Introduction. Stroke survivors use more energy than healthy people during activities such as walking, which has consequences for the way exercise is prescribed for stroke survivors. There is a need for wearable device that can validly measure energy expenditure (EE) of activity to inform exercise prescription early after stroke. We aimed to determine the validity and reliability of the SenseWear-Armband (SWA) to measure EE and step-counts during activity <1 month after stroke. Materials and Methods. EE was measured using the SWA and metabolic cart and steps-counts were measured using the SWA and direct observation. Based on walking ability, participants performed 2x six-minute walks or repeated sit-to-stands. Concurrent validity and test-retest reliability were determined by calculating intraclass and concordance correlation coefficients. Results and Discussion. Thirteen participants walked; nine performed sit-to-stands. Validity of the SWA measuring EE for both activities was poor (ICC/CCC < 0.40). The SWA overestimates EE during walking and underestimated EE during sit-to-stands. Test-retest agreement showed an ICC/CCC of <0.40 and >0.75 for walking and sit-to-stand, respectively. However, agreement levels changed with increasing EE levels . The SWA did not accurately measure step-counts. Conclusion. The SWA should be used with caution to measure EE of activity of mild to moderate stroke survivors <1 month after stroke. Cardiorespiratory fitness levels are low early after stroke , with fitness levels of stroke survivors ranging from 44% to 76% that of age- and sex-matched sedentary healthy adults . In stro2) using breath-by-breath analyses and has been shown to be a valid measure of VO2 uptake during different workloads in sedentary adults, moderately trained individuals, and athletes [Indirect calorimetry using a metabolic cart is commonly used to measure the EE of physical activities. The metabolic cart measures the volume of oxygen uptake (VOathletes . The devunaffected), was highly correlated with doubly labelled water (gold standard) in chronic stroke survivors  [unaffected showed good agreement (ICC > 0.70) and the SWAaffected showed fair agreement (ICC < 0.60) with a metabolic cart in chronic stroke survivors, suggesting that the SWA worn on the unaffected arm gives a more accurate estimate of EE during walking [unaffected to measure EE during activity in people with acute stroke, that is, within the first month after stroke.In a recent systematic review 60 different devices to measure physical activity in stroke were identified . Of thes walking . In the  walking . It is uunaffected. Using a mobile metabolic cart as a gold standard we sought to determine the concurrent validity and reliability of SWA measures of EE during a physical activity in stroke survivors whose stroke onset was less than 1 month ago. We hypothesised that the SWAunaffected is a valid and reliable tool to estimate EE during activity in acute stroke survivors.This study is a part of a larger study in which we aimed to compare EE of acute stroke survivors to healthy controls. In this paper we will only discuss the result regarding the validity and reliability of the SWAStroke survivors admitted to the acute stroke ward at the Austin Hospital in Melbourne, Australia, were eligible to participate if they met the following criteria: (1) >18 years of age, (2) being within 1 month after stroke, (3) clinically diagnosed with stroke, (4) cognitively able to consent as assessed by the treating clinician, (5) sufficient English language command to follow complex instructions, and (6) medically cleared to participate by their treating clinician.Stroke survivors were excluded from participating in the study if they had (1) comorbidities that impaired their ability to either walk or perform sit-to-stands  for six minutes  or (2) other neurological comorbidities that might affect the EE of activity .All stroke survivors who participated in this study provided written informed consent. The study was approved by the Austin Health Human Research Ethics Committee (reference number H2011/04447).Each participant's age, height (measured using a stadiometer), weight while clothed but without shoes , and smoking status were recorded. Additionally, we obtained date of stroke, severity of stroke on admission, and stroke subtype from the participant's medical records. The National Institutes of Health Stroke Severity (NIHSS) scale was used to measure stroke severity with scores ranging from 0 to 42. Stroke severity was categorised into mild (<8), moderate (8–16), and severe (>16) .2 of less than 2.0 mLO2/kg/min over the last three minutes of activity [To measure EE of activity in both ambulatory and nonambulatory stroke survivors, we used two different test protocols: walking and sit-to-stand. Stroke survivors able to ambulate under supervision or with light manual support for balance and coordination  performed the walking protocol. Stroke survivors who were unable to walk independently and needed manual support from at least two people performed the sit-to-stand protocol. We consulted with the participants' treating clinician  to determine which protocol was most appropriate. All participants started with a three-minute resting period in a seated position followed by two bouts of six minutes of continuous activity . Between the two bouts, participants rested for 30 minutes in different positions , allowing EE to return to baseline levels before commencing the second six-minute bout. We aimed for participants to reach steady-state during the six-minute activity bouts, with steady-state defined as variability in VOactivity . not to move as fast as possible during the six minutes of activity.Ambulatory stroke survivors walked back and forth along a 30-metre corridor at a self-selected pace. Participants performing the sit-to-stand protocol started in a seated position on a height-adjustable physiotherapy plinth, which was set at a height that required moderate effort and minimal assistance only for balance from the researchers. Before every test we emphasised to the participant that the goal was to move at a steady pace andaffected—and the other arm—unaffected). We only reported and discussed data regarding the SWAunaffected; the data of the SWAaffected can be found in the supplementary materials.EE was measured using a metabolic cart  and the SWA. For completeness, we applied one SWA on each arm  per minute using proprietary algorithms of the manufacturer.2 continuously using breath-by-breath analyses, averaged over three breaths; the readout of the data was in five-second epochs. Calibration of the metabolic cart was performed according to the manufacturer's operational instructions prior to testing. Gas calibration was performed against gas with a ratio of 16% O2 and 4% CO2 in Nitrogen. The ambient conditions were automatically measured by the unit. The participants were fitted with a facemask and a harness that carries the Oxycon units that allow telemetric transmission of data to a laptop.The metabolic cart measured VOStep-count was recorded by direct observation using a manual counter and by the SWA. The accelerometer output of the SWA is converted to step-counts using proprietary algorithms of the manufacturer. Sit-to-stand counts were also recorded by direct observation using a manual counter; a single sit-to-stand was counted when the participant stood up and sat back down.The test protocol was terminated if a participant did not meet the following criteria assessed in a sitting position before the six-minute activity bouts: systolic blood pressure between 120 to 220 mm Hg , oxygen saturation > 92%, heart rate between 40 and 100 bpm , and temperature < 38.5°C . The test was also terminated if the participant requested stopping or felt unwell.unaffected.Purposeful sampling was used to recruit participants and we aimed to include stroke survivors who were able and not able to walk. We considered it feasible and practical to recruit 20 stroke survivors. We continued recruitment until 20 stroke survivors completed both activity bouts and had complete data sets including EE data measured by the metabolic cart and SWA2 in ml/kg/min which is automatically converted to METs/min, where 1 MET is equal to a VO2 of 3.5 ml/kg/min.After the test had been completed the data of the metabolic cart and SWAs were downloaded on to a computer. In this study we expressed EE in METs. The Oxycon expresses EE in VOThe output of the metabolic cart data is averaged over five-second epochs; we averaged the metabolic cart data for each minute to match the SWA data, which is collected in METs/min. EE under steady-state conditions was calculated by averaging the EE output over the last three minutes of the six-minute bouts for each participant for both the data acquired by the metabolic cart and the SWAs. We used descriptive statistics, calculated medians and interquartile ranges (IQR) for demographic data, anthropometric data, walking speed, step-counts, and sit-to-stand counts, and we calculated means and standard deviations for the EE of steady-state activity.We confirmed that all EE data were normally distributed using the Shapiro-Wilk test. We calculated intraclass correlation coefficients (ICC) and Lin's concordance correlation coefficients (CCC) to assess agreement between measures. Both ICC  and CCC Additionally we employed reduced major axis (RMA) regression, which is appropriate in the setting of this study, where both measurement tools produce readings that are susceptible to measurement error . The reg concurrent validity of the SWAunaffected, we determined agreement between the metabolic cart and the SWAunaffected for the following measures: (1) EE of steady-state walking and (2) EE of steady-state sit-to-stand. Test-retest reliability for both the metabolic cart and the SWAunaffected was assessed by determining agreement between EE measurements taken during the 1st and 2nd bout during walking and sit-to-stand.To test our hypotheses regarding concurrent validity of the SWA by determining agreement between the observed step-counts and step-counts recorded by the SWAunaffected. Test-retest reliability of the SWAs was determined by agreement between the step-counts recorded during the 1st and 2nd bout of walking.Step-count measurements of the SWA utilise accelerometer data from the movement of the wearer's arm. When the wearer's arms are fixed on walking frames or supported by people walking with physical assistance, the recorded accelerometry data are likely to lack validity. We therefore excluded step-count data of participants who walked with a 4-wheel frame or required physical assistance. We tested our hypothesis regarding poor, between 0.40 and 0.59: fair, between 0.60 and 0.74: good, and between 0.75 and 1.00: excellent [We used the following ICC and CCC cutoff points to interpret the strength of agreement: less than 0.40:xcellent . All anaaffected). We included the average of EE values of both arms for this participant in the analyses; hence the difference in numbers of SWA measures in the group that performed sit-to-stand  and a marked higher median of 94 (IQR 40 to 121) during the 2nd bout (n = 7).We analysed the data of 22 participants. The median age was 78 (IQR 73–81) and all participants performed the test within the first seven days after stroke , except  poor agreement (ICC/CCC < 0.40) between EE measured by the metabolic cart and the SWAunaffected. There was evidence of proportional bias (slope = 1.42) during the 1st bout with agreement decreasing at higher levels of EE  between the EE of sit-to-stands measured by the metabolic cart and the SWAunaffected for both sit-to-stand bouts. During the 1st bout of sit-to-stands, there was little evidence for proportional bias; the SWA systematically underestimated EE (intercept = −0.57). During the 2nd bout there was some evidence of proportional bias (slope = 0.64) where agreement decreased at higher EE levels (see We foundvels see .n = 12). There was no evidence of proportional or fixed bias (slope = 1.05 and intercept = −0.19) indicating that participants performed at a similar level across bouts.We first assessed the test-retest reliability of our gold standard, the metabolic cart, and found excellent agreement between the EE measured during the 1st and the 2nd bout of walking . The RMA showed proportional bias (slope = 0.63 and intercept = 1.15) indicating that when EE levels increase just beyond 3 METs, agreement gets poorer ; however, there was some evidence of proportional bias (slope = 0.74 and intercept = 0.53) with agreement reducing at higher levels of EE. In the scatterplot (not shown) we identified one clear outlier. This individual moved substantially faster during the