In agricultural systems, interactions between plants and microorganisms are important to maintaining production and profitability. In this study, bacterial communities in floodwaters of rice fields were monitored during the vegetative and reproductive stages of rice plant development using 16S amplicon sequencing. The study was conducted in the south of Brazil, during the crop years 2011/12 and 2012/13. Comparative analyses showed strong differences between the communities of floodwaters associated with the two developmental stages. During the vegetative stage, 1551 operational taxonomic units (OTUs) were detected, while less than half that number (603) were identified in the reproductive stage. The higher bacterial richness observed in floodwater collected during the vegetative stage may have been favored by the higher concentration of nutrients, such as potassium, due to rhizodeposition and fertilizer application. Eighteen bacterial phyla were identified in both samples. Both communities were dominated by Gammaproteobacteria. In the vegetative stage, Alphaproteobacteria and Betaproteobacteria were more abundant and, in contrast, Bacilli and Clostridia were the more dominant classes in the reproductive stage. The major bacterial taxa identified have been previously identified as important colonizers of rice fields. The richness and composition of bacterial communities over cultivation time may contribute to the sustainability of the crop. Oryza sativa L.) is the most important cereal crop in the world, feeding more than 50% of the human population. In South America, Brazil is the main producer [Rice , outer coastal plain, Rio Grande do Sul (RS), Brazil, Twelve water samples were collected from the three locations above in the agricultural years 2011/12 and 2012/13, six in the vegetative and six in the reproductive stages. One sample from each location and developmental stage was collected during the 2011/12 growing season and another sample was collected during the 2012/13 growing season. The collections were organized as follows: the vegetative (tillering) (October/November) stage was defined by active tillering and coleoptile and radicle (2-3 mm) formation to the beginning of panicle differentiation, while the reproductive stage was defined from the formation until complete maturation of the panicle (December to March) . Each of−1).Total DNA was extracted from each of the 12 pellets collected from the floodwater samples from two life stages at three locations using the MoBio Power Soil® DNA Isolation Kit  following the manufacturer's instructions. The DNA yield was measured using a NanoDrop® ND-1000 spectrophotometer  and the DNA quality was verified on a 1.5% agarose gel stained with ethidium bromide (0.1 mg mLμL of DNA (5 ng μL−1) using HotStarTaq Plus Master Mix . DNA was initially denatured at 94°C for 3 min followed by 28 cycles of 94°C for 30 s; 53°C for 40 s and 72°C for 1 min; and a final extension at 72°C for 5 min. PCR products of approximately 300 bp were purified using Agencourt Ampure beads . The amplicons were sequenced using 454 GS FLX Titanium chemistry (Roche) following the manufacturer's guidelines. The raw 454 reads generated during the current study have been deposited in NCBI's Sequence Read Archive (SRA) under the accession SRP077313 and are associated with BioProject PRJNA326968.Purified DNA from the six replicates collected during the vegetative stage and the six replicates collected during the reproductive stage were pooled together to create a single DNA pool for the vegetative samples and a single DNA pool for the reproductive samples. These two DNA pools (one DNA pool from vegetative samples and one DNA pool from reproductive samples) were used as templates for amplicon pyrosequencing analysis, which was performed by MR DNA Laboratory , with the goal of investigating the bacterial community structure and composition. The V1–V3 variable regions of the 16S rRNA gene were amplified using primers ill27Fmod (5′-AGRGTTTGATCMTGGCTCAG-3′) and ill519Rmod (5′-GTNTTACNGCGGCKGCTG-3′) [Water parameters such as temperature, turbidity, and pH were measured from all 12 samples using the Intelligent Meter equipment (INSTRUTHERM PH-1300). In addition, nutrient levels, including total phosphorus (P) and potassium (K), were measured from each of the six water samples collected from the two developmental stages using standard methods . The avan = 1) and vegetative (n = 1) stages were analyzed using the mothur pipeline (version 1.32.1) [The 454 amplicon data from bacterial communities sampled from floodwaters from the reproductive ( 1.32.1) . In brie 1.32.1) . In addi 1.32.1)  using th 1.32.1) . Then, tThe remaining bacterial sequences were categorized into operational taxonomic units (OTUs) at 3% sequence dissimilarity using the average neighbor algorithm. The consensus taxonomy for each OTU was determined using the classify.otu command. Rarefaction curves were calculated using the rarefaction.single command at a genetic distance of 0.03, while the summary.single command was used to compute various community metrics including the Shannon-Wiener (H′) and Simpson indices, the Chao 1 richness estimator , which e2 transformed, z-scale standardized, and used for clustering and heatmap analysis. Venn diagrams depicting OTUs found in floodwaters from both developmental stages and and OTUs unique to each developmental stage were constructed using the venn command in mothur.Heatmaps and cluster dendrograms based on Euclidean dissimilarity metrics were built according to the relative abundances of the most common genera in the two communities using the “vegan,” “heatmap.2,” and “cluster” packages  in the R−1), and potassium (K) (mg L−1). Prior to analysis, all variables were log10 transformed in order to facilitate comparisons among variables expressed in different units. The correlation matrix with the varimax rotation was set and only autovalues greater than 1 were utilized as criteria for extraction of the principal components. The analysis was performed in the R statistical computing environment (version 3.1.0) using the princomp and biplot functions. The differences among physicochemical parameters between the rice stages were tested using Student's t-test in SYSTAT 12 . p values of <0.05 were considered statistically significant.The physical and chemical characteristics of the floodwater from the rice fields sampled during two agricultural years were analyzed by principal components analysis (PCA) using the following variables: temperature (°C), turbidity (NTU), pH, total phosphorus (P)  using Libshuff. In the vegetative stage, 1,551 OTUs were identified, while less than half the number of OTUs (603) were detected in water samples collected from the reproductive stage , Actinobacteria (7.84%), and Firmicutes (5.52%). In total, 83.50% of the total OTUs detected in these two communities was associated with these four phyla. Several phyla with lower richness estimates were also detected in these samples, defined as phyla containing less than 1% of the total OTUs detected in this analysis. Based on this criterion, a total of twelve rare phyla were detected including Chloroflexi, Deferribacteres, Deinococcus-Thermus, Fusobacterium, Gemmatimonadetes, OP10, Planctomycetes, SR1, Spirochaetes, TM7, Tenericutes, and Verrucomicrobia, which were detected in both stages of crop development. The phyla Acidobacteria (1.94% of the OTUs) and Cyanobacteria (1.25%) were also present, but at low frequencies (≤2% of the OTUs).In total, 27 different bacterial classes were identified in both developmental stages. In general, the class level abundances of the 88 OTUs shared between the floodwaters from the vegetative and reproductive stages were very similar and were both dominated by Betaproteobacteria, Actinobacteria, and Alphaproteobacteria Figures . In cont Beijerinckia, Curvibacter, Pelomonas, and Rhodoferax occurred in high counts in both phenological stages. In general, the abundances of Beijerinckia, Pelomonas, and Rhodoferax were similar in flood waters from both communities; however, the relative abundance of Curvibacter was slightly higher in vegetative stage. The abundances of the genera Aeromonas, Bradyrhizobium, Emticicia, Massilia, Methylibium, and Spirosoma were lower and did not differ significantly between vegetative and reproductive stages  occurred in both stages . Beijerie stages .Polynucleobacter (29.5%), Curvibacter (23%), and Rhodoferax (10.3%) represented the most abundant genera in the vegetative stage, while Curvibacter (18.3%), Alcaligenes (11.8%), and Flavobacterium (11.3%) were more abundant in the reproductive stage , while 822 OTUs were predicted in the reproductive bacterial community (27% more OTUs than what were detected). Considering these results, it is clear that a larger number of rare species were associated with water collected from the vegetative stage compared to the reproductive stage (The Shannon-Wiener (H′) index was approximately 25% higher in the water sample collected from the vegetative stage compared to the reproductive stage, suggesting that the vegetative community is richer. In addition, the Simpson index, which represents the probability that two reads selected at random belong to the same OTU, was higher in the sample collected from the reproductive stage. This finding is probably due in part to the lower richness associated with the reproductive stage, the higher number of rare OTUs found in the vegetative stage, and the overabundance of three genera,ve stage .Reflecting the higher richness and higher numbers of rare OTUs, the rarefaction curve of the vegetative stage did not reach complete saturation considering the number of sequences sampled, indicating that additional OTUs will likely be detected with additional sequencing. On the other hand, rarefaction curve of the reproductive stage showed stabilization, indicating that the sequencing effort was sufficient to capture the majority of the OTUs associated with this community .In flooded rice, the water source used for cultivation is a key component in the management of the crop. Nutrient levels in these water sources can have significant effects on plant nutrition  while vaIn the current study, the water samples retrieved from vegetative and reproductive stages showed variations in physical and chemical parameters. The abiotic parameters most strongly associated with the vegetative stage were pH, P, and K and in the reproductive stage, the parameters were temperature and turbidity . Of thesA total of 2,066 OTUs at 3% dissimilarity were identified in floodwaters collected from the reproductive and vegetative developmental stages of rice. In flooded rice systems, the short spacing between the plants, the low depth of the floodwaters (10 cm deep), and soil management regimes can increase the levels of suspended soil particles in the floodwaters , which iDespite the presence of several common phyla in floodwaters sampled from both developmental stages, only 88 (4.08%) of the OTUs were common between vegetative and reproductive stages, suggesting that the dynamics of the bacterial communities in floodwaters change throughout the growing season. These changes could reflect temporal variations in carbon availability. For example, in a previous study, Aulakh and coworkers  documentThe richest bacterial classes found in the vegetative stage were Gammaproteobacteria (276 OTUs), Alphaproteobacteria (237 OTUs), and Betaproteobacteria (211 OTUs), while richness was the highest in Gammaproteobacteria (24 OTUs), followed by Bacilli (46 OTUs) and Clostridia (18 OTUs) during the reproductive stage. In flooded plains, the adherence of Betaproteobacteria representatives to particles can play an important role in their transport from the terrestrial environment to floodwater , while t Escherichia, Salmonella, Yersinia, Vibrio, and Pseudomonas characterized by their metabolic plasticity that can be adapted to varying levels of temperature, oxygen supply, and nutritional requirements. Hence, members of the class Gammaproteobacteria often dominate microbial assemblages after nutrient enrichment [Gammaproteobacteria were present in high richness and abundance in floodwaters associated with both rice developmental stages. Overall, members from the phylum Proteobacteria are often the most abundant freshwater prokaryotes, but Gammaproteobacteria are usually present in low or transient abundances , althougrichment . In addirichment . Furtherrichment .Beijerinckia, Curvibacter, Pelomonas, and Rhodoferax were the four most abundant genera in both stages of the crop. These bacterial