). We hypothesized that these may represent grouped small dilated vessels associated with white matter injury and cavitation . In this study, we describe the prevalence and characteristics of these possible clusters of small dilated vessels on SWI, their associations with patient demographics, SVD lesions, and measures of vascular reactivity, in patients with sporadic SVD or with monogenetic SVD .Susceptibility-weighted imaging (SWI) is a gradient-echo–derived MRI sequence that is commonly used to identify hemorrhagic features in SVD ,s on SWI  but did 9 The study was approved by the local ethics committee in each participating institution, and informed consent was obtained from all participants. The full protocol of the study is available elsewhere.9This was a secondary cross-sectional analysis from a prospective multicenter observational study .9 Patients with other causes of stroke such as ≥50% luminal stenosis, major-risk cardioembolic source of embolism , and other specific causes of stroke identified  were not enrolled in the study.9 The patients with sporadic SVD were recruited from centers in Edinburgh (UK) and Maastricht (the Netherlands), and patients with CADASIL from Munich (Germany). We excluded patients with unusable SWI acquisition. We collected a full medical history, including demographic factors (age and sex), vascular risk factors , alcohol intake, history of ischemic heart disease and peripheral vascular disease, previous stroke, prescribed statins, and antihypertensive and antithrombotic treatment. Systolic blood pressure and diastolic blood pressure were recorded at MRI visit.The INVESTIGATE-SVD study cohort included patients with symptomatic sporadic (a lacunar ischemic stroke in the past 5 years or vascular cognitive impairment with SVD) or genetic SVD (diagnosis of CADASIL).9 In brief, images were acquired using 3 Tesla MRI scanners  in all 3 centers using a standardized protocol, within 3 months after enrollment . The study protocol included T1-weighted, T2-weighted, fluid-attenuated inversion recovery (FLAIR), and SWI sequences. Cerebrovascular reactivity (CVR) was assessed using an established validated blood-oxygen-level-dependent (BOLD) MRI with CO2 challenge.9The full image acquisition protocol has been previously published.2 and graded using validated qualitative scales for WMH 10 and PVS load scale in basal ganglia and centrum semiovale.11 All image analysis was centralized and conducted by an analyst not involved in the clinical assessments and masked to patient characteristics and CVR results and performed before the assessment of this study.9Radiologic markers of SVD, that is, lacunes, PVS, microbleeds, and WMH, on structural MRI sequences were identified according to STRIVE criterialinks.lww.com/WNL/C29).12 Hence, the definition of “vessel-cluster” is based on its radiologic appearance, although the nature of such finding may need further confirmation in pathology studies.We defined as “vessel-clusters” the presence on SWI of grouped low-signal small tubular-like structures  in white matter  that appp < 0.001) and the number of single–vessel-like structures in each cluster . Details of visual identification of vessel-clusters and interobserver reliability analysis are available in the Supplemental material .An experienced stroke neurologist (S.R.) and a trained medical student (E.C.) independently evaluated the SWI acquisitions to identify vessel-clusters and the number of vascular-like structures per cluster . Disagreements were resolved by discussion with a third expert neuroradiologist (J.M.W.). Interrater agreement was substantial for both the presence of the clusters  and shape  of the regions of the brain covered by each vessel-cluster were assessed after segmentation of the regions of interest . The features of the brain areas corresponding to the vessel-clusters were assessed on structural sequences  and classified on the basis of previous work14: normal appearing white matter, noncavitated WMH, partially cavitated WMH , and completely cavitated lesions containing CSF-like liquid. Some examples of clusters and related tissue appearance on structural sequences are shown in We described the location of the vessel-clusters referring to the spatial relationship with anterior and posterior horns of the lateral ventricles , as previously described,15 We obtained intracranial, normal appearing white matter, WMH volumes, and calculated standardized WMH volume . For the analysis of CVR magnitude and delay within normal appearing white matter and WMH, we eroded the outer margin of the original white matter mask by 2.5 mm (1 voxel) to reduce the influence of partial volume effects. In addition, we masked the images with a dilated ventricle mask to exclude contamination from ventricular CSF and normal vessels running along the ventricle walls.Normal appearing white matter and WMH were segmented using a validated semiautomated pipeline.16 was used for the mask processing.Finally, to assess CVR magnitude and delay in the vessel-clusters and in the surrounding tissue, from the original vessel-cluster segmentations, we generated 3 additional concentric circumferential 3D expansions (shells) around the vessel-clusters, each 2 voxels thick in T2-w space (which approximates 1 voxel in CVR data) limited to the white matter, as presented in t test or Mann-Whitney U test as appropriate, while a Pearson 2 test or Fisher exact test was used for categorical variables. Interrater agreement between the 2 readers for the vessel-cluster assessment was assessed with kappa statistics . No missing values were detected among variables, except for systolic and diastolic blood pressure at the time of MRI that were not available in 6 of the 76 patients (8%), and CVR data were not usable in 7 of the 76 patients (9%).Variables were described as mean and SD, median and IQR, or absolute and relative frequency as appropriate. Continuous and ordered variables were compared between patients with and without vessel-clusters in univariable analysis using Student p < 0.1) were assessed in multivariable logistic regression analysis adjusted for specific preselected clinically relevant variables  and other according to the p < 0.1 significance level in univariable analysis , excluding CVR values for not being available in 7 patients. Continuous variables without normal distribution  were log10 transformed in the multivariable analysis. Collinearity according to variance inflation factor (VIF) was identified between visual SVD scale/quantitative measures and log10-normalized WMH volume/type of SVD. In the multivariable analysis, log10-normalized WMH volume and type of