second bout compared to the 1st bout, completing 139 sit-to-stands versus 116, respectively. Post hoc analysis of the data excluding the outlier resulted in an ICC of 0.99 (95% CI 0.93 to 1.00) and a CCC of 0.99  and a slope and intercept of 1.12 and −0.25, respectively, indicating that the remaining participants performed at a similar rate between bouts.The metabolic cart was reliable between bouts of sit-to-stands, similar to our finding for walking, with excellent for EE measures between the 1st and 2nd bout of sit-to-stands recorded by the SWAunaffected . The RMA showed proportional bias (slope = 0.80 and intercept = 0.65) with agreement improving up to an EE level of 3.5  than the average step-counts measured via direct observation  between EE measured by the metabolic cart and the SWA during a six-minute walk [ poor (CCC < 0.40). It has been suggested that slower walking speeds might lead to decreased accuracy of EE estimation by the SWA [We set out to determine if the SWA wearable device could provide a valid measure of EE during physical activity in acute stroke patients. Our results indicated that the SWA worn on the unaffected arm did not accurately measure EE during a bout of physical activity; rather, it seemed to overestimate EE compared to the metabolic cart during walking and underestimate EE during sit-to-stands. Our findings regarding EE during walking are in contrast with the findings in the Manns and Haennel (2012) study. Their study, which included 12 chronic stroke survivors, foundute walk . In our  the SWA . Our resunaffected and excluded data from participants that walked with physical assistance or with a walking aid from the step-count analyses. It is possible, though, that the included participants also had lower-than-normal arm swing during walking. The results of our study confirm that the SWA is not a valid tool to measure step-counts in stroke survivors. A recent review highlights that there are other accelerometer based tools available that validly and reliably measure step-counts [We found that the SWA greatly underestimated step-counts by approximately 300 steps on average within two weeks of stroke onset. This difference was larger than the difference found in the Manns and Haennel study in which step-counts measured by the SWA were compared to step-counts measured by the StepWatch Activity Monitor (SAM) in stroke survivors > 6 months after stroke . The SAMp-counts .It is however important that our results are interpreted with caution; no formal calculations were performed to predetermine sample size and the sample size of this study was small. On the other hand we did compare the data of the SWA to the metabolic cart, which is regarded as a gold standard, and showed that in our sample it was a reliable measure of EE of activity and we are confident that the metabolic cart is a true measure of EE. Including a broad variety of stroke survivors in a clinical study like this is challenging. We were able to include ambulatory and nonambulatory stroke survivors within 14 days of stroke onset. Almost all of our participants, except one, were able to reach steady-state (22/23), regardless of their ability to ambulate. Exercising under steady-state conditions is one of the methods to improve cardiorespiratory fitness and we showed that some stroke survivors have the potential to start performing cardiorespiratory exercise early after stroke. It is important, however, that all of the included stroke survivors had mild to moderate stroke severity.unaffected measuring EE showed mixed results regarding agreement, but most importantly the RMAs for both walking and sit-to-stands showed that agreement changed when levels of EE change. This suggests that SWA EE output during therapy sessions with mixed activities would be highly variable, and using the SWA would not be a reliable method to track EE over time.The differences between SWA and metabolic cart were systematic and small. During walking, EE was overestimated by less than 1.0 MET and during sit-to-stands it was underestimated by less than 1.1 METs. Considering that the range of activity intensity is 3 METs, that is, light intensity is <3 METs and moderate intensity is 3–6 METs, a difference of less than 1.1 METs is relatively small. It is a concern, however, that the direction of the estimation error was not consistent across the two activities we tested. Therapy session in stroke rehabilitation can consist of different exercises including walking and sit-to-stand amongst other activities. Furthermore the test-retest reliability of the SWABased on the results of our study the SWA does not accurately measure EE and therefore should be used with caution when measuring EE during activities early after stroke.


Protein-repellent dental composite with crack-healing abilityN,N-dihydroxy ethyl-p-toluidine (DHEPT). The bioactivity of the resulting MPC-microencapsulated TEGDMA-DHEPT was evaluated on protein adsorption through early bacterial attachment. Its mechanical properties were also investigated, including self-healing assessment. Microcapsules of poly  (PUF) were synthesized by incorporating a TEGDMA-DHEPT healing liquid. A set of composites that contained 7.5% of MPC, 10% of microcapsules, and without MPC/microcapsules were also prepared as controls. The two distinct characteristics of strong protein repellency and load-bearing recovery were achieved by the combined strategies. The novel dual composite with a combination of protein-repellent MPC and PUF microcapsules for restoring microcracks is a promising strategy for dental restorations to address the two main challenges of fracture and secondary caries. The new dual composite formulation design has the potential to improve the longevity of dental restorations significantly.Fracture and secondary caries are the primary reasons for the failure of dental restorations. To face this omnipresent problem, we report the formulation design and synthesis of a protein-resistant dental composite composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) that also can self-repair damage and recover the load-bearing capability via microencapsulated triethylene glycol dimethacrylate (TEGDMA) and  In a range of materials available for the restoration of the tooth cavity, composites are the predominantly selected choice as they offer advantages in their aesthetics and less invasive preparation techniques ,2. ProspThe predominant reasons for composite restoration failures are secondary caries and restoration fractures, which represent more than 90% of recorded failures . Short-tOne step on this path addresses the inhibition of nonspecific adsorption of proteins at the surface of dental restorative materials . Since pN,N-dihydroxy ethyl-p-toluidine (DHEPT) were synthesized and incorporated into dental resin good self-healing efficacy ), where load P divided by displacement d is the slope in the linear elastic region of the load-displacement curve [Following previous studies, composites specimens were placed in metal molds, photo-cured  for 1 min on each side and then smoothed, which produced bar specimens with dimensions of 2 mm× 2 mm× 25 mm (n = 6) ,34. Twennt curve .IC) was measured using a single edge V-notched beam (SEVNB) method [IC of the specimens (KIC-virgin).Fracture toughness (K) method . The SEV) method ,29. AfteIC healed) was calculated [IC-healed KIC-virgin × 100).Before testing the self-healing assessment, the two halves of the specimen were attached to the metal mold and placed in a humidor at 37 °C for 24 h. After the fracture, the healing process was trigged . In thislculated . The selp < 0.05.Statistical evaluations were performed with SigmaStat 3.5 . The normality distribution of the data and equality of variances were checked using the Kolmogorov–Smirnov test and Levene’s test, respectively. As the data were normally distributed, analysis of variance (ANOVA) and the Tukey test were applied at a significance level of n = 6). Adding MPC to composites significantly decreased the protein adsorption (p < 0.05). The resin composite with 7.5% MPC had the lowest amount of protein adsorption, which was nearly 5% that of the control and the composite without MPC (p < 0.05).The amounts of protein adsorption on composite disc surfaces are plotted in n = 6). The flexural strength and elastic modulus of a composite containing 10% microcapsules and 7.5% MPC were not significantly different from controls (p > 0.5).The flexural strength and elastic modulus of composites with combined and isolated tested components were evaluated after one day of water storage, and are plotted in IC were expressed in MPa-m1/2.The results for all tested composites after one day of water storage are shown in IC and the percentage of damage recovery of the composites are expressed for each material. The virgin KIC of the composite to which the microcapsules were added was about 36% higher than that at 0% microcapsules . The healed KIC were similar for the composites containing 10% microcapsules with and without the addition of MPC. The MPC incorporation did not compromise the healed KIC (p < 0.05). The self-healing efficiency of 57–71% in damage recovery was obtained in both composites where the microcapsules were present.The critical stress-intensity value, or plane-strain fracture toughness, denoted KSignificant advancements in mechanical strength and surface roughness have been achieved in dental composites. Nevertheless, microcracking induced by fatigue and biofilm accumulation are long-standing problems in dental resin composites, and current strategies are non-responsive. In this study, we present an innovative strategy that involves the combination of a protein-repellant approach and the simultaneous targeting healing of the microcracks that eventually lead to the failure of restorations. More specifically, we aimed to design a composite that can fight the recurrence of dental caries around the composite, as well as help elicit mechanisms for the self-correction of many initial micro-cracks without a dentist’s operative intervention. The facets of this approach complement each other for optimal efficacy and increased clinical life service.The self-healing approach has been investigated in previous reports, which indicate that healing efficiency is relative to the matrix used, and can vary tremendously due to different matrix-healed network interactions ,30,31. BThe incorporation of additives with different purposes into a resin matrix would inevitably affect its intrinsic properties. The fraction of each element is critical for the current system to achieve the highest healing efficiency. One major goal of this study was to combine the best performance of those two elements  without detrimental effects on their highlighted properties. High healing efficiency can be acquired at 10% capsules content so that the fundamental mechanical properties of the matrix are insignificantly affected. Previous studies showed a positive correlation between microcapsule content incorporation and the fracture toughness of the polymer matrix ,27. In aOur results highlight composites for dental restorations that satisfy the characteristics of autonomous recovery after damage and ultra-low protein adsorption, and also, that low bacterial adhesion thereon was successfully achieved. The incorporation of 2-Methacryloylothelxyethyl-phosphorylcholine (MPC) into dental composites displays antibiofilm potential properties regulated by reduced protein adsorption, since the attachment of oral bacteria to a material’s surface is moderated by adsorbed proteins . A recenThe polymer MPC has been shown to be highly hydrophilic and capture the surrounding water that is credited to detach proteins efficiently, thereby repelling protein adsorption ,37. BaseThe set of tests performed here significantly revealed reduced oral biofilm attachment and a triggered a self-healing response, which prove that our approach was feasible and efficient in vitro, and suggest that this combined strategy may indeed help overcome recurrent caries and material fracture in dental composites.This preliminary study demonstrates the feasibility of preparing a self-healing dental composite formulation by embedding the TEGDMA-DHEPT healing agent into microcapsules combined with an antibiofilm surface. High healing efficiency can be acquired at optimal capsules content so that the basic mechanical properties of the matrix are insignificantly affected. In this research, the design and development of a protein-repellent dental composite formulation with autonomous crack-healing ability was successfully performed. The dual strategies for overcoming the most commons problem of composites can provide a new platform for anticaries dental materials.