groups are often associated with high availabilities of sugar  [ Beijerinckia are reported as growth promoters in plants [ Curvibacter spp. often code for large numbers of sugar transporters [ Pelomonas sp. and Rhodoferax sp., were found in approximately the same proportion in both rice stages. Previous rDNA 16S amplicon analysis showed that Rhodoferax spp. are common in diverse freshwater systems [ Rhodoferax are nitrate-reducers [ Pelomonas spp. often code for machinery required for nitrogen fixation (nifH gene) [ Polynucleobacter differed significantly in floodwaters from the two life stages. Its abundance was significantly higher in the vegetative stage (29.5%) compared to reproductive stage (4.6%). This result could suggest that the OTUs derived from this genus are not well adapted to environmental conditions associated with floodwaters collected from reproductive stage plants. Polynucleobacter members often dominate planktonic freshwater communities [ Polynucleobacter in freshwater environments [glucose) . In agrin plants , while tsporters , enhanci systems . Some spreducers , while PfH gene) . Thus, tmunities , where pmunities . Also, fronments , 61.Rises in temperature can increase the abundance of prokaryotes of the natural communities by enhancing their growth rates . The abuRhizodeposition occurs continuously during the lifetime of plants in the form of water soluble exudates, secretions, and plant dead cells. This process contributes as a primary source of energy and nutrients for microbes in the floodwaters . In futu4+, which is the main form of available N in the soil [Heterotrophic microorganisms, which represent a large portion of the rice field microbiome , 71, arethe soil . These fThis study revealed that the communities associated with rice floodwaters were variable and differed between floodwaters from the vegetative and reproductive stages of rice. The higher bacterial richness observed in floodwaters associated with vegetative plants may have been favored by the higher concentration of nutrients  due to changes in rhizodeposition and crop management. OTUs from the phylum Proteobacteria were present in floodwaters collected from both developmental stages, indicating the persistence of this phylum in flooded rice ecosystems. Moreover, the predominance of the class Gammaproteobacteria and the occurrence of the phylum Firmicutes in the reproductive stage demonstrated that the environment was favorable to microbes that can persist longer in paddy fields where they are able to utilize the degradable fraction of organic materials and survive in waterlogged and dry conditions. However, further investigations regarding the composition and quantification of root exudates and the impacts of rice exudates on microbial communities in rice fields are essential to decipher the linkage between the microbial community dynamics and rice plant over cultivation time.


Our study aims to explore the effect of total parathyroidectomy (PTX) with forearm autotransplantation (FAT) on the quality of life and recurrence of secondary hyperparathyroidism (SHPT) in chronic kidney disease patients.A total of 104 chronic kidney disease patients with SHPT were enrolled and divided into the PTX (n = 62) and PTX + FAT (n = 42) groups. The operation efficacy was evaluated by analyzing preoperative and postoperative values, including levels of intact parathyroid hormone (iPTH), serum phosphorus, serum calcium, alkaline phosphatase (ALP), calcium-phosphorus product, signs and symptoms, and MOS 36-item short-form health survey (SF-36) scores. Moreover, complications and recurrences were followed up for 12 months after the operation. Binary logistic regression was to present the risk factors for the recurrence of chronic kidney disease patients with SHPT.Compared with the preoperative values, the PTX and PTX + FAT groups showed decrease postoperative levels of iPTH, serum phosphorus, serum calcium, calcium-phosphorus product, bone pain, and skin pruritus at all time periods. The PTX and PTX + FAT groups demonstrated decreased ALP, fracture or deformity, and coronary artery calcification at 1 month, decreased short stature at 3 months after the operation but increased SF-36 score after operation. Compared with the PTX group, the level of iPTH decreased and the levels of serum calcium, calcium-phosphorus product increased at 3, 6, and 12 months after the operation in the PTX + FAT group. The levels of ALP, fracture or deformity, short stature, and SF-36 decreased separately at 1 week and 6 and 12 months after the operation, along with the decrease of coronary artery calcification and the recurrence rate, respectively, at 6 and 12 months after the operation in the PTX + FAT group when compared with those in the PTX group. Logistic regression analysis evidenced that the preoperative iPTH level, SF-36 score, and operation type were the risk factors for the recurrence of chronic kidney disease with SHPT.Total PTX combined with FAT is more effective in improving the quality of life and reducing the recurrence of chronic kidney disease with SHPT than PTX alone. Fundamentally, the etiology of SHPT is known as long-term parathyroid hyperplasia, which results in the formation of functionally independent adenoma. SHPT in patients can lead to high-turnover bone disease, interstitial and vascular calcifications, and as well as cardiovascular mortality and morbidity.–7 The diagnosis of SHPT in patients mainly relies on high-resolution ultrasonography with color Doppler imaging (US/CD) and 99 mTc-methoxyisobutylisonitrile (MIBI) scintigraphy. Owing to the high occurrence and recurrence of SHPT, the patients’ quality of life is seriously affected. Therefore, multiple efforts have been made for the search of better solutions for SHPT.Secondary hyperparathyroidism (SHPT) is a common severe complication in patients with end-stage renal disease (ESRD), and often persists after successful renal transplantation. Although conventional drug treatment has improved a lot in recent years, the parathyroidectomy demand still remains high, particularly in patients depending on long-term dialysis and patients with medically refractory SHPT. Remnant-conserving techniques for parathyroidectomy consist of subtotal parathyroidectomy (PTX), which removes all parathyroid tissue, leaving a small portion of a normal appearing gland in the neck, and total PTX with forearm autotransplantation (FAT), which can be helpful in removing all parathyroid tissue and also implanting small pieces of tissue at a distance in well-vascularized muscular structures.–14 Although both techniques are now under criticism due to the high recurrence rate of SHPT, they still remain the most preferred and widely performed surgical procedures. Recently, total PTX without autotransplantation is regarded as a reliable surgical solution for patients suffering from severe SHPT, especially patients who have low perspective for renal transplantation. Herein, the study aims to explore the role of total PTX combined with FAT in the quality of life and recurrence of SHPT in chronic kidney disease patients by comparing the use of total PTX + FAT and total individual PTX.Cinacalcet and Vitamin D are the commonly used drugs, and they are used in combinations to treat SHPT in patients on dialysis.22.1The study was approved by the Ethics Committee of Yinzhou Hospital of Ningbo University Medical College and written informed consents were obtained from all patients.2.22.,17 The patients were included into this study following the criteria: patients were with good adherence for systematic treatment and observations; and patients were diagnosed with SHPT by preoperative clinical symptom, signs, and biochemical and X ray manifestations (patients were included into SHPT patients group when patients having intact PTH (iPTH) levels of >600 pg/mL accompanied with obvious calcium and phosphorus metabolic disorders; patients unresponsive to available medical therapy; patients suffering from severe osteoporosis and skeletal deformity observed by radiological examination; patients showing severe clinical symptoms, such as bone pain, cutaneous pruritus, and coronary artery calcification (CAC). Patients with the following symptoms were excluded from the study: patients suffering from congenital, genetic, autoimmune, or cardiovascular diseases; patients with a history of heavy smoking, alcohol abuse, drug abuse, psychiatric problems, and patients in pregnant or suckling period; patients suffering from severe anemia, bleeding, coagulation dysfunction, and patients unsuitable for general anesthesia; patients having deformities in neck bone or soft tissues and patients suffering from morbid obesity; patients underwent neck operation, radiological treatment, and with hypertrophic scar; patients who had large thyroid nodules, thyroiditis, substernal ectopic parathyroid, and parathyroid carcinoma patients; patients who had a high PTH >35 pmoL/L and serum calcium >2.60 mmoL/L; and patients who had bone hunger syndrome. All study subjects showed no obvious side reaction after heparin dialysis.From January 2010 to May 2014, a total of 104 uremic patients suffering from SHPT diagnosed and treated in the Yinzhou Hospital of Ningbo University Medical College were divided into PTX groups  and PTX + FAT groups . Diagnosis criteria were as below: Nuclide scanning showed 1 or more hot nodules with highly concentrated 131I, and low iodine intake capability in thyroid tissue other than nodules was evaluated; size of the hot nodules was inspected by ultrasonography or computerized tomography scan to confirm the nodular goiter; clinical manifestations presented decreased arrhythmia, limb numbness, and limb proximal muscle strength; serum 25-hydroxyvitamin D (25OHD) < 30 ng/mL or estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2.3Before the operation, the cardiopulmonary function of patients was estimated by routine blood examinations, blood biochemistry, X-ray chest radiographs, electrocardiographs (ECG), and cardiopulmonary function tests, aiming to assess patient tolerance for the operation. Adequate dialysis and strict dry weight control were confirmed to ensure a cardiothoracic ratio of < 60%. Hemodialysis was performed a day before the operation using low molecular weight heparin anticoagulatant . B ultrasound positioning was adapted to reveal the exact location of parathyroid glands and its correlation with other organs around it. The operation procedures were as follows: marker pen, 0.9% NaCl ice cube, hammer, steel ruler, small Warburg retractor, microneedle holder, sterile thermos flask, balance-type scale, 5-0 suture, and self-made small instruments of table pad were prepared.3 sections. After that, 20∼30 slices were separately incubated into the patients’ forearm muscles without arteriovenous and with fistula. The sarcolemma was sutured with a nonabsorbable suture .Totally, 62 SHPT patients underwent total PTX. Patients were general anesthetized and laid supinely. Then, a cervical collar incision (6 ∼ 8 cm) was made in the anterior muscles in the middle of the neck to fully expose the thyroid. Later, 4 parathyroid glands were entirely removed after they were identified around the thyroid gland and breast bone. Particular attention was paid in order to protect the recurrent laryngeal nerves during the above procedures. A combined PTX and FAT operation was performed on 42 SHPT patients. During this operation, the parathyroid glands were resected, sent for pathological examination. After confirmed to be diffuse hyperpolarized, the diffuse hyperpolarized parts were frozen, sliced into 1∼2 mm3  orally taken. At 1 ∼ 2 days after the operation, half or total heparin anticoagulation hemodialysis was performed to patients according to the wound condition. Vital signs, incision bleeding, and drainage tube were carefully monitored. Close attention was paid to the vascular care to prevent the occurrence of phlebitis.Serum calcium, phosphorus, and alkaline phosphatase (ALP) levels were monitored dynamically and reviewed once a day from the first week to the third week after operation. If serum calcium level was less than (<) 1.8 mmoL/L or convulsions ensued, calcium gluconate  would be intravenously injected to patients with active vitamin D2.4 CAC was evaluated by the calcification in patients after the operation. The SF-36 questionnaire was divided into the following 8 dimensions: physical functioning, mental health, social activity, emotional health, physical influence, vitality, physical pain, and general health to measure the quality of life. Accumulation