SVD were both maintained  for the assessment of the effect of other variables, while log10-normalized WMH was excluded to assess the association between vessel-cluster presence and CADASIL type of SVD, and vice versa.In per-patient analysis, we analyzed the factors associated with the presence of vessel-clusters (1 or more vessel-clusters) in univariable analysis. Then, the relevant factors identified in the univariable analysis . Graphpad Prism 8.4 for Mac was used for graphing.We considered as statistically significant Data not provided in the article because of space limitations may be shared (anonymized) at the reasonable request of any qualified investigator for purposes of replicating procedures and results.Seventy-seven patients were recruited in the INVESTIGATE-SVD study from November 2017 to September 2019. Patients with sporadic SVD were recruited in Edinburgh (n = 25) and Maastricht (n = 20), and patients with CADASIL in Munich (n = 32). All patients completed the main structural sequences. However, SWI sequences from 1 patient with CADASIL were seriously affected by movement artifacts, and that patient was excluded from the analysis. The mean age (SD) of the selected patients was 57 years (12) (64 years [SD 11] in sporadic SVD and 53 years [11] in CADASIL patients), and 35 of the 76 patients (46%) were male (26 [58%] in sporadic SVD and 15 [48%] in CADASIL patients). Current smoking and alcohol use were frequent . The most common vascular risk factors were history of hypertension and hyperlipidemia . None of the patients had atrial fibrillation, and 80% of the patients were on antiplatelet treatment (no patients were on anticoagulant treatment). On MRI evaluation, the SVD structural features were more severe in patients with CADASIL than in those with sporadic SVD. Detailed information on clinical and radiologic features of the study cohort is summarized in links.lww.com/WNL/C29). In the univariable analysis, a diagnosis of CADASIL, alcohol use, and increased severity of SVD on structural imaging were associated with the presence of vessel-clusters, whereas no associations were found for age, sex, other vascular risk factors, or concomitant antithrombotic treatment (p = 0.018) and normalized log10 WMH volume value  remained significant (p = 0.040) and a trend for lower CVR in WMH  (Thirty-six of the 76 patients (47%) showed at least 1 vessel-cluster  . The detp value), age , male sex , the number of lacunes , and normalized log10 WMH volume value  remained significantly associated with the number of vessel-clusters after adjusting for covariates, whereas CVR (per %/100-mm Hg) in both normal appearing white matter  and WMH  had a nonsignificant negative trend .In the ordinal regression analysis for the per-patient number of vessel-clusters . An example of the traceability of vessel-like structures on SWI and MIP through contiguous axial planes is shown in p = 0.005), were more likely to be associated with completely cavitated lesions , were more likely to appear as a linear rim , but with no difference in location in the white matter (p = 0.186). In the 12 vessel-clusters corresponding to noncavitated regions, we observed 1 main dilated vessel inside an area of WMH , whereas vessel-clusters associated with partial and full cavitation  had a linear low-signal rim appearance on SWI in the edges of a cavity  were located in the left hemisphere, 22 of 94 (23%) were located in the anterior, 55 (58.5%) in the middle, and 17 (18%) in the posterior deep white matter. The median (IQR) volume of the region containing the vessel-cluster was 0.15 mL (0.08–0.26), which had a round shape in 45 (48%) vessel-clusters, ovoid in 32 (34%), irregular in 6 (6%), and linear in 11 (12%). Among the 94 vessel-clusters, 27 of them showed clustered or linked low-signal dots or lines that seem to correspond to 1 main vessel-like structure (29%), while clusters of multiple vessel-like structures (up to 5) were observed in the remaining 67 (71%), with a median of 2 (1–3) vessel-like structures per vessel-cluster (examples in a cavity . More det test). The white matter surrounding these vessel-clusters (up to 4-voxel shells) also had lower CVR compared with mirrored volumes. These differences in CVR magnitude were not significant in vessel-cluster volumes corresponding to partial or no cavitation (links.lww.com/WNL/C29).For each vessel-cluster , we assessed CVR magnitude in the vessel-cluster volume and in concentric shells in the surrounding tissue. CVR magnitude from the vessel-cluster volumes was available in 73 of 94 (76%) after excluding small volumes that were poorly coregistered with CVR maps, and expanded volumes and penumbral shells were available . In vessel-cluster volumes (including volumes up to 4-voxel expansion) corresponding to complete cavities, the CVR magnitude was lower compared with the contralateral volumes (2-sample Student p < 0.001), but this gradient was not present in clusters without full cavities , interaction p < 0.001 .Finally, in the group of vessel-clusters with complete cavitation, there was a significant linear gradient for increasing CVR magnitude values from the vessel-cluster volume through concentric penumbral shells  but are likely to be more subtle than those identified as vessel-clusters . Finally, the SWI signal may overestimate the true size of a paramagnetic structure due to blooming artefact, meaning that the actual size of the vessel-like structures in the SWI vessel-clusters may be much smaller than depicted on MRI.4 Nevertheless, the traceable appearance of small tubular structures through contiguous axial planes, some of them finally draining to the deep venous system  if they contain deoxygenated hemoglobin due to the latter's paramagnetic distortion of the magnetic field.r cohort . Howevers system , makes t21 However, further studies are needed to confirm whether and to what extent vessel-cluster may be assessed on 1.5 Tesla SWI-MRI. Low-definition MRI or the use of other gradient-echo–based techniques could in part be the reason why these findings have not been described before or might have been overlooked or considered as tortuosity of the deep medullary veins or hemorrhagic features in SVD.The results of this study should not be extrapolated to images obtained using 1.5 Tesla MRI, but it is likely that at least the more apparent vessel-clusters might be visible similarly to deep medullary veins despite lower resolution compared with 3 Tesla MRI.22 For this reason, we assessed the values of CVR measures in per-cluster analysis, using the corresponding volume in contralateral brain white matter area as a reference. This showed that