Gastrointestinal stromal tumors (GISTs) are rare, and patients usually present with vague and non-specific abdominal symptoms. This report illustrates how point-of-care ultrasound performed in the emergency setting in the evaluation of such patients helped in management of two undiagnosed GIST patients. Gastrointestinal stromal tumors (GISTs) are rare gastrointestinal tumors though they are the most common mesenchymal neoplasms of the gastrointestinal tract. Patients may present with non-specific symptoms  or may be asymptomatic. Computed tomography of the abdomen and pelvis (CTAP) are crucial in the diagnosis and staging of GISTs but are not universally available in all emergency settings worldwide. Point-of-care ultrasonography (POCUS) has the potential of being the first-line modality in the evaluation of acute patients with such non-specific symptoms. To date, there is no published article on the use of POCUS in the identification or management of GISTs. And presented here are two cases of GIST with different clinical presentations where POCUS had an impact on the management.A 64-year-old woman presented to the emergency department (ED) after three episodes of syncope. She also had an increasing abdominal girth with epigastric fullness and discomfort for the prior few months. Clinically her vital signs were blood pressure (BP) 146/97 millimeters of mercury (mmHg), pulse rate of 121 beats per minute (bpm), respiratory rate 18 breaths per minute (BPM), pulse oximetry of 96% on room air and temperature at 36.6 degrees Celsius (C). Her abdomen, though distended, was soft and non-tender. A point-of-care ultrasound (POCUS) was performed to evaluate for possible aortic pathology but revealed a large left hypochrondrial mass of mixed echogenicity ; intra-aCTAP was performed, which showed a large heterogeneous exophytic mass (9.2.× 9.5 × 9 cm) arising from the greater curve of the stomach suspicious of GIST , with heIntra-operative finding revealed a thin-walled stomach GIST with recent bleeding and intraperitoneal hematoma adjacent to the GIST. Histopathological report was that of GIST of intermediate to moderate risk of behavior.A 53-year-old man presented to the ED with a month of lower abdominal discomfort, which was more pronounced when the bladder was full. He had already undergone a cystoscopy and colonoscopy, which did not reveal any significant finding to account for his symptoms. At the ED, his vital signs were BP 122/71 mmHg, pulse rate of 92 bpm, respiratory rate 18 bpm, pulse oximetry of 99 % on room air and temperature at 36.2ºC. Clinical examination of the abdomen revealed a left lower quadrant mass that was mildly tender on palpation. POCUS showed a suprapubic mass of mixed echogenicity with a marginal halo .CPC-EM CapsuleWhat do we already know about this clinical entity?Gastrointestinal stromal tumors (GISTs) are rare malignancies of the gastrointestinal tract, which are often incidental findings on computed tomography (CT). Symptomatic patients often present late and the diagnosis would usually involve CT and endoscopic ultrasonography.What makes this presentation of disease reportable?Point-of-care ultrasound (POCUS) has never been previously described in the literature as a modality for the diagnosis of GIST. This case report illustrates the usefulness of POCUS in the evaluation of patients with non-specific abdominal symptoms and/or findings, leading to the diagnosis of GIST.What is the major learning point?POCUS may be a potential screening modality for patients with non-specific abdominal symptoms and/or findings, leading to the diagnosis of GIST.How might this improve emergency medicine practice?POCUS can help emergency physicians to expedite investigations and management plans for patients with non-specific abdominal symptoms and/or findings. This leads to an earlier diagnosis of GIST and the potential to improve the patient’s prognosis.He underwent a CTAP, which revealed a large centrally necrotic mass (9.3.x 11.6 × 9.3 cm) that was closely related to the pelvic small bowel loops , highly GISTs accounts for 90% of mesenchymal tumors in the gastrointestinal tract and 2–3% of all gastric malignancies.6The most common symptom at presentation is bleeding,Trans-abdominal sonography can help to characterize the internal content of both the primary and metastatic GIST. It usually appears as a homogenously hypoechoic mass in close relation to the gastrointestinal tract, but a variable degree of heterogeneity is seen in larger GISTs representing necrosis, cystic changes and hemorrhages. The site of origin is often hard to detect on US especially for a large tumor.Although we are not able to confidently diagnose a GIST based on a point-of-care ultrasound, certain sonographic features, e.g., heterogenicity and marginal halo of an abdominal mass that is not within an intra-abdominal organ such as liver, spleen or kidneys, are suggestive of GIST and would prompt us to arrange for urgent definitive imaging modalities. Our two cases indicate that point-of-care ultrasound may be useful in screening for patients with vague abdominal pain.


Soil respiration induced by biological soil crusts (BSCs) is an important process in the carbon (C) cycle in arid and semi-arid ecosystems, where vascular plants are restricted by the harsh environment, particularly the limited soil moisture. However, the interaction between temperature and soil respiration remains uncertain because of the number of factors that control soil respiration, including temperature and soil moisture, especially in BSC-dominated areas. In this study, the soil respiration in moss-dominated crusts and lichen-dominated crusts was continuously measured using an automated soil respiration system over a one-year period from November 2015 to October 2016 in the Shapotou region of the Tengger Desert, northern China. The results indicated that over daily cycles, the half-hourly soil respiration rates in both types of BSC-covered areas were commonly related to the soil temperature. The observed diel hysteresis between the half-hourly soil respiration rates and soil temperature in the BSC-covered areas was limited by nonlinearity loops with semielliptical shapes, and soil temperature often peaked later than the half-hourly soil respiration rates in the BSC-covered areas. The average lag times between the half-hourly soil respiration rates and soil temperature for both types of BSC-covered areas were two hours over the diel cycles, and they were negatively and linearly related to the volumetric soil water content. Our results highlight the diel hysteresis phenomenon that occurs between soil respiration rates and soil temperatures in BSC-covered areas and the negative response of this phenomenon to soil moisture, which may influence total C budget evaluations. Therefore, the interactive effects of soil temperature and moisture on soil respiration in BSC-covered areas should be considered in global carbon cycle models of desert ecosystems. Howevestation ,4. Soil estation ,5. Thus,2 net uptake rates recorded in desert ecosystems [Biological soil crusts (BSCs) are widespread communities that consist of cyanobacteria, green algae, lichens, mosses and other organisms, and they are closely integrated with particles of the soil surface in arid and semi-arid regions . These cosystems . Previouosystems . Anotherosystems . TherefoAutomated soil respiration systems have been widely used to provide continuously dense datasets that reveal a complex relationship between soil respiration and temperature, which is not easily illustrated using less frequent survey measures. Numerous studies have analyzed the data from automated soil respiration systems and found significant diel hysteresis, which is supported by the semielliptical shapes observed in the regression analyses between temperature and soil respiration –19. StudThis experiment was conducted at the Shapotou Desert Research and Experimental Station, Chinese Academy of Sciences , a department of the Northwest Institute of Eco-Environment and Resources, Chinese Academy of Science. The study was approved by the Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences. No specific permissions were required for sample collection in the Shapotou-Yiwanquan region , and the field work did not involve any endangered or protected species.This study was conducted in the Shapotou-Yiwanquan region , which is located in Zhongwei County in the Ningxia Hui Autonomous Region at the southeastern edge of the Tengger Desert in northern China. This area is a transitional zone between desertified steppe and sandy desert . The meaCaragana korshinskii and Artemisia ordosica, and the total plant cover is approximately 38.6%. The predominant natural herbal species are Echinops gmelinii, Artemisia capillaries, Allium mongolicum and Salsola ruthenica, which present coverage of approximately 29.1%. In addition, open areas between plant patches, which consist of moss-dominated and lichen-dominated crusts, have a cover of approximately 51.2% and 33.6%, respectively. The moss species include Didymodon constrictus (Mitt.) Saito., Bryum argenteum Hedw., Tortula desertorum Broth., and Tortula bidentata Bai Xue Liang [Endocarpon pusillum Hedw., Collema tenax (Sw.) Ach., and a number of Diploschistes muscorum (Scop.) R. Sant and Squamarina lentigera (G.H. Weber) Poelt [A field investigation of the vegetation was conducted in July 2015. The dominant natural shrub species are ue Liang . The licr) Poelt . The phyIn July 2015, intact BSC samples (moss-dominated crusts and lichen-dominated crusts) were collected using PVC collars (20 cm in diameter and 20 cm in height) in the Shapotou-Yiwanquan region. To avoid terrain and vegetation influences on BSC development, all samples were randomly collected from undisturbed soil in the spaces between shrubs. The BSC samples were taken to the Shapotou Desert Research and Experimental Station and randomly buried in the soil (still in the PVC collars) of six plots (10 m × 10 m each), and the BSC surfaces were placed at the same level as the soil surface. To ensure that the test conditions closely resembled the natural environment, we maintained the natural soil water and air cycles by keeping the bottoms of the PVC collars open for drainage. Each type of BSC had three replicates in each plot, and they were incubated for four months before the first measurements.BSC, μmol m-2 s-1) were measured using an Automated Soil Respiration System  from November 2015 to October 2016. A permanent opaque chamber  was set on each collar. The measurement time for each chamber was 3 min and 10 s, including a 45 s pre-purge, a 45 s post-purge, a 90 s observation period, and a 10 s dead band. During the measurements, one-third of the samples were randomly measured on the first day, and the other samples were measured on the following two days. All plant seedlings present within the measurement collar were manually removed. The half-hourly soil temperature (T) and volumetric soil water content (VWC) were measured at a 5 cm soil depth outside of each chamber using the 8150–201 soil temperature sensor and 8150–204 ML2x soil moisture sensor . Rainfall was measured at a distance