Responses of sympathetic nerve activity and arterial blood pressure are augmented during activation of the exercise pressor reflex in rats with femoral artery occlusion. The present study examined the role played by proinflammatory tumor necrosis factor-α (TNF-α) in regulating augmented sympathetic responsiveness induced by stimulation of muscle metabolic receptors and static muscle contraction following 72 h of femoral artery occlusion. We first observed that the levels of TNF-α and protein expression of TNF-α receptor type 1 (TNFR1) were increased in the dorsal root ganglion (DRG) of hindlimbs with femoral artery occlusion. Note that TNF-α was observed within DRG neurons of C-fiber afferent nerves. Capsaicin (TRPV1 agonist) and AITC (TRPA1 agonist) were injected into arterial blood supply of the hindlimbs to stimulate metabolically sensitive thin-fiber muscle afferents. The effects of these injections on the sympathetic and pressor responses were further examined in control rats and rats with femoral artery occlusion. As TNF-α synthesis suppressor pentoxifylline (PTX) was previously administered into the hindlimb with femoral artery occlusion, sympathetic, and pressor responses induced by capsaicin and AITC were attenuated. In occluded rats, PTX also attenuated the exaggeration of blood pressure response induced by muscle contraction, but not by passive tendon stretch. Overall, the results suggest that TNF-α plays a role in modulating exaggerated sympathetic nervous activity via the metabolic component of the exercise pressor reflex when the hindlimb muscles are ischemic in peripheral arterial disease. Exercise activity increases sympathetic nervous activity (SNA) and decreases parasympathetic activity and thereby amplifies blood pressure (BP) and heart rate (HR), myocardial contractility and peripheral vasoconstriction . A mechaMechanically sensitive Group III afferents and chemically sensitive Group IV afferents are primarily engaged in the exercise pressor reflex . Group IWith an atherosclerotic tissue peripheral arterial disease (PAD) decreases blood flow to the arteries of the lower extremities. Intermittent claudication is the most common symptom in PAD, and this is worsened by intense exercise as a result of muscle ischemia; however, this symptom can subside at rest with a lower metabolic demand of the muscles . During The augmented exercise pressor reflex might be due to in part to inflammation, specifically pro-inflammatory cytokines (PICs), associated with PAD. Numerous cells  produce and release PICs , which i+  channels are highly expressed in group IV afferents -resistant Nafferents . The rolfferents . There iy nerves . A priory nerves .Accordingly, the emphasis of the current study was to determine the role for TNF-α in engagement of the augmented sympathetic responsiveness induced by the exercise pressor reflex. We hypothesized that femoral artery occlusion increases TNF-α in the DRG and inhibition of TNF-α production in the hindlimb muscles with femoral artery occlusion alleviates overactive sympathetic and pressor responses induced by stimulation of metabolically sensitive muscle afferents nerves. Our general hypothesis was that TNF-α contributes to the exaggerated SNA induced by muscle contraction via the metabolic component of the exercise pressor reflex when arterial blood supply to the hindlimb muscle is deficient in PAD.the Institutional Animal Care and Use Committee of Pennsylvania State College of Medicine and complied with the National Institutes of Health guidelines.All animal experimental procedures were approved by Male Sprague–Dawley rats (250–300 g) were anesthetized with an isoflurane–oxygen mixture (2–5% isoflurane in 100% oxygen). For the immunofluorescence and Western blotting experiments, the femoral artery on one limb was surgically exposed, dissected, and ligated ∼3 mm distal to the inguinal ligament as previously described . In contn = 6) and ischemic limbs (n = 14) were removed after the rats were anesthetized and decapitation. Total protein was extracted by homogenizing DRG samples in ice-cold radioimmunoprecipitation assay buffer with protease inhibitor cocktail kit. The lysates were then centrifuged for 15 min at 4°C, and the supernatants were collected for measurements of protein concentrations using a bicinchoninic acid assay reagent kit. Briefly, polystyrene 96-well immunoplates were coated with affinity-purified polyclonal goat anti-TNF-α antibody (Signosis) and incubated overnight. Then, the diluted samples and TNF-α standard solution were distributed in each plate and incubated for 1 h at room temperature. After incubation, the plates were washed and then incubated with streptavidin-conjugated HRP for 45 min at room temperature. Then, substrate solution (100 μl) was added to each well and incubated for 30 min. The optical density was measured using an ELISA reader (BioTek).We analyzed the levels of TNF-α in the DRGs instead of its levels in the circulation since TNF-α in sensory nerve system closely indicate its effects on the exercise pressor reflex evoked by activation of muscle afferent nerves. The DRGs (L4–L6) of control limbs  followed by 500 ml of 4% ice-cold paraformaldehyde in phosphate-buffered saline (PBS). DRGs (L4–6) were dissected out and immersed in the same fixative at 4°C for 2 h. The tissues were then stored in PBS containing 30% sucrose overnight. Then, 10 μm of DRG sections were obtained using a cryostat.DRG sections were fixed in 4% of paraformaldehyde in PBS for 10 min at room temperature. After being washed with PBS, the tissue were permeabilized, blocked in 0.3% Triton X-100 in PBS supplemented with 5% goat serum for 1 h, and then incubated with rabbit anti-TNF-α antibody  overnight at 4°C. After being washed in PBS, the sections were incubated with goat anti-rabbit fluorescein isothiocyanate (FITC) labeled secondary antibody  for 2 h at room temperature.To examine TNF-α within DRG neurons of C-fiber and/or A-fiber, the sections were incubated with the second primary antibody  overnight. After the sections were washed and incubated at room temperature with secondary antibody (Alexa Fluor-594 conjugated goat anti-mouse IgG at 1:200), they were cover slipped. A Nikon Eclipse 80i microscope was used to examine FITC- and Alexa Fluor-594-labeled DRG neurons, and the images were stored digitally on a computer.We used six rats to examine expression of TNF-α receptor type 1 and 2 (TNFR1 and TNFR2) in the L4–6 DRGs of control limbs and ischemic limbs. Western blot was performed as previously described . In brieAfter being denatured by heating at 95°C for 5 min in an SDS sample buffer, the supernatant samples with 20 μg of protein was loaded onto 4–20% Mini-Protean TGX Precast gels (Bio-Rad Lab) and then electrically transferred to a polyvinylidene fluoride membrane. The membrane was blocked by 5% nonfat milk in 0.1% Tween-TBS buffer for 1 h and was then incubated overnight with primary antibodies (1:500): rabbit anit-TNFR1 (Cat#: Ab19139) and rabbit anit-TNFR2 (Cat# Ab109322).Then, the membrane was incubated with horseradish peroxidase-linked anti-rabbit secondary antibody  and visualized for immunoreactivity using an enhanced chemiluminescence system . The membrane was stripped and incubated with anti-β-actin. The densities of TNFR1/TNFR2 and β-actin bands were examined using the NIH Scion Image Software.The rats were anesthetized with a mixture of 2–5% isoflurane and oxygen and ventilated as described previously . The jugA bundle of the renal nerves on the left side was dissected from other connective tissues. A piece of laboratory film was placed under the isolated nerves, and two tips of a bipolar electrode for recording neural activity were placed between the nerves and the film; these were embedded in a silicone gel. Once the gel hardened, the silicone rubber was fixed to the surrounding tissue. The renal SNA (RSNA) signal was amplified with an amplifier  with a band-pass filter of 300 Hz in low-cut frequency and of 3 kHz in high-cut frequency and recorded as previously described .Decerebration was performed in order to minimize the effects of anesthesia on the reflex pressor response. Prior to the procedure, dexamethasone  was injected to minimize brain stem edema. A transverse section was made anterior to the superior colliculus and extending ventrally to the mammillary bodies. All tissues from rostral to the section were removed. Following this procedure, the anesthesia was withdrawn from the rats and rats were on a ventilator, and 60 min were allowed before the experiment began.n = 18), occluded rats (n = 14), and occluded rats with prior TNF-α synthesis suppressor pentoxifylline . Capsaicin and AITC were given in random order. PTX (10 mg/kg/day for 3 days) was previously administered into the hindlimb muscles following femoral artery occlusion. The concentrations of capsaicin and AITC were selected on the basis of the results of the prior studies ; and TRPA1 agonist, AITC  were injected into arterial blood supply of hindlimb muscles of control rats , occluded rats (n = 10), and occluded rats with prior injection of PTX (n = 8). This experiment was to examine if inhibition of TNF-α altered the effects of femoral artery occlusion on the pressor responses evoked by muscle contraction . Although the muscle mechanoreflex component of the exercise pressor reflex is not largely involved in its overactivity in occluded rats, it was unclear