method was applied to score each dimension and the lower score represented a lower quality of life.Venous blood of all subjects after 12 hours of fasting was collected in the morning before operation, and at 1 week, 1, 3, 6, and 12 months after operation, and routine blood tests were detected by the XE-2100 type automatic blood cell analyzer. Moreover, serum calcium, phosphorus, and alkaline phosphatase (ALP) levels were detected by the Hitachi-7600 automatic biochemical analyzer and the calcium-phosphorus product was calculated. Finally, iPTH levels were detected using chemiluminescent enzyme immunoassay. The signs, symptoms, and the MOS 36-item short-form health survey (SF-36) score, complication, and recurrence condition were recorded. An iPTH level > 300 pg/mL 12 months after operation was defined as recurrence.2.5A 12-month follow-up was conducted for all patients once every month. General information of patients, blood biochemical indicators, symptoms, remission of signs, complications, and recurrences were recorded and the SF-36 questionnaire was conducted. The follow-up ended by May 2015.2.6T test was adapted for the comparison between 2 groups, and repeated measurement analysis of variance (ANOVA) for comparison between 2 groups at different time points. Count data were expressed as percentage or rate, and the Chi-square test was used to compare the data between groups. The risk factors for the recurrences of the uremic patients with SHPT were analyzed by binary logistic regression analysis. P < .05 signifies a statistical difference.G∗Power3.1 was used to estimate the sample size. The previous study showed that the overall numerical rating scale (NRS) standard deviation was 2. NRS score difference of 1 represented a practical significance, and a = 0.05 revealed 80% efficiency with a total sample size of 95 cases or more. Data analyses were performed using the SPSS 20.0 . Measurement data were expressed as mean ± standard deviation (SD). 33.1P > .05). There was no statistical significance in dialysis type, dialysis age, primary disease, symptoms and signs, fracture site and blood biochemical indicators , and SF-36 score between the PTX and PTX + FAT groups .As presented in Table 3.2P < .05), among which iPTH, serum phosphorus, and calcium-phosphorus product showed relatively small changes, and serum calcium levels initially decreased to the lowest level at the first week after the operation, and which subsequently increased thereafter. The blood biochemical indicators had no significant difference before the operation and 1 week after the operation . Compared with the PTX group, the iPTH levels decreased significantly, but serum calcium levels and the calcium-phosphorus product were significantly elevated in the PTX + FAT group at 3, 6, and 12 months after the operation . ALP levels increased and then declined in the PTX group. One week after the operation, the PTX and PTX + FAT groups showed a significant decreased ALP levels, and moreover, the PTX + FAT group showed a more prominently decreased ALP levels . The serum phosphorus levels showed no remarkable difference 1 week after the operation.As summarized in Table 3.3P < .05); fractures or deformities and CAC decreased significantly upon measurement at 1, 3, 6, and 12 months after the operation . Short stature decreased significantly at 6 and 12 months after the operation in the PTX group, while it decreased significantly at 3, 6, and 12 months after the operation in the PTX + FAT group . Compared with the PTX group, the bone pain and skin pruritus in the PTX + FAT group showed no significant differences at any given time periods , and the differences among fractures, deformities, or short stature between the 2 groups were not statistically significant at the 1 week, and at 1 and 3 months . Fractures, deformities, and short stature in the PTX + FAT group decreased significantly at 6 and 12 months compared with the PTX group . Differences in CAC between the PTX and PTX + FAT groups were not statistically significant at 1 week, and at 1, 3, and 12 months. However, CAC decreased significantly at 6 months after the operation in the PTX + FAT group (P > .05).The signs and symptoms of each group are summarized in Table 3.4P < .05). Compared with the PTX group, the SF-36 scores in the PTX + FAT group were not statistically significant at 1 week, and at 1 and 3 months , but the SF-36 scores increased significantly at 6 and 12 months .SF-36 scores in 3 groups are shown in Fig. 3.5P > .05), but the recurrence decreased significantly (P < .05). A total of 14 patients in the PTX group had recurrent laryngeal nerve injury , who slowly recovered within 7 to 10 days; 6 patients had severe low calcium convulsion, and 8 had pulmonary infection during the perioperative period. In the PTX + FAT group, 6 patients had blockage in internal fistula due to hypotension; 8 patients had unilateral recurrent laryngeal nerve injury, and 4 had severe low calcium convulsions.Postoperative complications and recurrences 12 months after the operation are shown in Fig. 3.6P < .05). Also, the results showed that the patients treated with PTX individually had a higher recurrence rate of SHPT than patients treated by PTX + FAT. Moreover, an increased preoperative iPTH and a decreased preoperative SF-36 score signified an increased recurrence rate of SHPT.Logistic regression analysis of recurrence-related factors for chronic kidney disease patients with SHPT is summarized in Table 4 an American surgeon, in 1967 reported that PTX was proved to be an effective treatment for SHPT, although the patients suffered severe hypocalcemia and metabolic bone disease after the operation. It is reported that total PTX + FAT procedures had a high success rate and low recurrence rate, which was thus considered as the classic surgical procedure in the treatment of SHPT. Our study revealed the advantages of total PTX + FAT procedures in terms of postoperative life quality and recurrence of uremic patients with SHPT.With the increasing number of patients suffering from SHPT, the need for parathyroidectomy increases as well. The search for better treatments for SHPT is imperative. Ogg, He et al also demonstrated that total PTX + FAT procedure was a safe, feasible, and effective surgical option for SHPT patients owing to the results that reveal no deaths occurred among the 47 study subjects. The results of SF-36 questionnaire indicated that the quality of life in the PTX and PTX + FAT groups significantly increased after the operation compared with those preoperatively. Moreover, the SF-36 score at 6 and 12-month time periods after the operation in the PTX + FAT group was very close to that in the control group. From the results, we can say that total PTX and PTX + FAT procedures are effective in promoting the quality of life of patients suffering from SHPT, but PTX + FAT is far more advanced and effective than PTX. Rao et al demonstrated in their study that among the 283 patients suffering from mild asymptomatic primary hyperparathyroidism, 53 of them chose PTX, whereas the rest chose to go with the conservative treatment, and the SF-36 score in the 53 patients was similar to the normal population. In a previous study, the risk of reaching overly low iPTH value appears to increase after total PTX, which predisposes the patient to adynamic bone disease, increasing the risk of fractures and lowering the patient's quality of life.It was found that no deaths occurred among patients suffering from SHPT during the perioperative period of PTX or PTX + FAT procedures. It is reported that total PTX without autotransplantation could be the viable choice for treating patients with SHPT with a relative low recurrent rate and short operative time. which conforms to increased level of serum calcium in patients after PTX + FAT operation. Jing et al found that calcitriol was in need for postoperative patients suffering from uremia SHPT, and also the muscle weakness and bone pain muscle weakness were much relieved in patients after PTX + AT operation. It was revealed by previous research that the serum bone-specific ALP level presented the bone turnover and the bone scintigraphy was seen high in patients with SHPT, thus making the decreased ALP meaningful for patients after PTX + FAT operation. An improved bone fracture, bone pain, and tendon rupture were demonstrated in patients after PTX + AT operation.,29 As been revealed by former evidence, the calcium levels of patients after operation were lower in the PTX operation group, while higher in the PTX + AT group. A lower CAC score was detected in patients after PTX + AT operation. From all the change of biological plausibility occurred to patients after PTX + AT operation, the advantage of PTX combined with FAT was shown obviously.Besides, the levels of serum calcium, calcium-phosphorus product increased; the ALP, fracture or deformity, short stature; and the CAC decreased in the PTX + FAT group. According to the former study, patients were diagnosed to be hypoparathyroidism with a low serum calcium level, and the intravenous calcium levels were required for patients from performed the PTX + AT operation, Recurrence may be associated with residual parathyroid tissue, hyperplasia of transplanted parathyroid tissue, or ectopic parathyroid. Although ultrasound and radionuclide scanning technology have matured a lot over the last few years, the failure in locating the entire parathyroid tissue still remains. Tissue traction often results in parathyroid ectopic. Therefore, preoperative parathyroid localization is imperative. The PTX group had 14 cases of recurrent laryngeal nerve injury , 6 cases of severe low calcium convulsion, and 8 cases of pulmonary infection during perioperative period. In the PTX + FAT group, 6 cases of blockage in internal fistula due to hypotension, 8 cases of unilateral recurrent laryngeal nerve injury, and 4 cases of severe low calcium convulsions were observed. Recurrent laryngeal nerve injury may be associated with intraoperative traction of recurrent laryngeal nerve, and therefore, the requirement to entirely expose the recurrent laryngeal nerve is necessary and it is not only favorable for the progression of the operation, preventing recurrent laryngeal nerve injury, but also beneficial for the resection of parathyroid and adipose tissue around lymph node. Hypocalcemia was supposed to be associated with the decreased secretion of vitamin D. It is reported that the secretion of 1, 25 (OH) 2D3 (a member of vitamin D) was significantly decreased in patients with renal failure, resulting in hypocalcemia. A majority of the patients felt that their symptoms were significantly relieved after the operations in both groups. Patients with bone pain and skin pruritus revealed obvious improvements in both groups. A previous study demonstrated significant association between serum calcium level and skin pruritus, and in our study, it was suggested to be related to decreased calcium levels after the operation. Fractures or deformities, extraskeletal calcification, and short stature decreased significantly in both groups. Moreover, patients with the aforementioned symptoms recovered faster and more efficiently in the PTX + FAT group than the patients in the PTX group. In addition, we discovered that serum phosphorus levels decreased significantly and maintained a relatively low level in both groups. Dhingra et al reported that a higher serum phosphorus level is associated with an increased risk of chronic kidney disease. The results in our study showed that the iPTH levels decreased significantly but persisted, and there was no significant difference in the postoperative values of each group, suggesting it would not cause loss of iPTH level.Most importantly, compared with the PTX group, the recurrence in the PTX + FAT group decreased significantly. As shown in a previous study, the recurrence rates in patients with SHPT were lower after total PTX + FAT than individual PTX.To conclude, our study proves that PTX + FAT procedures are comparatively safer and more effective in the treatment for uremic patients suffering from SHPT owing to its advantages in improving the quality of life and inhibiting the recurrence of the patients. However, the sample size of our study was too small in order to determine the most appropriate approach for SHPT and the follow-up period was only a short span of 12 months; therefore, further studies with larger sample size and longer follow-up durations are needed to support the findings.The authors acknowledge all the reviewers who had given supports for our article.


