Ligasure hemorrhoidectomy for thrombosed external hemorrhoids in pregnancy has been rarely studied.The purpose of this article is to study the efficacy and safety of Ligasure hemorrhoidectomy comparing with conservative treatment for thrombosed external hemorrhoids in pregnancy.This was a retrospective cohort study.The patients were treated at a tertiary referral center in China.94 pregnant patients hospitalized for thrombosed external hemorrhoids from September 2020 to December 2021.Ligasure hemorrhoidectomy treatment or conservative treatment according to the patient’s wishes.Symptom relief, recurrence and satisfaction of thrombosed external hemorrhoids in pregnancy with different interventions.There were no differences between groups in maternal age, gestational age, body mass index, parity, constipation and a prior history of thrombosed external hemorrhoids. The pain scores were less in surgical group than in conservative group in post-treatment days 1 and 7. Time to return to normal activities was shorter in surgical group than in conservative group . Post-treatment complications were mild in surgical group and there were no significant differences concerning the rate of abortion, preterm birth, cesarean delivery and weight of fetus. Recurrence rate was significantly lower in surgical group . The patient satisfaction scores were significantly higher in surgical group than in conservative group .This was a retrospective study with a limited number of patients, the data was obtained from only one center.Comparing with conservative treatment, Ligasure hemorrhoidectomy for TEH in pregnancy results in more rapid pain relief, shorter time to return to normal activities, lower incidence of recurrence, and better patient satisfaction. This type of surgery has low and mild postoperative complications, is not attended by any risk to the mother or her fetus. Thrombosed external hemorrhoid (TEH) in pregnancy is the manifestation of the acute attack of hemorrhoid due to the increased intra-abdominal pressure from uterine growth, constipation and diarrhea. TEH, can involve both the external and internal hemorrhoidal plexuses, being the first more frequently affected, a certain degree of hypertonicity of the internal anal sphincter may trap the hemorrhoidal mass outside the anus leading to strangulation, necrosis, and gangrene –3. From Hemorrhoidectomy is only indicated in strangulated or extensively thrombosed hemorrhoid in pregnancy . The surSurgical intervention can be made more effectively and reliably because of development in surgical technique. The usage of the Ligasure technique results in a significantly shorter operative time and less postoperative pain without any adverse effect on postoperative complications, compared to conventional hemorrhoidectomy . MoreoveIn fact, sufficient data existing on the efficacy and safety of Ligasure hemorrhoidectomy for TEH is acquired according to the application of this technology in nonpregnant patients. Ligasure hemorrhoidectomy for TEH in pregnancy has been rarely studied. In this article, we aim to study the efficacy and safety of Ligasure hemorrhoidectomy for TEH in pregnancy.94 pregnant patients with TEH between their 6th and 35th week of pregnancy treated from September 2020 to December 2021 were included. Patients were offered Ligasure hemorrhoidectomy or conservative treatment, and they were allowed to choose between treatment options by themselves. The inclusion criteria were as follows: (1) consistent with clinical manifestation of thrombosed external hemorrhoids (TEH): involve both the external and internal haemorrhoidal plexuses; the haemorrhoidal mass outside the anus leading to strangulation, necrosis, and gangrene; (2) Clinical symptoms: acutely intense pain, painful anal swelling, bleeding, and a visible perianal/anal lump; (3) less than 35 weeks of gestation. The exclusion criteria were as follows: (1) more than 35 weeks of gestation; (2) patients with anal fissure, perianal abscess, fistula, inflammatory bowel disease; (3) immunocompromised patients; (4) cardiac disease, diabetes, cirrhotic patients or after pelvic radiotherapy; (5) patients under anticoagulant therapies; (6) patients with colorectal or anal neoplasia; (7) previous anal surgical procedures.All patients who underwent therapy performed preoperative laboratory, electrocardiographic testing and fetal heart monitoring. Patients’ characteristics included demographics and clinical characteristics  were obtained. Pre- and post-treatment pain scores, time to