if TNF-α can alter muscle metabolites in ischemic muscles and affect this component of the reflex. Thus, in order to activate the mechanical component of the exercise pressor reflex the passive tendon stretch was performed in the right hindlimb of control rats (n = 8), occluded rats (n = 6), and occluded rats with PTX (n = 6). Passive tendon stretch (500 g of tension) was produced manually over ∼5 s by using a rack and pinion attached to the Achilles’ tendon of rats. Each bout of muscle stretch was maintained for 30 s after 500 g of tension was achieved.In the second group of experiments, a laminectomy was performed to expose the lower lumbar and upper sacral portions of the spinal cord and peripheral ends of the transected L4 and L5 ventral roots were placed on platinum bipolar stimulating electrodes . Static Mean arterial pressure (MAP) was obtained by integrating the arterial signal with a time constant of 4 s. HR was calculated on a basis of beat to beat from the arterial pressure pulse. The peak responses of MAP and HR were determined by the peak change from the control value. RSNA signals were transformed into absolute values, integrated over 1 s interval, and subtracted by 1 s of integrated background noise. To quantify RSNA response to capsaicin and AITC injection, baseline values were obtained by taking the mean value for the 30 s immediately before each injection and by ascribing the mean value of 100%, and relative change from baseline during the injection were then evaluated.post hoc tests as appropriate. All values were presented as mean ± SEM. SPSS for Windows version 21.0 was used for all statistical analyses, and differences were considered significant at P < 0.05.Two-way repeated measures analysis of variance (ANOVA) was used to analyze experimental data with Tukey’s n = 6 in each group). Thus, 72 h of femoral artery occlusion was used for other experiments in this report. In another group, we further determined the effects of PTX treatment on TNF-α level 72 h after femoral artery occlusion (n = 8). Figure 1A shows that TNF-α was amplified in the DRG by femoral artery occlusion and PTX administered previously into the hindlimb muscles attenuated increases of TNF-α in the DRG induced by 72 h of femoral artery occlusion .Seventy-two hours of femoral artery occlusion significantly increased the levels of TNF-α in the DRG tissues of ischemic limbs as compare with control limbs (n = 3). The appearance of TNF-α and peripherin/NF200 within DRG neurons is characterized by fluorescent green and red color, respectively . The photomicrographs show that TNF-α largely appears within C-fiber of DRG neurons, but TNF-α barely is seen within A-fiber of DRG neurons.We also examined if TNF-α exists within DRG neurons projecting C- and/or A-fiber afferents. Peripherin was used to label DRG neurons that project thin C-fibers. NF200 was used to identify A-fibers of DRG neurons. The dual immunofluorescence techniques were employed to determine co-localization of fluorescent TNF-α and peripherin/NF200 immunoreactivity in the DRG neurons of rats without femoral artery occlusion . Figure 1C also demonstrates that no differences were observed in the expression of TNFR2 in the DRG tissues of control limbs and ischemic limbs.The protein expression of TNF-α receptors, namely TNFR1 and TNFR2, in the DRG tissues of control limbs and ischemic limbs was examined. Typical bands and average data of n = 10); 91 ± 3 mmHg and 383 ± 18 bpm in occluded rats (n = 8); and 93 ± 5 mmHg and 386 ± 22 bpm in occluded rats with PTX (n = 8). No significant differences in basal MAP and HR were observed before injections among three groups (P > 0.05 among groups). Figure 2A demonstrates that femoral artery occlusion amplified the responses of RSNA and MAP evoked by capsaicin . This figure also shows amplified RSNA and MAP responses to capsaicin in occluded rats were attenuated after inhibition of TNF-α with PTX, that is, when 0.25 μg/kg capsaicin was given, the RSNA and MAP responses were 44 ± 4% and 26 ± 2 mmHg in control rats; 73 ± 8% and 37 ± 4 mmHg in occluded rats; and 51 ± 5% and 29 ± 2 mmHg in occluded rats with PTX treatment .Baseline values for MAPs and HRs before arterial injections of capsaicin are 90 ± 4 mmHg and 388 ± 22 bpm in control rats (n = 8) and 92 ± 2 mmHg; 385 ± 15 bpm in occluded rats (n = 6) and 92 ± 4 mmHg; 391 ± 21 bpm in occluded rats with PTX (n = 8) before injection of AITC . Figures 2B demonstrates that femoral artery occlusion significantly increased the responses of RSNA and MAP evoked by 20, 40, and 60 μg/kg of AITC. Also, this figure shows RSNA and MAP responses to arterial injection AITC after PTX. PTX attenuated amplification of RSNA and MAP responses induced by injection AITC, that is, with 40 μg/kg AITC, the RSNA and MAP responses were 62 ± 6% and 32 ± 3 mmHg in control rats; 91 ± 8% and 45 ± 2 mmHg in occluded rats; and 75 ± 8% and 34 ± 4 mmHg in occluded rats with PTX treatment .Baseline MAPs and HRs were 89 ± 4 mmHg; 392 ± 23 bpm in control rats (n = 8); 93 ± 5 mmHg, 377 ± 20 bpm in occluded rats (n = 10) and 92 ± 5 mmHg, 376 ± 19 bpm in occluded rats with injection of PTX (n = 8) . Figure 3A demonstrates that femoral artery occlusion significantly increased MAP and HR responses evoked by muscle contraction. Also, this figure illustrates MAP and HR responses to muscle contraction after inhibition of TNF-α with injection of PTX in occluded rats. PTX inhibited increase of MAP response induced by muscle contraction in occluded animals. The MAP responses were 25 ± 2 mmHg in occluded rats and 17 ± 2 mmHg in occluded rats with PTX treatment . Note that there were no significant differences in muscle developed tension among three groups. Furthermore, Figure 3B shows that femoral artery occlusion amplified MAP response induced by passive tendon stretch, but PTX failed to attenuate amplification of MAP response .Baseline MAPs and HRs were 96 ± 4 mmHg, 379 ± 20 bpm in control rats (The current study examined the role played by TNF-α in augmented sympathetic responsiveness during activation of the exercise pressor reflex following 72 h of femoral artery occlusion. Our data showed that TNF-α was increased in the DRG of limbs with occluded femoral arteries and that TNF-α was present in DRG neurons supplying C-fiber afferents. Femoral artery occlusion further upregulated protein expression of TNF-α subtype receptor TNFR1. Blocking production of TNF-α by prior administration of PTX in ischemic limbs significantly attenuated enhancement of sympathetic and BP responses induced by stimulation of metabolic receptors of thin fiber muscle afferent nerves, as well as by static muscle contraction, but not by muscle stretch. These data suggest that TNF-α plays a role in modulating augmented sympathetic and BP responses in occluded femoral arteries via the metabolic component of the exercise pressor reflex.Previous studies have demonstrated that inflammation contributes to the development of intermittent claudication in PAD by adversely affecting skeletal muscle function . ElevateTNF-α plays a role by stimulating TNF-α receptor subtypes, TNFR1 and TNFR2 . TNFR1 iPTX is a phosphodiesterase inhibitor found in small clinical trials by blocking cytokine expression . Besides+ current in sensory neurons (The mechanisms by which increased TNF-α enhances the exercise pressor reflex are likely associated with changes in channels on muscle afferent nerves. A previous study demonstrated that TNF-α increased the current densities of Nav1.8 in DRG neurons . A block neurons  are alsoTRPV1 and TRPA1 in muscle afferent nerves are also likely a part of the mechanism leading to the enhanced exercise pressor reflex by activation of TNF-α. TRPV1 and TRPA1 are present in sensory nerves and respond to chemical stimuli in the processing of inflammation . It shouIn addition, we examined BP response evoked by static muscle contraction and passive tendon stretch. Muscle contraction was performed to simultaneously activate muscle mechanoreflex and metaboreflex whereas passive stretch was used to activate the mechanoreflex only. Our data demonstrated that prior administration of PTX attenuated amplification of BP response by muscle contraction, but not by tendon stretch, further suggesting involvement of TNF-α in the metabolic component of the exercise pressor reflex.A possibility cannot be ruled that TNF-α was increased in the glial cells. In addition, TNFR1 in the DRG tissues was examined by Western blot analysis. Using this method, it was unlikely to determine if the upregulation of TNFR1 of the PAD rats was specific in C-fibers.Femoral artery occlusion amplifies the levels of TNF-α in the DRG and thereby results in heightened expression of TNFR1. We further observed that increased TNF-α is present within DRG neurons of C-fiber afferent nerves. Moreover, inhibition of TNF-α production in hindlimb muscles with femoral artery occlusion alleviates exaggeration of SNA and pressor responses during stimulation of metabolically sensitive muscle afferents nerves and during activation of the metabolic component of the exercise pressor reflex.JX and JLi participated in the design of the experiment and drafting the manuscripts. JX and JLu contributed to the data collection and analysis. JLi contributed to revising the article critically for important intellectual content. All authors approved the final 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.


