Novel oral poliovirus vaccine type 2 (nOPV2) was used to control an outbreak of type 2 circulating vaccine derived poliovirus (cVDPV2) in Tajikistan, in 2021. We measured seroconversion and seroprevalence of type 2 polio antibodies in children who were reported to have received two doses of nOPV2 in outbreak response campaigns.In this community serosurvey, children born after Jan 1, 2016 were enrolled from seven districts in Tajikistan. Dried blood spot cards were collected before nOPV2 campaigns and after the first and second rounds of the campaigns and were sent to the Centers for Disease Control and Prevention  for microneutralisation assay to determine presence of polio antibodies. The primary endpoint was to assess change in seroprevalence and seroconversion against poliovirus serotype 2 after one and two doses of nOPV2.2 p=0·010). Total seroconversion after two nOPV2 doses was 77% .228 (97%) of 236 enrolled children were included in the analysis. The type 2 antibody seroprevalence was 26%  before nOPV2, 77%  after one dose of nOPV2, and 83%  after two doses of nOPV2. The increase in seroprevalence was statistically significant between baseline and after one nOPV2 dose , but not between the first and second doses . Seroconversion from the first nOPV2 dose, 67% , was significantly greater than that from the second nOPV2 dose, 44% (20/45; 30 to 60; χOur study demonstrated strong immune responses following nOPV2 outbreak response campaigns in Tajikistan. Our results support previous clinical trial data on the generation of poliovirus type 2 immunity by nOPV2 and provide evidence that nOPV2 can be appropriate for the cVDPV2 outbreak response. The licensure and WHO prequalification of nOPV2 should be accelerated to facilitate wider use of the vaccine.World Health Organization, Centers for Disease Control and Prevention, and Rotary International. Substantial progress has been made in the past several years to eradicate wild poliovirus. In 2021, only five cases of poliomyelitis caused by endemic wild poliovirus were detected; four from Afghanistan and one from Pakistan—the last two remaining endemic countries.VDPVs result from the use of live Sabin-based oral poliovirus vaccine (OPV), which in rare circumstances regains neurovirulence following prolonged circulation in under-immunised populations.Response to cVDPV2 outbreaks typically includes vaccination campaigns with OPV2-containing vaccines because inactivated poliovirus vaccines (IPV) induce insufficient intestinal mucosal immunity required to prevent infection and halt virus transmission.nOPV2 is a modified version of mOPV2, which was purposefully engineered to be more genetically stable than mOPV2, making it significantly less likely to revert into neurovirulent forms.14Evidence before this studyOn Nov 13, 2020, a novel live type 2 oral poliovirus vaccine (nOPV2) was recommended for use under WHO Emergency Use Listing (EUL). As part of the clinical development, phase 1 and phase 2 clinical trials have been completed for nOPV2 in Belgium demonstrating the safety, tolerability, and immunogenicity of the vaccine. A larger phase 2 study conducted in Panama confirmed the safety, tolerability, and immunogenicity of nOPV2 in the target population for polio outbreak response in children aged 1–4 years and infants aged 18–22 weeks. Because the authors are part of the research group on nOPV2 development, we did not conduct a formal literature search.Added value of this studyBetween Nov 13, 2020, and Oct 14, 2022, approximately 500 million nOPV2 doses have been administered in outbreak response to circulating vaccine-derived type 2 poliovirus (cVDPV2) in 23 countries. This is the first study to provide estimates of immunogenicity of nOPV2 in an outbreak response setting. We observed that vaccination with nOPV2 induced a strong immune response in children younger than 5 years in Tajikistan, in agreement with previous results from phase 1/2 clinical trials. Of note, the first nOPV2 dose resulted in significantly higher seroconversion rates than the second one.Implications of all the available evidenceWe demonstrate that high coverage campaigns provide sufficient immunity against cVDPV2 to interrupt transmission, supporting the use of nOPV2 under EUL. Phase 3 trials and longer-term evaluation of safety and genetic stability of nOPV2 are needed to receive full licensure and WHO prequalification for nOPV2.In Tajikistan, an outbreak of cVDPV2 was detected in January, 2021. A total of 36 patients with paralytic poliomyelitis have been reported in Tajikistan, with onset of paralysis between Nov 1, 2020, and July 31, 2021 .1 An env16The government of Tajikistan responded to the outbreak by conducting a series of vaccination campaigns: initially with IPV in February, 2021, to provide protection against type 2 paralysis in children who had not previously received IPV; followed by two national campaigns of nOPV2 to interrupt cVDPV2 transmission and targeting all children born after Jan 1, 2016, between May 31 and June 5, 2021, and between June 29 and July 5, 2021 .15 An adIn the routine immunisation programme in Tajikistan, children born before April 30, 2016 received trivalent OPV . From May 1, 2016, the schedule changed to bivalent OPV , which does not provide protection against type 2 poliovirus, and was administered at birth and at age 2, 3, 4, and 12 months, and one dose of IPV at age 4 months. IPV introduction into the routine immunisation schedules in Tajikistan was delayed until June 1, 2018, with the IPV catch-up campaign between Feb 15 and 20, 2021, targeting children born between May 1, 2016, and June 30, 2018. WHO/UNICEF estimates that the polio vaccine immunisation coverage in Tajikistan has been consistently higher than 90% in recent years.To provide rapid evaluation of nOPV2 immunogenicity achieved in vaccination campaign settings, we conducted a serological survey of polio antibodies in children in Tajikistan before and after they received nOPV2 during the two national outbreak response campaigns. The study was conducted in areas that were actively affected by the cVDPV2 outbreak, where exposure to circulating virus could not be controlled.This was a community-based, serological survey carried out between May 1 and July 31, 2021. Children born on or after Jan 1, 2016, residing in one of the following districts were eligible for enrolment.Epi Info (version 7) without replacement: Jaloliddin Balkhi (n=1), Dushanbe (n=10), Faizabad (n=3), Kushoniyon (n=1), Tursunzoda (n=1), Vakhdat (n=4), and Vakhsh  were selected for the study, including the capital city Dushanbe . These dhsh n=1; . ChildreThe study received ethical clearance from WHO (ERC.0003599) and the Committee of Biomedical Ethics, Ministry of Health and Social Protection of the Population, Tajikistan.The study procedures were carried out during three health centre visits. Visit 1 was in the days before the first nOPV2 campaign, visit 2 was 1 month after the first campaign of nOPV2 concluded (just before the second nOPV2 campaign), and visit 3 was 1 month after the second campaign concluded. At the first visit, after obtaining written informed consent from the child's parents or guardians, children were enrolled, and a simple demographic questionnaire was taken, which included age, gender, and poliovirus vaccination history; vaccination history of IPV (through routine immunisation or catch-up campaign) and bOPV (through routine immunisation) was recorded through vaccination cards where available, or parental recall. At the second and third visits, inclusion in the preceding nOPV2 campaign was recorded through parental recall. Trained phlebotomists generated dried blood spots (DBSs) on Whatman 903tm cards using a finger prick technique at each of the three health centre visits for each child. A third subnational nOPV2 campaign was conducted after the last visit, due to continued detection of cVDPV2, but this campaign did not affect the analysis in this study because it occurred after all blood samples were collected and the timeframe was outside of the approved protocol.The DBS cards were sent to the Centers for Disease Control and Prevention in Atlanta, GA, USA and were tested for the presence of poliovirus neutralising antibodies using standard microneutralisation assays.The primary endpoint of the study was to assess change in seroprevalence and seroconversion against poliovirus serotype 2 after one and two doses of nOPV2 administered as part of outbreak response campaigns. The secondary endpoint was to describe seroprevalence against poliovirus serotypes 1 and 3.Seropositivity for each serotype was defined as the reciprocal titre of poliovirus neutralising antibodies of 8 or more. Seroconversion was defined as the change from seronegative to seropositive  in children with antibodies at baseline. Boosting was defined as at least a 4 times increase in reciprocal titres in children that were seropositive at the initial timepoint of comparison. Analyses of seroconversion and boosting are both restricted to children aged 6 months or older at enrolment ; boosting analysis was further restricted to children with initial antibody titres of 362 or less to show a 4 times increase given the maximum reported titre of 1/1448.The total sample size was calculated to be 215 children. We assumed a 30% dropout rate during follow-up (estimated higher due to the COVID-19 pandemic) and laboratory analysis and a baseline seroprevalence of 40% to provide 90% power to detect a change in seroprevalence of at least 15%.2 test is used to determine if there was a statistically significant difference between seroprevalence across different timepoints. For univariate risk analysis of seroconversion, binomial generalised linear models were generated to calculate statistical significance.Seroprevalence, seroconversion, and boosting are presented in percentages with binomial 95% CIs. Median antibody titres are provided with IQR. Pearson's χWHO employees participated in the study design, data collection, data analysis, data interpretation, and writing of the report.%) were enrolled  at enrolment, with 19 (8%) of 228 children born between Jan 1 and April 30, 2016, who received tOPV instead of bOPV in their routine immunisation . 222 (97At visit 1 (baseline visit), 53 of 204 children (26% [95% CI 20–33]) with analysable samples were seropositive against poliovirus type 2 . BaselinAfter one dose of nOPV2 the seroprevalence against type 2 poliovirus increased to 77%  and after two doses of nOPV2 the seroprevalence against type 2 poliovirus increased to 83%  at visit 1, 7·77 (3·17–10·50) at visit 2; and 7·17 (3·83–10·17) at visit 3. The limits of detection of the assay are 2·50 log2 and 10·50 log2.The reverse cumulative antibody titres against serotype 2 at each of three visits are shown in ; 68–83; vs 44%; p= 0·010). The proportion of children that were boosted between visit 1 and 2 was 58% ; between visit 2 and 3 was 9% , and between visit 1 and 3 was 54% .Rates of seroconversion were calculated for children older than 6 months with three analysable samples (n=182). The proportion of children that seroconverted between visit 1 and 2 was 67% , between visit 2 and 3 was 44% , and between visit 1 and 3 was 77% . There was no significant difference in the rate of seroconversion between boys  and girls  or in children who were reported as receiving a dose of IPV  or those who did not receive IPV . We hypothesise that some children had been immunologically primed by one IPV dose but had not seroconverted and that these children readily responded to the first nOPV2 dose.In our study, we observed that the first nOPV2 dose resulted in significantly higher seroconversion rates than the second dose  and does not correspond with the reported IPV coverage in our study (88%), or with WHO/UNICEF estimates (97%). Type 2 seroconversion after a bOPV schedule with a single dose of IPV administered at 3–4 months has been measured between 50% and 80%; therefore, our results suggest that IPV in Tajikistan achieved a lower seroconversion or that the coverage was overestimated.% and doeWe observed that seroconversion rates were higher in the capital city Dushanbe (>90%) than in other districts (around 66%) and that this difference was borderline significant (p=0·048). This difference could indicate that the cVDPV2 circulation in Dushanbe was more intense than in the other areas surveyed, resulting in higher seroconversion in Dushanbe from a combination of the nOPV2 vaccine and natural infection with cVDPV2. The environmental surveillance site in Dushanbe consistently detected positive samples for cVDPV2 during the study period; however, environmental surveillance was not established elsewhere for comparison. Demographic or health disparities  between the capital city and other districts that affect vaccine performance could also be considered.We demonstrated uniformly high seroprevalence against poliovirus types 1 and 3, indicating successful implementation of routine immunisation with bOPV in Tajikistan . There were also no significant differences in type 1 and 3 seroprevalence rates following nOPV2 vaccination as expected.Our study had some limitations. The study was conducted under extraordinary circumstances in Tajikistan during a peak of the COVID-19 pandemic and active poliovirus outbreak, which affected availability of health-care personnel for this study. The study used simple random sampling and was conducted in areas that were affected by the active cVDPV2 outbreak; therefore, it is possible that individuals might have serologically converted in response to infection with cVDPV2. Although no cases of acute flaccid paralysis were reported in study participants, the study did not monitor shedding of cVDPV2 in participants or household contacts; however, evidence suggests that in areas with active transmission, only a small proportion of children in the community excrete poliovirus at any time.The cVDPV2 outbreak in Tajikistan has been officially closed by WHO and the Tajik Ministry of Health, with the last detected isolate in an environmental sample from Dushanbe on Aug 13, 2021. Our study demonstrates that high coverage campaigns with nOPV2 provide an immune response against cVDPV2 and probably interrupted transmission in Tajikistan, providing evidence that nOPV2 vaccine is an appropriate tool to interrupt cVDPV2 transmission during outbreaks. Phase 3 clinical trials and long-term evaluation of safety and genetic stability of nOPV2 need to be performed, to enable consideration of full prequalification from WHO for the nOPV2 vaccine.Individual participant data will not be made publicly available. Contact the corresponding author for inquiries regarding data.We declare no competing interests.