Type I methanotrophs, particularly Methylobacter, dominated all communities, however there was a clear taxonomic separation between the two landscape types, consistent with environmental control of community structure. In contrast, methanotrophic potential activity, measured by pmoA transcript concentrations, did not vary with landscape type, but correlated with conductivity, phosphorus and total suspended solids. Methanotrophic potential activity was also detected in low-oxygen bottom waters, where it was inversely correlated with methane concentrations, suggesting methane depletion by methanotrophs. Methanotrophs were present and potentially active throughout the water column regardless of oxygen concentration, and may therefore be resilient to future mixing and oxygenation regimes in the warming subarctic.Lakes and ponds derived from thawing permafrost are strong emitters of carbon dioxide and methane to the atmosphere, but little is known about the methane oxidation processes in these waters. Here we investigated the distribution and potential activity of aerobic methanotrophic bacteria in thaw ponds in two types of eroding permafrost landscapes in subarctic Québec: peatlands and mineral soils. We hypothesized that methanotrophic community composition and potential activity differ regionally as a function of the landscape type and permafrost degradation stage, and locally as a function of depth-dependent oxygen conditions. Our analysis of  Methane emissions to the atmosphere are the net result of microbial methanogenesis and methanotrophy, and in many environments, the activity of aerobic methanotrophic bacteria (methanotrophs) may impose a strong control on methane emission flux rates . MethanoMethanotrophs require the presence of oxygen as an electron acceptor for methane oxidation , and in pmoA), is a phylogenetically useful marker for enzymes involved in bacterial methane oxidation, and can be used to assess methanotroph community composition and activity [Methylobacter-related) and Type Ib (Methylococcus-related) based on their phylogenetic affiliation [Crenothrix, which belongs to the gamma-proteobacteria but has a phylogenetically divergent pmoA [The application of functional genomics to ecosystems provides a means to establish links between microbial diversity and biogeochemical processes , includiactivity –21. Methactivity . Type I iliation . Recentlent pmoA ; the verent pmoA ; and theent pmoA .Lakes and ponds, which are a characteristic feature across northern landscapes, are strong emitters of methane to the atmosphere ,27. EmisIn the Quebec subarctic region, permafrost thaw ponds have different geomorphological origins that may influence methanotrophic community composition and activity. First, the quality and the quantity of the carbon pool in the ponds change as a function of permafrost degradation and the landscapes surrounding the ponds, with two distinct landscape types: peatlands with raised mounds of organic material  and shrub-tundra landscapes with raised mounds of inorganic soils . These different landscapes affect the community composition of methanogenic Archaea  and alsopmoA transcripts in ponds across a gradient of permafrost degradation gradient, and in the two ecosystem types: organic-rich palsa ponds and mineral-rich lithalsa ponds. We hypothesized that the methanotrophic community and their potential activity differ as a function of landscape type, extent of permafrost degradation and depth in the ponds. We surmised that methanotrophic activity down the water column would be determined by oxygen availability, limiting pmoA transcription to the oxygenated surface waters. We additionally tested the difference in pmoA phylogenies between free-living and attached methanotrophic communities, given the importance of particles for bacterial activity in these waters.Given the heterogeneity of conditions in subarctic thaw ponds at local and regional scales, our goal in the present study was to identify the factors that influence the composition and potential activity of their methanotrophic communities. We conducted this research by way of phylogenetic and quantitative PCR (qPCR) analysis of Water samples were collected during two field expeditions, 1 to 13 August 2012 and 31 July to 19 August 2013, from four subarctic valleys in northern Québec . The Kwa-1. Bottom water samples were collected using a horizontally mounted Van Dorn bottle (Wilco) and immediately transferred to acid washed 4-L Cubitainers™ that were rinsed with sample water prior to filling. The Cubitainers™ were overfilled to avoid oxygenation, capped, placed in coolers and brought back to the laboratory. Sub-samples for methane concentrations were measured with the headspace technique as described in Matveev et al. [Profiles of temperature, dissolved oxygen (DO), and pH were taken with a YSI 600R multiparameter probe (Yellow Springs Instruments); the detection limit for dissolved oxygen with this probe is 0.2 mg Lv et al. , while sv et al. .™ polycarbonate (PC) filter and a 0.2 μm Sterivex™ unit (Millipore) to separate large  and small  fractions of planktonic micro-organisms. After one hour of filtration, the filtration was stopped and filters were conserved in RNAlater (Life Technologies) and subsequently stored at -80°C until extraction. RNA was extracted with the AllPrep DNA/RNAMini Kit (Qiagen) modified to include an additional step using polyvinylpyrrolidone  to minimize potential PCR inhibition [The water for RNA analysis was prefiltered through a 20 μm mesh (Nitex) and then sequentially filtered through a 3 μm Nucleporehibition . RNA waspmoA was performed with a two-step dual-indexed PCR approach modified for Illumina® sequencing, with two consecutive sets of primers , 0.25 μM of each primer, 200 μM of each dNTPs (Life Technology), dimethylsulfoxide  at a final concentration of 3%, 1 U of Phusion® High-Fidelity DNA polymerase (NEB) and 1 μL of template cDNA. To minimise primer bias, two more reactions with 5 and 10 fold diluted template were also carried out for each sample. Temperature and duration of thermal cycling were started with an initial denaturation at 98°C for 30 s followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 56°C for 30 s, extension at 72°C for 30 s and a final extension at 72°C for 300 s. The three dilution reactions were pooled together and purified using the Axygen PCR cleanup kit. The quality and quantity of the purified PCR product were checked on a 1% agarose gel. Then 50 to 100-fold dilutions of this purified product was used as a template for a second PCR step to add barcodes . The pmoA marker was not successfully amplified from some cDNA samples and these were excluded from further processing and sequence analysis (™ 1000 (Thermo Fisher Scientific). Barcoded amplicons were pooled in equimolar concentrations for pair-end sequencing on the Ilumina MiSeq at the Plateforme d’Analyses Génomiques . The raw Illumina reads have been deposited in the NCBI sequence read archive (SRA) under accession number SRP091008.Amplification of analysis . This se™ spectrometer. Each reaction (standard and samples) was run in triplicate. Standards for calibration were diluted 10 times to ensure measurements from 107 to 101 copies μL-1. Potential inhibition was checked by running 10 and 100 fold dilutions of the sample, which covered the expected copy number range. qPCR reactions for standards and samples were performed in 20 μL reactions containing 5 μL of template, 500 μM of each primer (PmoA169F and PmoA661r), 1X Ssofast EvaGreen™ Supermix and nuclease free water on a Chromo4 thermal cycler (Bio-Rad) with the following steps: initial denaturation (30 s at 95°C) and then 40 cycles of denaturation (5 s at 95°C), annealing (30 s at 55°C) and elongation (20 s at 72°C). At this step no transcripts were detected in some samples  PCR purification kit. Amplicons were cloned using a Stratagene cloning kit following the manufacturer’s instructions, with the following modifications: a polyA tail was added to the amplicons using Feldan polymerase and buffer and dATPs  and 26 to 30 ng were used for the ligation reaction. Transformed cells were incubated 1 h in SOC medium at 37°C for recovery before plating on agar plates. Positive clones were incubated again in LB media containing 7% glycerol for 16h at 37°C. To verify that the target gene was amplified, the T3/T7 PCR reaction was performed on the clones and 11 of them were sent for Sanger sequencing at the Sequencing Center of Laval University Hospital Center . Standards were then prepared by amplifying a positive clone with T3 and T7 primers. Amplicons were checked on a 1% agarose gel and the band corresponding to the size of the target amplicon (around 700bp) was cut and purified with a gel purification kit (Qiagen). Another T3/T7 PCR was performed on the purified amplicon and was purified with a PCR purification kit (Feldan). Concentrations were measured with the NanoDrop samples .PmoA reads from the Illumina amplicon sequencing were first assembled using the usearch command “fastq_mergepairs” from UPARSE [pmoA reference sequence with FrameBot for detecting frameshift errors and sequences with inframe STOP codons, which were removed. Remaining high quality sequences were aligned with HMMER3 and then clustered at 93% similarity, which has been identified as the threshold that corresponds to 97% sequence similarity for 16S rRNA [pmoA database was constructed by downloading pmoA sequences from the Functional Gene Repository v.8.0. Reference sequences were checked against the NCBI nr database and the reference database was manually inspected to ensure all types of known methanotrophs were represented. Taxonomic affiliation of the representative sequences of pmoA operational taxonomic units (OTUs) was determined in QIIME using the assign_taxonomy.py command [m UPARSE  and thenm UPARSE . Reads sm UPARSE . Sequenc16S rRNA ,45. A cu command . For una command . A princ command  and miss command . VariablTesting for differences in the qPCR data required a non-parametric test since the values did not meet normality assumptions even after log transformation. The Kruskal-Wallis test was then used to assess differences in methanotrophic potential activity between valleys, depth and size fraction. To compare with the β-diversity pattern, 15 samples  that had corresponding Illumina reads were used in this analysis . SurfacepmoA gene. The relationship between the small fraction of the qPCR data and CH4 concentration was assessed for the bottom samples. The qPCR data were log transformed to meet normality assumptions. Pearson correlation values were then calculated using the Vegan package in R [To evaluate which environmental variables and groups of methanotrophs (X-variables) contributed to the potential activity of methanotrophs (Y-variable), we used partial least squares regression (PLS) in the R package mixOmics , which tage in R .