The aim of this study was to compare the performance of these technologies to each other and to a capnography-based reference device. Subjects were asked to decrease their RR from 14 to 4 breaths per minute (BPM) and then increase RR from 14 to 24 BPM. The effects of physiological noise, ambient noise, and head movement and shallow breathing on device performance were also evaluated. The test devices were: (1) RRa, Radical-7 (Masimo Corporation), (2) RRoxi, Nellcor™ Bedside Respiratory Patient Monitoring System (Medtronic), and (3) reference device, Capnostream20p™ (Medtronic). All devices were configured with their default settings. Twenty-nine healthy adult subjects were included in the study. During abrupt changes in breathing, overall RRoxi was accurate for longer periods of time than RRa; specifically, RRoxi was more accurate during low and normal RR, but not during high RR. RRoxi also displayed a value for significantly longer time periods than RRa when the subjects produced physiological sounds and moved their heads, but not during shallow breathing or ambient noise. RRoxi may be more accurate than RRa during development of bradypnea. Also, RRoxi may display a more reliable RR value during routine patient activities.Respiration rate (RR) is a critical vital sign that provides early detection of respiratory compromise. The acoustic technique of measuring continuous respiration rate (RRThe online version of this article (10.1007/s10877-018-0222-4) contains supplementary material, which is available to authorized users. Respiration rate (RR) is a critical vital sign that provides early detection of patient distress and respiratory compromise, a state in which there is a high likelihood of deterioration into respiratory insufficiency, respiratory failure or death. However, during respiratory compromise, specific interventions  might prevent or mitigate this deterioration , 2. CurrIncreased RR (i.e. hyperventilation) may reflect microcirculatory failure induced by underlying medical conditions as sepsis, pulmonary embolism and congestive heart failure . The mic2) monitoring  during both moderate and deep sedation . Values are reported as mean ± SD unless otherwise noted and statistical significance was set at p < 0.05 for all analyses.For the analysis of the accuracy of the evaluated devices during monitored RR changes , a Mixed Model analysis based on the differences between the two accuracy errors were used. The non-parametric related samples Wilcoxon test with α level of 0.05 was used to analyze the data other than accuracy error rates and absolute value error. Accuracy error rates were computed for each test device using the following: accuracy error rate = |RRThe study population included 29 healthy subjects (21 men and 8 women), with an age of 23.6 ± 4.4 years, a BMI of 24.6 ± 3.8, and a neck circumference of 36.0 ± 7.4 cm. Subject skin color ranged from white to olive skin tone. Five subjects reported a prior history of smoking and six subjects were current smokers (4.67 cigarettes per day on average). One subject was treated with insulin for type 1 diabetes and one subject had a history of childhood asthma.All the devices were well tolerated by all subjects and no adverse events were reported.oxi was more accurate than RRa. When examining only periods of bradypnea, RRoxi indicated similar values to the reference device for a significantly longer time than RRa. The absolute value error and the accuracy error rate during bradypnea of RRoxi was significantly lower than that of RRa. However, during tachypnea, RRoxi indicated similar values to the reference device for shorter time than RRa but no differences in accuracy error rate or absolute value error were observed. The accuracy estimation with 95% confidence intervals (CI) for proportions of misdetection of bradypnea at the default alarm setting was 0.99% (95% CI 0.17–1.82%) for RRoxi and 5.41% (3.13–7.69%) for RRa, resulting in a significant difference between devices (p = 0.002).Comparing the overall accuracy errors rate and absolute value errors, RRThe results are detailed in Table oxi was more accurate than RRa, with significant differences during bradypnea and normal breathing and non-significant differences during tachypnea. The estimation of the proportion of misdetection of bradypnea was 0.57% (95% CI 0–1.23%) for RRoxi and 10.25% (7.08–13.42%) for RRa, resulting in a significant difference between devices (p < 0.001).Overall, throughout the session, RRThe results are detailed in Table a had significantly more episodes in which it did not display a value and a false alarm was triggered, as compared to RRoxi , but there were no significant differences in the percentage of time that the devices showed a value . The total recorded time from all subjects was 3418 s. Of the 569 s that RRoxi did not display a value, RRa displayed the same value (± 2 BPM) as the reference device for 285 s (50.1%). Of the 996 s that RRa did not display a value, RRoxi displayed the same value (± 2 BPM) as the reference device for 285 s (29.5%).During 2 min of shallow breathing (24 BPM), RRa had significantly more episodes in which it did not display a value and a false alarm was triggered, as compared to RRoxi, and the percentage of time that RRoxi displayed a value was significantly longer than the percentage of time that RRa presented a value. Analysis of specific physiological noises indicated that during groaning, talking and coughing, RRoxi displayed a value for longer time than RRa while no significant differences were seen for snoring.During the physiological noises session, RRThe results are detailed in Table oxi = 98.16%, RRa = 98.30%).Ambient noises had no effect on the devices’ ability to display a value and both devices displayed a value for more than 98% of the time . Also, the percentage of time that RRoxi displayed a value was significantly longer than RRa displayed a value . For head movements in lateral directions and circular movement, but not for anterior/posterior directions, RRoxi displayed a value for a longer time than RRa did.During the head movement session, RRThe results are detailed in Table oxi device utilized is not intended for use in identifying apnea and therefore was not part of the apnea analysis. Compared to the reference device, RRa detected 26 of 63 (41.3%) breath cessation events. Of the breath cessation events, RRa did not detect 4 of 14 (28.6%) events that were over 60 s and did not detect 2 of 8 (25.0%) events that were over 90 s. Apneas were detected on average 13.5 s after the reference device. For the sensor removal evaluation, the overall time to detect inactivity was 44.5 ± 3.2 s in the first session and 47.7 ± 2.4 s in the second session. Changes to the position of the RRa neck sensor had no effect on the device working time .The RRa and RRoxi was compared during a wide variety of subject breathing rates, using capnography as a reference standard. The different breathing rates and changes in these rates were intended to simulate respiratory compromise patterns which may appear in patients with bradypnea  or in patients with tachypnea (e.g. patients with sepsis or heart failure). The impact of both physiological and ambient noise on the ability of the tested devices to display an accurate RR were also evaluated. While the overall accuracy of both methods was relatively high, the findings of the study suggest that RRoxi may be more accurate than RRa during the development of bradypnea. Our results also indicate that physiological noises and certain head movements are more likely to adversely impact the performance of RRa as compared to RRoxi, suggesting that RRa may be more sensitive to noise artifacts and patient movement than the pulse-oximetry-based RRoxi technology.In this study, the accuracy of RRa compared favorably to capnography, with modest but statistically higher accuracy and precision [a method [a device was seen to be reduced compared to that of the RRoxi device during physiological noise challenges, and also in response to certain head movements.Our study results are different from a 2013 study of 33 post-surgical patients, which found that RRrecision . Howevera method . Indeed,a failed to detect almost 60% of breath cessation events lasting more than 30 s and 25% of events lasting more than 90 s. In our analysis, RRoxi successfully detected bradypnea (< 9 BPM) over 99% of the time as opposed to a detection rate of slightly < 90% for RRa. This data suggest that RRoxi may be better suited than RRa for the continuous monitoring in patients at risk of respiratory depression, such as post-operative patients receiving opioid analgesia.A study by Kitsiripant et al.  comparedoxi may be preferred over RRa for patients who are able to talk and cough, and for patients with abrupt changes in breathing and multiple bradypnea sessions (such as patients who have received opioids), but in patients with normal RR or tachypnea, the differences were not clinically significant.The evaluation of Nellcor™ Bedside Respiratory Patient Monitoring System PM1000N and Radical-7® during a controlled wide range of breathing patterns and rates, and in the face of physiological and ambient noise challenges, provides insight into the technical performance of these devices and may give the clinician better understanding about the functioning and accuracy of the devices in different types of patients. The study indicates that the RRa and RRoxi were clinically significant, although these differences were found to be statistically significant. Secondly, the study used artificial, subject-controlled changes in breathing rate that mimic bradypnea or tachypnea in patients but do not reflect the natural variations in breathing rate which would be observed in a patient population, including both sedated and non-sedated patients. Finally, the physiological noises evaluated, including groaning, snoring, and coughing, were generated by the subjects on demand rather than in response to actual physiological stimuli. Despite these limitations, our results serve to expand our understanding of the accuracy and real-world performance of RRa and RRoxi for the continuous monitoring of respiratory rate while addressing some of the noted limitations of prior studies.However, controlled assessment has its limitations. First, the study employed healthy volunteers, thus the accuracy and performance of these devices in distinct patient populations, including patients predisposed to respiratory depression  remains unknown. Similarly, our analysis of non-hospitalized subjects did not and could not address whether or not the observed differences between RRoxi was more accurate and reliable than RRa during the development of bradypnea and during routine patient activities. While the clinical implications of these observed differences are unknown, further study into the accuracy and real-time performance of these methods for continuous monitoring of respiratory rate in a true clinical setting is warranted.RRBelow is the link to the electronic supplementary material.Supplementary material 1 (DOCX 26 KB)