return to normal activities, post-treatment complications, pregnancy outcomes, recurrence, and patient satisfaction were all assessed. Patients were followed up in 1 day, 1 week, and 2 weeks post-treatment and had a final follow-up visit in 3 months postpartum.Clinical evaluation was performed by the surgeons. The Rome IV criteria were used to define constipation and the Bristol Stool Chart (BSC) was used to determine stool type. Patients were asked to complete pain surveys using a visual analogue scale (VAS) ranging from 0 cm (no pain) to 10 cm (the worst pain ever experienced) during the peri-treatment period in days 0, 1, 7, and 14. Patients were advised to return to normal activities as soon as they felt able. Abortion was defined as fetal loss before 28 weeks of gestation. Preterm birth was defined as delivery in the range of 28 to 36 weeks of gestation. Recurrence was defined as the onset of symptoms (pain and bleeding) or new prolapse at follow-up to 3 months postpartum. Patient satisfaction was assessed with 4 categories: excellent (score = 4), good (score = 3), fair (score = 2), and poor (score = 1), in 1 week, 2 weeks post-treatment and had a final follow-up visit in 3 months postpartum. The local Institutional Board of Ethics approved the study (2022IEC057).Patients in surgical group received spinal anesthesia with 0.5% lidocaine in right lateral decubitus. They were placed in the lithotomy slight left lateral position to prevent uterine compression of the vena cava and left iliac vein and supplemented by monitored anesthesia care and fetal heart care. Ligasure hemorrhoidectomy consisted of excision of both external and internal components using the Ligasure handset up to the anorectal junction without ligation of the pedicle, retaining enough anal margin skin at the same time to avoid postoperative stenosis. When they were freed from anal pain and meanwhile evacuation had occurred, the patients were discharged from the hospital. Patients received regular laxatives for 1 weeks (Polyethylene glycol 4000 10 mg po qd or Lactulose 10 ml po qd) after discharge. Fetal heart monitoring was crucial and done throughout the whole therapy period.Patients in conservative group received fiber and oral fluid intake, stool softeners (Polyethylene glycol 4000 10 mg po qd or Lactulose 10 ml po qd), assisting by hand for the swelling return, warm sits bath, topical analgesia treatment (0.5% lidocaine cream) when the patients felt severe anal pain, consultation and frequent communication with obstetrical colleagues to help pregnancy refrain from straining and anxiety.P < 0.05 was considered significant.The statistical analysis was conducted using IBM Statistical Package for the Social Sciences (IBM SPSS), version 23.0, software. Analysis of the distribution of the data using the Shapiro–Wilk test, and Levene’s test was applied to confirm the homogeneity of variability between groups. Descriptive quantitative data were displayed in the form of mean, median, standard deviation, range, and interquartile range (IQR), while numerical and percentage values were chosen to describe qualitative data. An independent t-test was performed to compare quantitative data and parameter distributions between two groups. The non-parametric distribution was performed by using the Mann–Whitney test. For the qualitative data, Chi-square tests were carried out to compare between the groups. 94 pregnant patients with TEH between their 6th and 35th week of pregnancy treated from September 2020 to December 2021 were included. 13 patients were excluded for several reasons  of all patients had constipation, and 38.27% (31/81) of all patients had a prior history of TEH.Demographics and characteristics were summarized in Table There was no difference in pre-treatment pain score of two groups. The median pain scores were less in surgical group than in conservative group in post-treatment days 1 and 7. In post-treatment days 14, there was no obvious difference in two groups Table .Table 2VTime to return to normal activities averaged 6.51 days in surgical group vs. 13.52 days in conservative group . There was obvious difference in two groups. (Table The rate of post-treatment complications was 14.29% (5 cases) in surgical group and 26.09% (12 cases) in conservative group in Table Pregnancy outcomes were shown in Table 2 = 5.73, P = 0.017, Fig. The overall recurrence rate at follow-up to 3 months postpartum was 20.99% (17 cases). The rate of recurrence in conservative group was 30.43% (14 cases), whereas only 8.57% (3 