In the published article, there was an error. Due to an error during production, references in the text relating some tables were incorrect.Abstract, “Methods.” This sentence previously stated:A correction has been made to “A total of 40 nurses from four primary and secondary hospitals in Jiangxi Province and 100 patients treated with…”The corrected sentence appears below:“A total of 40 nurses from four primary and secondary hospitals in Jiangxi Province and 80 patients treated with…”Subjects and methods, “Patients,” paragraph 1–4. This sentence previously stated:A correction has been made to “A total of 100 patients in need of enteral nutrition treatment admitted from January 2020 to December 2021 were selected as the research objects. These data were obtained from four primary and secondary hospitals.Inclusion criteria: (1) Patient age >18 years; (2) indication of enteral nutrition; (3) nutritional risk screening 2002 (NRS2002) score >3; (4) normal cognitive ability; (5) hospital stay >1 week.Exclusion criteria: (1) The presence of intestinal diseases, such as intestinal ischemia, intestinal obstruction, and intestinal perforation; (2) patients with very severe craniocerebral injury or patients at a terminal stage of disease with a short life expectancy.P > 0.05).”A total of 50 patients from January to December 2020 were selected as the control group, and 50 patients from January to December 2021 were selected as the study group. There were 25 male patients and 25 female patients in the control group, with an age range of 18–75 years (average age 43.58 ± 10.53 years). The cases in the control group included 15 cases of cerebral infarction, 12 cases of acute pancreatitis, 12 cases of tumor, and seven cases of craniocerebral trauma. There were 27 female patients and 23 male patients in the study group, with an age range of 18–75 years (average age 44.25 ± 9.86 years). The cases in the study group included 13 cases of cerebral infarction, 13 cases of tumor, 12 cases of craniocerebral trauma, and 12 cases of acute pancreatitis. The general data of the two groups were comparable  Patient age >18 years; (2) indication of enteral nutrition; (3) nutritional risk screening 2002 (NRS2002) score >3; (4) normal cognitive ability; (5) hospital stay >1 week.Exclusion criteria: (1) The presence of intestinal diseases, such as intestinal ischemia, intestinal obstruction, and intestinal perforation; (2) patients with very severe craniocerebral injury or patients at a terminal stage of disease with a short life expectancy.P > 0.05).”A total of 40 patients from January to December 2020 were selected as the control group, and 40 patients from January to December 2021 were selected as the study group. There were 21 male patients and 19 female patients in the control group, with an age range of 18–75 years (average age 43.58 ± 10.53 years). The cases in the control group included 15 cases of cerebral infarction, 12 cases of acute pancreatitis, 6 cases of tumor, and seven cases of craniocerebral trauma. There were 23 female patients and 17 male patients in the study group, with an age range of 18–75 years (average age 44.25 ± 9.86 years). The cases in the study group included 13 cases of cerebral infarction, 3 cases of tumor, 12 cases of craniocerebral trauma, and 12 cases of acute pancreatitis. The general data of the two groups were comparable (The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The first sentence of the subsection Secondary malignancies (in Results) should read as follows: Three patients were diagnosed with a secondary malignancy during follow-up (Table 2).(2)the percentage of patients with seizures specified in the last column should be 21.9 (for n = 28). the total number of patients specified in the last column for Neuroendocrine sequelae should be qualified to include two patients with transient endocrine alterations.the percentage of LGG patients with Grade 0 hearing deficits should be 84.8 (for n = 39).In Table 1, the following corrections were required:


To this end, using induced neurons (i3Neurons) derived from human iPSCs lacking MAPK8IP3, we demonstrate that loss of hMAPK8IP3 affects endocytic uptake in neurons but does not affect the proteolytic activity of lysosomes in neuronal cell bodies. Our findings indicate that MAPK8IP3 may be a regulator of bulk endocytosis in neurons and that altered endocytic uptake may play a role in MAPK8IP3-linked neurodevelopmental disorders.Perturbations in endo-lysosomal trafficking pathways are linked to many neurodevelopmental and neurodegenerative diseases. Of relevance to our current study, MAPK8IP3/JIP3, a brain enriched putative adaptor between lysosomes and motors has been previously implicated as a key regulator of axonal lysosome transport. Since  De novo variants in MAPK8IP3, a putative adaptor protein believed to link cargo to dynein and kinesin motors , demonstrate that loss of this protein results in altered axonal lysosome abundance in all these models , also examined the effect of some of these variants on axonal lysosome abundance in C. elegans  derived from iPSCs lacking MAPK8IP3 , they did show defects in endocytic uptake of BSA and Dextran, cargoes that serve as markers for fluid phase endocytosis. The finding that loss of MAPK8IP3 does not adversely affect the proteolytic activity of lysosomes in neuronal cell bodies is as important as its potential role in regulating fluid-phase endocytosis. Given variants of MAPK8IP3 are linked to a neurodevelopmental disorder, identifying more neuronal cellular processes that are regulated by MAPK8IP3 will help us understand the neurodevelopmental pathology associated with the different variants.The study by Platzer and colleagues that identified 13 3Neurons were differentiated from iPSCs as described previously  coated with 0.1 mg/ml poly-L-ornithine (Sigma Aldrich) and 10 μg/ml mouse Laminin (Gibco). i3Neurons were differentiated for 14 days in Cortical Neuron Culture Medium containing KO DMEM F12 (Gibco) B27 supplement (Thermo Fisher), 10 ng/ml BDNF and NT3 (PeproTech), and 1 μg/ml mouse Laminin. i3Neurons were supplemented with Cortical Neuronal Culture Medium every 3–4 days during differentiation.The JIP3 KO/MAPK8IP3 KO iPSC line was previously generated using CRISPR-Cas9 gene editing  for a period of either 5–7 h or 2 h, followed by gentle washes [two times with warm imaging media (IM)]. They were then imaged live in IM at 37°C using a Zeiss 880 confocal microscope in Airyscan mode with a 60× objective (NA 1.4). Healthy i3Neurons were identified using brightfield mode and imaged using the 561 and 488 lasers to capture fluorescence of DQ-Red BSA and BSA-488. Fluorescence intensity for each of the two channels was measured following outlining of individual cells using Image J software and the normalized ratio of DQ-Red BSA to BSA-488 intensity was computed for each cell  to label endo-lysosomes , avacuolar ATPase inhibitor, Bafilomycin A , was used , number of cells (n), statistical test used, and 3Neurons, as read out by fluorescence of DQ-Red BSA normalized to fluorescence of BSA-488 for each cell, revealed that the MAPK8IP3 i3Neurons do not have reduced lysosomal proteolytic activity compared to Control i3Neurons  was altered in these MAPK8IP3 KO i3Neurons compared to Control i3Neurons. We found a similar decrease in Alexa-647 Dextranfluorescence in MAPK8IP3 KO i3Neurons compared to Control i3Neurons  represent loss of function mutations. For instance, the study by Platzer and colleagues which used the CRISPR-Cas9 system to target six conserved positions in C. elegans, found that two of the six human alterations resulted in elevated axonal lysosome density while five affected locomotion  could also result from reduced axonal transport to the soma of cargo-loaded endocytic vesicles. However, similar BSA uptake experiments in the hippocampal neurons suggest that the number of BSA-loaded vesicles in axons is about ten percent of those in dendrites  into cells normally occurs In conclusion, our studies reveal a role for MAPK8IP3 in regulating endocytic uptake in human neurons. Future work will focus on elucidating other molecular machinery that acts in concert with MAPK8IP3 in this pathway, as well as whether MAPK8IP3 variants linked with intellectual disability affect this pathway in neurons.The original results presented in the study are included in the article/AS performed experiments and analyzed data. AS and SG designed experiments and wrote the manuscript. SG helped with data analysis and supervised the project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A sphygmomanometer cuff was inflated to a pressure 30mmHg above diastolic pressure until StO2 reached a plateau and deflated to 0mmHg. Tissue oxygen saturation parameters were divided into resting StO2 (r-StO2) and minimal StO2 (m-StO2) at the end of the venous occlusion test. In patients, the cuff was inflated to a pressure 30mmHg above diastolic pressure for 5 min (volunteers’ time derived) or until a StO2 plateau was reached. Tissue oxygen saturation parameters were divided into r-StO2, m-StO2, and the mean time that StO2 reached ScvO2. The StO2 value at the mean time was compared to ScvO2.Observational study in intensive care unit patients. Tissue oxygen saturation was monitored  with a multiprobe (15/25mm) in the thenar position. A venous occlusion test in volunteers was applied in the upper arm to test the tolerability and pattern of StO2 plateau was 7 ± 1 minutes. We studied 22 patients. The mean time for StO2 equalized ScvO2 was 100 sec and 95 sec (15/25mm probes). The StO2 value at 100 sec  was then compared with ScvO2 (75 ± 6%). The StO2 value at 100 sec correlated with ScvO2  without discrepancy (Bland Altman).All 9 volunteers tolerated the venous occlusion test. The time for tolerability or the StO2 during a venous occlusion test.Central venous oxygen saturation can be estimated from StO Measurement of SmvO2 is an established form of monitoring global DO2/VO2 balance (oxygen extraction) in adult intensive care.Failure of oxygen delivery leads to an increase in the extraction ratio of oxygen from the blood, resulting in a decrease in mixed venous saturation  has been advocated as a simple method to evaluate changes in the relationship between supply and demand oxygen in various clinical settings.,3 Therefore, ScvO2 measurements have become an established form of monitoring systemic tissue oxygenation in critically ill patients.,5 Its measurement requires a central venous line that should reside in the superior vena cava. Thus, blood sampling collected in this location reflects systemic oxygenation mainly from the upper part of the body. Venous oxygen saturation differs among several organ systems since they have different metabolic rates.,7However, the measurement of SmvO2 are not without hazards and can be infeasible in certain scenarios. Therefore, it would be useful to find a noninvasive technique to estimate ScvO2. Near-infrared spectroscopy (NIRS) offers a technique for continuous, noninvasive, bedside monitoring of tissue oxygenation. Near-infrared spectroscopy uses the principles of light transmission and absorption to measure the concentrations of hemoglobin noninvasively in tissues and provides a global assessment of oxygenation in all vascular compartments . It has been used to assess forearm skeletal muscle oxygenation during induced reactive hyperemia in healthy adults, and it produced reproducible measurements of tissue oxygenation during both arterial and venous occlusive events.