Objective: Resveratrol, a safe and multitargeted natural agent, has been linked with inhibition of survival and invasion of tumor cells. Tumor Necrosis Factor-β (TNF-β) (Lymphotoxin α) is known as an inflammatory cytokine, however, the underlying mechanisms for its pro-carcinogenic effects and whether resveratrol can suppress these effects in the tumor microenvironment are poorly understood. Methods: We investigated whether resveratrol modulates the effects of 5-Fluorouracil (5-FU) and TNF-β on the malignant potential of human colorectal cancer (CRC) cells (HCT116) and their corresponding isogenic 5-FU-chemoresistant derived clones (HCT116R) in 3D-alginate tumor microenvironment. Results: CRC cells cultured in alginate were able to migrate from alginate and the numbers of migrated cells were significantly increased in the presence of TNF-β, similar to TNF-α, and dramatically decreased by resveratrol. We found that TNF-β promoted chemoresistance in CRC cells to 5-FU compared to control cultures and resveratrol chemosensitizes TNF-β-induced increased capacity for survival and invasion of HCT116 and HCT116R cells to 5-FU. Furthermore, TNF-β induced a more pronounced cancer stem cell-like (CSC) phenotype  and resveratrol suppressed formation of CSC cells in two different CRC cells and this was accompanied with a significant increase in apoptosis (caspase-3). It is noteworthy that resveratrol strongly suppressed TNF-β-induced activation of tumor-promoting factors  and epithelial-to-mesenchymal-transition-factors  in CRC cells. Conclusion: Our results clearly demonstrate for the first time that resveratrol modulates the TNF-β signaling pathway, induces apoptosis, suppresses NF-κB activation, epithelial-to-mesenchymal-transition (EMT), CSCs formation and chemosensitizes CRC cells to 5-FU in a tumor microenvironment. The worldwide incidence of colorectal cancer (CRC) has risen to approximately 1.2 million new cases and 0.6 million deaths annually [A key feature to support and modulate colon cancer progression is the result of complex interaction of tumor cells with their microenvironment ,4,5. It Whereas TNF-α acts as an initiator and modulator of tumorigenesis has been intensively studied , the rolIt is believed that tumor progression, resistance and metastasis are driven by a subpopulation of cells with stem cell like characteristics ,21. ThesBesides surgery, adequate chemotherapy with 5-Fluorouracil alone or in combination with other chemotherapeutics  is the gold standard of CRC therapy ,28. LimiThe natural polyphenol resveratrol  is a phytoalexin produced in several plants such as grapes, peanuts and various herbs, protecting them against UV-radiation, oxidative stress and fungal infections ,37,38. PThus, in the present study we investigated the modulatory effect of resveratrol in a TNF-β-mediated inflammatory tumor microenvironment on malignity of 5-FU resistant and non-resistant CRC cells during early stages of tumorigenesis in monolayers and 3D-alginate culture model. Antibodies to ALDH1, CD44, CD133 were purchased from antibodies online and antibody to β-Actin was from Sigma-Aldrich Chemie . Antibodies to MMP-9 and activated caspase-3 were purchased from R&D Systems . Antibodies to CXCR4 were purchased from Abcam PLC  and to phospho-specific p65 (NF-κB) were from Cell Technology . Anti-vimentin, anti-slug and anti-E-cadherin were purchased from Santa Cruz Biotechnology . Secondary alkaline phosphatase–linked sheep anti-mouse and sheep anti-rabbit antibodies for immunoblotting were from EMD Millipore  and secondary antibodies for fluorescence labeling from Dianova . The antibodies were applied at dilutions suggested by the manufacturers.Cell culture growth medium was prepared as previously described ,44. All 2 and the medium was changed every two to three days. Cells were passaged at 80–90% confluency using trypsin/EDTA.The human colon cancer cell line HCT116 was purchased from the European Collection of Cell Cultures . From this cell line, a 5-Fluorouracil (5-FU) resistant cell line (HCT116R) was generated as previously described . Cell cu6/mL, added drop wise into a 100 mM CaCl2 solution at ambient temperature  and washed with 0.15 M NaCl solution and with serum-starved medium before starting treatment. All subsequent treatments were carried out in serum starved medium.Alginate tumour microenvironment culture for 3-dimensional cultivation of CRC cells has been previously described ,41,44. BStarved HCT116 and HCT116R cells were cultured in 3-dimensional alginate culture as described above to investigate invasion and migration capacity. In an additional set of experiments, CRC cells were left untreated, treated with 10 ng/mL TNF-α, 10 ng/mL TNF-β, treated with 5 µM resveratrol alone, 5-FU (0.1 or 1 nM) alone, or a combination of 1 nM 5-FU and either 10 ng/mL TNF-α or 10 ng/mL TNF-β, or a combination of 5 µM resveratrol and 1 nM 5-FU alone or in combination with either 10 ng/mL TNF-α or 10 ng/mL TNF-β. At day 10, cells migrated through the alginate matrix and adhered at the bottom of the petri dish were fixated with Karnowsky and stained with toluidine blue for 10 min. The number of adhered colonies was quantified by counting all colonies under a light microscope .p < 0.05 are designated by an asterisk (*); p < 0.01 by two asterisks (**).Immunofluorescent investigation was performed on HCT116 and HCT116R monolayer cultures that migrated from alginate cultures after 10 days with anti-ALDH1 and anti-CD44. Alginate cultures were left untreated or treated with either 5 µM resveratrol alone, 10 ng/mL TNF-α, 10 ng/mL TNF-β or a combination of 5 µM resveratrol and either 10 ng/mL TNF-α or 10 ng/mL TNF-β. Immunofluorescence labelling was performed as previously described . Brieflyp < 0.05 (*); p < 0.01 (**).DAPI  nuclear staining assay was performed to assess the number of apoptotic changes induced by TNF-α, TNF-β, 5-Fluorouracil (5-FU) and resveratrol and their combination in HCT116 and HCT116R cells as previously described . Brieflyp < 0.05 (*); p < 0.01 (**).In an additional set of experiments, alginate beads from HCT116 and HCT116R CRC cells were either left untreated, treated with 5 µM resveratrol alone, 10 ng/mL TNF-α, 10 ng/mL TNF-β, 1 nM 5-FU or a combination of 1 nM 5-FU with either 10 ng/mL TNF-α or 10 ng/mL TNF-β, or a combination of 5 µM resveratrol and 1 nM 5-FU alone or additionally with either 10 ng/mL TNF-α or 10 ng/mL TNF-β for 10 days. Subsequently, cells were fixed with Karnowsky fixative and the ultrastructure of cells were evaluated as described previously ,44. StatHCT116 and HCT116R CRC cells were cultured in alginate bead culture and either left untreated, treated with either 5 µM resveratrol alone, 10 ng/mL TNF-α, 10 ng/mL TNF-β, 0.1 and 1 nM 5-FU or a combination of 0.1 and 1 nM 5-FU with either 10 ng/mL TNF-α or 10 ng/mL TNF-β, or a combination of 5 µM resveratrol and 1 nM 5-FU alone or with either 10 ng/mL TNF-α or 10 ng/mL TNF-β for 10 days and immunoblotting performed as previously described .p value of <0.05 was considered to establish statistically significant differences.Experiments were performed three times as individual experiments with three individual replicates. For statistical analysis, a Wilcoxon–Mann–Whitney test was applied. Data were shown as mean values ± SD or SEM and were compared by one-way, or two-way or a three-way ANOVA using SPSS Statistics, if the normality test passed (Kolmogorov–Smirnov test). A The aim of this study was to examine the potential role of TNF-β to induce an inflammatory microenvironment to promote CRC cell malignancy alone or during treatment with 5-FU in human CRC cells (HCT116 and HCT116R) in a 3D-alginate tumor microenvironment. We used a well-characterized 3D-alginate tumour microenvironment culture model that allows examination of the early, initial steps of tumorigenesis such as invasion and migration of cancer cells. Furthermore, we investigated the modulatory effects of resveratrol on TNF-β-mediated inflammatory signaling in the treatment of CRC either alone or in combination with 5-FU.6/mL) were cultured in an alginate-based matrix, treated as described in detail in Material and Methods and the capacity of migration and invasion was determined through evaluation of colony-formation with toluidine blue staining after 10 days. Treatment of the CRC cells with 5-FU  alone significantly blocked migration of HCT116 cells through the alginate in a dose dependent manner (p < 0.05) or resveratrol (5 µM) and co-treatment with 5-FU (1 nM) and TNF-β (10 ng/mL) or TNF-α (10 ng/mL) enhanced dramatically inhibition of the invasion ability of HCT116 and HCT116R cells through the alginate-based matrix compared to untreated cells  were cultured in an alginate-based tumor microenvironment, treated as described in detail in Material and Methods and cells that had migrated through the alginate beads and formed adhered monolayer culture on the bottom of the glass dish were examined. These monolayer cultures were subjected to immunofluorescence labeling with primary antibodies for colon CSC markers (CD44 and ALDH1). We found, that moderate expression of CD44 and ALDH1 was detected in basal control in HCT116 and HCT116R cells  and whether resveratrol could modulate TNF-induced CSC formation. Furthermore, a large body of literature indicates that formation and proliferation of CSC-like cells are induced by inflammatory cells, cytokines and growth factors in the tumor microenvironment ,50,51,526R cells A,B. Inte6R cells A,B, indi6R cells A,B, demo6R cells A,B.Resveratrol blocks TNF-β-induced CSC-formation and chemosensitizes CSC to 5-FU in pro-inflammatory alginate tumor microenvironment cultures.To confirm the immune-cytochemical observations that TNF-β-induced CSCs formation in an alginate tumor microenvironment and that resveratrol can modulate this effect and additionally to demonstrate the chemosensitization effect of resveratrol to 5-FU on CSC markers  expression, western blotting analysis was performed C,D. AlgiTo examine whether the suppressing effect of resveratrol and/or 5-FU on TNF-β-induced cell viability is related to the promoting of apoptosis, HCT116 I,II and Because resveratrol can reduce (modulate) the colony formation and invasion effects of TNF-β and enhance 5-FU-effects in 3D alginate beads, HCT116 I,II and To explore the underlying molecular mechanism of how resveratrol suppresses TNF-β-induced malignancy of CRC cells and chemosensitizes to 5-FU, we investigated whether the effects of resveratrol on CRC cells in TNF-β-induced pro-inflammatory alginate tumor microenvironments was associated with the inhibition of NF-κB activation and NF-κB-regulated gene products involved in tumor metastasis. Indeed, it has been reported that cytokines, like TNF-α, execute their pro-inflammatory effects primarily through activation of NF-κB, regulating the expression of inflammatory genes involved in invasion and metastasis ,53. SeruWe further investigated whether resveratrol can modulate NF-κB-dependent gene products involved in apoptosis (cleavage of caspase-3) of CRC cells in alginate tumor microenvironment. As shown in Resveratrol inhibits TNF-β-induced epithelial-to-mesenchymal transition of CRC cells and chemosensitizes to 5-FU in alginate microenvironment culturesIn order to get more insights into the functional suppressive roles of resveratrol in TNF-β-induced tumor malignity in CRC cells and in the process of the synergistic effect with 5-FU in relation to epithelial-to-mesenchymal transition (EMT), we have examined the expression of EMT-associated signaling molecules, such as E-cadherin, vimentin and transcription factor slug . It has been reported that EMT is related to CSC-like cell formation, up-regulation and development of resistance to chemotherapeutic agents  and thatThe current study was designed to examine, on one hand, the potential pro-inflammatory role of TNF-β (lymphotoxin α) to promote tumour inflammatory microenvironment and thus enhance the malignant potential of CRC cells individually or during treatment with the chemotherapeutic drug 5-FU. On the other hand, it was designed to investigate the potential of resveratrol  to blockIn this report, we have found the following novel findings: (1) Resveratrol suppressed the TNF-β-, similar to TNF-α-induced invasion and viability in with 5-FU-treated CRC cells in 3D-alginate tumor microenvironment; (2) Resveratrol suppressed TNF-β-, similar to TNF-α-enhanced survival in with 5-FU-treated CRC cells by promoting apoptosis ; (3) Moreover, resveratrol inhibited TNF-β-induced formation of CSC-like cells and