-1) to anoxic at the bottom. Ponds from the northern BGR and NAS valleys had higher pH values (from 7 to 8), while pH was more acidic in the southern valleys (from 5 to 6). Bottom waters of the ponds in the KWK and SAS valleys had higher CO2, CH4 and nutrient concentrations, with highest concentrations of total nitrogen and phosphorus in the bottom of KWK23: 2.7 mg N L-1 and 170.5 μg P L-1. The TSS values followed the same trend, with the highest value at the bottom of the KWK1 pond of 140.8 mg L-1. The difference between lithalsa and palsa ponds was most pronounced in terms of DOC concentration: ponds from the SAS peatland valley contained higher DOC concentrations, which ranged from 9.6 mg L-1 at the bottom of SAS1A to 18.9 mg L-1 for the bottom of SAS2A  and Type Ib (< 1% of the reads for most sites), and to other genera such as Methylovulum, Methylosoma and Methylococcus, which represented less than 1% of reads in each sample and were then grouped with unclassified and other Methylococcaceae. The genus Methylomonas had a low relative abundance in the lithalsa ponds. It was better represented in the palsa ponds, and abundant in the surface of both the small and large fractions in SAS2B . The genus Methylomicrobium was present in each sample, ranging from less than 1% in the large fraction of BGR2 bottom to 11% of the reads in the large fraction of SAS1B bottom. Unlike Methylomonas, the genus Methylosarcina was better represented in the lithalsa sites and reached its highest abundance in the small and large fractions from the bottom of NASH ponds .All of the pond communities were dominated by Type I methanotrophs . The mosMethylocystis had highest transcript contribution to the potentially active community (5% of the reads) in the large fraction from the SAS1B bottom, but was absent from several samples in the palsa and lithalsa valleys. Methylosinus was present in lower abundance, with a maximum of 2% of the reads in the small fraction from the SAS2B surface waters. Finally, Methylocapsa was found in SAS1B, SAS2A and SAS2B, always at less than 1% of the reads.Type II methanotroph transcripts were poorly represented in the potentially active community and showed less diversity relative to Type I taxa. Only three Type II genera were identified. pmoA transcripts per mL of sample water , but measurements were higher in 2012 compared to 2013. The results showed potential methanotrophic activity not only in the oxygenated surface waters but also in the hypoxic to anoxic bottom waters of the ponds. This included the bottom waters SAS2B and SAS2Ab, where oxygen concentrations were below our limit of detection  of Methylocystis. The relationship between CH4 concentration and potential methanotroph activity varied with cell fraction and depth. The large fraction at both depths and the surface samples for the small fraction showed no clear trend. However the small fraction in the bottom samples showed a strong, negative, nonlinear relationship between pmoA transcripts (log transformed) and CH4 concentrations  and again during the fall (September). However, such mixing does not always extend to the bottom ,53. KWK pmoA transcripts to determine the composition of the potentially active methanotroph community. This method enabled us to identify the methanotrophs that were most actively participating in methane oxidation in thaw ponds and that were unlikely to be dormant, inactive or dead cells [Here we used ad cells . This coad cells , and in Our results strongly indicate that the main driver of community composition of potentially active methanotrophs in permafrost thaw waters was landscape type, with marked differences between palsa and lithalsa ponds. This effect was greater than either depth or the extent of permafrost thawing. The overriding influence of landscape is consistent with previous work highlighting its importance for overall bacterial community structure ,34, and pmoA community profiles in the surface and bottom waters suggest similar potentially active methanotrophs, despite large differences in physico-chemical conditions, including oxygen. This was unexpected relative to results from experimental studies. Microcosm experiments on sediments from Lake Washington showed that different species of methanotrophs predominated under different oxygen tensions [The tensions , with a tensions . This miMuch of the total bacterial production in thermokarst lakes and ponds is associated with suspended particles , and thepmoA sequences from DNA and cDNA [Methylobacter tundripalidum are psychrotolerant [Methylobacter psychrophilus, are truly psychrophilic [-1  is present in almost all methanotroph genera [methane mono-oxygenase (sMMO) on the other hand, is restricted to certain species [pmoA transcripts are verified for known alpha- and gamma-proteobacteria methanotrophs but would not have detected pmoA related genes from the NC10 phylum [Crenothrix [Crenothrix, in low abundance, has been recorded. For the Verrucomicrobia that account for 1 to 6% of the reads of the total bacterial community in the KWK and SAS valleys [The h genera  and its h genera ,77. Solu species  and is r species , making 0 phylum ,80, the enothrix , nor forenothrix ,81. We n valleys ,82, the pmoA transcripts in natural environments, but those published to date imply that the values here could be considered a reasonable proxy of methanotrophic activity. In a boreal wetland, the number of pmoA transcripts was positively correlated with CH4 oxidation rates [pmoA transcripts per cell [pmoA copy numbers from DNA analysis have also found a positive correlation with methane oxidation rates [pmoA transcripts, as measured here.Few previous studies have estimated the concentration of on rates , and in per cell . Studieson rates ,85, and Potential methanotrophic activity in the thaw ponds did not follow the pronounced landscape pattern that we observed in community structure. Only ‘year of sampling’ had a significant effect on methanotrophic potential activity, consistent with the large interannual variability in certain limnological properties of these aquatic ecosystems. This means that the functional trait of methanotrophy was expressed independently of species composition, suggesting a certain functional redundancy amongst the different phylotypes of methanotrophs. This is in accord with studies indicating that there can be a high level of functional redundancy in bacterial communities and that gene expression strongly depends on the environment ,87. It c4 oxidation in soil [The PLS analysis indicated that three environmental factors primarily influenced methanotrophic potential activity: total phosphorus concentration, conductivity and total suspended solids. Phosphorus has been shown to enhance microbial CH in soil , and cou in soil . The lin in soil  with oxi in soil  that wouMethylocystis as a factor associated with pmoA expression, yet this genus represented only a small fraction of the reads . Conversely, the most abundant genus, Methylobacter, showed no statistical relationship with putative methanotrophic potential activity. This might reflect how taxa with low proportional abundance in the “rare biosphere” may have a disproportionate effect on key the ecosystem functions [Methylocystis is also known to be acid-tolerant [Methylocystis to potential activity might also be due to an over-representation of pmoA transcripts, since some strains have two isozymes of the pmo enzyme [The PLS analysis also identified the relative abundance of unctions –95. Modeunctions , and thatolerant ,97,98 witolerant . These fo enzyme .pmoA transcripts were recorded in the hypoxic to anoxic bottom waters in addition to the surface of the ponds, suggesting an unexpected degree of metabolic flexibility. This is consistent with increasing evidence that methanotrophs are micro-aerobic and occur even in anoxic waters. For example, Blees et al. [Contrary to our original hypothesis, methanotrophic potential activity as estimated by transcript concentrations was not closely linked to oxygen concentration. High concentrations of s et al.  found mes et al. ,102 or ds et al. ,104. Phys et al. , and mets et al.  creating4 fluxes and number of pmoA transcripts was observed at a peatland site [4 in a boreal lake, suggesting that the methanotrophs were limited by CH4 concentrations at that site. These contrasting results indicate that the relationship between methanotrophic activity and methane concentration is still poorly understood and may vary among environments.The relationship between methanotrophic potential activity and methane concentration depended on depth of sampling: while no trend was observed at the surface, a negative relationship was found between methane concentration and methanotrophic activity in the small fraction for the bottom samples, suggesting depletion of methane by free-living methanotrophs. A negative relationship between CHand site , but in and site  showed apmoA transcripts did not follow the landscape pattern, and was mainly influenced by total phosphorus, conductivity and total suspended solids, which varied greatly within each pond type. High methanotroph potential activity from pmoA transcripts was attributed to the Type II genus Methylocystis, which was rare, implying a disproportionate, functional importance of certain taxa. Contrary to expectation, pmoA transcripts occurred in hypoxic and anoxic bottom waters as well as in surface oxygenated waters. This implies that methanotrophs are active under a wide range of oxygen conditions and may be resilient to future changes in stratification, mixing and oxygenation.The permafrost landscape has a major influence on the type of thaw ponds that develop, and this in turn has a selective effect on the relative contribution of potentially active methanotrophs in the communities, with a marked difference between organic palsa and mineral lithalsa ponds. In both landscape types, the community was dominated by Type I methanotrophs that are characteristic of low-temperature environments. However, methanotrophic potential activity as measured by S1 FilepmoAFig A in S1 File. Rarefaction curve of the . OTUs were clustered at 93%. pmoA primers fused with the Trueseq sequencing primers. Table C in S1 File. Physico-chemical properties of the surface and bottom (0.5 m above the sediments) of the sampled pondsTable A in S1 File. Ponds sampled during the 2012 and 2013 field campaign and the availability of data. Table B in S1 File. Properties of the . Temperature (T°C), Conductivity in μS cm-1 (Cond), dissolved oxygen in mg L-1 (O2), pH, total nitrogen in mg L-1 (TN), total phosphorus in μg L-1 (TP) dissolved organic carbon in mg L-1 (DOC), total suspended solids (TSS) in mg L-1, Chlorophyll a in μg L-1 (Chla), concentration of carbon dioxide in μM (CO2) and concentration of methane in μM (CH4). -: no data.(DOCX)Click here for additional data file.