CVR magnitude depended on the degree of cavitation: In the subgroup of vessel-clusters with complete cavitation, as expected, CVR in the vessel-cluster volume was lower than contralateral white matter due to CSF content. However, the surrounding white matter up to 4-voxel concentric shells also had lower CVR than the corresponding contralateral volumes. The presence of a linear gradient increasing centrifugally from the vessel-cluster through the penumbral shells confirmed that CVR was impaired around cavities with vessel-clusters. In clusters with incomplete cavitation, we did not find reductions in CVR across the volume. These results suggest that CVR is reduced in the tissue surrounding cavities because of exhausted vasodilatory reserve, while the visibility on SWI indicates that they contain deoxyhemoglobin resulting from maximum oxygen extraction,24 representing concurrent factors contributing to the blooming effect on SWI enabling the cluster visualization. On the other hand, it is possible that the noncavitated and early-cavitated vessel-clusters still maintain CVR but are approaching a stage where vascular function fails. Hence, the results from the CVR analysis have to be interpreted cautiously because of the cross-sectional design, the small sample size and variability of vessel-clusters in size, and corresponding tissue characteristics and require confirmation in longitudinal and larger cohorts.In per-patient multivariable analysis, we observed a weak relationship between decreased CVR in the white matter  and the presence of vessel-clusters. However, changes in CVR are subtle, and the influence of different factors might not be easy to control in global analysis.25 However, incident lacunes in CADASIL predominantly appear on the edges of WMH rather than following a distal vascular distribution, thus supporting the concept of alternative mechanisms.26 Lacunes have also been related to progressive pathologic changes in deep venule walls in venous collagenosis.28 As suggested in the current analysis, microvascular dysfunction could lead to gradual tissue damage ending in lacune formation that could be less symptomatic due to its gradual occurrence than in acute infarctions.30 In this scenario, vessel-clusters might represent over-dilated vessels  that cannot respond further to vasoactive stimuli in regions showing tissue degeneration. Unfortunately, the cross-sectional design of our study precludes the assessment of causal or longitudinal relationships between the vessel-clusters and white matter cavitation. However, the association of vessel-clusters with low CVR is consistent with focal loss of homeostasis and worthy of future longitudinal studies to determine the pathophysiologic significance and hypothetical role in cavitation processes leading to lacune formation.Vessel-clusters were associated with a range of stages of cavity formation in the white matter, adding further complexity to the theories of lacune formation. It is generally believed that most lacunes are the result of ischemic necrosis after infarction due to a perforating arteriolar occlusion.31The main associations of some structural radiologic SVD markers such as lacunes and WMH with vessel-cluster presence were confirmed in ordinal regression analysis for the number of vessel-clusters, while CVR magnitude was no longer significant but still showed a negative trend. Interestingly, we observed a significant association between male sex and the number of vessel-clusters, which might reflect different severity and SVD patterns depending on specific genetic and hormonal profiles. However, the differences in SVD radiologic and clinical manifestations attributable to sex are still largely unknown.The homogeneity of the imaging acquisition and processing across all enrolling centers represent major strengths of this study. This study also included patients with sporadic and genetic SVD, which allowed us to assess the new feature on SWI in patients believed to have different SVD mechanisms.This study also has some limitations. First, the size sample might not have been sufficient to detect subtle differences in vascular function measures. However, the patients of this cohort presented moderate-to-severe SVD, thus mitigating the effect of the small sample and increasing the likelihood of SVD-related findings. Second, the interrater agreement was “good,” not excellent, and external validation in independent cohorts is warranted. Third, the nature of the vessel-clusters is open to many interpretations, some of them discussed above. However, the main aim of this study was to describe the prevalence, associations, and characteristics of this new radiologic sign. Further correlates including on pathology can now be examined in further studies. Fourth, the limited z definition (3 mm) of the SWI sequences precluded a 3-dimensional analysis of the findings, and the spatial resolution of the CVR BOLD sequence that limits precise geographical localization and values within the cavitated vessel-clusters may have been affected by CSF in cavities. However, CVR magnitude values were also lower in the tissue surrounding cavities, demonstrating the impairment in vascular reserve in these penumbral areas. Finally, the presence of vessel-clusters has not been assessed in a control group without SVD. However, the likelihood of finding vessel-clusters in brains without small vessel lesions is expected to be extremely low because none of the vessel-clusters in our series corresponded to normal appearing white matter, their presence was highly dependent on burden of lacunes and WMH, and furthermore, we have not observed similar findings on SWI in any of our healthy research volunteers over many years of MRI scanning.Vessel-clusters may be observed in up to half of the patients with moderate-to-severe SVD, including both sporadic and genetic types. The vessel-clusters are related to damaged tissue showing different grades of cavitation in WMH. The properties of deoxygenated blood on susceptibility-weighted sequences and the association with lower CVR in the white matter and in the surrounding tissue in clusters with cavities suggest that the vessel-clusters represent maximal of small deep vessels and oxygen extraction in white matter that is approaching terminal injury and cavitation. The pathophysiologic significance of this new feature warrants confirmation in imaging techniques with higher resolution , pathology studies, and further longitudinal investigation that should assess whether vessel-clusters predict cavitation in SVD and even contribute to lacune formation, or are an epiphenomenon of white matter injury.