chemosensitized CSC to 5-FU; (4) Finally, we showed for the first time, that resveratrol abrogated TNF-β-, similar to TNF-α-mediated expression of NF-κB activation, NF-κB-dependent tumorigenic gene products  and EMT-associated signaling molecules  of HCT116 and HCT116R in alginate tumor microenvironment cultures; highlighting resveratrol as a potent inhibitor of TNF-β-induced tumor malignancy and increasing thereby the chemosensitivity of the CRCs to 5-FU mediated, at least in part through the suppression of NF-κB transcription factor.We found that TNF-β, similar to TNF-α significantly induced migration and invasiveness of both CRC cell lines from the 3D-based alginate beads matrix. Interestingly, the migration of HCT116R cells was clearly increased compared to the HCT116 adhered cells on the bottom of petri dishes, indicating an increase in cancer malignancy, highlighting, inflammatory cytokines in tumor microenvironment support cancer cell viability and metastasis. Moreover, we have shown that resveratrol alone or in combination with 5-FU suppressed or potentiated the effects of 5-FU through its effects as an anti-metastasis drug on HCT116 and HCT116R cells metastasis in alginate-based 3D culture model. It was noted that 5-FU resistant cells were more sensitive to chemotherapeutic agents, such as resveratrol or the resveratrol and 5-FU combination, represented a potential treatment strategy for 5-FU resistant colon cancer. Indeed, multiple pieces of evidence indicate that development and progression of carcinomas is the result of complex interaction of tumor cells with their immediate microenvironment ,5. ActivNext, we investigated the expression of surface and intercellular molecules linked to cancer stem cell (CSC) markers in human colorectal cancer cells, including CD133, CD44 and ALDH1, which are most widely used to characterize CSC ,67. We fWe have shown that resveratrol in combination with 5-FU had a synergistic effect, in increasing the anti-tumor effects of 5-FU including inhibition of invasion and increasing apoptosis in parental HCT116 and HCT116R cells, highlighting that resveratrol chemosensitizes HCT116R cells to 5-FU-based drug regimens. These findings are also in agreement with previous findings that resveratrol exhibits synergistic activity with 5-FU against tumor cells . DiverseSpecific tumor cell death related to the synergistic effects of CRC treatment with a combination of resveratrol and 5-FU was evaluated by multiple parameters, such as DAPI nuclear staining, ultrastructural electron microcopy and apoptosis-associated protein biomarkers. We found that resveratrol significantly induced apoptotic nuclear chromatin condensation and apoptotic bodies in TNF-β-, similar to TNF-α-treated cells. We also further demonstrated that a significant increase in apoptosis and nuclear chromatin condensation with the combination of resveratrol with 5-FU and/or TNF-β or TNF-α compared with either treatment alone in both CRC cell lines. Thus, these results revealed that the resveratrol and 5-FU combination represents a potential treatment option for TNF-β-mediated inflammatory microenvironment on survival and malignity of 5-FU resistant colon cancer.Several lines of evidence have shown that inflammation stimulates chemokines in the tumor microenvironment, which plays an essential role and is a requisite of both cancer cell promotion and cancer progression ,63,76. AWe found major gene products associated with activation of EMT and tumorigenesis were up-regulated (vimentin and slug) and down-regulated (E-cadherin) through TNF-β and resveratrol decreased significantly TNF-β-induced expression of vimentin and this was accompanied with increasing of E-cadherin expression via inhibition of NF-κB and slug activity. These results correlated with the activation of viability and migration and CSC formation induced by TNF-β and 5-FU in tumor microenvironment in both CRC cells. Furthermore, we have found that TNF-β additionally caused dramatically morphological changes on the ultrastructural level, which clearly pointed out a mesenchymal phenotype . Moreover, these findings revealed that deficit of E-cadherin and increase of vimentin causes disruption of cell-cell contact and migration of cancer cells from epithelial assembly to surrounding tissue. Resveratrol promotes cell-cell junction, apoptosis and thus blocks EMT, which is consistent with the results from our laboratory that resveratrol induced an epithelial conversion in CRC cells in tumor microenvironment with decreasing of vimentin and slug and increasing of E-cadherin . InteresOverall, our results suggest that resveratrol may suppress multiple pathways activated by TNF-β, modulate the NF-κB-regulated gene expression, CSC formation and increase apoptosis in CRC cells and chemosensitizes CRC cells to 5-FU in TNF-β-induced inflammatory tumor microenvironment . This po


All aspects of an argument need to be evaluated in evidence-based scientific pursuit to prevent the development of self-reinforcing cohorts of studies through the cross-referencing of selected subsets of studies (citation bias). Controling populations of unowned cats using Trap–Neuter–Return and its variants (hereafter TNR) is an example of how a scientific topic develops and is communicated academically and publicly. We found 145 TNR publications between 2002–2019, with the last two years seeing a rapid increase in studies. Publication clusters occur, focusing on: population control, interactions with wildlife, disease transmission , free-roaming cats, and feral and domestic cat management. A quarter of all studies were published in Open Access formats, which have the highest potential social impact because they are free to download, share and read. While it is encouraging that diverse views are expressed, the development of clusters of introspective topics risks reduced dialogue between groups of authors with differing views. Journal editors could encourage exchanges between groups by choosing reviewers from different camps to assess manuscripts and by asking authors to acknowledge alternative views.Trap–Neuter–Return and its variants (hereafter TNR) aims to control unowned cat populations. Papers on this topic form a useful case study of how how an area of literature grows, papers become influential, and citation networks form, influencing future study as well as public perceptions of the science. We analysed 145 TNR studies published 2002–2019. Common topics, identified by frequently used language, were population control, interactions with wildlife, disease transmission , free-roaming cats, and feral and domestic cat management. One or more papers on each of these topics was judged influential because of high citations overall, high average citations/year, or frequent mentions in social media. Open Access papers were more influential in social media, raising greater public awareness than studies published in journals that were less accessible. While divergent views exist on a range of topics, the network analysis of the TNR literature indicated potential for forming self-reinforcing groups of authors. While it is encouraging that diverse views are expressed, there is a risk of reduced dialogue interactions between groups, potentially constraining dialogue to refine arguments, share information, or plan research. Journal editors could encourage communication by choosing reviewers from different camps to assess manuscripts and by asking authors to acknowledge alternative views.  Su,3. Su2,3 to it.’ . Howeveridence’  p.480). idence’ ,7,8), wi,8, wi‘I 0. idenceidence’  provides a case study of how knowledge, understanding and authority develop in a scientific field and are transferred to wider public understanding via social media. In TNR, unowned, free-roaming cats are trapped, desexed, sometimes vaccinated or provided with other health care, and  preferentially returned to colonies (that may or may not include other cats) with supplemental feeding sometimes provided. The ultimate aim of such programs is to reduce or eliminate populations of unowned cats with minimal use of euthanasia, thereby mitigating the problems they cause. The topic is important because free-roaming cats, owned or otherwise, may hunt or otherwise disturb wildlife ,16, spreDescribe citation networks in the TNR literature based on text data from titles and abstracts, shared references between TNR papers in different journals, and the bibliographic coupling of authors .Describe the TNR literature in terms of the increasing number of studies over time, the journals in which the studies were published, the countries where the authors were based, the institutional affiliations of the authors  and identify the most influential papers based on citations and on non-citation altmetrics.Document the topics of major concern in the TNR literature, and test whether the relative proportions of positive, neutral or negative assessments of TNR vary depending on the topic under discussion.Document the role of Open Access publication  in distributing TNR findings.Since 2002, there has been a growing scientific literature on TNR, covering population turnover and size e.g., ,24,25), ,25, 24,2We specifically did not aim to evaluate papers critically nor to pass judgement on the applicability or otherwise of TNR in any situation. We searched the Web of Science Core Collection (WoSCC) (coverage since 1900) on 13 January 2020 using the search terms ‘trap neuter release’, OR ‘trap neuter return’, OR ‘trap neuter vaccinate’, to locate relevant studies. While WoSCC is a highly selective database  requirinhttps://www.vosviewer.com/) to visualise connections between TNR publications [We used VOSviewer version 1.6.8 , co-citations for journals , co-authorship for all authors, and co-authorship for authors with at least two publications in the network (indicating which authors tend to be cited together).We listed the journals in which relevant papers were published, noting whether or not the journal was Open Access (OA) or, if not, whether individual papers were published OA in subscription journals. We also tallied the number of papers published in each journal. All authors on each paper, not just the first author, were grouped into one of three broad affiliation categories by examining the address lines in the papers: non-government organisation or private researcher (hereafter NGO), government (address indicated a government agency or department) and academia (a university or college address). If an author had multiple affiliations, only the first mentioned affiliation was entered. To assess possible changes in the distribution of authors in different affiliation categories over time, we grouped papers into four time periods-  and tested for any association between author affiliations and time period using a chi-square contingency table with expected values calculated assuming an equal proportion of the overall distribution of the categories represented across each time period. We also noted the country named in the address lines of all authors. If an author indicated two countries, we included only the first-named. To assess changes in the international profile of the TNR literature over time, we grouped papers into the same time periods as used for assessing author affiliations. Within those periods, we noted the distribution of countries of authors, as well as the number of ‘international’ papers, defined as those having authors from more than one country. We tested for any association between author countries and time period using a chi-square contingency table with expected values calculated assuming an equal proportion of the overall distribution of authors publishing from each country were represented across each time period.Influential publications were first assessed using conventional citation statistics taken