Brassica napus L.) fields along a large geographical gradient in Jiangxi province in China. In each field, oilseed rape plants were grown in closed cages that excluded pollinators and open cages that allowed pollinator access. The pollinator community was sampled by pan traps for the entire oilseed rape blooming period.Insect pollinators play an important role in crop pollination, but the relative contribution of wild pollinators and honey bees to pollination is currently under debate. There is virtually no information available on the strength of pollination services and the identity of pollination service providers from Asian smallholder farming systems, where fields are small, and variation among fields is high. We established 18 winter oilseed rape  contains supplementary material, which is available to authorized users. A wide range of agricultural crops depend on pollination by insects . The decThe vast majority of the studies focussing on the interplay between wild and managed pollinators in providing agricultural pollination services originates from Europe and North America, where industrialization of agriculture has resulted in agroecosystems dominated by monocultures in large fields. In contrast, Chinese agroecosystems, particular in South China, are characterised by relatively small fields, leading to a high heterogeneity in terms of crop species, field management and field edges . This hiBrassica napus L.), of which China is one of the world’s largest producers with more than 7.5 million ha cultivated area for the production of cooking oil, feed and biofuel [A globally important crop benefitting from pollination services is oilseed rape  and all fields were sown between the middle and the end of October 2014 with the same traditional open-pollinated winter oilseed rape cultivar YangGuang-2009.We selected 18 oilseed rape fields across a large geographical area in Jiangxi Province, China . The mean distance between fields was 36.9 km (range: 5.8–75.2 km). As the maximum foraging range of most pollinator species is less than 2 km , individ2, a height of 2.0 m and were entirely covered with 1 × 1 mm2 mesh to exclude pollinators. This mesh size has only a limited influence on the microclimate in the cage [In the centre of each field, eight oilseed rape plants at a similar growth stage were selected, spaced 4 m apart. Each plant was covered by an individual cage . Closed cages had a base of 0.6 × 0.6 mthe cage . The opeAfter harvest, the number of pods and total number of flower stalks were counted for each plant. Seeds were removed from pods, weighed and counted using an automatic seed counter . The following yield parameters were measured and calculated per plant: seed set (number of seeds per pod), number of pods (siliques) per plant, number of flowers per plant, fruit set (pod/flower ratio), seed weight  and yield . Plants were dried for 30 days in the greenhouse and the total aboveground dry vegetative biomass excluding seeds and pods (referred to as straw) was assessed.The pollinator community in each experimental oil seed rape field was sampled by pan traps, which is a suitable method for sampling pollinators such as bees , hoverflApis mellifera and Apis cerana). Asynchronous flowering times of the oilseed rape crops prevented the separation of insect communities that were visiting the oilseed fields during and after flowering  and honey bees  and plants without pollinator access (closed cages), and included (1) seed set, (2) number of pods per plant, (3) number of flowers per plant, (4) fruit set (pod/flower ratio), (5) thousand seed weight, (6) plant yield, (7) straw biomass, and (8) yield/straw ratio. Plant yield/straw ratio is the ratio between total seed weight and total dry vegetative biomass. It expresses the ratio of assimilates to seeds or vegetative growth, and provides a useful indicator for limitation in active seed sinks on the plant , 36 as aP < 0.001) and Fisher’s alpha , and is a robust indicator for mobile insects and uneven sample sizes [The second analysis focused on the effect of pollinator abundance and diversity on yield parameters. Generalized linear mixed effect models were used with study field as a random factor. Data from the open and closed cage treatments were analysed separately. The purpose of the analysis on open cages was to assess the role of different pollinator taxa in pollination, while the analysis on closed cages was conducted to verify that pollinator abundance and community composition did not affect plant yield parameters in closed cages. Response variables included (1) seed set (Gaussian error distribution with identity-link function), (2) fruit set (gamma error distribution with log-link function), (3) thousand seed weight (gamma error distribution with log-link function), and (4) yield/straw ratio (gamma error distribution with log-link function). Explanatory variables included (a) wild pollinator abundance, (b) honey bee abundance, (c) wild pollinator diversity, and (d) plant straw biomass. Study field was included as a random factor. Wild pollinator diversity was characterised in terms of the back-transformed Shannon entropy index , the rarle sizes –42, we sP > 0.05). All calculations and analyses were conducted in R (v3.1.2) [All models were validated by checking residuals according to the protocol of Zuur et al.  and devi(v3.1.2)  using th(v3.1.2) , the “lm(v3.1.2) , and the(v3.1.2) . Means aPieris rapae), two wild bee species Eucera chinensis and Lasioglossum proximatum, and two honey bee species A. mellifera and A. cerana, accounting for 21.6, 20.8, 16.5, 9.1 and 8.1% of the overall specimens, respectively , and 1165 Lepidoptera (9 species). The top five most abundant species were cabbage butterfly  from 1181 individuals were reported in Wiltshire, UK , 36 flowInsect pollinated plants showed higher seed set and overall higher yields than plants deprived from pollinators. Therefore, our study contributes to a body of evidence that insect pollination matters for oilseed production despite its capacity for self-pollination , 50–52. The higher straw biomass of oilseed rape plants in closed cages points to an increased allocation of assimilates to the above ground vegetative plant parts, which supports a lack of sink strength resulting from a pollination deficit. The positive effect of pollinator exclusion on straw weight may also in part be due to the high energetic cost of producing fatty acids in seeds as compared to the lower energetic conversion costs to leaf and stem dry matter . OverallWild pollinator abundance and diversity were positively associated with oilseed rape seed set and yield/straw ratio, but not with fruit set and seed weight, suggesting that their benefits to oilseed rape yield per plant mostly result from an increased number of seeds per pod. The control analysis, which showed no relationship between plant yield parameters and pollinator abundance and diversity for closed cages, confirmed the effectiveness of the exclusion treatment and the overall consistency of the experimental setup. This also suggested that pollinator collections from pan traps can be used as a proxy in reflecting the pollinator communities and pollination service at the landscape scale , 56.We assessed the contribution of insect pollination on isolated plants where neighbouring plants were removed. The focus on isolated plants may have also resulted in an reduction of plant-to-plant pollen transfer  and a rePieris rapae) might be an important pollinator [Surprisingly, our analysis gave no support for the contribution of honey bees to oilseed rape yield, even though there were large differences in honey bee abundance between fields. While the contribution of honey bees to crop pollination is widely documented [see review in llinator . As theiOur study demonstrates that wild pollinators play an important role in the pollination of oilseed crops in small-holder farming systems in China. Wild pollinator abundance and diversity contribute to oilseed rape yield by mediating increased allocation to seeds rather than above-ground straw biomass, but oilseed rape plants suffering from a pollination deficit can compensate to some extent by generating heavier seeds, more flowers and higher straw biomass. This study highlights the importance of conserving wild pollinators in order to maximise oilseed rape production, especially in heterogeneous landscapes where their pollination service is exceeding the service provided by managed pollinators.