of 1.6 km from the research location using a manual rain gauge during the experiment.The soil respiration rates of the BSC .All Rdata set . Instrumerformed ,25. A re3/m3 in the moss-dominated crusts and from 0.001 to 0.189 m3/m3 in the lichen-dominated crusts .Because of the marked seasonal variations in soil temperature and moisture, the half-hourly soil respiration rates in the moss-dominated crusts and lichen-dominated crusts presented a temporal variation from November 2015 to October 2016. The relationship between the half-hourly soil respiration rates and soil temperature at a depth of 5 cm in the two types of BSC-covered areas showed daily hysteresis loops with semielliptical shapes each month over the daily cycles, and the hysteresis loops varied at a temporal scale among months Figs . Over thContinuous measurements of half-hourly soil respiration rates in both the moss-dominated and lichen-dominated crusts were conducted with an automated soil respiration system in a desert ecosystem and provide new insights into the control of soil respiration in BSC-covered areas base on environmental variables at diurnal and seasonal time scales. Over the daily cycles, the half-hourly soil respiration rates in both types of the BSC-covered areas were commonly related to soil temperature, and soil temperature often peaked later than the half-hourly soil respiration rates in the BSC-covered areas.13CO2 values, and most of the depletion was observed on nights when the atmospheric CO2 showed strong increases [13CO2 values was hypothesized to be the result of the non-steady state process “diffusive fractionation”, which is induced by the increasing atmospheric CO2 concentrations at nighttime, especially in arid ecosystems [2 efflux is strongly coupled with the temperature and moisture of the surface BSC layer in the BSC interspaces, whereas it is less strongly correlated with the temperature and moisture of the deeper soil layers [2 production [Over the course of the diurnal cycles, our results demonstrate that a significant time lag occurred between the half-hourly soil respiration rates and soil temperature at a depth of 5 cm in both types of BSC-covered areas, with the soil respiration peaking earlier than the soil temperature Figs  and 3. Ancreases . This obosystems . Lastly l layers . In addil layers ,15,39. Toduction –42. Thus2 produced at the topmost soil layer compared with that produced at lower depths, thereby reducing lag times. Therefore, further investigations of the lag times between soil respiration and soil temperature under differences in soil water content are warranted.Soil respiration and soil temperature present a time lag, and these variables are plotted against each other in a semielliptical form, and vary seasonally with the soil moisture , 19, 28.2 production. Lag times between soil respiration rates and soil temperatures in the BSC-covered areas were negatively related to the soil moisture, which is possibly because water transfers heat more efficiently than air. This finding suggests that global carbon cycle models should account for the interactive effects of soil temperature and moisture on soil respiration in BSC-covered desert ecosystems. Moreover, the diel hysteresis phenomenon between soil respiration and soil temperature should be considered to accurately evaluate the total C budgets in BSC-dominated areas at the ecosystem level.In this study, high-frequency soil respiration measurements were performed in a BSC-dominated ecosystem at the southeastern edge of the Tengger Desert in northern China to identify the temporal variations between soil temperature and soil moisture over the diel and seasonal cycles. Over the daily cycles, the proximate cause of the diel hysteresis between soil respiration rates and soil temperatures in the BSC-covered areas is likely a mismatch between the depth of the soil temperature and moisture measurements and the depth of COS1 Table(DOCX)Click here for additional data file.


To evaluate the relative cost-effectiveness of introducing the RTS,S malaria vaccine in sub-Saharan Africa compared with further scale-up of existing interventions.A mathematical modelling and cost-effectiveness study.Sub-Saharan Africa.People of all ages.The analysis considers the introduction and scale-up of the RTS,S malaria vaccine and the scale-up of long-lasting insecticide-treated bed nets (LLINs), indoor residual spraying (IRS) and seasonal malaria chemoprevention (SMC).Plasmodium falciparum cases averted in all age groups over a 10-year period.The number of Assuming access to treatment remains constant, increasing coverage of LLINs was consistently the most cost-effective intervention across a range of transmission settings and was found to occur early in the cost-effectiveness scale-up pathway. IRS, RTS,S and SMC entered the cost-effective pathway once LLIN coverage had been maximised. If non-linear production functions are included to capture the cost of reaching very high coverage, the resulting pathways become more complex and result in selection of multiple interventions.RTS,S was consistently implemented later in the cost-effectiveness pathway than the LLINs, IRS and SMC but was still of value as a fourth intervention in many settings to reduce burden to the levels set out in the international goals. We sought to evaluate the relative cost-effectiveness of introducing the RTS,S malaria vaccine in sub-Saharan Africa compared with further scale-up of existing interventions. We did not identify any studies modelling the impact and costs of intervention packages with respect to scaling-up coverage.We find that implementing the RTS,S malaria vaccine generally only enters the optimal pathway of scale-up of interventions once very high coverage of vector control interventions, along with seasonal malaria chemoprevention in settings where it is recommended, has been achieved.While the RTS,S malaria vaccine can be an effective tool for reducing burden, enhancing coverage of vector control should generally remain of higher priority across sub-Saharan Africa. This is particularly the case within settings where universal coverage of long-lasting insecticide-treated nets has not yet been achieved.Following the adoption of the Millennium Development Goals (MDGs) in 2000, the concurrent targets of increasing the proportion of children under 5 years that sleep under a bed net and that have access to appropriate antimalarial drugsIn early 2015, the final results from the large phase III trial of the RTS,S malaria vaccine across 11 sites in Africa were published.5Existing malaria interventions are also highly cost-effective.9Plasmodium falciparum malaria and its associated interventionsHere, we use a well-established transmission model for P. falciparum malaria transmission that incorporates the full suite of interventions.P. falciparum malaria from bites of infectious mosquitoes. The rate at which susceptible individuals become infected is influenced by mosquito density and infectivity and is moderated by immunity. Infants are partially protected for the first 6 months of their life by passively acquired maternal immunity and individuals acquire natural immunity with repeated exposures through time. Infected individuals may be treated, after which follows a period of prophylaxis before returning to the susceptible class. Infected individuals can develop clinical disease,14We used an established model of PfPR2_10) was simulated in 17 bands between 0.1% and 80% to capture the range of transmission levels observed prior to intervention scale-up.Anopheles gambiae s.s./Anopheles funestus to those associated with Anopheles arabiensis. The combination of parasite prevalence bands, seasonality of transmission and vector bionomics resulted in 272 baseline strata. Further details are provided in the online To capture the range of transmission settings across Africa, we generated a baseline set of ‘strata’. These were characterised by the parasite prevalence in the absence of interventions other than treatment, the annual seasonal pattern of transmission and the mosquito vector species present and their associated bionomics (which affect the predicted impact of LLINs and IRS). The mean parasite prevalence over the full year in 2–10 year-olds  at a range of coverage levels  or full correlation . Throughout we assumed that 60% of those with clinical disease received prompt and effective first-line treatment.In the absence of detailed country-level data for all interventions, we adopted a unit costing approach. These were derived from the literature and inflated to 2015 US$ .et al,We considered two approaches for costing increasing coverage of the four interventions. The first approach assumed increases in coverage were associated with linear increases in cost, while in the second approach, we derived non-linear relationships between coverage and unit costs. For this second approach, the number of nets required to achieve a given coverage level (defined by usage) was obtained from Bhatt τ, above which the delivery costs increase logarithmicallyThe commodity cost remains fixed per person  with respect to coverage (C). The delivery cost per person is fixed at a baseline amount, N, until coverage reaches a given threshold, CWe considered a number of outcome measures to evaluate the incremental cost-effectiveness of each intervention. The primary outcome measure presented throughout is the number of cases averted over a 10-year period. Other outcome measures considered include the DALYs averted and the number of cases averted in children aged 6 months–5 years old and over a 10-year period  start with 0% usage/coverage of all interventions, (ii) for each available intervention, calculate the ICER of scaling up to the next usage/coverage level, (iii) implement the step with the lowest associated ICER (the most cost-effective), (iv) repeat the process (i–iii) until the scale-up of all interventions has been maximised or elimination is achieved. This process is summarised in We assessed the sensitivity of the order of scale-up to uncertainty in the costs of interventions and their associated production functions. The analysis was repeated with 100 random draws from the posterior predictive interval for the IRS, SMC and RTS,S production functions. LLIN costs were randomly drawn from the interval between the least optimistic  and most optimistic  net-allocation models.The sensitivity of the scale-up order to the assumed $5 per dose cost of the vaccine was also examined by incrementally decreasing its price . At each step, the proportion of settings where the vaccine appeared in the pathway before LLINs, IRS and SMC was then recorded.PfPR2–10 . For the majority of settings, across the full-range of baseline PfPR2–10, for vector bionomics characteristic of the three main species found in Africa  and for seasonal and non-seasonal settings, LLINs appear first in the most cost-effective pathway. At baseline PfPR2–10 <5%, LLINs and RTS,S are predicted to reduce ongoing transmission to pre-elimination levels. If scale-up of these interventions is unable to achieve pre-elimination transmission levels, our results suggest that LLINs should be scaled up to