In this article we summarize the current state of knowledge about one particular chaperone family, that of the HSP70, and highlight their importance, function, and potential co-chaperone interactions. This is then discussed with regard to the suitability of these proteins and interactions for drug development.As part of their life-cycle, malaria parasites undergo rapid cell multiplication and division, with one parasite giving rise to over 20 new parasites within the course of 48 h. To support this, the parasite has an extremely high metabolic rate and level of protein biosynthesis. Underpinning these activities, the parasite encodes a number of chaperone/heat shock proteins, belonging to various families. Research over the past decade has revealed that these proteins are involved in a number of essential processes within the parasite, or within the infected host cell. Due to this, these proteins are now being viewed as potential targets for drug development, and we have begun to characterize their properties in more detail Plasmodium falciparum  which caused 241 million cases in 2020. The African continent accounted for up to 95% of these cases. Children under 5 years of age represent the most vulnerable group to the disease and account for 80% of the 627.000 deaths reported in 2020  a major role in the survival, virulence and pathogenicity of the parasite. As they lie at the heart of proteostasis, they assist in protecting parasite proteins in several situations of proteotoxic stress, including the temperature spikes caused by febrile episodes of the human host, temperature transitions taking place during transmission from the mosquito vector to the human host and vice-versa, and exposure to cytotoxic drugs. Due to their central role in such a diverse number of essential biological processes, these proteins have gained interest as potential targets for development of small molecule inhibitors. Several HSP have been shown to be upregulated in response to various drug treatments, and may play a role in helping parasites survive these stress situations . The focus of this mini-review is to collate what is currently known about the biology of PfHSP70 and PfJDP, their interactions, and what progress has so far been made in developing specific inhibitors of this important parasite Achilles Heel.Chaperones of the HSP70-class are crucial elements of the cellular protein surveillance network. They are a highly conserved family of proteins that share a very similar structure. In general, they comprise an N-terminal nucleotide-binding domain (NBD) that is able to bind ATP. Following this is a protease-sensitive linker domain leading to a substrate-binding domain to which the corresponding substrate polypeptides bind . HSP70 aJ-domain proteins  are generally co-chaperones for HSP70. They perform several tasks including recruitment of substrates to HSP70 and then stimulating the ATPase activity of HSP70. They can thus be viewed as adapters which allow a limited number of HSP70 to work on highly diverse substrates, and JDP are one of the most diverse co-chaperone families. In agreement with this, most organisms encode a higher number of JDP than HSP70 .HSP70 act as molecular chaperones. As such, they are able to bind and hold exposed hydrophobic peptide-sequences of other, aggregation-prone proteins. Beyond this “holdase” function, HSP70 are able to refold denatured proteins. A catalytic ATP-dependent interaction cycle enables the folding or refolding of substrate proteins. This essential cycle couples the ATPase activity of HSP70 to its affinity for substrate proteins . In the The enzymatic ability to hydrolyze ATP is essential for functional HSP70-substrate interaction . Based oin vitro cell culture but may be important in an infection situation  have reported inhibitors of PfHSP70 . Some ofE. coli DnaK structure  and or interfaces. In infectious disease research, this often involves finding differences between proteins found in host and pathogen. Recent research findings focused on crystallographic elucidation of the functional domains of PfHSP70 and JDP . A studytructure . The NBDtructure .While these results might lead to the conclusion that it is not viable to design inhibitors which specifically target PfHSP70-X while not affecting HsHSP70, the authors indicate that the NBD of PfHSP70-X does indeed contain potential binding sites for small-molecule activity-modulation which are structurally different in the human homologue. It is worth noting that crystallography can merely outline fixed protein structures whereas in the cell proteins  are often conformationally dynamic. This flexibility influences their binding affinity to small molecules, allosteric modulators or other proteins . Thus, tThe search for small molecule inhibitors of the Plasmodium HSP70 chaperones has identified several suitable compounds. Amongst others, these compounds are pyrimidinones, malonganenones, naphtaquinones, lipopeptides, and a catechin from green tea extract . The actP. falciparum, PfHSP70-1 is heavily associated with maintaining viability and proteostasis and is thus a prominent target of molecular inhibitory research.As the main cytosolic chaperone of β-lapachona (BBL) showed a concentration dependent inhibition of the aggregation suppression activity of PfHSP70-1  and the lipopeptide polymyxin B (PMB) were defined in the same way  and ursolic acid (UAA) feature anti-Plasmodium activity  in vivo . Both coficantly .Recently, the bis-indole violacein was tested for anti-malarial properties and possible inhibitory effects on PfHSP70-1. A significant and concentration dependent inhibition of the chaperone’s ATPase and aggregation suppression activity by violacein was observed . Thus, tin vitro binding assays, recently (Data on small molecule inhibitors of PfHSP70-2 is limited. Four commercially available GRP78 inhibitors, namely Gilvocarcin V, Apoptozole, MKT-077 and VER-155008, have been reported to exhibit broad specificity to members of the HSP70-family . They werecently . HoweverPfHSP70-X is of high interest in inhibitor research, as it is believed to assist the correct folding of exported proteins and thereby supporting parasite virulence. Similar to PfHSP70-1, the malonganenone-compounds did not modulate the basal ATPase activity of PfHSP70-X. However, napthaquinones, especially BBL, inhibited its ATPase and aggregation suppression activity in a concentration dependent manner . RegardiIn contrary to the benzothiazole rhodacyanines JG98 and JG231, the chalcone C86 did not inhibit the basal PfHSP70-X ATPase activity significantly . The autComparing the varying results of studies with multiple small molecules and particularly PfHSP70-1 and PfHSP70-X, a striking distinction regarding their susceptibility to these compounds is observable. Thus, and according to via SPR and in vitro activity assays showed an inhibitory effect of both compounds for the