Lysosomal dysfunction is associated with a number of age-related pathologies that affect all organ systems. While much research has focused on neurodegenerative diseases and aging-induced changes in neurons, much less is known about the impact that aging has on lower urinary tract function. Our studies explored age-dependent changes in the content of endo-lysosomal organelles  and age-induced effects on lysosomal degradation in the urothelium, the epithelial tissue that lines the inner surface of the bladder, ureters, and renal pelvis. When examined by transmission electron microscopy, the urothelium from young adult rats (~3 months), mature adult rats (~12 months), and aged rats (~26 months old) demonstrated a progressive age-related accumulation of aberrantly large endolysosomes (up to 7μm in diameter) that contained undigested content, likely indicating impaired degradation. Stereological analysis confirmed that aged endolysosomes occupied approximately 300% more volume than their younger counterparts while no age-related change was observed in multivesicular bodies or lysosomes. Consistent with diminished endolysosomal degradation, we observed that cathepsin B activity was significantly decreased in aged versus young urothelial cell lysates as well as in live cells. Further, the endolysosomal pH of aged urothelium was higher than that of young adult (pH 6.0 vs pH 4.6). Our results indicate that there is a progressive decline in urothelial endolysosomal function during aging. How this contributes to bladder dysfunction in the elderly is discussed. The endo-lysosomal system consists of interconnected organelles and pathways that are involved in internalization, recycling, and degradation of internalized membrane and fluid. Central to these pathways is the lysosome, a pleomorphic organelle that contains greater than 60 hydrolytic enzymes that allow for degradation of all macromolecules in the cell including proteins, lipids, carbohydrates, and nucleic acids . In the Lysosomal function is widely known to diminish with aging , and thuThe urinary bladder is an organ that is impacted in a significant and adverse manner during the aging process –21. MajoLysosome Associated Integral Membrane Protein2 (LIMP2), are known to cause significant alterations in urothelial morphology and function [Despite the strong correlation between lower urinary tract symptoms and aging, as well the potential increased burden imposed on urothelial lysosomes, how aging affects the endo-lysosomal organelles of the urothelium or what role these effects may have in the onset of lower urinary tract dysfunction in age and age-related disease is largely unknown. This is critical to understand as umbrella cells traffic massive amounts of membrane through the exocytosis and endocytosis of a subapical pool of vesicles that regulate membrane surface area during filling and voiding cycles . Importafunction –28.To our knowledge, only one study has explored age-related changes in lysosomal function within the urothelium . Using aAs lysosomes have been proposed to lie at the heart of many age-related pathologies, we first sought to characterize any morphological changes in the endo-lysosomal system of the urothelium, in particular, the umbrella cells. We used transmission electron microscopy (TEM), which allowed us to distinguish organelles by their ultrastructural features. Previous studies showed that in umbrella cells apically internalized cargoes, which are endocytosed by a clathrin-independent pathway, first enter small membrane-delimited structures that depending on species can have an ovoid to polygonal morphology when examined in cross section , 30–33. Temporally, endocytosed cargo is next observed in multivesicular bodies MVBs; , which aIn order for the cargo within the MVB to be degraded, the MVBs fuse with lysosomes, which contain hydrolytic enzymes for digestion. As in other cells, the lysosomes of umbrella cells are typically 0.5μm to 1μm in diameter and by TEM appear electron dense, have a single limiting membrane, and their lumen has a fine granular appearance , Lys. UnLastly, in addition to degrading endocytic cargo, lysosomes are also responsible for clearing spent or damaged organelles by autophagy. These cargoes are delivered to lysosomes via autophagosomes , which cWe used TEM to determine how aging impacted the endo-lysosomal system of the urothelium by comparing two age groups: young adult animals (~3 months), subsequently referred to as young, or aged ones (~26 months). While we did not detect any apparent differences in the morphology of the lysosomes (not shown) or MVBs of these two groups compare , we readCloser inspection of the aberrant endolysosomes of aged umbrella cells by TEM revealed a number of other notable features. First, the luminal content in aged endolysosomes was heterogeneous, usually containing a mixture of luminally disposed vesicles of wide-ranging number, size, shape, and content that were morphologically distinct from the ILVs, which as noted above are formed by invagination of the limiting membrane of the MVBs . The numOther luminal vesicles–especially the largest ones–generally exhibited lower electron density Figs . This woIn addition to luminal vesicles, we also noted other content in the endolysosome lumen. For example, many endolysosomes contained stacked membrane , which we subsequently refer to as “adult” rats, to determine whether the changes we observed were progressive. As a marker of endolysosomes we used an antibody against LIMP2, an integral membrane protein that resides on the limiting membrane of lysosomes, endolysosomes, and MVBs. In young rats, LIMP2-positive endosomes were found dispersed throughout the urothelium; however, we noted an accumulation of LIMP2-positive structures in the umbrella cell layer as the animals aged , arrows.v; volume of each organelle relative to the total cell volume) of MVBs, endolysosomes, or lysosomes in umbrella cells as a function of age. While there was no significant change in the volume fraction of endolysosomes in adult rats versus aged ones, the stereological analysis revealed that endolysosomes from aged rats occupied a significantly higher cytoplasmic fractional volume (~300% greater) than endolysosomes from young umbrella cells . The measured volume of endolysosomes significantly increased between young and aged rat umbrella cells (from ~ 83 μm3 to ~ 347 μm3)  and cathepsin B, a major cysteine protease. Each cell lysate was resuspended in buffer conditions optimized for maximal enzyme activity in order to nullify any age-related differences othelium . By contothelium . These dThe above assays measure enzymatic activity from cell lysates in a nominally “ideal” enzymatic environment to promote maximal activity. To determine whether cathepsin B activity is diminished in cells, we measured the hydrolysis of Magic Red Cathepsin B (MR-CB) in isolated urothelial cell clusters. MR-CB is a membrane permeant substrate that when cleaved by active cathepsin enzymes emits fluorescent light. When we compared the fluorescence intensity immediately after adding the reagent versus 12 minutes after incubation at 37°, we observed a significant increase in fluorescence of punctate structures consistent with lysosomal cathepsin activity in urothelium from young animals . By contLysosomes not only perform critical housekeeping functions such as degradation of endocytosed proteins and lipids, and turnover of spent organelles and select cytoplasmic proteins by autophagy, they also serve as hubs for metabolic adaptation and nutrient-dependent signaling , 39. CriPerse et al. previously reported that the umbrella cells of aged mice, but not young ones, accumulate “lipofuscin granules,” which are autophagosomal or autolysosomal in origin. Based on morphological grounds, these lipofuscin granules are identical to the endolysosomes that we describe in our aged rat model, including their size and accumulation of pleomorphic vacuolar elements and stacked membranes. Moreover, our studies indicate that endolysosomes are present even in young animals, and that in addition to autophagosomal content they also contain products of endosomal fusion, including MVBs. Thus, these organelles contain both endosomal and autophagosomal content. Additional novel findings of our studies include the observation that the endolysosome compartment of umbrella cells undergoes a dramatic expansion during aging. In contrast, there is no corresponding observable age-associated change in the morphology or volume of MVBs and electron-dense lysosomes, both of which act as precursors for the endolysosome. This may indicate that either the biogenesis of lysosomes and MVBs are unaffected by aging, or that their biogenesis  is increased, leading to enhanced production of endolysosomes.per se, but instead only change the average size of each organelle. Increased endolysosome-autophagosome fusion could also contribute to the expansion of the endolysosomal compartment. Indeed, we observed autophagosomes inside the lumen of the aged endolysosomes and a deficiency in autophagy has been reported to affect the number of MVBs and lysosomes in umbrella cells [vide infra). Nonetheless, any change in the total compartment volume of autophagosomes could affect endolysosomal volume and future studies could address whether increased fusion, versus decreased turnover might be occurring.Although we also observed that the endolysosomes of aged urothelium appeared to show increased fusion, this would not in itself account for the expansion of the endolysosome compartment we observed in aged animals. This is because fusion alone would not increase the total compartment volume la cells . Howevervide infra).To understand better why the endolysosome compartment, filled with undigested membrane and lipid, expanded with age, we also explored the possibility that endolysosome function was diminished during the aging process. In fact, we observed that the pH of acidified compartments in the urothelium was increased in aged animals, and we also observed a decrease in the activity of cathepsin B, a critical lysosomal enzyme , would have a significant negative impact on degradation, as most of the lysosomal hydrolases have optima in the pH 4.5–5.0 range. While the pH assay we employed does not distinguish among the acidified compartments in the cell, endolysosomes are reported to be the most acidified organelle in the cell . BecauseIn addition to impaired organelle acidification, our results indicate a defect in the activity of at least one hydrolytic enzyme, the cysteine protease cathepsin B.in vivo.Impaired cathepsin B activity has been implicated in neurodegenerative diseases such as Huntington’s, Parkinson’s and Alzheimer’s, all of which exhibit some morphological defect in lysosomes –50. CathFinally, an age-related decrease in lysosomal function in urothelium may have significant effects on the physiological function of the bladder. Importantly, in response to external stimuli  the urothelium releases mediators that transmit information to afferent nerve fibers that lie directly adjacent to the urothelial cells and relay information to the CNS , 54. LikIn summary, our results provide important information that sheds light on the relationship between lysosomal function and the deleterious effects of aging on the bladder urothelium. It is our hope that these findings will be of value in not only furthering our understanding of urothelial dysfunction with age but also in understanding better the mechanisms by which lysosomal dysfunction impacts cells throughout the body.2 inhalation at three months of age. Fisher F344 mature adult rats and Fisher F344 aged rats  were