very high usage levels (75%) prior to introducing additional interventions if cost-effectiveness is the single deciding factor. After this level has been achieved, in non-seasonal settings, IRS is the second-most cost-effective intervention at lower baseline PfPR2–10 (5%< PfPR2–10 <65%), while RTS,S is estimated to be the second most cost-effective intervention in settings with baseline PfPR2–10 ≥65%. In seasonal settings, SMC is generally the second most cost-effective intervention to introduce prior to IRS or RTS,S.The results shown in PfPR2–10<60%, IRS appears second with RTS,S also introduced once moderate levels of IRS coverage have been achieved. In seasonal settings, for baseline PfPR2–10<50%, LLINs remain the first most cost-effective intervention. However, in settings with higher baseline transmission, SMC and RTS,S appear earlier. Following this, the second and third most cost-effective additional interventions vary by setting, with IRS being favoured in locations where the vector bionomics are more amenable to insecticidal control, whereas RTS,S or SMC are favoured in settings with less favourable vector bionomics.Including the non-linear production functions shown in PfPR2–10 and seasonality. Across the majority of settings, LLINs are the first most cost-effective intervention. Using the results from PfPR2–10, other interventions are estimated to be more cost-effective at lower levels of LLIN usage. This threshold is similar to the levels of usage that have now been achieved in many parts of Africa (f Africa B.While the results vary for different assumed unit costs for each intervention, in a sensitivity analysis of these costs, we found that the order of scale-up is generally maintained A. Since Our analysis demonstrates that LLINs remain the most cost-effective first intervention to reduce malaria transmission across the broad range of transmission settings observed in sub-Saharan Africa. The high consumption and competitive market for LLINs has driven costs down over the last 10 years.While increasing usage of LLINs is identified as the most cost-effective first intervention, once these levels reach 50–60%, we estimate that the three alternative interventions—IRS, SMC and RTS,S—become increasingly competitive when comparing the relative cost-effectiveness. This level of LLIN usage is similar to the levels reported for many countries in sub-Saharan Africa in 2015,We explicitly excluded treatment scale-up options from the cost-effectiveness analysis for several reasons. First, treatment has been previously shown to be highly cost-effective.Systems-level inefficiencies,While cost-based uncertainties were explored in this analysis, we did not additionally explore the uncertainty in model structure or parameterisation due to the computational complexity in undertaking such an analysis. Clearly, model parameters and structures could affect the relative impact of interventions as well as the combinations of interventions needed to reach pre-elimination levels. Determining the cost-effective scale-up in a stepwise manner informed by the ICER always chooses the next most cost-effective option. This is analogous to a gradual scale-up of interventions over time, where future options are considered, given an established intervention landscape. However, for a given spend, the resulting intervention package estimated from a stepwise approach may differ from a global optima if all scale-up options were considered in unison.Context-specific challenges to scaling-up a given intervention will always be present and cannot be represented in this style of analysis. To this end, policy decisions must take into account such challenges when considering recommendations in a specific setting. While a number of countries have achieved the high levels of LLIN usage that our analysis suggests to be cost-effective,


FKBP5) gene as a sensitivity gene. However, previous GxE studies with FKBP5 have not measured the full environmental spectrum or applied statistical tests to discern whether the GxE interaction fits better with the differential-susceptibility or diathesis-stress hypotheses. This study examined whether single nucleotide polymorphisms (SNPs) on FKBP5 gene moderate the association of positive and negative recent life events (LEs) with depressive symptoms, state-anxiety, neuroticism, and social anxiety traits.Gene-environment interaction (GxE) research has highlighted the importance of investigating the FK506 binding protein 51 .A total of 86 nonclinical young adults were administered psychological measures and were genotyped for five Regression analyses indicated significant GxE interactions for social anxiety and neuroticism. The interactions predicting neuroticism fit different models for different SNPs, although the overall effect indicated by the haplotype was consistent with the differential-susceptibility hypothesis: the risk-haplotype group presented higher neuroticism in the presence of more negative LEs and lower neuroticism in the presence of more positive LEs. The GxE interactions for social anxiety were consistent with the diathesis-stress model. The lack of significance in the for-better side for social anxiety might be related to the fact that it mapped onto low extraversion, which is associated with a lower permeability to positive experiences.FKBP5 to adequately characterize its role in healthy and pathological developmental processes.Findings underscore the importance of testing the differential-susceptibility model in relation to  Gene-Environment interaction (GxE) research has been primarily guided by the diathesis-stress model –3, whichAlthough a fast-growing research has recently pointed to the critical role of positive environments , the difFKBP5), located on chromosome 6p21.31, is a highly interesting target for GxE as it is considered a shared etiological factor underlying stress-related disorders ).T-test comparisons revealed that none of the 2 nor X2Z (or their combination) were statistically significant , confirming the absence of nonlinear phenomena masked by the significant linear-interactions. No significant GxE interactions emerged for depressive symptoms or state-anxiety, although rs9296158, rs1360780 and the haplotype reached a trend towards significance for state-anxiety. The interaction of rs3800373, rs9296158 and the haplotype with LESms predicted both neuroticism and social anxiety, whereas the interaction of 1360780 with LESms only predicted neuroticism. Simple slopes analyses indicated that in all cases the effect of LESms on neuroticism and social anxiety was only significant for those carrying the risk alleles .Multiple regression main-effects analyses showed that genetic variables did not predict criterion measures . In contrast, positive LEs predicted lower levels of depressive symptoms , state anxiety , neuroticism  and social anxiety . Regarding GxE interactions, we found 7 significant results see . In all C/C or A/C) only differed from those carrying the non-risk alleles (A/A) in that they showed less neuroticism when experiencing more positive LEs. PoI > 0.60 and PA > 0.16 also supported the RoS on X analysis, highlighting the importance of the “for-better side” in this interaction. The interaction of LESms and rs1360780 on neuroticism was consistent with a diathesis-stress pattern evaluated with the RoS on X and the PoI indices (PoI 0.40 > 0.60), that is, the risk group showed higher neuroticism when experiencing more negative LEs. However, it was consistent with differential-susceptibility as evaluated with the PA, which indicated that 21% (criterion > 16%) of individuals were differentially affected by the interaction. Regarding rs9296158 and the haplotype, all DE/DS-indices converged to be highly consistent with a differential-susceptibility pattern. Individuals carrying either risk alleles or the risk-haplotype had higher neuroticism when experiencing more negative LEs as well as lower neuroticism when experiencing more positive LEs as compared with non-risk carriers  lost statistical significance (p = 0.26), whereas all the other interactions involving a for-better side  remained almost the same  was consistent with differential-susceptibility. The overall differential-susceptibility pattern was consistent with the cross-sectional study by Pluess et al. , who fout effect .DRD4 gene might be an interesting candidate as it is linked to extraversion and has been proposed as a sensitivity marker [Unlike the differential-susceptibility effect in neuroticism, all significant interactions for social anxiety were consistent with a diathesis-stress pattern. This discrepancy might be explained by the interplay of social anxiety with neuroticism, extraversion, and resilience. Social anxiety is a combination of high neuroticism and low extraversion; actually, the genetic factors that influence those dimensions account entirely for the genetic liability to social anxiety . Interesy marker , 47.In the diathesis-stress model, being affected by environmental adversities is equivalent to being vulnerable, whereas being unaffected is equivalent to being resilient. By contrast, in the differential-susceptibility model, putatively vulnerable individuals are considered to be plastic in a for-better-and-for-worse pattern, whereas the putatively resilient individuals are thought to be “unmalleable” to both supportive and adverse environments . It seemFKPB5 x adult LEs interactions on depressive symptoms found in this study is partially related to the fact that epigenetic changes that mediate the association of early-life adversity with adult depression are no longer  induced by the impact of negative (though not necessarily severe) LEs in adulthood. Regarding state anxiety, the measure used in our study is largely characterized by somatic complaints and might not be as sensitive to FKPB5 x LEs interactions as the trait and social component measures.Previous studies reporting GxE interactions on adult depressive outcomes mostly evaluated the effect of distant and severe childhood adversities . In thosThe strengths of this study include the estimation of a haplotype that increases the power to detect genetic associations  and the FKBP5 gene may have interesting implications. For instance, there have been efforts to develop drugs that block FKBP5 activity in order to modify the risk for the development of stress-related disorders [FKBP5 activity in individuals carrying ‘risk’ genotypes might have the undesirable effect of altering a natural resilience mechanism: the potential of benefitting from positive and supportive environments.From a clinical standpoint, this avenue of research with the isorders , 16 baseTo conclude, our results indicated that the interactions predicting neuroticism fit different models for different polymorphisms, although the overall effect indicated by the haplotype was consistent with the differential-susceptibility hypothesis. The interactions predicting social anxiety  were consistent with the diathesis-stress model, possibly reflecting that the low extraversion component of social anxiety is associated with a lower permeability to positive experiences.S1 Table(DOCX)Click here for additional data file.