-11 Using the venous occlusion test (VOT) method, NIRS can be applied to measure changes in tissue oxygen saturation (StO2) by following the changes in the concentrations of oxygenated and deoxygenated hemoglobin (HbO2 and Hb). In this method, a pneumatic cuff is inflated to a pressure above the diastolic and below the systolic pressure for a few seconds. Such pressure blocks venous outflow but does not prevent arterial inflow, unlike the standard vascular occlusion test. As a result, venous blood volume and pressure increase,,12 and the increased pooling of venous blood causes an increase in Hb.However, measurements of ScvO2 value. In view of these observations, the aim of this study was to test the hypothesis that NIRS with VOT could be used to estimate ScvO2 in a population of critically ill patients.Thus, it is reasonable to assume that NIRS will reflect this change by decreasing the StOThis prospective observational study was conducted in the intensive care unit (ICU) of a university hospital with 33 beds of the Erasmus Medical Center - Rotterdam, Netherlands. We enrolled consecutive adult (>18 years) critically ill patients within 24 hours of ICU admission who had undergone initial resuscitation and stabilization. All patients were mechanically ventilated and had a central catheter with the tip placed in the superior vena cava. The ICU has single-person closed rooms, and the ambient temperature in each patient’s room was individually and actively set at 22°C. None of the patients had elevated bilirubin levels. We also recruited healthy volunteers with no history of receiving any vasoactive medication. The volunteers were instructed not to consume caffeine-containing drinks until after the experiments. The institutional review board approved the study. Each patient (or his or her relative) and healthy volunteer provided written informed consent.2-derived tissue oxygenation was continuously monitored using an InSpectra Tissue Spectrometer Model 650  with a multiprobe (15 and 25mm) over the thenar eminence.StO2 changes during VO. Venous occlusion was performed by arresting forearm blood flow using a conventional sphygmomanometer pneumatic cuff. All volunteers were seated with their arm rested on a table at the heart level, and StO2-derived tissue oxygenation was continuously monitored with the probe over the thenar eminence. The cuff was placed around the upper arm and was inflated to a pressure approximately 30mmHg greater than diastolic pressure until StO2 reached a plateau line or compression was not tolerated. The plateau or tolerance was considered the completion of the occlusion period, and then the cuff was rapidly deflated.Venous occlusion (VO) in healthy volunteers was designed to investigate how long a person can tolerate venous pooling of VO and to study the pattern of StO2 parameters were divided into two components: resting StO2 (r-StO2) and minimal StO2 (m-StO2) at the end of venous occlusion.VO-derived StO2 before initiation of the VOT. All measurements were made using a cooximeter. All patients had to have arterial saturation greater than 92% (Maximo Pulse Oximetry). Following the recording of ScvO2, StO2-derived tissue oxygenation was continuously monitored with the probe over the thenar eminence, and the VO test was then performed by arresting forearm blood flow using a conventional sphygmomanometer pneumatic cuff. The cuff was placed around the upper arm and was inflated to a pressure approximately 30mmHg greater than diastolic pressure for 5 minutes  or until a plateau line on the screen was reached. On the completion of the occlusion period, the occluding cuff was rapidly deflated. VO-derived StO2 parameters were divided into three components: resting StO2 values (r-StO2), the minimum StO2 value at the end of venous occlusion (m-StO2), and the time that StO2 reached the same ScvO2 of the patient (StO2 = ScvO2). The StO2 value at the mean time, when StO2 equals ScvO2, was subsequently compared with ScvO2.A blood sample  was collected from the central line placed in the superior vena cava to measure ScvOThe first measurement was performed within 24 hours of intensive care admission after hemodynamic stability was obtained  every 24 hours thereafter until Day 3.2, m-StO2, 100-StO2 and ScvO2 were linearly related to each other. To compare StO2 and ScvO2, we calculated bias, systemic disagreement between measurements (mean difference between two measurements) and precision (the random error in measuring [standard deviation of bias]).A sample size of 19 patients was estimated for a correlation coefficient of 0.6 . The results are presented as the mean ± standard deviation, unless otherwise specified. A paired t test was conducted to estimate significant differences after a normality test (Kolmogorov-Smirnov test). Bivariate correlation was used to determine whether r-StO A p value < 0.05 was considered statistically significant .The 95% limits of agreement were arbitrarily set, in accordance with Bland and Altman, as the bias ± 1.96 standard deviations.2 in all volunteers in a linear pattern, and the release of the occlusion was followed by a rapid increase in StO2 (All volunteers (n = 9) tolerated the occlusion test well, and the average tolerance time was 7 ± 1 minutes. The VO using a pneumatic cuff resulted in an immediate decrease in StO in StO2 . Conside2 and VO-derived StO2 (15mm and 25mm) for all patients. In four patients, equalization of StO2 and ScvO2 was never obtained.We included 22 ICU patients . All pat2 15mm and 25mm, respectively. Using 100 seconds as a time reference, we used the StO2 descend line of VO to select the exact StO2 value after 100 seconds of VO (100-StO2). That value was then registered and compared with ScvO2. Table 2 shows ScvO2 and VO-derived StO2 parameters stratified by the StO2 probe type. 100-StO2 was significantly correlated with ScvO2, and Bland Altman analysis revealed relevant agreement for both probes  or m-StO2 . At rest, ScvO2 values were lower than StO2 values but higher than minimal StO2 values (Table 2).We found a significant correlation between ScvO2) after a VO test moderately correlates with ScvO2. In addition, we have shown that the time to equalization of both values was close to 100 sec. This may be a method that can assess peripheral StO2 noninvasively and which can be repeated every few minutes and help to estimate ScvO2 or its trend.,12,14,15. In particular, Yoxall et al. and Wardle et al. two decades ago investigated peripheral venous hemoglobin saturation measured by using near infrared spectroscopy after venous occlusion of the forearm with partial venous occlusion to track changes in the balance between global oxygen demand and consumption in preterm infants and adults.,12,15The main finding of our study is that peripheral venous oxygen saturation  to be used in the patients. We assumed that this time length was reasonable to obtain the equalization of StO2 and ScvO2 in our patients. Similar to Yoxall et al., we have shown that the VOT was safe and feasible.,12The first part of the study evaluated VOT tolerability in healthy volunteers. We observed that the VO test until a StO2 and ScvO2 after 100 sec of the VOT. The mean time required for the StO2 value to equalize with the ScvO2 for most patients value was rather short (~100 sec), much less than the predefined 5 minutes VO duration, which makes the VO test rather accurate and safe. Central venous oxygen saturation and 100-StO2 were well correlated without significant discrepancy for both probes, which strengthens the use of StO2 after a VOT as an estimation of ScvO2.We found a moderate correlation between StO2 and m-StO2 were significantly correlated with ScvO2, but the relationship was somewhat weak. At rest, other investigators have shown a similar finding, particularly in nonseptic patients in whom the oxygen extraction capabilities are preserved.,16 Experimental and clinical studies on hypovolemic shock show that StO2 levels correlate with systemic flow variables.,17-20 However, there is uncertainty regarding this correlation when the patient is septic due to altered oxygen extraction capabilities, as has been shown previously after stagnant ischemia. In fact, half of our patients were septic, which could explain the limited correlation found in our study.Both r-StO2 values were lower than r-StO2, which is explained by two mechanisms. First, the peripheral arterial compartment is intact before stagnant ischemia. Second, critically ill patients, mainly septic patients, do show high StO2 because reduced cellular extraction of oxygen is common.,23 In turn, after the VOT, the m-StO2 values decreased considerably. We speculate that the StO2 falls below the ScvO2 during VOT because StO2 is related not only to oxygen consumption but also to the reactive vasoconstriction that occurs during vascular occlusion. Therefore, the decrease in StO2 is due to two factors: muscle VO2 and reactive vasoconstriction, which induces the sharpest decline in StO2 .Prior to the VOT, ScvO2  were followed by similar changes in 100-StO2 or m-StO2. However, 100-StO2 tracked ScvO2 on Day 2. We could not evaluate this relationship on Day 3 because only two patients were followed on the third day. This result, although imperfect to evaluate acute changes in ScvO2, suggests that the 100-StO2/ScvO2 relation is not affected by the clinical condition of the patient. It could be interesting to study this relationship in patients during acute resuscitation, as our patients were partially or fully resuscitated, as one can see by normal ScvO2 and lactate mean values.We did not evaluate whether dynamic changes in ScvOIt is important to emphasize that the results of tissue saturation found in the thenar muscle of one hand may not be reproducible in the other if the conditions of demand and/or oxygen consumption are different.2 if the patients are to mirror at least the venous saturation of the upper part of the body.The regional or peripheral venous saturation value can only be extrapolated as being SvcO2 after a VOT would rapidly track the changes in ScvO2. It is well known that significant changes in StO2 could occur after an intervention that induces ischemia and reperfusion. Our focus, however, was to assess the relation between StO2 after a VOT and ScvO2 in a single moment. Second, changes in ambient temperature at each patient’s bedside were not measured. However, the ICU consists of one-person closed rooms, and the ambient temperature in each patient room was individually controlled at 22°C. Last, the duration of the VO test (5 minutes) was based on the volunteers’ results, but we do not know whether it is appropriate in heterogeneous disease conditions. There is no support in the literature to show which method of VOT is superior or more reliable to assess the relation between ScvO2 and peripheral SvO2. In addition, we arbitrarily chose to use the StO2 value after 100 seconds of VOT based on our findings in healthy volunteers. Our strategy must be tested in different settings. Therefore, the results of our study cannot be extended to other studies that have addressed the ScvO2/StO2 relation.This study has some limitations that should be acknowledged. First, measurements during changes in the oxygen extraction ratio were not made in this study to investigate whether StO2 after a venous occlusion test in critically ill patients. We found that ScvO2 and StO2 correlate, but StO2 levels are accompanied by alterations in the peripheral circulation, indicating that StO2 abnormalities are related to regional hemodynamics and macrohemodynamics. Thus, it is not surprising that the correlation between both parameters is imperfect but still of clinical usefulness.We established the usefulness of the monitoring of StOIn conclusion, this study has shown the feasibility of frequent noninvasive measurements of peripheral venous oxygen saturation as an estimation of central venous oxygen saturation after a venous occlusion test. However, it is not yet advisable to recommend predicting absolute values of venous oxygen saturation for any given patient based solely on the noninvasive measurement of tissue oxygen saturation after a venous occlusion test, as the correlation was only moderate. Further clinical studies are required before it is considered a useful adjunct to the clinical monitoring setting.