directly from the WoSCC and included the proportions of papers cited over time, the 10 most highly cited papers overall, and the 10 most highly ranked papers based on citations/year since the paper was published . Examples of key phrases paraphrased from papers that characterised these stances included ‘data support TNR’ (positive), ‘the evidence contradicts TNR claims’ (negative) and ‘studies provide at best only qualified support’ . We tested for associations between the topic and stance using a chi square test with expected values calculated assuming an equal proportion of the overall distribution of stances recorded were represented across each topic The search in WoSCC retrieved 162 papers published between 2002 and January 2020. We excluded 17 documents that were short letters, corrections or editorial material, as well as two that pertained solely to dog management. For the 145 papers remaining, the distribution of papers across years showed a rapid increase from four in 2002 to 10 in 2011, 24 in 2018 and 25 in 2019 . The ratOf the 684 terms identified in the titles, abstracts or keyword lists in the 145 papers, 51 terms appeared at least four times and were deemed relevant  . They weJournal of Feline Medicine and Surgery and focused on questions of disease, health and management options. A second (purple) had its largest node centred on Journal of the American Veterinary Medical Association (JAVMA), where a wide diversity of topics across disease, welfare and management were considered. Wildlife considerations predominated in the diffuse cluster of red journals, without a single journal being prominent. Welfare issues characterised a cluster of journals (blue) around the Journal of Applied Animal Welfare Science and, to a lesser extent, Preventive Veterinary Medicine. A small cluster (turquoise) developed around Anthrozoos and the Canadian Veterinary Journal; these journals covered human dimensions of cat management. The final, smallest cluster (olive) centred on the OA journals Animals and Frontiers in Veterinary Science, where recent papers, including a special issue in Frontiers in Veterinary Science, have reported the outcomes of TNR. Bibliographic coupling of the 56 journals based on shared citations revealed six clusters . One , with growing numbers of authors from Australia, Canada and Spain. The first international collaborations (where authors on a single paper come from more than one country) appeared in 2012. Overall, five papers included authors from more than one country and were classed as international. Authors came from 24 different countries, with most from the USA . However2χ6 = 6.93, p = 0.33).Across the period 2002–2019, academic authors remained the largest category , and theJournal of Feline Medicine and Surgery, Journal of the American Veterinary Medical Association (JAVMA), Animals, Frontiers in Veterinary Science and Journal of Applied Animal Welfare Science) published nearly half (46%) the contributions. Two of these journals  are OA, as are a further 10 journals publishing fewer TNR papers  yet to be cited (probably because of insufficient time since publication for citations to accumulate) . Although we intended to identify the 10 most highly cited papers, two papers tied for the 10th spot, so 11 papers from five journals were represented, ranging from 57–120 citations. These papers were contributed by 30 USA authors, seven Italian authors and three Dutch authors. There were 30 academic authors, seven government authors and three NGO authors. None of these top 11 papers was published OA . When ci2χ4 = 28.25, p < 0.01). USA authors predominated on all three lists, with no variation across them in the proportions of countries of authorship .The proportion of OA papers in the lists of top ranked papers by citations, citations corrected for time since publication and social media mentions varied significantly, with OA papers predominating in the ranking by social media mentions . The proportion of academic authors relative to NGO and government authors combined also varied significantly across the three ranking tables, with NGO and government authors more strongly represented in the social media ranking . Positive stances were more common for five of the nine categories, most strongly in studies of the influence of TNR on shelter intake and in studies of the demographics of TNR colonies. Negative stances were most prominent for cat behaviour studies. Nine broad topics were identified . Twenty-2χ1 = 12.66, p < 0.01). Papers published OA were more likely to take a positive attitude (pro) towards TNR .Our examination of key terms around TNR cat management indicates that there are five main groups of publications on the topic: 1. disease and health, 2. free-roaming cats, 3. feral and domestic cat management, 4. population control, and 5. wildlife and predation. These papers represent different topics or angles on the debate, approximating the interest groups of veterinarians (topics 1 and 4) the public, animal protection/welfare advocates (topics 2 and 3), and conservation advocates (topics 4 and 5). All these interest groups are identified by Farnworth et al.  as imporJAVMA or, if they are feline specialists, in Journal of Feline Medicine and Surgery. Similarly, prolific authors such as Levy, Slater, Lepczyk and Rand are nodes of citation for others contributing to the TNR literature.Citation groups also formed around journals and authors. In some cases, these reveal clear professional interests, such as veterinarians reading and publishing in There are also signs in the citation networks suggesting problems might develop in terms of communication across the full spectrum of the TNR literature. For example, the three largest nodes in the key terms analysis were ‘domestic cats’, ‘free-roaming cats’, and ‘feral cats’; but they fell into different clusters. ‘Free-roaming cats’ were perceived as distinct from the other cat categories. The language used to describe cats may have consequences for how they are perceived that ultimately translates into how they are managed ,73, so t‘... thepower of citation through the choice of which papers to cite and which to ignore (citation bias), by citing but distorting content (citation diversion), and by using citation to invent fact .’ We believe we have shown the potential for such biases to develop in the TNR literature. This could lead to sub-optimal management responses, especially if decision-makers are operating within a closed citation network. To address this problem, we suggest that editors of journals publishing on TNR cat management could aim to broaden scientific debate on the topic by: ‘Choose reviewers for all the manuscript’s agendas’  to generate a greater depth of scientific debate around the topic. Sourcing reviewers from the reference list of submitted papers would be problematic, as would be accepting all the authors’ suggestions for reviewers.Selecting reviewers who have complementary expertise; i.e., what Fletcher and Black Having stringent requirements to declare and publish financial conflicts of interest, insisting that authors include a section on the limitations of their study, querying if employers or funders restricted what could be published, and ensuring that the power balance in negotiations over revisions remains with the editors, not the authors .Publishing reviews and responses alongside the paper in cases of unresolved differences of opinion between reviewers and authors .‘in which a small number of these influential review papers and model papers containing no data on claim validity collected and focused citation (similar to a magnifying lens collecting light) on particular primary data papers supportive of the belief, while isolating others that weakened it.’Checking review papers to avoid what Greenberg  cChecking abstracts thoroughly to ensure that they are an accurate reflection of the content of the paper .Favouring systematic reviews that document clearly how the review was conducted, with decision rules for including or excluding specific studies , over deMisemer et al.  noted thThe TNR papers that have a high profile through either citations or social media indicate the central issues in the influential TNR literature. Citations reveal academic interest and use, while social media mentions are perhaps a better indication of the matters that occupy public attention. Excepting a small handful of very recent publications that have not had an opportunity to accrue citations, all TNR papers have been cited. The most highly cited papers, either by the total number of citations or as citations per year, indicate the range of concerns around TNR, including: human dimensions of people’s interactions with cats , the evaCitation data note only the use of publications for academic purposes and may not measure other influences, such as the extent to which papers may be consulted but not subsequently cited, or the uptake of the work by news outlets, practitioners who do not publish, or the public ,82. AlteOverall, the 25 unique papers that featured on one or more of the lists of influential papers included 17 papers taking a positive view of TNR, four that were neutral and four that were negative. This indicates a diversity of opinion at the influential level, but that most influential papers are positive towards TNR. A detailed meta-analysis would be needed to discriminate whether this is a true reflection of the data in the field, or an example of bias towards positive findings.Considering all the studies rather than just those ranked highly, studies taking positive and negative attitudes to TNR were present in all topics identified , indicating that divergent opinions are being expressed. Whether these opinions are being considered by the opposite camps is not revealed by our study. Positive studies were more likely to be published OA, so they may have a higher profile in the broader community. Since the first TNR papers were published in 2002, the literature remains dominated by academic authors. This is perhaps a logical extension of its connection to veterinary expertise, although academics from other disciplines also contribute. The imperative to publish for academic career advancement may also encourage substantial contributions from academic authors . HoweverWhile most TNR authors in the early years came from the USA, this predominance has reduced significantly over time, with growing contributions from 23 other countries. The breadth of international authorship shows the extent of problems with unowned cats internationally. A challenge is to evaluate robust findings that are likely to translate to many localities, as distinct from those that may have more limited regional application . Animals and Frontiers in Veterinary Science) just behind two veterinary subscription journals ) in numbers of TNR papers published. There is extensive and on-going debate as to whether or not papers available by OA are more highly cited than those that are not . ,86. AnimThis is a descriptive study, so it includes no assessment of the validity of the findings of any of the TNR papers in the sample. The literature sample was based on WoSCC, which is a selective database and will have missed some TNR studies, especially those published in languages other than English . Like anDespite these limitations of citation and alternative metric measures, this study offers specific strengths. While WoSCC is selective, it excludes studies published in potentially questionable journals. Examining the topics prominent in papers with a high profile by both citation and social media measures, coupled with descriptions of networks based on keyword analysis, gives a triangulated view of important topics in the TNR literature.The growth in the TNR literature indicates global concern to address the problems of stray cat population control, their interactions with wildlife, and disease transmission . Network analysis in the TNR literature drawing on the journals publishing the work and the connections between authors and journals established through referencing indicates the potential for self-reinforcing groups of authors to form. While, on the one hand, it is encouraging that diverse views are expressed, there is a risk of lack of dialogue between interest groups on the other hand. This could reduce the opportunity for constructive exchanges of differing views to refine arguments, exchange information, or develop research programs to provide empirical data for dispute resolution. This could compromise management of free-roaming cats that addresses the concerns of all stakeholders. Journal editors could encourage such exchanges by choosing reviewers from different camps to assess manuscripts and by asking authors to acknowledge alternative views.