basal PfHSP70-Z ATPase activity. Additionally, EGCG and PMB interfere with the capability of PfHSP70-Z to suppress the aggregation of heat stress-prone proteins (Because PfHSP70-Z acts as a NEF for PfHSP70-1, inhibitors of the chaperones’ interaction have become a research target. The binding of the small molecules EGCG and PMB to PfHSP70-Z has been confirmed proteins . Alternaproteins .via small molecule inhibitors (The explicated chaperone/co-chaperone interaction offers the possibility to inhibit a single part of the network and thereby achieving a loss of function in associated metabolic pathways . Especiahibitors . The fachibitors . A numbehibitors . Similarhibitors .First promising results were achieved by examining the human, Plasmodium and yeast HSP70 in combination with the human and yeast HSP40 co-chaperones Hlj1 and Ydj1, respectively . DistincSince the export of PfHSP70-X into the RBC has been shown, this particular chaperone gained further attention in small molecule compounds and JDP-interaction research . ChangesJ or PFE0055cJ, BBL did not decrease the ATPase activity of PfHSP70-X. Even broad-spectrum HSP70 inhibitors like methylene blue and MKT-077 showed little potency against PfHSP70-X that was stimulated by its in vivo co-chaperones (As more details on the interaction of PfHSP70 with specific PfHSP40 partners emerged, new experimental data on their inhibition was obtained . Upon thaperones .The chalcone C86 and the benzothiazole-rhodacyanines JG98 and JG231 were already identified as small molecule inhibitors of the human JDP/HSP70 system. Recently, they have been used on plasmodium proteins . A signiThese results imply that the development of parasite-specific chaperone/co-chaperone-based small molecule inhibitors is a complex task, but success can be achieved .P. falciparum, they represent a promising new and sustainable drug target. Several studies successfully targeted and inhibited the PfHSP70’s and PfHSP40’s molecular chaperone activity in in vitro assays with small molecule inhibitors. However, the specificity of potential inhibitors is a critical point in research as human and Plasmodium chaperone counterparts share high structural similarity. Recent studies suggest that it may be possible to design specific small molecule inhibitors for PfHSP. Results of high-throughput screenings with derivates of already identified small molecule activity modulators and entirely new compounds are expected in the near future.This mini-review summarizes the current findings of the search for Plasmodium HSP70 and HSP40 small molecule inhibitors. As these molecular chaperones are involved in multiple important molecular-biological processes of


Hepatocellular carcinoma (HCC) is aggressive cancer with a poor prognosis. It has been suggested that the aberrant expression of LOXL2 is associated with the development of HCC, but the exact mechanism remains unclear. This research is aimed at examining the expression level and prognostic value of LOXL2 in hepatocellular carcinoma and its relationship with immune infiltration and at predicting its upstream noncoding RNAs (ncRNAs).  The transcriptome data of HCC was first downloaded from The Cancer Genome Atlas (TCGA) database to investigate the expression and prognosis of LOXL2. Then, the starBase database was used to find the upstream ncRNAs of LOXL2, and correlation analysis and expression analysis were performed. Finally, the Tumor Immune Estimation Resource (TIMER) was used to explore the association between LOXL2 and immune cell infiltration.  CARMN was considered to be the potential upstream lncRNA for the hsa-miR-192-5p/LOXL2 axis in HCC. Furthermore, the level LOXL2 was markedly positively associated with tumor immune cell infiltration and immune checkpoint expression in HCC.  Higher expression of LOXL2 mediated by microRNA (miRNA) and long noncoding RNAs (lncRNA) is associated with poor overall survival (OS), immune infiltration, and immune checkpoint expression in HCC. Hepatocellular carcinoma (HCC) is the main type of liver cancer (LC). As the sixth most common cancer in the world, it is the second largest cause of cancer-related death and the most common primary liver cancer with poor prognosis , 2. The Lysyl oxidase (LOX), an extracellular enzyme, plays a key role in the covalent cross-linking of collagen fibers by oxidizing deamino-specific lysine and hydroxylysine in the telopeptide structural domain of the collagen molecule to form allantoin . In addiIn this study, the expression level of the LOXL2 and its relationship with prognosis were first analyzed in various common cancers. Next, we found some noncoding RNAs (microRNAs (miRNAs)) and long noncoding RNAs (lncRNAs) as regulatory molecules of LOXL2 by bioinformatics analysis, so as to establish the LncRNA-miRNA-mRNA regulatory network and explore the mechanism of HCC at a deeper level. Moreover, the correlation of LOXL2 expression with immune cell infiltration, biomarkers of immune cells and immune checkpoints was finally discussed.https://portal.gdc.cancer. gov/) is a collaboration between the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI) for cancer research. It provides a large and free reference for cancer research by collecting and organizing various cancer-related histological data. A total of 33 cancer types are currently included. Data in this research was obtained from the Liver Hepatocellular Carcinoma (LIHC) cohort, and then Log2 transformed. The expression levels of LOXL2 in tumor tissues were compared with normal tissues using Wilcoxon rank sum test in the eighteen cancers , and then visualized by box plots. The analysis was performed using the R software “limma” and “ggplot2” packages [The Cancer Genome Atlas (TCGA) database (packages .http://gepia.cancer-pku.cn/) integrates TCGA cancer data with GTEx normal tissue data, exploiting bioinformatics techniques to drill down into novel cancer targets and markers. In this research, we analyzed the expression LOXL2 in tumor and normal samples and prognosis in pan-cancer by the GEPIA database [The GEPIA database (database . The boxhttps://starbase.sysu.edu.cn/) is used to analyze data related to multiple cancers integrated from the TCGA project. It provides a platform for predicting miRNA targets by searching for miRNA targets through high-throughput CLIP-Seq experimental data and degradome experimental data which include lncRNAs, miRNAs, snoRNAs, and mRNAs [The starBase database  is a comprehensive resource for the systematic analysis of immune infiltrates in diverse cancer types [The TIMER database .The expression analyses of LOXL2 in unpaired samples, paired samples, and different clinical subgroups are shown in As for survival, we further performed subgroup analyses to assess the impact of LOXL2 expression on OS of patients with HCC according to age, gender, pathological stage, and differentiation grade. We found that high-expression of