Oral squamous cell carcinoma (OSCC) is particularly prevalent in Taiwan. The goal of this study was to determine the clinicopathological role of insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) proteins as an indicator of clinical outcomes in OSCC patients. In this study, immunohistochemical (IHC) analysis was used to examine IGF2BP2 protein expression in 244 OSCC patients. We investigated the relationships among IGF2BP2 expression, clinicopathological variables, and patient survival. Our results showed that IGF2BP2 cytoplasmic protein expression was significantly correlated with lymph node metastasis, cancer stage, and patient survival. Kaplan-Meier survival curves revealed that elevated cytoplasmic IGF2BP2 expression levels in OSCC patients were associated with poor overall survival. Moreover, multivariate cox proportional hazard models revealed that cytoplasmic IGF2BP2 expression, T status, and lymph node metastasis were independent prognostic factors for survival. In conclusion, IGF2BP2 protein was found to be a helpful predictive marker for OSCC patients, as well as a possible therapeutic target for OSCC treatment. Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the head and neck region Insulin-like growth factor (IGF) and IGF-binding protein play a vital role in the premalignant oral lesions and oral cancer IGF2BP2 has been shown to promote tumor growth in cases of solid tumors and leukemia Nonetheless, there is a pressing need to further elucidate the function of IGF2BP2 protein expression and other clinical variables in OSCC. In the current study, immunohistochemical (IHC) analysis was used to examine the expression of IGF2BP2 proteins tissue samples from 244 OSCC patients. We also examined the relationship between IGF2BP2 protein expression and OSCC clinicopathological variables and prognosis. Finally, we sought to identify potential prognostic markers to facilitate the early detection of OSCC.n = 244) were recruited from Changhua Christian Hospital in Taiwan. The most common forms of treatment included tumor removal and radical neck dissection followed by post-operative irradiation. A number of patients also received 5-fluorouracil (5-FU) and cisplatin chemotherapy. This study was also approved by the Changhua Christian Hospital's Ethics Committee in accordance with Institutional Review Board guidelines . Prior to surgery, all OSCC patients provided written informed consent.Patients . On the following day, the TMAs were tested for immune complex using a LASB 2 Kit . The TMAs were stained using aminoethyl carbazole followed by hematoxylin to detect enzyme activity. The experiment involved a positive control (pancreatic cancer tissue as a known positive case) IHC staining was performed in accordance with our previous studies The clinicopathological variables of cytoplasmic IGF2BP2 protein expression and OSCC were assessed using Fisher's exact test or the Chi-Square test. The Kaplan-Meier method was used to create overall survival curves for OSCC patients with low and high cytoplasmic IGF2BP2 protein expression, and the log-rank test was used to estimate cumulative survival rates. The Cox proportional hazard regression model was used to confirm prognostic variables of OSCC using univariate and multivariate analyses after adjusting the stage, tumor size, lymph node metastasis and cell differentiation status. A p-value of <0.05 was used to identify statistically significant results Table The expression of IGF2BP2 in OSCC cancer tissue was examined via IHC staining. As shown in Figure The role of IGF2BP2 in tumor prognosis was elucidated in terms of the relationship between IGF2BP2 expression and the overall survival of OSCC patients. In Kaplan-Meier analysis, the survival curves of OSCC patients with high IGF2BP2 expression were lower than those with low IGF2BP2 expression (p=0.003) using log-rank tests (Figure Univariate and multivariate analysis based on the Cox proportional-hazards model were used to determine the degree to which independent prognostic factors of IGF2BP2 expression affect overall survival of OSCC patients Table . UnivariCancer has become one of the most common causes of mortality among middle-aged and elderly individuals. OSCC is among the most common malignant tumors of the head and neck. The high recurrence and metastasis of the disease pose a severe threat to human health and welfare Note that the specific mechanism underlying OSCC tumorigenesis has yet to be elucidated, and there are currently no reliable early indicators for the diagnosis or prognosis of OSCC in vitro experiments Researchers have reported that IGF2BP2 is elevated in cases of malignancy. IGF2BP2 levels can also use as a prognostic indicator of acute myelocytic leukemia in vitro and in vivo testing, will be required to corroborate our data and assess the efficacy of IGF2BP2 as a therapeutic target.In the current study, we used clinical tissue samples from OSCC patients to characterize the connection between IGF2BP2 and clinicopathologic indicators. High cytoplasmic IGF2BP2 expression was strongly linked to disease stage and survival. The connections between positive IGF2BP2 protein expression and lymph node metastases, as well as between IGF2BP2 and AJCC cancer stage, suggest that IGF2BP2 may play a role in OSCC metastasis Table . Our finIn conclusion, our findings demonstrate that IGF2BP2 protein expression is prevalent in OSCC tissues, and that protein expression levels were associated with histological grade, T status, lymph node metastasis, disease stage, and survival. Our results from 244 OSCC patients show a strong link between IGF2BP2 protein levels and survival rates. Our results also show that IGF2BP2 protein expression could potentially be used as an independent OSCC prognostic predictor and/or therapeutic target for OSCC treatment.