Published in 2019, a new addendum to the ICH E9 guideline presents the estimand framework as a systematic approach to ensure alignment among clinical trial objectives, trial execution/conduct, statistical analyses, and interpretation of results. The use of the estimand framework for describing clinical trial objectives has yet to be extensively considered in the context of patient-reported outcomes (PROs). We discuss the application of the estimand framework to PRO objectives when designing clinical trials in the future, with a focus on PRO outcomes in oncology trial settings as our example.“What is the effect of treatment X on patient’s quality of life?”, we build up an example estimand statement and also use a previously published phase III oncology clinical trial to illustrate how an estimand for a PRO objective could have been written to align to the estimate framework.We describe the components of an estimand and take a naïve PRO trial objective to illustrate how to apply attributes described in the estimand framework to inform construction of a detailed clinical trial objective and its related estimand. We discuss identifying potential post-randomization events that alter the interpretation of the endpoint or render its observation impossible  in the context of PRO endpoints, and the implications of how to handle intercurrent events in the construction of the PRO objective. Using a simple objective statement, The use of the estimand framework, as described in the new ICH E9 (R1) addendum guideline will become a key common framework for developing clinical trial objectives for evaluating effects of treatment. In the context of considering PROs, the framework provides an opportunity to more precisely specify and build the rationale for patient-focused objectives. This will help to ensure that clinical trials used for registration are designed and analysed appropriately, enabling all stakeholders to accurately interpret conclusions about the treatment effects for patient-focused outcomes. Patient input and incorporating the patient’s perspective within clinical trials is increasingly being recognized as a critical component of achieving patient-centricity in quality health care . As such“...the target of estimation to address the scientific question of interest posed by the trial objective” and has five attributes – population, treatment, variable (endpoint), intercurrent events (defined as events that can occur post-randomization and preclude or affect the interpretation of the variable) and the summary measure. An example of an intercurrent event in a clinical trial context could be discontinuation of treatment.The International Committee for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) is a global organisation that brings together regulatory authorities and pharmaceutical industry in a mission to ensure development and registration of high-quality medicines. ICH issues and maintains numerous guidance documents used internationally in the design and conduct of clinical trials. A key guideline relating to design and analysis of clinical trials is ICH E9 “Statistical Principals for Clinical Studies”. ICH has recognised that a precise definition of the scientific question of interest is required to ensure alignment between objectives, trial design, data collection, analysis and interpretation and that this alignment has not always been observed in the past. In November 2019 the ICH issued the final version of an addendum (R1) to ICH E9, ICH E9(R1) . Simply referring to a “PRO score” is not specific. In our naïve example, we have selected pain severity as the PRO variable of interest.It is important to clearly define a specific patient outcome of interest, i.e., to define the “variable of interest”  and treatment comparisons  are presented next and thus to build this estimand:Defining the parts of the estimand relating to the population of interest . It is important to note that in this context the summary measure refers to the statistical summary approach, rather than just the measure or PRO instrument used. The summary measure should be defined; this could be the difference in the mean score between the two groups, or the proportion of patients in each group who have had meaningful improvement in symptoms. In this example, we suggest comparing the difference in mean scores between treatment groups.The ICH E9 terminology uses the phrase “a summary measure” , and then we can clarify our intercurrent event approaches ; let us assume a treatment policy approach to handle treatment discontinuation and while on treatment to handle death were used Therefore, our estimand becomes:To return to the example estimand, let us add in potential subsequent treatments and any concomitant treatments to the treatment attribute [> 7 points) [It is important to ensure that the final clinical trial objective reflects required specificity and details that explain whether it is intended for the PRO objective to demonstrate statistical superiority or non-inferiority of treatment X compared with treatment Y . Additio points) .In the context of the estimand framework, the primary objectives relating to the GALLIUM trial have been presented by Rufibach (PharmStats 2019) , along wClinicalTrials.gov ID NCT01332968). The primary variable of interest was investigator-assessed progression-free survival [GALLIUM was an open-label, randomised phase III clinical trial of obinutuzumab-based and rituximab-based chemo-immunotherapy in patients with previously untreated advanced indolent non-Hodgkin lymphoma . This subscales measures “additional concerns” relating to symptoms relevant to patients with lymphoma, and a change from baseline score > = 3 points for a patient is recognised as minimally important difference. In this trial there was a 6 months induction treatment period where patients received chemo-immunotherapy, followed by up to 2 years of immunotherapy maintenance in patients who responded to induction therapy .One of the PRO endpoints of interest stated on Taking the patient-reported lymphoma-related symptoms, as measured by the FACT LymS score as the key PRO measurement, along with information from the trial design, we can propose an estimand as follows (when considering treatment discontinuation and deaths as intercurrent events):Estimand 1:In order to propose this estimand we have had to consider carefully the patient-reported outcome of interest – in this example we have made clear that we are focusing on the lymphoma-related symptoms reported by the patient as measured by the FACT-LymS score. We have also clearly stated the timeframe we are interested in considering – in this case it is all FACT-LymS scores for the planned full duration of therapy . In this estimand interest centres on the “While on Treatment” scores only.An alternative estimand of interest could be considering FACT-G LymS score up to the planned 30 months treatment period, irrespective of whether patients continued to take active treatment or not (a treatment policy approach). From a practical point of view this means that FACT-G LymS score would need to be collected even after treatment discontinuation . The collection of PRO data regularly post treatment discontinuation is not currently a common approach in oncology clinical trials. There are other considerations highlighted by this proposed estimand: what is the summary measure of interest – in this case the estimand describes the proportion of patients with an improvement – but is the timescale chosen of 30 months appropriate for this summary measure? An alternative could a summary measure relating to a mean change from baseline in score over time (up until maximum of 30 months) in each treatment group, as shown in Estimand 2.Estimand 2:The estimand framework provides guidance on the key attributes for defining study objectives, including PRO objectives, that should be thoughtfully considered when designing a phase III clinical trial. A clear and specific PRO objective should be defined, enabling a precise estimand statement. Attributes of the estimand described in the framework help to ensure that a detailed PRO objective can be constructed. Even with one “naïve” objective, the use of the estimand framework provides a way to clearly discuss several very feasible estimands of interest, as illustrated above. One estimand could be chosen as the key approach; other ways of handling an intercurrent event could lead to different estimands and additional supplementary analyses. Using this staged approach to specify estimands should then enable clearer specification of PRO objectives in protocols and analysis plans of what sensitivity analysis should be considered, together with the data that needs to be collected to enable this.These discussions with a case-study example, aim to highlight how the use of the attributes of the estimand provide potential ways to consider the detailed PRO objective of interest and build the related estimand. It also highlights with just one case-study example how different strategies can change how the estimand is written and how to more clearly interpret study results.It is recognised that different stakeholders  have different objectives to answer from clinical trials conducted and that this may influence how trials should be designed and analysed. As examples, the long-term impact of adverse events on patient symptoms or PROs after radiological disease progression, or patient outcomes collected post-end of treatment, would each require consideration of different specific estimands and clear specification about what was needed to be measured in order to evaluate each PRO endpoint.Once an estimand has been specified, we then need to move on to focus on the estimator which is the statistical technique with which you will derive your ‘estimate’ of the treatment effect. A well-defined estimand allows study teams to work constructively with statisticians to take a well-thought-out objective and to choose an appropriate analysis that need to be applied. However, the specifics and appropriateness of these estimators and a thorough discussion of their selection is out of the scope of this work.Experience with the estimand framework is growing in clinical research across various therapeutic areas , 14–18, However, the estimand framework is not just a new language for defining trial objectives – it will change the way in which clinical trials are designed, analyzed, and interpreted, as well as how the data collection strategy is informed. By developing precise objectives relating to assessing the patient’s perspective of treatment and embracing the estimand framework, we can use the estimand framework to ensure useful collection of data in clinical trial protocols and provide clear interpretation and conclusions to all stakeholders, thereby lending to clear evaluation on the impact of treatment on patients’ lives.