LOXL2 resulted in poor survival in patients older than 60 years old and in male patients .p value was less than 0.05. Finally, only hsa−miR−192−5p met the conditions  and positively correlated with LOXL2  , and it was concluded that LOXL2 was significantly differentially expressed in HCC. The next survival analysis using GEPIA online tool revealed that LOXL2 overexpression leads to a poorer prognosis in patients with HCC. Wu et al. constructed LOXL2-small interfering RNA using a lentiviral vector and investigated the effect of LOXL2 on the proliferation of HCC cell lines by reverse transcription-quantitative polymerase chain reaction and other experimental methods . The resκB signaling [The starBase database contains seven programs that can be used to predict miRNAs, including TargetScan, miRmap, miRanda, PicTar, RNA22, PITA, and microT. Through these programs, twenty-two potential miRNAs were found for LOXL2. Among all these 22 possible miRNAs, only hsa-miR-192-5p showed a significant negative correlation with LOXL2. Subsequently, the data of miRNAs were downloaded from the TCGA database, and the differential analysis showed that hsa-miR-192-5p was lowly expressed in the tumor samples, and its low-expression was related to the poor prognosis of HCC. During the past decade, more and more studies are focusing on the role of miR-192-5p in cancers. For example, in Wang et al.'s experiment, they found that miR-192-5p-modified tumor-associated macrophages-derived exosome suppressed endometrial cancer progression by targeting IRAK1/NF-ignaling . The rolignaling . PreviouAccording to the ceRNA theory, it is known that ceRNA inhibits the inhibitory effect of miRNA on mRNA by binding to it. Therefore, it is essential to find the upstream lncRNA of hsa-mir-192-5p because it plays a role in cancer development. StarBase bioinformatics software predictions showed that CARMN may be the upstream lncRNA of miR-192-5p. The expression analysis also showed that CARMN was highly expressed in the tumor samples. There is a paucity of research on CARMN in cancers. Sheng et al. found that overexpression of CARMN can promote the prognosis and chemosensitivity in breast cancer . Other rThe tumor microenvironment is a hot topic of research in recent years. The immune microenvironment, consisting of tumor-infiltrating lymphocytes  and other immune cells , is an important part of the tumor microenvironment and is considered as the “seventh hallmark feature” of tumors, so more research is urgently needed to focus on the link between immune cell infiltration and tumors –40. In oThe limitation of this study should be mentioned. First, this is a bioinformatics study based on an online database that lacks experimental validation. Second, in identifying CARMN as a potential upstream LncRNA for hsa-miR-192-5p in HCC, serval extreme values may leverage the true correlation and may lead to overestimation of the correlation of CARMN with hsa-miR-192-5p. Third, the results need to be interpreted carefully because many of the data do not have high-correlation values.In short, CARMN was identified as a possible upstream lncRNA for miR-192-5p, which affects LOXL2 expression and promotes the progression of HCC. Our study suggests that LOXL2 may exert its tumorigenic effects by potentiating tumor immune cell infiltration and immune checkpoint expression. However, in the future, more relevant studies are still needed to verify these predictions by bioinformatics.


The impact of COVID-19 on population health is recognised as being substantial, yet few studies have attempted to quantify to what extent infection causes mild or moderate symptoms only, requires hospital and/or ICU admission, results in prolonged and chronic illness, or leads to premature death. We aimed to quantify the total disease burden of acute COVID-19 in the Netherlands in 2020 using the disability-adjusted life-years (DALY) measure, and to investigate how burden varies between age-groups and occupations. Using standard methods and diverse data sources , we estimated years of life lost (YLL), years lived with disability, DALY and DALY per 100,000 population due to COVID-19, excluding post-acute sequelae, stratified by 5-year age-group and occupation category. The total disease burden due to acute COVID-19 was 286,100  DALY, and the per-capita burden was 1640 (95% CI: 1620–1670) DALY/100,000, of which 99.4% consisted of YLL. The per-capita burden increased steeply with age, starting from 60 to 64 years, with relatively little burden estimated for persons under 50 years old. SARS-CoV-2 infection and associated premature mortality was responsible for a considerable direct health burden in the Netherlands, despite extensive public health measures. DALY were much higher than for other high-burden infectious diseases, but lower than estimated for coronary heart disease. These findings are valuable for informing public health decision-makers regarding the expected COVID-19 health burden among population subgroups, and the possible gains from targeted preventative interventions.The online version contains supplementary material available at 10.1007/s10654-022-00895-0. As in most other European countries, SARS-CoV-2 infection was first detected in early 2020 in the Netherlands. The COVID-19 epidemic during 2020 and early 2021 was characterised by two waves, the first of which peaked in mid-March then subsided to a very low level by the end of June; the second wave was distinguished by a slow increase during the summer months that transitioned into a steep rise in positive cases from about mid-September, reaching a peak in the last week of December and then dropping to a relative low by the first week of February 2021   , 5. In aBesides the strain on the healthcare system, the COVID-19 epidemic has had a large direct effect on population health. The loss of healthy life years in those affected—whether with self-limiting mild symptoms, or requiring hospital and/or ICU admission, or leading to premature death – is recognised as being substantial . In thismild/moderate (acute symptomatic illness), severe , and critical (requiring ICU care) [mild/moderate symptomatic COVID-19 can progress to severe disease and then to the critical disease state. Due to insufficient data we do not estimate the disease burden attributable to post-acute long-term sequelae ('long-COVID'). Death due to COVID-19 is assumed possible following any of these three disease states  , 6, withsevere and critical health states, respectively), data were provided by National Intensive Care Evaluations (NICE) [Because data on the incidence of each clinical severity category was not available from a single data source, we drew upon data from several sources. For non-ICU hospital admissions and ICU admissions (informing the numbers of persons in the s (NICE) . As compFor the number of COVID-19 deaths, we used data on registered COVID-19 deaths, as