Special AT-rich sequence-binding protein 2 (SATB2) is a new marker that could identify the colonic origin, but whether its expression is preserved in metastatic colorectal carcinomas (CRCs) remains unclear. This study was designed to investigate SATB2 validity in the identification of CRC either alone or in combination with caudal-type homeobox 2 (CDX2) and/or cytokeratin 20 (CK20). Moreover, we examined the concordance of SATB2 expression in primary CRC and paired metastatic specimen. Immunohistochemical expression of SATB2, CDX2 and CK20 was evaluated in primary CRC, 50 paired metastatic CRC and 80 non-CRC specimens. This study demonstrated that the ideal SATB2 cut-off value for recognising colonic from non-colonic origin was 10%. SATB2 was more sensitive and specific than CK20. However, it was more specific but less sensitive than CDX2. Analysing the combined markers expression, SATB2 and CDX2 combination revealed better sensitivity, specificity and larger area under curve compared to SATB2 alone, CDX2 alone and combined CDX2 and CK20. Moreover, SATB2 was able to retain its expression at the metastatic sites. SATB2 was totally concordant between primary CRC and their paired metastatic sites (concordance rate = 100%) with perfect level of agreement. SATB2 could be considered as an accurate diagnostic marker of primary and metastatic CRC. SATB2 and CDX2 is the best combination serving the highest sensitivity and specificity in detection of CRC. Colorectal cancer (CRC) is the third most prevalent cancer in both men and women and the fourth most prominent cause of cancer-related deaths worldwide . Patholoet al [It is well-established that CRCs consistently express cytokeratin 20 (CK20) and caudal-type homeobox 2 (CDX2) . Howeveret al . Taken tThe special AT-rich sequence-binding protein 2 (SATB2) is a transcription factor that binds to the nuclear matrix attachment region of DNA and hence regulates transcription and chromatin remodelling . SATB2 wThis study was designed to investigate the validity of SATB2 in the identification of CRCs either alone or in combination with CK20 and/or CDX2. Moreover, we examined the concordance of SATB2 expression in primary CRC and their paired metastatic CRCs specimens.This is a retrospective study that was carried out at the Pathology Department, Faculty of Medicine, Tanta University, during the period from April 2019 to December 2021. It included 70 cases of primary CRC as well as 80 cases of non-CRC. In addition, 50 paired metastatic specimens of the primary CRC cases  were obtained so as to analyse the concordance of SATB2 expression between primary and metastatic CRCs. The included non-CRC cases exhibited either an intestinal morphology or were poorly differentiated carcinomas. They were incorporated as follows: lung adenocarcinoma (10 cases), oesophageal adenocarcinoma (3 cases), gastric adenocarcinoma (7 cases), small intestine adenocarcinoma (7 cases), pancreatico-biliary carcinoma (8 cases), hepatocellular carcinoma (8 cases), female genital tract adenocarcinoma (19 cases), prostatic adenocarcinoma (6 cases), breast carcinoma (6 cases) and urinary bladder adenocarcinoma (6 cases). The study was approved by the Institutional Research Ethics Committee (reference # 32895).For primary CRC cases, clinical data as well as tumour-related features  were gathered from the accompanying pathology reports and medical records. Primary CRC cases were graded following the 2-tiered grading system into low and high grades, as recommended by the World Health Organization classification . PatholoFor each studied specimen, areas that adequately represent tumour tissues with appropriate preservation were identified. Necrotic areas and those with crushing artefacts were excluded. Tissue microarray (TMA) recipient blocks (6×4 arrays) were produced using the TMA builder mold . This is followed by insertion of two tissue cores from areas of interest on paraffin blocks of the studied specimens into the holes on the recipient blocks to form TMA Blocks.TM FLEX Target Retrieval Solutions was used reaching 97°C for 20 minutes. Immunostaining was accomplished using Dako Autostainer Link 48. Antibodies included in this study were SATB2 rabbit monoclonal antibody , CDX2 mouse monoclonal antibody  and CK20 rabbit monoclonal antibody . In brief, slides were kept in Peroxidase-Blocking Reagent for 5 minutes, incubated with primary antibodies for 20–30 minutes, horseradish peroxidase polymer reagent for 20 minutes and diaminobenzidine chromogen/substrate working solution for 10 minutes. Lastly, counterstaining with haematoxylin was performed.Sections (5 µm thick) mounted on positively charged slides were left to dry for 30 minutes at 37°C. Deparaffinisation and antigen retrieval were carried out using Dako PT Link unit. High pH EnVisionPositive staining was identified as brownish nuclear staining for both SATB2 and CDX2, whereas cytoplasmic and/or membranous staining was considered for positive CK20 expression. For markers scoring, percentages of positive tumour cell were considered regardless of the staining intensity. Receiver operator characteristic (ROC) curve was plotted to identify the best SATB2 cut-off point for diagnosis of CRC. The percentage located close by the point that provides maximum sensitivity and specificity was selected as the cut-off point . CDX2 anData were statistically analysed using the Statistical Package for the Social Sciences software version 23. Categorical variables were expressed as frequencies whereas numerical variables were expressed as mean ± SD. Accuracy, specificity, sensitivity, positive and negative predictive values (NPVs) were used to assess diagnostic values of the tested markers. The histopathologic diagnosis was considered the gold standard.ROC curve was used to select the best cut-off point for SATB2 through assessing the diagnostic values of different percentages of SATB2 expression. Areas under the ROC curve (AUC) of each marker were calculated; higher AUC values indicate better test performance. For evaluating the level of agreement between marker expression in primary CRC and their paired metastatic specimens, concordance rate and Kappa coefficient (κ) were applied.This study included 70 primary CRC cases with a mean age of 55.47 ± 12.78 years. Forty-five cases (64.3%) were male. Tumours were located in left colon in 31 cases (44%) and exhibited fungating appearance in 26 cases (37%). Conventional adenocarcinoma constituted the predominant histologic type (42 cases (60%)) and high-grade features were identified in 42 cases (60%). Vascular and perineural invasion were detected in 22 cases (31.5%) and 17 cases (24.2%), respectively. Tumours were associated with nodal involvement in 53 cases (75.8%) whereas distant metastasis was present in 24 cases (34.3%). As regard TNM staging, Stage III was the most frequent (29 cases (57%)). Clinicopathologic data of the studied cases are represented in Representative figures for the expression of SATB2, CDX2 and CK20 in primary CRC, paired metastatic CRC and non-CRC cases are demonstrated in p < 0.001). SATB2 had 97% positive predictive value (PPV), 95% NPV and diagnostic accuracy of 96%. The ideal SATB2 cut-off value for distinguishing colonic from non-colonic cases, in this study, was 10%.ROC curve analysis of SATB2 expression alone provided 94.3% sensitivity, 97.5% specificity and 0.975 AUC  but weaker specificity (87.5%) whereas CK20 positivity revealed the lowest sensitivity and specificity for detection of primary CRCs versus non-CRCs (sensitivity 91.4% and specificity 86.5%) as demonstrated in Next, we evaluated whether combinations of these three markers could improve the identification of a CRC origin. It was demonstrated that combining SATB2 and CDX2 revealed better sensitivity (98.5%), specificity (98.8%) and larger AUC (0.998) compared to SATB2 alone , CDX2 alone  and combined CDX2 and CK20 .When combining SATB2 and CK20, the same sensitivity as combined CDX2 and CK20 (97.1%) was obtained, but it was better than that of SATB2 alone (94.3%) and CK20 alone (91.4%). The specificity of this combination was the same as SATB2 alone (97.5%) but better than CK20 alone and combined CDX2 and CK20 .Combining the three markers provided better sensitivity (98.5%) and specificity (98.8%) than combining SATB2 and CK20 (sensitivity 97.1% and specificity 97.5%) and combined CDX2 and CK20 (sensitivity 97.1% and specificity 96.3%). Whereas it had the same sensitivity and specificity of SATB2 and CDX2 combination. Both showed the highest sensitivity (98.5%) and specificity (98.8%) as illustrated in In order to evaluate the concordance of SATB2 expression between primary and metastatic CRCs, achievable paired metastatic specimens (50 cases) were obtained. SATB2 expression was totally concordant between primary CRCs and their paired metastatic specimens (concordance rate 100% and κ coefficient = 1 denoting perfect level of agreement). As regard CDX2 and CK20, two cases lost CDX2 expression (concordance rate 96% and κ coefficient = 0.648 indicating good level of agreement) and three cases lost CK20 expression (concordance rate 94% and κ coefficient = 0.638 reflecting good level of agreement) in the paired metastatic specimens, whereas combining CDX2 and CK20 revealed concordance rate of 98% and κ coefficient of 0.790 denoting excellent level of agreement. Combining SATB2 to either CDX2 or CK20 improved the concordance (concordance rate 100% and κ coefficient = 1 denoting perfect level of agreement) as outlined in CRC metastases often simulate the histological patterns of other adenocarcinomas; differentiation between these metastases can be difficult especially in poorly differentiated adenocarcinomas . There iAnalysing SATB2 expression in primary CRC cases, the current study demonstrated that SATB2 was detected as a brownish nuclear staining in 66 94.3%) primary CRC specimens and only in 2 2.5%) non-CRC specimens. SATB2 sensitivity obtained in this work (94.3%) was close to other studies in which the sensitivity ranged from 90% to 99% % primary, 15–18. % non-CRCet al [et al [et al [From ROC curve analysis, in this study, the best SATB2 cut-off value that afforded the highest sensitivity and specificity for identifying the colonic origin was 10%. Lin et al  and De Ml [et al  reportedl [et al  consideret al [The cases included in this work were mostly conventional adenocarcinoma, as well as mucinous and signet ring cell carcinomas. Li et al  focused et al [et al [Considering the validity of the examined markers individually, this study achieved that SATB2 was more sensitive than CK20 but less sensitive than CDX2 in detection of CRC. However, SATB2 was more specific than CDX2 and CK20 similar to the findings of other reports , 26, 28.l [et al  who concCombination of different markers could improve their validity in the identification of the colonic origin. Therefore, this work was extended to investigate the validity of SATB2 in combination with CDX2 and/or CK20 in the diagnosis of CRC. Combining SATB2 with CDX2 or CK20 provided better sensitivity, specificity and larger AUC compared to SATB2, CDX2 and CK20 individually.Moreover, it was found that combining SATB2 with CDX2 provided better sensitivity, specificity and larger AUC than the commonly used combination in practice (CDX2 + CK20). It worth noting that the triple combination of SATB2, CDX2 and CK20 offered the same sensitivity, specificity and AUC as the double SATB2 and CDX2 combination. So, the current work suggested that the double combination of SATB2 and CDX2 was the best combination serving the highest sensitivity and specificity in detection of CRC.et al [et al [et al study [Similar results were detected by Dabir et al  and Salil [et al  who repoal study  reportedTo the authors’ knowledge, this is the first study to analyse the level of agreement of SATB2, CDX2 and CK20, either individually or combined, between primary CRCs and their paired metastatic specimens. In the current study, SATB2 was able to retain its expression in the corresponding metastatic lesions with a perfect level of agreement as all SATB2 positive primary CRC specimens retained SATB2 expression in their metastatic specimens. Whereas either CDX2 or CK20 alone provided only a good level of agreement as CDX2 positivity was lost in two paired metastatic specimens while three metastatic specimens lost CK20 expression. Moreover, adding SATB2 to CDX2 and/or CK20 improved their ability to detect the colonic origin in the metastatic specimens.SATB2 provides high sensitivity and specificity for establishing or ruling out the diagnosis of CRC. The optimal SATB2 cut-off value that afforded the highest sensitivity and specificity for discriminating colonic from non-colonic origin is 10%. SATB2 and CDX2 is the best combination for identifying CRC. Adding SATB2 to CDX2 and/or CK20 improves their ability to detect the colonic origin in metastatic specimens. SATB2 is able to retain its expression at the metastatic sites.The authors have no conflicts of interest to declare.The authors did not receive support from any organisation for the submitted work.The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.MSE: Data collection and interpretation of the histopathologic and immunohistochemical stained slides, captured the figures provided in this study, interpretation of the statistical analysis, drafting and writing the manuscript. DMG: Participated in interpretation of the histopathologic and immunohistochemical stained slides, statistical analysis and its interpretation, drafting and writing the manuscript. YAZ: Study design, participated in interpretation of the histopathologic and immunohistochemical stained slides, revision of the draft of the manuscript. AGN: Study design, supervision of the study, revision of the draft of the manuscript. FAE: Study design, supervision of the study, revision of the draft of the manuscript. All authors read and approved the final version of the manuscript.Protocol was approved by the Research Ethics Committee in Faculty of Medicine, Tanta University (reference# 32895/01).