Dental care seeking behaviour is often driven by symptoms. The value of oral health related quality of life (OHRQoL) measures to predict utilisation of dental services is unknown. This study aims to explore the association between OHRQoL and problem-oriented dental attendance among adults.We analysed cross-sectional data for 705 adults, aged 16 years and above, living in three boroughs of Inner South East London. Data were collected during structured interviews at home. The short form of the Oral Health Impact Profile (OHIP-14) was used to assess the frequency of oral impacts on daily life in the last year. Problem-oriented attendance was defined based on time elapsed since last visit (last 6 months) and reason for that visit (trouble with teeth). The association between OHIP-14  scores and problem-oriented attendance was tested in logistic regression models adjusting for participants’ sociodemographic characteristics.Problem-oriented attenders had a higher OHIP-14 total score than regular attenders . In regression models, there was a positive association between OHIP-14 total score and problem-oriented attendance. The odds of visiting the dentist for trouble with teeth were 1.07 greater  per unit increase in the OHIP-14 total score, after adjustment for participants’ sociodemographic characteristics. In subsequent analysis by OHIP-14 domains, greater scores in all domains but handicap were significantly associated with problem-oriented attendance.This study shows that oral impacts on quality of life are associated with recent problem-oriented dental attendance among London adults. Six of the seven domains in the OHIP-14 questionnaire were also associated with dental visits for trouble with teeth. Dental care-seeking behaviour is mainly motivated by symptoms , 2. The Earlier studies on the relationship between OHRQoL and dental services utilisation were conducted in children –11. WhilA population-based oral health survey was carried out in Inner South East London to identify perceived dental needed and dental care seeking behaviour of adults in Inner South East London. King’s College London Research Ethics Committee approved the survey protocol. A representative, multi-ethnic sample of the general population aged 16 years and above living in Lambeth, Southwark and Lewisham  was recruited using stratified multistage random sampling. A list of all valid addresses for postal delivery in each borough (stratum) organised by postcode sectors (primary sampling units) was the sampling frame. Addresses were selected in two stages. Fifteen postcode sectors were first selected from each borough randomly and a sample of addresses was subsequently selected from each sector randomly. Household rosters were generated in every eligible household, from which only one adult was invited to participate. A target of 500 addresses per borough was initially set out . Of them, 1244 were valid and 770 (62% response rate) agreed to take part in the survey [n = 32), OHRQoL (n = 22) and social grade (n = 14). Our study sample included 695 adults (56% of the eligible sample). A power calculation showed that our analytical sample had 90% power to detect a difference in the OHIP-14 total score of 2 units or greater between regular and problem-oriented attenders (using a 5 to 1 ratio), at 5% significance level and assuming a common standard deviation of 6 units.For the present analysis, 65 participants were excluded because of missing values on ethnicity , dental care seeking behaviour and perceived dental needs, all based on previous national surveys –18. PartParticipants’ OHRQoL was assessed with the short form of the Oral Health Impact Profile (OHIP-14) , which mParticipants also reported the time since last dental visit  and the reason for that visit . Based on the answers to both questions, recent problem-oriented attendance was defined as having visited the dentist in the past 6 months for trouble with teeth. The 6-month threshold was selected to mimic a temporal ordering between exposure  and outcome  and identify dental services use that most likely occurred after the experience of oral impacts .All analyses were weighted to correct for differences in the probability of selection because of non-response and non-coverage, and to adjust for differences in the age-by-sex-by-ethnicity distribution between the sample and the general adult population 16 years or older in the three London boroughs included in the study, according to the 2001 UK Census , 20.t-test. The association between OHIP-14 total score and recent problem-oriented attendance was assessed in crude and adjusted logistic regression models. The adjusted model controlled for sex, age groups, ethnicity, borough of residence and social grade. A similar set of models was used when testing the association between each OHIP-14 domain score and problem-oriented attendance. Multilevel modelling (participants nested within boroughs) was not considered due to the limited number of clusters. Instead, we included borough of residence as an explanatory variable in regression models as it is often done for geographical variables with few groupings such as rural/urban status.We first present the characteristics of the sample and compare them against those excluded due to missing values with the Chi-square test to evaluate the impact of missing data. We then compared the proportion of problem-oriented attenders by sociodemographic characteristics with the Chi-square test. OHIP-14 total and domain scores were compared between problem-oriented and regular attenders using the The study sample consisted of 705 adults 51% women) residing in Inner South East London. Table % women rThe mean OHIP-14 score was 4.19 , with the highest score for physical pain  and the lowest score for social disability (mean: 0.22 SD: 0.77). Problem-oriented attenders had a significantly higher OHIP-14 total score than regular attenders (6.73 versus 3.73). Problem-oriented attenders also had significantly higher OHIP-14 scores than regular attenders in all domains except for handicap . Second, the study sample represented 56% of the eligible sample, which some may view as non-representative. However, we used analytical weights to correct for key demographic differences between the study sample and the local population living in Inner South East London, thus allowing for the generalisation of findings to the study population. That said, the present findings are not generalizable beyond the study population . Third, participants’ utilisation of dental services was obtained through self-reports, which could be affected by information bias. However, self-reported dental visits have shown high levels of agreement with data from dental records , 27. FouGoing back to our findings, the magnitude of the association between oral impacts on quality of life and problem-oriented attendance was such that the odds of visiting the dentist for trouble with teeth increased by 7% per every unit increase in the OHIP-14 total score. There was a clear dose-response relationship, with greater odds of using dental services at higher OHIP-14 scores. Our finding disagrees with the only previous study that used the OHIP-14 to measure OHRQoL [Six of the seven domains in Locker’s model of consequences of oral health , spanninThere are some implications for policy and research. There might be some benefit in using OHRQoL measures for planning and predicting utilisation of dental services. In this regard, the socio-dental approach is a way forward in the complementary use of clinical and perceived measures for needs assessment, planning and prioritisation of services , 35. InfThis study shows that oral impacts on quality of life in the last year are associated with recent problem-oriented dental attendance among adults in Inner South East London. Six of the seven domains in the OHIP-14 questionnaire were also associated with dental visits for trouble with teeth.


Objective: To study the effects of intravitreal anti-Vascular Endothelial Growth Factor (VEGF) therapy with Avastin for wet Age-Related Macular Degeneration (AMD) on Benign Prostatic Hyperplasia (BPH)-related symptoms. Methods: An exploratory trial was conducted from August 1, 2013 to February 1, 2014, that included 14 male patients previously diagnosed with BPH, who were aged between 59 and 69 years. The trial was performed in Bucharest and involved two medical institutions: the Clinical Hospital of Eye Emergencies and the “Prof. Dr. Theodor Burghele” Hospital. This prospective study utilized both objective and subjective indicators to analyze the link between intravitreal anti-VEGF therapy for wet AMD and BPH. The evaluations consisted of uroflowmetry and International Prostate Symptom Score (I-PSS) assessments. Results: The maximum flow rate (Qmax) improved by an average of 5.05 ml/ sec in 9 patients, whereas the remaining 5 patients showed a slight decrease in Qmax (mean 1.6 ml/ sec). The I-PSS score improved, with an overall decrease of 1.18 points at follow-up compared to the initial score .Conclusion: The analysis revealed that anti-VEGF therapy for wet AMD had a significant positive effect on all BPH-related symptoms; patients reported improved urinary streams and decreased nocturia.Abbreviations: BPH = benign prostatic hyperplasia, AMD = age-related macular degeneration, VEGF = vascular endothelial growth factor, I-PSS = international prostate symptom score, Qmax = maximum flow rate, TSP-1 = thrombospondin-1, FGF-2 = fibroblast growth factor, mRNA = precursor messenger ribonucleic acid, PSA = prostate-specific antigen, DRE = digital rectal examination, AUR = acute urinary retention, COX2 = cyclooxygenase 2, QoL = quality of life Much research has focused on the key molecules that regulate new vessel formation. One of the most important angiogenic molecules is VEGF , also known as VPF (Vascular Permeability Factor), a potent and specific angiogenesis-related cytokine that is responsible for endothelial cell differentiation, migration, and proliferation as well as tubular formation and vessel assembly [2].Recent reports in literature have addressed the importance of the VEGF system in the development of the normal prostate and prostatic hyperplasia. VEGF is one of the most potent regulators of angiogenesis and has been shown to act on two tyrosine kinase family receptors: c-fms-like tyrosine kinase (Flt-1) and fetal liver kinase[3]. This pro-angiogenic activity depends primarily on Fibroblast Growth Factor (FGF-2) and/or VEGF, the secretion of which is increased in BPH. During disease progression in the prostate, the production of the major inhibitor TSP-1 is down-regulated, whereas that of stimulatory FGF-2 and/or VEGF is increased, leading to the induction of new vessels[4].Normal prostate epithelial cell secretions are anti-angiogenic due to the inhibitory effects of thrombospondin-1 (TSP-1), whereas this inhibitor is decreased in the pro-angiogenic secretions derived from benign prostatic hyperplasia (BPH)[5], support the hypothesis that VEGF is synthesized predominantly by prostatic hyperplastic epithelial cells. The majority of the staining for endothelial cells could be accounted for by the VEGF’s binding to specific endothelial cell receptors. Stromal