published by Statistics Netherlands .mild/moderate (symptomatic) infection, we used two principal data sources. The first was the third round of the national-level seroprevalence survey, the PIENTER Corona study (PICO-3), conducted in late September 2020 [For the cumulative incidence of ber 2020  to estimber 2020  (derivedber 2020  and PICOber 2020 ), where ber 2020 .mild/moderate infection consisted of age-group specific notified positive cases reported in Osiris [The second source informing the cumulative incidence of n Osiris . We thenn Osiris , which cn Osiris .Estimation of the cumulative incidence of symptomatic infection (SI) in 2020 required a two-step approach. We used PICO-3 age-specific seroprevalence to first estimate the cumulative incidence of infection in the period until the third week of September, and then integrated several data sources to estimate the cumulative incidence of infection from this date until the end of the year.For the period from 27 February until 24 September 2020 (which covers the first wave and the early part of the second wave), the cumulative SI incidence was estimated based on age-group specific seroprevalence from the PICO-3 study conducted between 22 September and 12 November 2020 . Observed seroprevalence was weighted by sex, age, ethnicity, and urbanisation to match the Netherlands population distribution in 2020, corrected for test performance  and seroFor this period, defined as 25 September until the end of 2020, we used an alternative approach to estimate cumulative SI incidence. We based this on the number of notified positive cases (in Osiris) in this period, adjusted for underascertainment. We pooled nine estimates of the ascertainment of all infected persons by notified cases based on population-level survey data from England  between 18–24 September and 22–28 November 2020). Using these data entailed making two strong assumptions: (i) testing policy, availability of tests, and willingness to be tested in England are broadly similar to the Netherlands over this period, and (ii) ascertainment does not vary with age. The pooled age-independent ascertainment estimate is 38.7% (95% CI: 36.1–41.4%). We then estimated cumulative infection incidence for the period 25 September through 31 December 2020 by synthesising estimates using this approach (while adjusting the precision of estimated ascertainment for multiple age-groups) with those from a second approach. This approach, for age-groups 30–34 and older only, was based on the observation of a relatively constant ratio between infections and hospital admissions, and involved multiplying age-group specific cumulative hospital admission ratios by the cumulative incidence as of 24 September 2020.We stratify disease burden estimates by age-group and by occupation category, and present both absolute DALY and DALY per 100,000 persons . In addition, we calculated DALY per 1000 infected persons comparing the first and second analysis periods  due to premature mortality, and years lived with disability (YLD) . YLD is To estimate YLL, conditional life expectancy values were adopted from the GBD 2019 study –23 whichFor the per-capita DALY estimates stratified by occupation category, estimates of the denominator–the total number of persons in each category (from CBS), stratified by age-group–are required. As the available information from Statistics Netherlands  containsWe first defined occupation categories according to notified case data in Osiris (Table S1), and then plotted the distribution over occupation category, stratified by 5-year age-group Fig. . EstimatWe then applied the distribution over occupation category  to both YLD and YLL. Note that deriving a single occupation distribution from the full analysis period conflates impacts from: the evolution of testing policy over time, closures of certain parts of the economy, the various (sector-specific) preventative measures in place, and the periods in which lockdown was imposed. Because a substantial proportion of notifications (14%) had occupation 'Not known', we applied simple univariate imputation to re-distribute the not-known category among the observed occupation categories.We next mapped the occupation category distribution (determined on the basis of 10-year age-groups) from Osiris to 5-year age-groups used for DALY calculation; e.g., the distribution inferred for 25–34 years was applied to both 25–29 and 30–34 years, and the assumed denominator populations for each of these two 5-year age-groups is the 10-year age-group denominator population weighted according to the national population sizes of the 25–29 and 30–34 years age-groups. Importantly, the occupation distribution is calculated separately within each age-group and applied to the DALY within each age-group. All estimates of DALY per occupation category are restricted to the 'working population' age range (defined as age 20 through 69 years).In supplementary analysis we compared the per-capita COVID-19 disease burden among individuals aged 80 years or older receiving long-term institutional health care , based on COVID-19 deaths and the total number of persons receiving long-term care published by Statistics Netherlands , 25, to Total burden of acute COVID-19 in 2020 was estimated at 286,100  DALY, of which 0.6% was contributed by YLD  between 27 February and 31 December, which corresponds to 14.8% (95% CI: 14.0–15.6%) of the total population. The cumulative SI incidence over the same period was estimated at 893,300   over this period was 31% (95% CI: 30–33%). Starting from 1 December 2020, testing was expanded to include asymptomatic persons who had travelled abroad or were identified via contact tracing. As this testing policy change affects only one month of notified case data, an unknown, though likely small, percentage of the total positives in this period were asymptomatic.The estimated age-aggregated ascertainment of cumulative incidence of infection by the cumulative number of Osiris notified positive cases . This reduction in severity over time was due to the estimated total disease burden in the second analysis period  decreasing while the cumulative incidence of infection increased .healthcare appears to bear a disproportionally high relative burden . The higher relative burden for this category also held when calculated separately per age-group, as the relative disease burden was notably higher than seen for other occupation categories starting from age-group 45–49 years . For 2020, the estimated burden for persons aged 80+ years receiving long-term care was 68,700 DALY per 100,000 persons; this was about ten times greater than the estimated 6800 DALY/100,000 persons for individuals aged 80+ years living in the community.The total disease burden due to acute COVID-19 in the Netherlands was overwhelmingly determined by premature mortality (> 99% of DALY is YLL), in particular from age 35 and up Fig. . The