Sal1 encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3′ (2′), 5′-bisphosphate nucleotidase activity and has been shown to alter abiotic stress tolerance in plants when disrupted. Precise gene editing techniques were used to generate Sal1 mutants in hexaploid bread wheat. The CRISPR  Cas9 system with three guide RNAs (gRNAs) was used to inactivate six Sal1 homologous genes within the Bobwhite wheat genome. The resulting mutant wheat plants with all their Sal1 genes disabled had slimmer stems, had a modest reduction in biomass and senesced more slowly in water limiting conditions, but did not exhibit improved yield under drought conditions. Our results show that multiplexed gRNAs enabled effective targeted gene editing of the Sal1 gene family in hexaploid wheat. These Sal1 mutant wheat plants will be a resource for further research studying the function of this gene family in wheat.The highly conserved  It provides on average 19% of the total calories in our diet and is one of the most important sources of plant protein. Bread wheat is unique among cereal crops in that it contains gluten which enables the production of leavened bread and is a major ingredient in numerous other foods [Wheat is a crucially important food source and one of the most widely grown crops in the world comprising approximately 17% of the world’s arable land and a global production of ~700 million tons , has emerged as a powerful tool for the precise manipulation of eukaryotic genomes including those of important crop plants ,12,13,14Arabidopsis and tobacco [The CRISPR/Cas system was initially shown to efficiently mediate targeted double stranded breaks in model plants such as  tobacco ,16, but  tobacco ,21,22,23 tobacco ,13,14,24Sal1 gene encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3′ (2′), 5′-bisphosphate nucleotidase enzyme activity that is hypothesized to be a plastid-localized oxidative stress sensor [Arabidopsis has shown that mutant plants lacking a functional Sal1 , exhibit drought stress tolerance [Sal1 plants exhibit a reprograming of their metabolism resulting in an increase in osmoprotectant compounds and the stress hormone abscisic acid, which manifests itself as abiotic stress tolerance [Sal1 gene also has been implicated in controlling reactive oxygen species levels and leaf senescence in Arabidopsis [Sal1 expression in wheat using a viral-based expression system has suggested that wheat lacking or with reduced Sal1 activity also exhibits drought tolerance under short-term water stress [The s sensor ,27,28,29olerance . The Salbidopsis . The trar stress .Sal1 in hexaploid bread wheat, CRISPR gene editing was used to introduce mutations within the gene family. The generation of transgenic Bobwhite wheat plants, their molecular characterization including the identification of targeted mutations and the characterization of the growth, morphology and response to abiotic stress in the mutants is presented.To investigate the role of Sal1 is a highly conserved single copy gene in the genomes of the model plants Arabidopsis thaliana and Brachypodium distachyon [Sal1 genes in wheat. stachyon . Modern Sal1: 5ABD, 7AD, and two genes on 4A . The pair of genes on 4A are less than 50 kilobase pairs (kb) apart and are highly similar except for a single base pair deletion in the seventh exon that causes a frameshift and premature stop codon in the 4A-1 allele. Although this is near the end of the coding sequence, the high degree of evolutionary conservation in this region implies that 4A-1 may be a pseudogene. All other Sal1 homeologs appear to encode a functional protein sequence. In Chinese Spring, preliminary analysis found seven different homeologs of Sal1 5D allele covering conserved amino acids in the fourth exon was BLAST searched against several other available wheat genomes , the uncultivated subspecies dicoccoides (accession Zavitan) has three copies on chromosomes 7A, 5A, and only a single functional copy on 4A (4A-2), while the cultivated subspecies durum (cv. Svevo and Kronos) also have 5B and the 4A-1 genes. Triticum spelta (accession PI190962) and Triticum aestivum cv. Norin 61 have the same seven alleles as Chinese Spring, however, most other cultivars of Triticum aestivum have lost the 5A homeolog of Sal1. The recently bred Canadian cultivars CDC Landmark and CDC Stanley have five Sal1 homeologs; they are missing 4A-2 and instead have a 4A-1 homeolog that does not have the single base pair deletion found in other wheat genomes.To investigate the evolution of this small gene family in other wheat varieties, a 203 base pair conserved section of the Chinese Spring  genomes . The AegSal1 homeologs have expression data except 7D, which was not annotated in the available datasets. Moreover, 5D is the most highly expressed gene and 5A has the lowest expression, with others being intermediate    targetinweb tool . These t alleles A, with on plants , mutatioSal1 targeting construct, the guide RNAs are flanked by transfer RNA (tRNA) spacers and are expressed using the switchgrass Ubiquitin1 (PviUbi1) promoter with the nopaline synthase (nos) transcription terminator [Cas9 gene is constitutively expressed under the control of the maize Ubiquitin 1 promoter (ZmUbi1) and nos terminator  assay to identify introduced mutations  and five of these events generated T1 progeny where the mutations were inherited. Three events carried mutations within both CAPS assays . These selected events were then propagated to the T2 and T3 generations for further characterization  in phenotyping experiments.One of the heritable events appeared to be a chimeric TSal1 target site, with the exception of the 7D site that contained a single mismatch with the 5′ gRNA. Several major rearrangements in these two events included the insertion of transgenic DNA  and 6KO2, carried mutations in every sequenced enic DNA . The incmed rice ,44,45. ASal1 5B and 5D alleles, but otherwise carried small indels in most target sites. The T0 plant was notably smaller and less fertile than other transformants, possibly as the result of somaclonal variation or the presence of unique mutations. Unlike 6KO1 and 6KO2, not all Sal1 target sites were mutated in this line; however, all sequenced genes appear to produce a nonfunctional protein. The only mutation in this line that did not result in a frameshift occurred within 4A-1 (which may be a pseudogene) and resulted in the loss of a single highly conserved amino acid that may also compromise function. This mutant line was called MM (for multiple mutant).A third independently transformed event had several missing regions in the Sal1 mutations (based on the CAPS assay screening), but were herbicide sensitive and lacked detectable transgenes. As Cas9 was no longer present to create further mutations, we used these lines to investigate the effect of specific mutations within this portion of the Sal1 gene family. These nontransgenic Sal1 mutant lines were named PM1  and PM2. PM1 has two different mutations in the 7A alleles and the 4A-1 gene targets could not be amplified. The PM2 event was missing both the 4A-1 and 4A-2 target sites and the 7D gene had a 1 bp insertion within the 5D 3′ target site. Thus, the 7A and 5B genes were the only unmutated homeologs in PM2, while PM1 had intact 5B, 5D, 4A-2, and 7D Sal1 genes  and their growth was measured and monitored during development  and a regeneration control line  were not segregating in these plants. Given that PM1 only has target mutations in the 7A and 4A-1 genes, and the PM2 line lacks the phenotype , this suggests that the Sal1 7A homeolog may have a role in stem growth and morphology in Bobwhite wheat. Although this developmental phenotype was consistently observed . Both the mutant and control lines also grew similarly well under well-watered conditions  stayed green longer than the control lines and the soil in their pots appeared to dry out more slowly .Drought in the field most often occurs during wheat flowering and seed development, so we examined the performance of the e slowly . All line slowly . These rSal1 mutant lines, their growth was examined when grown in the same pot as an equal number of wildtype Bobwhite plants, both under well-watered and drought conditions. Under these conditions the plants within the pot are competing for the same resources and should be subjected to the same degree of water stress. The 6K01, 6KO2, MM and PM1 lines co-grown with wildtype Bobwhite in a shared pot had significantly less aboveground biomass than when grown in their own pot, while Bobwhite’s biomass levels were unchanged in shared or single pots , which is an encouraging sign that CRISPR-Cas9 sequence recognition in the research presented was still highly specific.Creating gene knockouts is far more difficult in large, complex polyploid genomes such as bread wheat than in plant model systems. The multiple-targeting CRISPR-Cas9 gRNA strategy we used was successful in creating a variety of heritable Sal1 7A may have an important role in stem growth in Bobwhite. Interestingly, although Sal1 7A is not strongly expressed in shoot apical meristem compared to some of the other homeologs  showed that at least six of the seven alleles were expressed; 7D was not annotated as a gene and there is no evidence of its transcription. The expression data (www.genevestigator.com) on 19 April 2022. A 216 base pair fragment of the T. aestivum Chinese Spring Sal1 5D allele covering Exon 4, including the 5′ gRNA target site, was used to interrogate the high-quality wheat genome sequences available at GrainGenes . This region is highlighted in the Bobwhite Sal1 5D sequence shown in Bioinformatics analysis of the publicly available wheat genome sequence initially revealed seven ion data  was accehttps://primer3.ut.ee/; accessed 5 October 2021; Sal1 covering the three CRISPR/Cas9 target sites [Sal1 alleles and a potential Bobwhite 5A homeolog (which was not detected). Sanger sequence reads were aligned with the Chinese Spring sequence and annotated using Snapgene . Genomic DNA was extracted from Bobwhite wheat leaves using a modified Puregene kit protocol . Chinese Spring genomic sequences were used in Primer3 , and A. thaliana (AT5G63980) proteins were manually trimmed to remove the regions that were not sequenced in the Bobwhite Sal1 homeologs. The protein alignment was performed using Clustal Omega (CLUSTAL O (1.2.4)) at https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed 11 April 2022) with the default parameters. Note that the Sal1 5B sequence had a gap starting at residue 60, since we were unable to amplify and sequence that portion of the Bobwhite 5B sequence, likely due to the presence of a large intron nearby that contained a sequence we could not amplify across.For the lignment , T. aestSal1 transformation construct was constructed using standard restriction enzyme cloning techniques. The map and sequence of the plasmid is shown in Sal1-targeting guide RNAs  flanked by transfer RNA (tRNA) spacers, which are expressed using the switchgrass Ubiquitin 1 promoter and the nopaline synthase transcription terminator. The Cas9 gene is constitutively expressed in wheat using the maize Ubiquitin 1 promoter and nos transcriptional terminator.The CRISPR Ubiquitin 1 promoter and nos terminator controlling the expression of the bar selection gene which confers resistance to the bialaphos herbicide [® herbicide  diluted in water 1:128 . A paintbrush was used to apply a thin layer of the diluted herbicide to a 2 cm long area of a fully expanded young wheat leaf. A wildtype Bobwhite plant of the same age was painted at the same time as a control. The edges of the painted sections were marked with permanent marker, and the leaves were monitored for 3-5 days. The painted sections of transgenic herbicide-resistant leaves remained green, while wildtype sensitive leaves yellowed and/or became necrotic. Thirty-two independent Bobwhite spring wheat transgenic events carrying the CRISPR/Cas transformation construct were generated from these experiments out of 2,945 bombarded immature embryos.Transformation experiments were performed with a mixture of the CRISPR transformation plasmid construct and the pAHC20 plasmid which carries the maize erbicide . Bobwhiterbicide ,50,51. Ierbicide . After aerbicide ,50,51. Nerbicide  and geno0 wheat plants were screened using a Cleaved Amplified Polymorphic Sequences (CAPS) assay of the 3′ target site  and 8 h nights (minimum 55 °F). Plants were grown in Sunshine Mix #1 (Sungro Horticulture) in equal-sized pots (9.5 × 9.5 inches) and watered twice daily with fertilizer water (Peter’s 20-20-20 diluted as per instructions) administered by 2 drip lines per pot. During the first three weeks of growth, plants were sprayed with a baking soda solution twice weekly to control powdery mildew . Sal1 gene expression was normalized relative to the expression of the housekeeping gene CJ705892 [Sal1-specific primers were designed in Primer3 [For RNA experiments, 6 seeds of each line were planted per pot, then thinned to 5 per pot 7 days after planting. The youngest leaf on each plant was harvested 21 days after planting (approximately Zadoks stage 21-22) and snap frozen in liquid nitrogen and stored at −80 °C until processing. RNA was extracted using the Zymo Quick-RNA Plant miniprep kit , and the TURBO DNA-free kit was used to remove DNA . Sample RNA concentrations were measured using a DeNovix spectrophotometer (USA); 3 ng/µL RNA was used for each qPCR reaction. Next, 10 µL qRT-PCR reactions were performed with the iTaq Universal SYBR Green One-Step Kit  in an Applied Biosystems Quantstudio 3 Realtime PCR System With 96 well 0.1 mL block  using the standard conditions recommended in the iTaq manual. Control reactions with no reverse transcriptase were run for each sample using the CJ705892 primers to confirm that there was no DNA contamination . Sal1 geCJ705892  using th Primer3 . SamplesSal1 mutant line were planted on one side, and three Bobwhite seeds were planted on the other, then thinned to two plants per side at 12–14 days. All pots in an experiment were randomly arranged (interspersed with each other) in a 5ft. × 5ft. area on a single greenhouse bench and rearranged weekly to minimize bench position effects. Plant growth  was recorded weekly. Stomatal conductance was measured with an SC-1 Leaf Porometer (Meter Group Inc.) multiple times during two independent drought experiments.Wheat was grown in a greenhouse as described in Terminal drought experiments were started 5.5 weeks after planting. At the start of the drought experiments, all pots were hand watered to saturation (maximum soil capacity), then irrigation was halted, and they were not watered again for the duration of the experiment. Two pots were grown per line per experiment, and those placed under the drought treatment was determined by a coin flip. Control plants were watered normally with drip irrigation until their primary stems began senescing, at which point the drip lines were removed and the plants were allowed to naturally senesce. Primary stems were removed intact at harvest; their height was measured with a ruler and internodal diameters were measured with a digital caliper. The total aboveground plant biomass was then harvested into paper bags. Drought treated plants were harvested in the same way once they were fully senesced and dry. Biomass was dried in a drying oven 4–5 days at 30 C, then weighed. Afterward, the seeds were threshed and counted by hand and weighed. Statistical significance was determined by ANOVA. Three independent experiments were performed for the shared pot and drought experiments.