cases) in surgical group . Three patients who recurrence in surgical group were in remission with conservative treatment, 5 patients who recurrence in conservative group received Ligasure hemorrhoidectomy after postpartum. The result showed that conservative treatment might prolong the duration of symptoms and increased recurrence rates, and many patients would require subsequent radical operative treatment. Ligasure hemorrhoidectomy can confer protection against a further recurrence of TEH in pregnancy.There was significant difference in recurrence between two groups  risks of anaesthetics to the developing fetus .The majoHowever, surgery is always very stressful and anxiety producing for the pregnant woman, even if the outcome is successful. It is important to keep in mind that physiologic changes during pregnancy alter the maternal response to stress. A thoughtful and comprehensive preoperative counsultation on the dangers to the fetus is an important component of therapy. Consultation and frequent communication with obstetrical colleagues and professional psychology are crucial. Fetal monitoring should be done throughout the perioperative period .Constipation and a prior history of TEH may be the most common predisposing factors for TEH during pregnancy. The pathophysiology of constipation during the pregnancy is that progesterone causes a decreased small bowel and colonic motility and subsequent slow transit constipation, pressure on the rectosigmoid colon from the gravid uterus can cause an obstructive constellation of constipation symptoms . As womeOur study had some limitations. First, this was a retrospective study with a limited number of patients, the data was obtained from only one center. Thus, selection bias was inevitable. Another limitation was the short length of follow-up. The result could not accurately evaluate recurrence rate. Finally, two anorectal surgeons took part in the management of these patients, and their individual decisions and surgical techniques might affect the results. Therefore, further well-designed randomized studies are required.Compared with conservative treatment, Ligasure hemorrhoidectomy of TEH in pregnancy results in more rapid pain relief, shorter time to return to normal activities, lower incidence of recurrence, and better patient satisfaction. This type of surgery has low and mild postoperative complications, is not attended by any risk to the mother or her fetus.


Glycine max (L.) Merr.] yields in Ontario, Canada. Many studies of genetic variation for drought tolerance compare commercial lines with exotic, unadapted germplasm. We hypothesized that even current commercial cultivars adapted to Ontario would differ significantly for traits related to drought tolerance. In a greenhouse experiment, we grew fifteen soybean cultivars in field soil amended with sand in 1-m rooting columns, which allowed for simulation of field-like soil water profiles and rooting depths. Two watering treatments were imposed from the first flower until maturity by daily restoration of soil water to either 100% (control), or 50% (drought stress) of the maximum soil water holding capacity. Throughout the experiment, we measured volumetric soil water content at different depths in the soil profile, but found no evidence at any developmental stage that the cultivars differed for their ability to extract soil water from different depths. Drought stress reduced seed yield by 51% on average. Similar to the effects of drought in the field, pod number was the yield component most affected, with effects on seeds per pod and single-seed weight being comparatively minor. There were significant cultivar × treatment interactions for seed yield, pod number, shoot dry matter, and water use. We identified two drought-sensitive (Saska and OAC Drayton) and three drought-tolerant  cultivars based on their ratios of seed yield under drought stress to seed yield under control conditions . Regression and principal component analyses revealed that drought-tolerant (high-SYR) cultivars were consistently those that maintained relatively high values for water use, biomass accumulation and pod number under drought stress; high water use efficiency under drought stress was also associated with a high SYR. One of the cultivars, OAC Lakeview, displayed a distinct mode of drought tolerance, maintaining a very high fraction of its control pod number under drought stress. This study helps define the physiological basis of soybean cultivar differences in drought tolerance, and provides