sacrificed by CO2 inhalation at 12 months and 26 months of age, respectively.Animal experiments were performed under the approval of the Animal Use and Care Committee of the University of Pittsburgh. Urinary bladders were excised from Fisher F344 young adult rats  after sacrifice by COReagents used included: mouse monoclonal anti LIMP2 ; Magic Red Cathepsin B ; Lysotracker Red DND-99, Lysosensor DND-160, Rhodamine phalloidin and TOPRO-3 were obtained from Fisher Scientific .4, 2 mM CaCl2, 1.2 mM MgSO4, 11.1 mM glucose, pH 7.4) gassed with 95% O2/5% CO2 gas, then cut open lengthwise through the urethral opening and pinned down  on a rubber mat. The bladders were equilibrated in Krebs for 20–30 min, the Krebs was removed and then replaced by EM fixative  paraformaldehyde, 2% (v/v) glutaraldehyde, 1mM CaCl2, 1mM MgCl2) and incubated for 2–3 h at RT. The samples were osmicated 1–2 h with 1.5% (w/v) reduced OsO4 in 100 mm cacodylate, pH 7.4, washed several times with distilled water, and then block stained overnight at 4° C in 0.5% (w/v) aqueous uranyl acetate . Tissues were dehydrated in a graded series of ethanol, embedded in the epoxy resin LX-112 (Electron Microscopy Sciences) and sections  were cut with a Diatome diamond knife (Electron Microscopy Sciences). Sections were counterstained with uranyl acetate and lead citrate and viewed on a JEOL 1011 transmission electron microscope with a side mount AMT 2K digital camera . Images were imported into Photoshop CC , adjusted for brightness and contrast, and then assembled in Adobe Illustrator CC.Urinary bladders from each age group  were excised from euthanized rats and placed in Krebs solution . A minimum of 16 electron micrographs from randomly chosen umbrella cells were acquired for each of 3 separate bladders for each treatment group . The images were captured at 5000x then were imported into Photoshop and adjusted for contrast. For larger cells that did not fit within the field at that magnification, multiple overlapping images of the same cell were captured and combined using the photomerge function (File → Automate → Photomerge). A point grid  was copied from the ImageJ program (available at imagej.nih.gov) and pasted as a transparent layer on to each image. The average volume fraction of each organelle  was determined by point counting and calculating the ratio of counts on the organelle relative to the total counts on the cell as described previously [The tissue was sectioned perpendicular to the length of the epithelium to obtain vertical sections necessary to determine organelle volume fraction (Veviously .vide infra), we used its overall mean and the individual volume fractions to estimate the total compartment volume by multiplying each Vv by The data were summarized using means and standard deviations by organelle  and age group . Within each organelle type, the age effect was examined using ANOVA, followed by a post-hoc test using Holm-Sidák correction. Since the volume-weighted mean cell volume  paraformaldehyde) and incubated for 1 h at room temperature. Bladders were cryo-protected in PBS containing 30% (w/v) sucrose overnight at 4° C. The PBS/sucrose was exchanged with OCT compound  and frozen in plastic cryo-molds (Fisher) filled with OCT. The frozen tissue was mounted in a Leica Microsystems CM1950 cryostat , oriented to position the urothelium in a vertical axis perpendicular to the long axis of the tissue, and excess OCT was trimmed from the block. The bladder was then sampled by continuously sectioning the organ and collecting 10 random slices through the whole of the organ. The distance between slices was equidistant, but varied depending on the size of the bladder , and the first slice was uniform random in the interval between the start of the tissue and the distance between the slices. Three bladders from each age group  were used.l = intercept length.Each section was stained with Rhodamine phalloidin to outline cell borders and viewed on a Leica Microsystems DM6000 B wide-field fluorescence microscope using a 63x objective. Gray-scale images (2048 x 2048 pixels) were captured by a Retiga 4000R digital camera  and were imported into the Photoshop program, and the image inverted so that brightly fluorescent staining appeared black in the final images. The average volume of the umbrella cell was determined by the point/sampled intercept method . Brl3 estimates were averaged, and then multiplied by π/3 to determine 3; n = 9) to estimate the compartment volume for each organelle.The number of intercepts that were measured varied by section but a minimum of 100 measurements were recorded across the entire bladder for each animal . For each bladder, the Urinary bladders were excised from euthanized animals and placed in Krebs. A solution containing 10 μM Lysotracker Red diluted in Krebs (~500μl) was injected into the lumen of the bladder by securing one end of five-cm long piece of plastic Tygon tubing (Fisher) to the urethral meatus of the bladder using a suture wire and purse-string closure , and fitting the other end of the tubing over a 22½ gauge needle  connected to a 1-ml syringe (Becton Dickinson) containing the Lysotracker Red solution. Following instillation of the solution, the bladder was incubated for 30 min at 37° C before voiding the bladder by removing the urethral suture and incubating for an additional 2 h in Krebs at 37° C. The bladder was transferred to ice-cold Krebs, cut open lengthwise through the urethral opening and pinned down  on a rubber mat. The bladder was washed 3 times with ice-cold Krebs and then fixed with 100 mM sodium cacodylate, pH 7.4 containing 4% (v/v) paraformaldehyde. Bladders were cryo-protected in 30% (w/v) sucrose in PBS by incubating overnight at 4° C. The PBS/sucrose was exchanged with OCT compound and frozen in cryo-molds filled with OCT solution before sectioning in a cryostat to a thickness of 4μm per section.4Cl and 20 mM glycine, pH 8.0 dissolved in PBS containing 0.1% (v/v) Triton X-100. The tissue was incubated with block solution (PBS containing 0.6% (w/v) fish skin gelatin, 0.05% (w/v) saponin) for 60 min at RT. The block was aspirated and replaced with LIMP2 antibody and incubated 2 h at RT. The slides were washed with PBS and then incubated with Alexa 488-labeled secondary antibody  for 60 min at RT along with rhodamine phalloidin (to label actin) and TOPRO-3 to stain nuclei. The tissue was then washed with PBS and post-fixed in 4% (w/v) paraformaldehyde in 100mM cacodylate buffer, pH 7.4 for 10 min. The slides were washed with PBS and a drop of SlowFade Diamond Antifade (ThermoFisher) was added to the tissue and topped with borosilicate coverslips . The edges of the coverslip were sealed with nail polish and dried before viewing in a Leica TCS SP5 CW-STED confocal microscope using a 40X objective and the appropriate laser lines . Eight-bit images were collected using 8 line averages combined with 6 frame averages and imported into Volocity 4-D software . Images were exported as TIFF files, corrected for contrast in Photoshop, and assembled in Illustrator.Tissue sections were collected on Superfrost Plus glass slides (ThermoFisher), washed with PBS at RT and then unreacted fixative was quenched by incubating tissue slices for 10 min at RT with Quench buffer , covered with Minimal Essential Medium (MEM) containing 3 mg/ml Dispase  and placed in a 37°C incubator for 2 h. The urothelium was then recovered by removing the MEM/dispase solution by aspiration and then gently scraping the bladder using a sterile metal spatula. Scraped cells were placed in 2 ml StemPro Accutase Cell Dissociation Reagent (ThermoFisher) solution for 10 min and triturated to obtain single-cell suspensions. Each suspension was then centrifuged at 230 x g for 10 min and the cells resuspended in 500 μl of Krebs solution containing 2 μM Lysosensor DND-160 dye (Thermo Fisher). After incubation with the dye for 10 min at room temperature, the cells were centrifuged at 230 x g for 5 min to collect the cells, which were then resuspended in 500ul of normal Krebs solution. The cells were then aliquoted into wells of a black-walled 96-well plate  using 50 μl each for 10 wells and keeping young/aged cells separate. Fluorescence of the Lysosensor DND-160 dye was measured using a dual emission protocol  using a Spark 20M plate reading system  and calculating the 450/550 ratio. Ratios were converted to pH values by means of a standard curve using cells from a separate young rat. These cells were collected and treated with Lysosensor dye as described above, but they were aliquoted and resuspended in Krebs solution containing the ionophores Nigericin (20 μM) and Monensin (10 μM), with the pH adjusted to 4.0, 5.0 or 6.0 for 5 min prior to reading. Linear regression and extrapolation of unknowns was performed using GraphPad Prism 7.0 .NAG activity from urothelial lysates was measured by a NAG activity kit  following the manufacturer’s instructions. Enzymatic activity was measured by incubation of cell lysates with the synthetic p-nitrophenol derivative (R-pNP) as a NAG substrate, which releases pNP when cleaved and emits fluorescence. NAG activity was determined by measuring absorbance at 400nm in a Biophotometer Plus Spectrophotometer  and was expressed as nmol pNP/μm protein.Cathepsin B activity from urothelial cell lysates was measured by a Cathepsin B activity kit  following the manufacturer’s instructions. Enzymatic activity was determined using the cathepsin-B substrate Arg-Arg labeled with amino-4-trifluoromethyl coumarin, which when cleaved emits fluorescence. Fluorescence values were quantified in a Glomax multi-detection Systems fluorometer  equipped with a 400-nm excitation filter and a 505-nm emission filter. Activity was expressed as relative fluorescence units (RFU)/μg of protein.Urinary bladders were excised from the animals and placed in Krebs, the bladders were cut open, lengthwise, and pinned  to a rubber mat. The urothelium was treated for 2 h at 37° with 2.5 mg/ml Dispase diluted in MEM in a tissue culture incubator. The dispase solution was removed and the urothelium was gently scraped with a 16-cm cell scraper  into a microfuge tube with MEM containing 10% v/v Fetal Bovine Serum (Hyclone) and rotated for 10 min at 4° to inactivate the dispase. The cells were centrifuged for 2 min at 1500x g, the supernatant was carefully removed by pipette and the cells were washed twice with Krebs containing 10 mM HEPES, pH 7.4 and resuspended in a final volume of 200 μl of Krebs-HEPES solution. Forty μl of the cell suspension was added to Mat-Tek glass bottom microwell dishes , which were placed in a Tokai-HIT model GSI heated stage . Four μl of a 10 x stock (prepared according to manufacturer’s recommendations) of Magic Red Cathepsin B (MR-CB) was added to the cell suspension and 1024 x 1024 pixel images were taken using a 40 x oil objective on a TCS SP5 CW-STED confocal microscope (Leica Microsystems) with a line average of 8, a frame average of 4, and using a zoom of 2. Images were captured at t = 0 min and t = 12 min after addition of MR-CB. At least 2 images were taken per bladder, using 3 bladders for each age group (young and aged). Images were imported into Volocity  and the Quantitation module used to select objects with an intensity threshold > 40 and an object size > 4 pixels. Measurements were then exported to Microsoft Excel. The sum of the intensity of objects was determined at each time point for each image and the total change in intensity between time points (within the same image) was recorded. The final value for each bladder was determined by averaging the change in intensity from 0 to 12 min from all of the images taken within the same bladder.