N = 27) including (n = 16) cloth masks (CMs), (n = 7) surgical masks (SMs), and (n = 4) N95 filtering facepiece respirators (FFRs), of which SMs and N95 FFRs taken as a standard for efficiency comparison were all tested against ambient aerosols (PM2.5 and PM10 μg/m3). The prototype cloth masks (PTCMs) (N = 5) design was tailored, and their performance was assessed and compared with that of standard commercial masks. The filtering efficiency tested against ambient coarse particulates (PM10) ranged from (5% to 34%) for CMs with an average of 16%, (37% to 46%) for SMs with an average of 42%, (59% to 72%) for PTCMs with an average of 65%, and (70% to 75%) for N95 FFRs with an average of 71%, whereas against fine particulates (PM2.5), efficacy ranged from (4% to 29%) for CMs with an average of 13%, (34% to 44%) for SMs with an average of 39%, (53% to 68%) for PTCMs with an average of 60%, and (68% to 73%) for N95 FFRs with an average of 70%, respectively. The efficiency followed the order N95 FFRs > PTCMs > SMs > CMs showing poor exposure reduction potential in CMs and high exposure reduction potential in N95 FFRs and PTCMs. Amendment in existing CMs using eco-friendly cotton fabric with better facial adherence can protect human health from exposure to fine particulates <2.5 μm and can reduce the risk of micro-plastic pollution caused by polypropylene (PP) facemasks.Inexpensive cloth masks are widely used to reduce particulate exposures, but their use became ubiquitous after the outbreak of COVID-19. A custom experimental setup (semiactive at 5.1 m/s airflow rate) was fabricated to examine the efficiency of different types of commercial facemasks collected randomly from street vendors. The sample ( The bowl-shaped topographic structure in Kathmandu Valley is surrounded by mountains that are impediments to wind movement that retains particulates in ambient air , 2, whicLong-term policies such as shifting to clean energy and short-term policies such as population-level interventions such as the use of respiratory protective devices (RPDs) might be the two effective approaches to reduce particulate exposures and other harmful airborne contaminants , 17, 18.Anecdotal evidence showed that during the Manchurian epidemic, handmade masks of cotton gauze became useful for military barracks and healthcare workers when quality commercial masks were inaccessible , 36. SimVery few studies have been done in Nepal regarding evaluating the efficacy of facemasks. The study carried out by Shakya et al.  evaluateThis experiment was conducted in 2020 from February to March and August to November, consecutively, at the open ground of North Valley School, Kathmandu. As humans are naturally exposed to the ambient atmosphere, the experiment was conducted by extracting natural ambient aerosols in sealed setups .2.5) and coarse particulates (PM10) in real-time concentration (μg/m3) were extracted at 5.1 m/s (stagnant air flow rate) and tested with two calibrated portable hand-held detectors  simultaneously between filtrate air (with facemask) and nonfiltrate air (without a facemask).This experimental setup was fabricated using a normal plyboard, polyvinyl chloride (PVC) pipe, computer fan, revolutions per minute (RPM) controller, and a 12 V (volt) direct current (DC) charger. Ambient fine particulates (PMN = 27), which includes CMs (n = 16), SMs (n = 7), and N95 FFRs (n = 4), were randomly collected from street vendors. The SMs and N95 FFRs were used as standards in this study for efficiency comparison.Twenty-seven nasopharyngeal masks  fabric or tissue paper as per the user's convenience.10 ranged from 37% to 46%, with the lowest efficacy found on SM4 (37%), and the highest efficacy found on SM6 (46%)  of 54%. The average filtering efficacy of SMs against ambient PM10 was found to be 42%.The filtering efficacy of tested surgical masks (SMs) against PMM6 (46%) . The hig2.5 ranged from 34% to 44%, with the lowest efficacy found on SM4 (34%), and the highest efficacy found on SM6 (44%)  of 56%. The average filtering efficacy of SMs against ambient PM2.5 was found to be 39%.The filtering efficacy of tested SMs against PMM6 (44%) . The hig10 ranged from 5% to 34%, with the lowest efficacy found on CM13 (5%) and the highest efficacy found on CM14 (34%)  of 66%. The average filtering efficacy of CMs against ambient PM10 was found to be 16%.The filtering efficacy of tested CMs against PM14 (34%) . The hig2.5 ranged from 4% to 29% with the lowest efficacy found on CM 13 (4%) and the highest efficacy found on CM 14 (29%)  of 71%. The average filtering efficacy of CMs against ambient PM2.5 was found to be 13%.The filtering efficacy of tested cloth masks against PM14 (29%) . The hig10 ranged from 70% to 75%, with the lowest filtering efficiency found on N95 (i), N95 (ii), and NIOSH respirator (70%) and the highest filtering efficiency found on N95 (iii) (75%) at an airflow rate of 5.1 m/s  of only 25%. The average filtering efficacy of N95 FFRs against ambient PM10 was found to be 71%.The filtering efficiency of N95 filtering facepiece respirators (FFRs) against PM 5.1 m/s . The hig2.5 ranged from 68% to 73%, with the lowest filtering efficiency found on N95 (ii) (68%), and the highest filtering efficiency found on N95 (iii) (73%)  of 27%. The average filtering efficacy of N95 FFRs against ambient PM2.5 was found to be 70%.The filtering efficiency of N95 FFRs against PMi) (73%) . The hig10 ranged from 59% to 72%, with the lowest efficacy found on PTCM4 (59%) and the highest efficacy found on PTCM1 (72%)  of 28%. The average filtering efficacy of PTCMs against ambient PM10 was found to be 65%.The filtering efficacy of prototype cloth masks (PTCMs) against PMM1 (72%) . The hig2.5 ranged from 53% to 68%, with the lowest efficacy found on PTCM4 (53%), and the highest efficacy found on PTCM1 (68%)  of 68%. The average filtering efficacy of PTCMs against ambient PM2.5 was found to be 60%.The filtering efficacy of prototype cloth masks (PTCMs) against PMM1 (68%) . The hig10 were found at 42%, 16%, 65%, and 71%, whereas against ambient PM2.5 average efficiency of SMs, CMs, PTCMs, and N95 FFRs were found at 39%, 13%, 60%, and 70%, respectively  for particulate filtration at the stated airflow rate. The efficiency followed the order N95 FFRs > PTCMs > SMs > CMs showing poor exposure reduction potential in CMs and high exposure reduction potential in N95 FFRs and PTCMs.r) analysis between variables showed a significant positive correlation  between number of mask layers and efficiency (PM10) and correlation  between number of mask layers and efficiency (PM2.5), respectively (Karl Pearson's correlation (ectively .10 and PM2.5 are found to be significantly different from each other (p < 0.01), which rejected the null hypothesis of this study.One-way analysis of variance (ANOVA) test showed that the efficiency of all face masks against PM10 aerosols than PM2.5 due to the size and shapes of the aerosols [The efficacy of nasopharyngeal masks was estimated using different methods and techniques in previous studies, which contradict each other because different studies used different methods and experimental approaches whose findings varied from study to study although studied for the same subject . The appaerosols .Poor facial fit increases the particulate penetration level known as total inward leakage (TIL) , 52 at dThe efficacy of SMs was found better than that of CMs because it has similar surface characteristics as N95 FFRs embedded with a complex network of polypropylene (PP) nanofibers forming web-like structures interconnected with each other . HoweverN95 FFRs, characterized by a complex network of multiple layers of nanofibers forming a web-like structure, melt-blown filters, and better facial adherence over the face , 47, 62 The filtering efficacy of PTCMs against fine particulates ranging from 53% to 68% in our study has a close agreement with the filtering efficacy ranged from 20% to 60% against fine NaCl particles . The aveThese modified reusable CMs featured adjustable ear loops, pockets for installing replaceable filters, a nose pin for better facial fit, and some fabric layers for better filtration of particulate exposures, resulting in an efficacy almost equivalent to N95 FFRs. Such designs can be embraced by people when commercial facemasks are shortened or not available , 66.The filtering performance of CMs was found to have poor exposure reduction potential and is marginally beneficial to human health compared to SMs and N95 FFRs. However, few amendments in inexpensive cloth mask materials and design that fits replaceable filters inside, whether PP filter or tissue paper as per that user's convenience, with the installation of a few accessories like a nose pin and adjustable ear straps for better adherence to the human face resulted in efficacy almost equivalent to standard N95 FFRs. The findings suggest that PTCMs can be potential alternatives to expensive standard masks and can play a pivotal role in reducing harmful ambient particulate exposures. The findings of this study can be helpful for the government to formulate policies and guidelines for better use of eco-friendly facemasks as well as it can also help the public regarding the proper selection of facemasks. Eco-friendly facemasks with better efficacy should be brought into mass production to replace plastic-based facemasks to protect both the environment and human health.In the semiactive measurement experiment setup , the air