VEGF immunoreactivity could be attributed to the binding of VEGF, which is a heparin-binding growth factor, to extracellular matrix proteins[6] or to the production of VEGF by stromal cells. The widespread distribution of the VEGF receptor Flt-1 in BPH specimens suggests that the VEGF function in the prostate is not restricted to endothelial cells and angiogenesis[7].Immunolocalization studies have confirmed that the changes detected in vitro also occur in vivo. The localization of immunohistochemical staining, combined with published reports on VEGF precursor messenger ribonucleic acid (mRNA)[7]. Interestingly, androgens seem to regulate the VEGF expression in the prostate because castration acts through the VEGF system to inhibit angiogenesis and thereby induce apoptosis . Consistent with most reports, there is no significant VEGF expression in the normal prostatic epithelium. Anti-VEGF therapy has two primary effects: anti-angiogenic and anti-permeable  .All the 14 subjects from the final group underwent a complete urological evaluation after signing the informed consent, which included submitting an I-PSS questionnaire and undergoing uroflowmetry the day prior to their first Bevacizumab (Avastin 0.05 ml) injection. Subjects were also followed up 30 days later, on the same day as the administration of their second Bevacizumab injection.The subjects were selected from a group of patients diagnosed with wet AMD for whom no prior treatment was administered. Patients were selected to undergo intravitreal anti-VEGF therapy with Avastin®. Bevacizumab  is a VEGF inhibitor that hinders the formation of new blood vessels. It is licensed only for intravenous administration for a number of different types of cancer at doses typically in excess of 200 mg per infusion. Bevacizumab is also administered intravitreally at a dose of 1.25 mg (0.05 ml) as an off-label treatment for a wide range of ocular diseases, such as AMD . We selected Qmax (maximum flow rate) as the primary variable, as it is capable of providing the most clinically relevant and convincing evidence directly related to the primary objective of the trial. The data collection and analysis focused on one primary variable and several secondary variables.None of the patients included in our trial suffered from significant side-effects after the administration of anti-VEGF therapy . A Qmax value of over 15 ml/sec is usually considered normal, while a Qmax below 7 ml/sec is generally accepted as low. Of the patients included in the trial, 3 registered low Qmax values. After administrating the treatment, 2 of the patients with a low initial Qmax value did not respond to the treatment as expected, possibly due to the advanced disease. Qmax is considered one of the best measurements for diagnosing and monitoring BPH. Therefore, the positive change registered in nocturia after Bevacizumab treatment was considered highly relevant in the context of this study. Patients who reported the highest decrease in the number of voids per night consistently reported the largest improvements in the QoL indicator. Patients reported improvements in all I-PSS indicators. We focused on nocturia as this symptom was one of the most disturbing and frequently reported by patients with BPH. In addition, this indicator is sensitive and easily quantifiable by the patients compared with the others included in the I-PSS score. However, the associated systemic conditions were mild and well compensated through medical therapy alone. Medical conditions such as diabetes or cardiovascular diseases did not show any significant influence upon the outcomes of the study.Additionally, we were only able to perform a limited array of investigations  questionnaire) (2-7].Identifying the angiogenic factors involved in prostate growth and understanding their regulation may yield useful prognostic markers that can lead to the development of anti-angiogenic strategies and therapies. However, understanding the direct effects of Bevacizumab on BPH remains challenging for specialists in the field, as there is no mention of clinical research in this area. A review of literature showed that the data regarding the therapeutic effects of anti-VEGF therapy on BPH is only based on in vitro experiments [Whether anti-VEGF therapy could represent a future therapy for BPH, the exact mechanism of its action on the hypertrophied prostate gland has yet to be established. None of the professionals involved in this exploratory trial expected the results to be representative of all future users. We acknowledge that the methodology used for this experimental trial was limited and subjected to bias; further research must be apply a more rigorous methodological approach. So far, the results could be questioned due to the lack of control group for comparison. Additionally, although the results were statistically significant, the data should be interpreted with caution due to the very low number of subjects. This type of trial cannot act as the basis of a formal proof of efficacy, but it may contribute to the total body of relevant evidence. We believe that a confirmatory trial that can provide a firm support for the efficacy and safety of anti-VEGF therapy for BPH-related symptoms is mandatory before any definite conclusions are made.Conflicts of interestThe authors confirm that there are no conflicts of interest.


Drosophila melanogaster the same pathway maintains the hematopoietic precursor cells of the lymph gland, the hematopoietic organ that develops in the larva. Using Drosophila as an in vivo model, we investigated the mode of action of PF-04449913, a small-molecule inhibitor of the human Smo protein. Drosophila larvae fed with PF-04449913 showed traits of altered hematopoietic homeostasis. These include the development of melanotic nodules, increase of circulating hemocytes, the size increase of the lymph gland and accelerated differentiation of blood cells likely due to the exit of multi-potent precursors from quiescence. Importantly, the Smo inhibition can lead to the complete loss of hematopoietic precursors. We conclude that PF-04449913 inhibits Drosophila Smo blocking the Hh signaling pathway and causing the loss of hematopoietic precursor cells. Interestingly, this is the effect expected in patients treated with PF-04449913: number decrease of cancer initiating cells in the bone marrow to reduce the risk of leukemia relapse. Altogether our results indicate that Drosophila comprises a model system for the in vivo study of molecules that target evolutionary conserved pathways implicated in human hematological malignancies.The efficient treatment of hematological malignancies as Acute Myeloid Leukemia, myelofibrosis and Chronic Myeloid Leukemia, requires the elimination of cancer-initiating cells and the prevention of disease relapse through targeting pathways that stimulate generation and maintenance of these cells. In mammals, inhibition of Smoothened, the key mediator of the Hedgehog signaling pathway, reduces Chronic Myeloid Leukemia progression and propagation. These findings make Smo a candidate target to inhibit maintenance of leukemia-initiating cells. In  The dipteran Drosophila melanogaster has previously provided significant insights into oncogenic mechanisms. More recently, this well-established genetic model has been employed to study solid and hematological human malignancies, including their onset and progression. It has additionally been used in genetic screens and in the context of drug discovery platforms [Drosophila hematopoiesis is a developmental process that populates embryos, larvae and adults with circulating myeloid-like cells, known as hemocytes that regulate innate immunity and other processes. Three myeloid cell types circulate in the fly blood, the hemolymph: i) plasmatocytes, that are the most abundant, regulate the secretion of antimicrobial peptides and are provided with phagocytic ability to engulf small pathogens (i.e. bacteria) or cell debris; ii) crystal cells, that control the melanin production and the melanization process to inactivate big pathogen (i.e. eggs of parasite wasp); iii) lamellocytes, that are rarely seen in the hemolymph of healthy animals but that increase in number in case of big pathogen infestation since they are involved in the encapsulation of the pathogen that precedes melanization [Drosophila [in vivo genetic model based on Drosophila hematopoiesis to determine the impact at the cellular level of the small molecule PF-04449913, an inhibitor of the human Smo. Here we show that PF-04449913 impacts fly hematopoietic homeostasis, leading to increased and premature hemocyte differentiation and to the formation of melanotic nodules. The appearance of these nodules results from an excess of circulating lamellocytes and most likely to the formation of cell aggregates that are encapsulated and inactivated by the melanization process. They provide an easy-to-score phenotype due to the associated excess of blood cells and lamellocyte in particular [Drosophila alters hematopoietic homeostasis likely by inducing multipotent precursors to exit from quiescence, and accelerating blood cell differentiation. Moreover our body of evidence strongly supports Drosophila as a relatively cheap and fast genetic model to study and validate in vivo, at cell level, the way of action of small molecules targeting evolutionary conserved proteins and pathways implicated in hematological diseases and malignancies.A key challenge in the treatment of solid tumor and hematological malignancies with targeted therapeutic agents is the induction of stable remission and attainment of functional cure. The persistence of the disease phenotype is attributable to the presence of minimal residual disease that can be maintained by Cancer Stem Cells (CSC) –3. Examplatforms . In a manization –26. In tnization . Under pnization , 28, 29.nization –33. Giveosophila , 35 and osophila , we deverticular , 37. FurDrosophila lymph gland, the absence of the PSC or the inhibition of the Hedgehog signaling pathway, reduces the number of multipotent hematopoietic precursors in the medullary zone and induces premature hemocytes differentiation [Drosophila Smoothened (Smo) activity and induces phenotypes that can be connected to loss of the Hh pathway function  to produce small interfering RNAs targeting smo (smo-RNAi) or the dominant negative smo5A allele (smoDN) [domeless-Gal4  driver construct [smo function knock-down or inhibition induced melanotic nodules with a penetrance ranging between 12.35% and 35.6%. The results were always significantly different to negative controls represented by larvae expressing GFP and GFP-RNAi under the control of the domeless-Gal4 driver or by larvae carrying the driver construct only  driver [hemolectin-Gal4 driver is active in differentiating hemocytes of the lymph gland CZ, in the circulating and sessile hemocytes. Phalloidin labels filamentous actin making easier to identify lamellocytes by their bigger size and flattened morphology [domeless-Gal4 driver is expressed in the sessile hemocytes population, we analyzed the GFP expression at the position where sessile hemocytes reside both in dome-Gal4, UAS-GFP and hemolectin-Gal4, UAS-GFP larvae. In