A wearable and deformable graphene-based field-effect transistor biosensor is presented that uses aptamer-modified graphene as the conducting channel, which is capable of the sensitive, consistent and time-resolved detection of cytokines in human biofluids. Based on an ultrathin substrate, the biosensor offers a high level of mechanical durability and consistent sensing responses, while conforming to non-planar surfaces such as the human body and withstanding large deformations . Moreover, a nonionic surfactant is employed to minimize the nonspecific adsorption of the biosensor, hence enabling cytokine detection  in artificial tears (used as a biofluid representative). The experimental results demonstrate that the biosensor very consistently and sensitively detects TNF-α and IFN-γ, with limits of detection down to 2.75 and 2.89 pM, respectively. The biosensor, which undergoes large deformations, can thus potentially provide a consistent and sensitive detection of cytokines in the human body. Human biofluids  are attractive clinical diagnostic bio-media containing numerous cytokines (with a molecular weight lower than 70 kDa) ,2,3. TheGraphene, an attractive two-dimensional nanomaterial, is extremely sensitive to its surface charge distribution, and widely used as the transducer for sensors due to its outstanding electrical properties ,10,11. EIn this paper, we present a wearable and deformable aptameric GFET biosensor that is designed to enable the sensitive, consistent and time-resolved monitoring of cytokines in human biofluids. The biosensor is fabricated on a biocompatible and ultrathin, polymer-supporting substrate a. Due to2-TGG TGG ATG GCG CAG TCG GCG ACA A-3′) and IFN-gamma (5′-NH2-GGG GTT GTT TGT GTT GGG TGT TGT GT-3′) was synthesized and purified by Sangong Biotech .The monolayer graphene sheet was ordered from Graphenea Inc. . 1-pyrenebutyric acid N-hydroxysuccinimide ester (PASE), ethanolamine, Tween 80, human interleukin-002 (IL-002), TNF-alpha and IFN-gamma were purchased from Sigma-Aldrich . Human growth hormone (GH) and human epidermal growth factor (EGF) were ordered from ACRO Biosystems . Ultrathin Mylar films  were ordered from Chemplex Industries . Artificial tears were ordered from Alcon Laboratories. The aptamer specific to TNF-alpha  solution for 5 h at room temperature. PASE was modified on the graphene surface through π-π stacking, which was used to link the aptamer. After incubating in the 1 µM aptamer solution for 12 h, the device was washed with phosphate buffer (PBS) to remove free aptamer. Ethanolamine was then used to quench the unreacted PASE on the graphene by soaking in 100 mM ethanolamine solution for 1 h. Finally, the biosensor was immersed in 0.05% Tween 80 solution to passivate the uncoated graphene area.During the operation, a volume of 40 µL of analytes  at a given concentration was added to a polydimethylsiloxane (PDMS) open well, which was mounted on the graphene conducting channel to hold the analyte solution. In the experiments, 20 µL of artificial tears was added to a 1 mL centrifugal tube with 980 µL of 1 × PBS. Then, the mixture solution was stored at 4 °C before protein solution configurations.VDirac,where ΔVDirac = VDirac – VDirac,0 with VDirac,0 the Dirac point obtained in the solution without biomarker, before and after PASE, aptamer and Tween 80 functionalization was measured with the gate voltage Vg increasing from −0.2 to 0.4 V at a fixed drain-source voltage Vds of 0.01 V . ΔVDirac/ΔVDirac,max was determined using the Hill–Langmuir equation [KD was calculated to be 8.43 ± 1.67 nM. The limit of detection (LOD) was estimated based on the three-sigma rule, and the sigma was obtained from the standard deviation of the experimental and fitted data. The LOD was calculated to be 2.75 pM, which is many times lower than that for existing biosensors for TNF-α detection.The equilibrium dissociation constant, defined nd TNF-α c. The noequation . Based oVDirac was 0.029 V, decreasing from 0.029 to 0 V. The sensing signal was also consistently shifted in the direction of the negative gate voltage with the increasing IFN-γ concentrations. This was expected from the electrostatic mechanism [KD was estimated to be 7.36 ± 2.76 nM  at the same concentration. Additionally, the dissociation constant KD for the control proteins was investigated. KD was estimated to be 1.43 × 1010 and 1.77 × 109 nM for EGH and GH, respectively, which is much larger than the KD for TNF-α (8.43 nM). Subsequently, the biosensor modified with the aptamer specific to IFN-γ was tested using these control proteins  at the same concentrations. Furthermore, the KD was calculated to be 1.55 × 106 and 2.73 × 106 nM for EGH and GH, respectively, which is tens of thousands of times larger than the KD for IFN-γ (7.36 nM). Hence, the biosensor has a high level of specificity for the target cytokine in artificial tears.To investigate the specificity of the biosensor, EGF and GH, two related proteins, were chosen as control proteins. The biosensor, modified with the aptamer specific to TNF-α, was firstly exposed to control proteins in artificial tears a. The seproteins b. The ΔVIds, and the maximum change is denoted as ΔIds, max. The sensing signal was observed to increase stepwise and significantly from 0 to 0.98 with increasing TNF-α concentrations from 0 to 500 nM. Moreover, it was found that equilibrium was reached after the binding of aptamer with TNF-α within 7 min at different concentrations. By contrast, the ΔIds/ΔIds, max—generated by related cytokines (IFN-γ and IL-002)—was less than 15% compared with that for TNF-α at the same concentration.To verify the feasibility of the biosensor for wearable applications, time-resolved measurements were employed, using the biosensor modified with an aptamer specific to TNF-α as a representative. As shown in Additionally, we present a potential capability for wearable applications using the biosensor mounted on a homemade diaphragm chamber for cytokine detection b. The diWe presented a wearable GFET biosensor for the time-resolved monitoring of cytokines in artificial tears. The modification of the graphene with the aptamer enabled the highly specific detection of the target cytokine. Tween 80, which occupies the uncoated area of the graphene, offers a capability of suppressing nonspecific adsorption; the biosensor hence affords a high level of sensitivity for cytokine detection in artificial tears, with a LOD down to 2.75 and 2.89 pM for TNF-α and IFN-γ, respectively. In addition, the time-resolved monitoring of TNF-α in artificial tears using the biosensor under different tensile strains has been demonstrated. The sensing response for the target cytokines is consistent with that found on the flat surface. These results demonstrate that this wearable GFET biosensor is a critical step toward the general application of sensors for the monitoring of disease biomarkers in the human body.