Glucose transporter 10 (GLUT10) is encoded by the SLC2A10 gene. Our recent investigations have shown that GLUT10 is not only involved in glucose metabolism but also involved in the body’s immune response to cancer cells. However, the role of GLUT10 in tumor prognosis and in tumor immunity has not been reported.We knocked down SLC2A10 and performed transcriptome sequencing to analyse the biological function of GLUT10 and found that GLUT10 may be involved in immune signaling. Then, we studied the expression level of SLC2A10 in cancers by the Oncomine database and Tumor Immune Estimation Resource (TIMER) site. We also evaluated the prognostic potential of SLC2A10 in different cancers using the Kaplan‒Meier plotter database and PrognoScan online software. The correlations between SLC2A10 expression and immune infiltrates were analysed by TIMER. In addition, correlations between SLC2A10 expression and gene marker sets of immune infiltrates were analysed by TIMER and Gene Expression Profiling Interactive Analysis (GEPIA). Immunofluorescence staining of cyclooxygenase-2 (COX-2) and GLUT10 in lung cancer tissue and adjacent tissue was performed to confirm our findings from the database research.Knocking down SLC2A10 widely activated immune and inflammatory signaling. SLC2A10 was abnormally expressed in several tumors. The expression level of SLC2A10 was closely correlated with cancer prognosis. Low SLC2A10 expression was related to poorer prognosis and increased malignancy of lung cancer. Lung cancer patients with low expression of SLC2A10 have a much shorter median survival time than patients with high expression of SLC2A10. SLC2A10 expression is closely related to the infiltration of different types of immune cells, particularly macrophages. Both database research and lung cancer sample research revealed that GLUT10 might modulate immune cell infiltration via the COX-2 pathway.By transcriptome experiments, database studies, and human sample studies, we found that GLUT10 is a new immune signaling molecule involved in tumor immunity, especially in the immune cell infiltration of lung adenocarcinoma (LUAD). GLUT10 may modulate the immune cell infiltration of LUAD via the COX-2 pathway. The expression of some glucose transporters is downregulated while others are upregulated to meet the energy needs of tumor cells. Of the 14 glucose transport proteins, the roles of glucose transporter 1 (GLUT1) have been well established in a wide range of cancer types. GLUT1 overexpression was shown to promote the proliferation, invasion, and migration of malignant cells . HoweverGLUT10 is encoded by the SLC2A10 gene, and mutations in SLC2A10 result in hereditary arterial tortuosity syndrome (ATS). To date, the mechanism of ATS is still poorly understood . MeanwhiSLC2A10 is most highly expressed in smooth muscle cells and smooth muscle cell-enriched organs such as the aorta, digestive organs, prostate, and thyroid .To exploThe impact of immune infiltration on tumors has been a particular focus in recent decades. Several established databases, such as SEER and TCGA, with detailed survival and gene expression data are available to investigate the involvement and upstream molecules of immune infiltration in tumors.In this study, we used the public databases Oncomine, PrognoScan, Kaplan‒Meier Plotter, TIMER and GEPIA and IFC (immunofluorescence) of lung cancer specimens to further investigate the potential prognostic value and immunological roles of SLC2A10 expression in tumors.22.1This animal experiment protocol was approved by the Animal Research Ethics Committee of Renmin Hospital of Wuhan University. Male Sprague‒Dawley rats weighing 150–180 g were purchased from the Hubei Province Center for Disease Control and Prevention (China). Euthanasia of study animals was performed by removal of vital organs under deep anaesthesia as stated in the euthanasia policy of the UCSF laboratory (POLICY - Euthanasia.pdf (ucsf.edu)). The experiment was carried out according to the following process: Rats were intraperitoneally anaesthetized with 2% pentobarbital sodium , and the aorta was isolated in a sterile environment. The aorta was cleaned of all connective tissue, cut into small pieces and placed in a 6 cm cell culture dish. Then the cells were inoculated with fresh culture medium  . After culturing in a 37 °C incubator with 5% CO2 for 30 min, the medium was replaced after the cells grew and then replaced every 2–3 days. After the VSMCs were fully spread in the culture flask, the original medium was removed, the cells were washed twice with PBS , 1 mL of 0.5% pancreatin (both from Gibco) was added, the cells were digested at 37 °C for 2–3 min, and complete medium was added to terminate digestion. Finally, VSMCs were inoculated in culture plates. Cells from passage 3 were used in the present study .This work was supported by Data will be made available on request.The authors declare no competing interests.


The authors found that reported rates ofmyocarditis or pericarditis were higher after vaccination with mRNA-1273 compared withBNT162b2 and that for both vaccines, the rate was higher following dose 2 of the primary2-dose series when the interdose interval (the timing between dose 1 and dose 2) was 30days or less. These findings add to the body of knowledge about the association of mRNACOVID-19 vaccination with myocarditis and pericarditis and offer additional insight intothe differential risk between the 2 mRNA COVID-19 vaccine products and the possibleassociation of the interdose interval with risk of myocarditis or pericarditis.Buchan and colleagues4Public health and regulatory bodies have been using passive surveillance systems incombination with data on doses administered, clinical reports, and population-basedelectronic medical record systems to evaluate the association of COVID-19 vaccinationwith myocarditis and pericarditis.4 The evidence gathered to date supportsan association between mRNA COVID-19 vaccination and myocarditis or pericarditis; therisk appears highest for adolescent and young adult male individuals following dose 2,with symptom onset usually occurring within several days of vaccination.7Global vaccine-safety monitoring of adverse events following COVID-19 vaccinationhas been ongoing since COVID-19 vaccines became available in December 2020.1 used aCOVID-19 vaccine registry that captures doses administered and an electronic reportingsystem that collects adverse events following immunizations to further investigate thisassociation. Reported rates of myocarditis or pericarditis were generated by vaccineproduct, age, sex, and dose number as well as by the interdose interval. In summary, thereported rates for myocarditis and pericarditis among male individuals were higher thanthose among female individuals for all but 1 age group (25 to 39 years). This findingwas consistent for both dose 1 and dose 2 of BNT162b2 and mRNA-1273. Reported rates ofmyocarditis or pericarditis were higher following dose 2 for both vaccines. The highestreported rate was observed for male individuals aged 18 to 24 years following dose 2 ofmRNA-1273; the rate in this age group was more than 6 times higher than the ratefollowing dose 2 of BNT162b2. These primary findings are generally consistent withfindings from other surveillance systems across multiple countries.4Buchan and colleagues1 is the finding that a longer timeinterval between dose 1 and dose 2 in the primary mRNA COVID-19 vaccination series maybe associated with a lower risk of myocarditis or pericarditis. The overall reportedrates of myocarditis or pericarditis following dose 2 were higher for both vaccineproducts when the interdose interval was 30 days or less compared with 31 to 55 days and56 days or more. Although the absolute numbers were small, there was a consistentreduction in the rates of myocarditis or pericarditis with increasing intervals betweendoses, with the lowest rates occurring among individuals with interdose intervals of 56days or more. In addition, data from other countries indicate that vaccine effectivenessmay be higher with an interdose interval for mRNA vaccinations of 6 to 8 weeks comparedwith the 3- to 4-week interval that is recommended in the US.5 Therefore, an 8-week interval may be optimal forsome people aged 12 years or older, especially for male individuals aged 12 to 39years.5An important new contribution from the study by Buchan and colleagues1 is the finding that a heterologous second dosewith mRNA-1273  was associatedwith higher reported rates of myocarditis or pericarditis than was a homologous seconddoes with mRNA-1273 . The reasons for andsignificance of this finding are unclear, but it merits further study and replication inother data systems. This finding was not replicated when comparing a heterologous vshomologous vaccine series for BNT162b2; the homologous series had slightly higherreported rates of myocarditis or pericarditis for dose 2.Another new contribution of the study by Buchan and colleagues4 Additional analysis of Vaccine Safety Datalink data indicatedthat the risk of myocarditis or pericarditis was higher for mRNA-1273 compared withBNT162b2.6 A study conducted inDenmark also found a higher risk for myocarditis or pericarditis following mRNA-1273vaccination compared with BNT162b2 when evaluating the risk after dose 2 in maleindividuals aged 12 to 39 years.7 Thecorresponding rates of myocarditis or pericarditis within 28 days of vaccination in theDenmark study was 1.8 per 100 000 vaccinated individuals  for BNT162b2 and 9.4 per 100 000 vaccinated individuals  for mRNA-1273; dose 1 findings were notreported.Studies of myocarditis or pericarditis following mRNA COVID-19 vaccination havetended to focus on younger age groups because of their higher risk as observed inseveral vaccine-safety surveillance systems. Based on an analysis from Vaccine SafetyDatalink, an electronic medical record-based monitoring system in the US, mRNAvaccination was associated with a substantially increased risk of myocarditis orpericarditis in persons aged 18 to 39 years, with the highest risk occurring in the 0 to7 days following dose 2 of mRNA-1273 or BNT162b2.COVID-19 vaccination has prevented substantial morbidity and mortality and hasbeen the most effective primary prevention strategy against COVID-19 infection andserious complications. As the epidemiology of the COVID-19 pandemic continues to evolveand as vaccination programs expand to include younger age groups and additional boosterdoses, vigilance in monitoring for myocarditis or pericarditis and other adverse eventswill be critical to ensuring that public health and regulatory authorities have timelyand accurate safety data to weigh the benefits and risks of vaccination and makeevidence-based recommendations to protect the public and mitigate the pandemic.