hml-Gal4, UAS-GFP larvae we could clearly observe GFP expressing cells at the subepidermal positions where sessile hemocytes are localized [domeGal4, UAS-GFP larvae   in the Monstruct . In eachy Figure . The VDRy Figure . All tog) driver  and stairphology . Then, wrphology , 36, 37.ocalized  but we cP larvae  We concldome-Gal4;UAS-GFP larvae fed with PF-04449913- or DMSO-containing medium and labeled with an antibody that recognize the intracellular domain of the Notch membrane receptor. Notch is expressed and required in differentiated crystal cells of the CZ [hh-RNAi in the PSC cells under the control of either one of two different Gal4 drivers: Serrate-9.6-Gal4, and Antennapedia-Gal4 [To evaluate if Smo inhibition can also induce increase of crystal cells differentiation, we dissected lymph glands from mid-L3 (m-L3) f the CZ , 43. Lymf the CZ  and withdia-Gal4   and a posterior (P) cell compartment  that drives Gal4 expression in the P compartment under the control of the en gene promoter. Wing imaginal discs were dissected from l-L3 larvae and immunolabeled with an antibody against the Ptc protein. In wing discs from DMSO treated larvae we could observe the high Ptc expression and the activation at low level of the en-dependent GFP-reporter in few anterior cells at the A/P border  with either DMSO- or PF-04449913-medium. Both reduction of smo gene dosage (smo3/+) and smo knock-down  induced melanotic nodules after DMSO-feeding in, respectively, 23% and 35.5% of larvae. This confirms the requirement of the Smo protein to maintain hemocytes homeostasis and indicates a partial aploinsufficiency of the smo gene function in hematopoiesis. The melanotic nodule penetrance increased to 47% and 50%, respectively, in smo3/+ and dome-Gal4;smo-RNAi larvae, after administration of the compound . In the absence of Hh: i) the three protein kinases form a complex with the scaffold kinesin protein Costal 2 (Cos2) and phosphorylate Ci leading to its proteolysis and conversion to the repressor form (CiR); ii) the conformation of the Fused (Fu) kinase permits Suppressor of fused (Su(fu)) to impede Ci nuclear translocation and activity thereby promoting its conversion to the repressor form  [ptc function in the MZ  or animals heterozygous for the Cos2k16101 null allele and expressing Su(fu)-RNAi in the MZ -RNAi). While after compound administration dome-Gal4;Cos2;Su(fu)-RNAi larvae had a less penetrant melanotic nodule phenotype compared to the wt (19% vs 35%), the penetrance was unchanged in larvae expressing ptc-RNAi as well as in larvae either heterozygous for the Cos2k16101 allele or expressing Su(fu)-RNAi in the MZ  until the stage l-L3 when the phenotype was evaluated. Both approaches induced the phenotype when the pathway activation was inhibited as from L1, L2 and e-L3 but not when it was inhibited as from m-L3  –53. We aZ Figure . The facdomeless-Gal4, UAS-GFP - Figure hemolectin-Gal4, UAS-GFP - Figure + region (either MZ or CZ) and of the whole lymph gland primary lobe . We analyzed and compared both the average absolute size of GFP+ MZ, or CZ, and the relative size by calculating the average ratio between each MZ (or CZ) and the corresponding lobe size. In compound treated animals, LG primary lobes showed a modest but significant size increase compared to controls in both experimental sets both in term of average size change , a marker of cell proliferation. Then we counted PH3 positive nuclei of m-L3 lymph gland primary lobes on Z-stack projections of the max fluorescence intensity. We confirmed that Smo inhibition induces a significant increase of cell proliferation in the CZ of m-L3 larvae ) could suppress the melanotic nodule phenotype induced by PF-04449913-mediated Smo inhibition, constitutive activation due to downregulation of ptc, that acts upstream smo, could not. Altogether these observations strongly suggest that the PF-04449913 compound inhibits Drosophila Smo that is expressed and required in the MZ to keep blood cells homeostasis. Reduction of the smo gene dosage or smo downregulation potentiated the compound effect, increasing the penetrance of the melanotic nodule phenotype up to 50%. Although residual Smo function may account for the incomplete phenotypic penetrance still observed in these animals after PF-04449913 feeding, this may also results from the action of other signaling pathways , active in the MZ and regulating blood cell homeostasis in response to different signals and environmental conditions [Drosophila blood cell precursors, our observations and the evolutionary conservations of the pathways suggests that the simultaneous inhibition of parallel pathways might more effectively prevent LSC maintenance and reduce the risk of leukemia relapse. This is confirmed by improved antitumor efficacy and survival observed in animal models following combined pharmacological inhibition of different pathways as those regulated by Hh and Notch among others [engrailed gene is not expressed in response to Hh signaling in few cells of the A compartment abutting the A/P border of the wing pouch. This is very similar to what has been observed in animals in which a LOF mutation of the fused gene blocked the Hh signaling [One of the key issues in the development of drugs that target hematological malignancies as AML, CML and MF, is to devise therapeutic strategies that reduce the risk of disease recurrence. CML is a hematological malignancy in which functional cure may be obtained using compounds (TKIs) that target the singular pathogenic mechanism driven by the chimeric Bcr-Abl kinase. However, although tyrosine kinase inhibitors are effective at eradicating Bcr-Abl expressing cells, they do not routinely eliminate LSCs. These comprise a disease reservoir and lead to the development of TKI-resistant leukemic cells and subsequent relapse –15. The patients , 19. The nodules , 36. The nodules . When ne nodules , 54. Thunditions , 55–57. g others . Gene exg others . Interessmo-RNAi expression at different stages of development showed that the e-L3 stage is the phenocritic phase, shortly after the PSC niche cells have begun to secrete the Hh ligand. Hh contributes to maintain the precursor cells of the lymph gland MZ that at this stage are quiescent or slowly cycling after the intense proliferative phase in L2 larvae [smo-RNAi in the lymph gland MZ, showed that Smo inhibition results in loss of blood cell precursors and increase of differentiating hemocytes of the CZ that might enter the circulating hemolymph. This is concurrent with increased cell proliferation in the lymph gland, especially in the cortical zone, and is consistent with the tendency of the cortical zone to occupy almost the whole lymph gland primary lobe as documented by the huge expansion of the GFP expression domain in the differentiating hemocytes of the CZ. This is very similar to the expected effect of Smo inhibition by PF-04449913 in patients: the inhibition of one of the principal pathway controlling the quiescence and maintenance of normal and leukemic hematopoietic stem cells. The expected consequence is that the LSCs are forced to exit from the bone marrow and to enter the bloodstream where they become sensitive to therapeutic agents [Drosophila as a simple model of hematopoiesis, we were able to directly observe in the hematopoietic organ the effect of pharmacological Smo inhibition and correlate it with the development of a phenotypic trait visible in living larvae. We observed that the human Smo inhibitor PF-04449913 or RNAi against Drosophila smo impairs hematopoietic homeostasis and induces the development of melanotic nodules. This phenotypic trait is associated to increase of hemocyte differentiation due to significant, and in some cases complete, loss of quiescent hematopoietic precursor cells. These features highlight the potential for using Drosophila as a model to facilitate the evaluation of the in vivo functional effectiveness of compounds targeting evolutionary conserved pathways involved in human hematological diseases.The drug administration or the 2 larvae , 33. Thic agents , 19, 21 c agents , 19. UsiTS mediated conditional Gal4/UAS dependent expression, crosses were maintained at the permissive temperature (18°C) and shifted to 29°C at the beginning of the stage of interest. Unless otherwise specified, larvae were analyzed for the melanotic nodule phenotype at late L3 instar. For further details see Supplementary Materials and Methods.Stocks and crosses were maintained at 24°C. Experimental progenies expressing transgenic constructs under the control of the Gal4/UAS system  were groIn order to establish control over the treatment timing and the concentration of compound administered to animals, we grew and treated the larvae in a defined volume of fresh yeast water suspension (liquid yeast-medium). PF-04449913 was dissolved in DMSO and diluted in the liquid yeast-medium to the indicated final concentration. Newly hatched and staged L1 larvae were harvested and divided into batches of 30 animals Figure . Since thml-Gal4 driver, were bled into 30 μl of Drosophila Ringer solution, labeled with Cy3 conjugated phalloidin  that binds filamentous actin, and mounted under a coverslip with Fluormount. In order to evaluate the number of circulating hemocytes, GFP expressing cells were counted in three random fields per coverslip, and the average number of hemocytes per field between the coverslips was calculated. To evaluate the number of lamellocytes, we counted the phalloidin/GFP positive cells that showed big size and flat shape  present under each coverslip and the average between samples was calculated.Three groups of ten l-L3 instar larvae treated with PF-04449913 or DMSO and expressing GFP in all hemocytes under the control of the domeless-Gal4, UAS-GFP larvae, or to CZ in hemolectin-Gal4, UAS-GFP larvae), the fluorescent signal generated by indirect immunolabeling and the Hoechst signal, were acquired along the Z-axis. The max fluorescence intensity of each Z stack was projected on a single image and the total lobe area (Hoechst) or the area occupied by GFP+ cells calculated using NIS Element software by outlining the area of interest. The total lobe area, the GFP+ area (MZ or CZ), the ratio between MZ (or CZ) over total area, and the difference between total area and the GFP+ area, were evaluated for each lobe and used to calculate the average and standard error .Lymph glands from experimental and control larvae were dissected, fixed, and processed for fluorescent indirect immunolabeling with the indicated primary antibodies  and for DNA Hoechst staining. For each experimental or control group, 11–21 lymph gland primary lobes from 9–15 larvae were scanned through their whole thickness using a Nikon A1R confocal laser-scanning microscope, equipped with a Nikon PlanApo 40× lens and captured using the NIS Elements AR 3.10 software (Nikon). For each lobe, the GFP (corresponding to lymph gland MZ in t-test to data from at least three independent experiments and calculating the p-value with the software GraphPad Prism 5. The software SPSS has been used to produce the Box Plots showed in Figure Comparison between samples and statistical analysis was performed by applying the two-tailed Student's