disUsing the relative disease burden measure , we could compare the per-capita burden between different strata of the population. The (age-aggregated) estimated burden experienced by healthcare workers  was an order of magnitude lower than the burden experienced by the oldest segment of the population  and stroke  have been previously estimated for the year 2015 . As thesIt is important to set the Netherlands estimates into the European and international context. To date DALY estimates using the COVID-19 burden protocol  have beeOur estimates covered the calendar year 2020, to facilitate comparison with the COVID-19 burden in other countries, and with the routinely reported burden of other infectious diseases in the Netherlands. However, although the peak of the second wave (based on notification data) was in December 2020, the end of this wave occurred around the end of January  we estimAs we have shown, for meaningful across-country and between-disease DALY comparisons, the same life-table must be used in the calculation of YLL. Furthermore, we have chosen to use the aspirational life expectancy approach, for which age at death is the only relevant factor. When DALY are used to inform public health decision-making, it is important that certain subpopulations  are not disadvantaged for receipt of prevention or treatment interventions because they have a lower expected remaining life expectancy than other subpopulations .Strengths of this study include making use of all relevant data sources to estimate the disease burden, and the adoption of a developed protocol for estimation of the COVID-19 disease burden. We have identified the following limitations. First, the total disease burden for the period until 31 December 2020 presented here is known to underestimate the true burden because health outcomes following the resolution of acute infection  have not yet been included. Current knowledge regarding post-COVID-19 syndrome is that it can be described as constellations of symptoms affecting different physiological systems that can vary in severity and duration , but earSecond, the estimated relative disease burden per occupation category must be interpreted with caution as there were limitations to the data sources and the consequent possible analyses. For a given occupation category, the relative disease burden was estimated for the entire analysis period and is not necessarily indicative of the recent burden; for instance, widespread availability of personal protective equipment and other risk-reducing measures may mean that the proportion of burden experienced by healthcare workers over the last half of the year was likely much reduced. Related to this point, DALY per occupation category was derived using the distribution of notified cases over the entire year, thus aggregating together periods of relatively 'open' society with periods in which strict measures were in place. The procedure also combined periods in which there was non-universal access to testing  and/or priority testing for certain occupations, such as healthcare workers and the education sector, and so the distribution of occupation categories among notified cases is influenced by access to testing; with periods in which there were minimal public health restrictions in place, with (near) universal access to testing.When strict measures were in place, some occupations could not be practiced; for others, contact patterns and ensuing transmission risk in the workplace setting might be quite different. As an example, the proportion in category 'education' will not be fully representative of the normal term-time situation with in-person teaching, due to the  continuation of online teaching after 1 June 2020, and the school holiday period. A further assumption is that the occupation provided in a notified case's Osiris record applied throughout the analysis period  inactive in their occupation, and did not become unemployed). In addition, our approach did not take into account possible variation in the risk of severe disease and/or mortality by occupation, because the occupation distribution (per age-group) is applied to the total burden (for that age-group). For instance, if (conditioning on age) healthcare workers have better underlying health and therefore better prognosis , or are Third, our estimate of the cumulative SI incidence depends on the age-specific attributable risk derived symptomatic proportion. This method estimates the proportion of infections for which symptoms can uniquely be attributed to SARS-CoV-2 infection and as such represents an lower bound for the true proportion; mild symptoms that also occur with other afflictions  are thus discounted. This would lead to an underestimation of YLD, but would have a very small impact on DALY due to the overwhelming contribution of YLL. Finally, disability duration post-hospital/ICU discharge (time until recovery) was not estimated or included, which would also lead to an underestimation of YLD, and improvements in treatment over time, potentially leading to short hospital stay, were not considered.We have presented the real-world disease burden estimates, i.e., as derived from infections that occurred during a period in which  lockdown measures were in place more often than not. We did not attempt to calculate counterfactuals – what would the disease burden have been if no measures were imposed? How much could the burden have been reduced if stricter measures were taken, or at earlier stages of the epidemic? Although such alternative scenarios are potentially useful for evaluation and future planning, for a number of infectious agents – whether the cause of large historical outbreaks or endemic situations—widespread population interventions were not been considered feasible, and so the best use of disease burden estimates is to inform planning and prioritisation based on the data generated by real-world situations.directly caused by SARS-CoV-2. The impact of health care displaced or delayed by COVID-19 patients  has been calculated to be on the order of 34,000 to 50,000 lost healthy-life years (QALY) up to 31 August 2020 [The primary focus of this work is the morbidity and mortality ust 2020 . In addiEstimates of the acute COVID-19 disease burden are important for establishing the direct impact of the SARS-CoV-2 pandemic on population health, for guiding the deployment of preventive measures, such as vaccination, and for informing economic evaluations. When examined in the context of other high-burden infectious and non-communicable diseases, it is clear that COVID-19 was responsible for a very high burden of disease in the Netherlands in 2020, despite extensive control measures. Furthermore, age was the main source of variation in estimated disease burden, driven by the sharply skewed age-specific mortality risk.Supplementary file1 (DOCX 453 KB)Below is the link to the electronic supplementary material.