direction for soybean breeders to select traits that could improve yield under drought stress.Drought stress significantly limits soybean [ Glycine max (L.) Merr.] is the most valuable oilseed crop grown in Ontario, Canada, where it is typically produced under rainfed conditions. Drought stress occurring during the critical stages of the crop’s development can significantly reduce its yield potential  whole-plant water use efficiency (WUE), (b) regulation of whole-plant water use in response to soil water content, and (c) leaf epidermal conductance to water vapor when stomata are closed, also known as dark-adapted leaf conductance which predicts WUE of soybean . The gen‘Sohoma’ that displayed a significantly lower reduction in its seed yield and yield components (100-seed weight and seed number) under soil water deficit conditions in the field, as compared to three other cultivars. In that work, the authors imposed the drought stress treatments at either the R2 (full flowering) or R4 (full pod) developmental stage; stress imposed at R2 resulted in a greater reduction (~31%) in seed yield than stress imposed at R4 (~21%).Drought stress affects soybean yield through a reduction of any of its yield components  e.g., . MoreoveMany studies of genetic variation for drought stress tolerance and related traits compare elite commercial lines with exotic, unadapted germplasm e.g., . We hypo;Plants were grown in the Crop Science Building’s greenhouses at the University of Guelph , Guelph, ON, Canada, in 2017 and 2018 using a culture system and drought stress simulation method developed and described in detail by The plants were grown in 1 m long 10 cm diameter PVC rooting columns (tubes) lined with polyethylene liners, and PVC end caps at the bottom, each with a drainage hole. The soil mixture was a blend of six parts by volume field soil, two parts granitic sand , and one part peat-based potting mix .London loam (Grey-brown Podzolic loam till), was collected from the topsoil (upper 15 cm) at the Elora Research Station  that had a prior history of soybean production. It was a silty loam  texture that contained 4.2% organic matter, 23.5 ppm P, 61.5 ppm K, 280 ppm Mg, 2375 ppm Ca, 15 ppm Na, 14.4 meq per 100 g CEC, and had a pH of 7.4 according to a soil test performed by A&L Laboratories Inc., London, ON, Canada. During the process of potting, the tubes were filled in a systematic fashion of loading and packing until the soil reached approximately 1 cm below the top of the tube. The total weight of each tube with its soil was then recorded. A commercial 20-20-20 N-P-K plus micronutrients fertilizer  at the rate of 0.8 g tube-1 dissolved in 500 mL of water was added at the top of the soil surface in each tube so that the first 0-30 cm of the soil profile could be thoroughly wetted with the fertilizer solution.The field soil used, classified as a °C during the day and 20°C during the night with an average relative humidity of 80%. The actual greenhouse daily minimum, maximum, and average temperatures from planting to physiological maturity  of the soil loaded in each tube was approximately 2.5 L (-1) again to free drainage to ensure maximum SWHC was achieved. Elastic bands were used to close the plastic liners at the tops of the tubes to prevent surface evaporation. The tubes were allowed to drain until a constant weight was achieved (tube weight at maximum SWHC). The weights of the dry soil and the tubes were then subtracted from this weight to determine the soil water content at maximum SWHC. Then, the target weight for each tube was calculated as the tube + soil dry weight, plus the water weight at maximum SWHC measured for that tube multiplied by the target fraction of the maximum SWHC .To determine the soil water content and mass of dry soil in each tube, soil samples were taken during the potting process and dried in a forced-air drier at 80ly 2.5 L . Thus, 2× 2 split-plot design (15 × 2 = 30 tubes), with the 15 soybean cultivars as the main-plot factor and the two watering treatments (watered daily to either 100 or 50% of the maximum SWHC) as the sub-plot factor. The experiment consisted of five sequential replicates (replicated in time). The 30 experimental units (tubes) in each replicate were placed on a wooden stand  = [total amount of water added to each tube from planting to harvest + (starting weight – end weight of each tube at harvest) + whole-plant fresh biomass at harvest].Until the R1 (beginning flowering) developmental stage, all tubes were weighed and watered daily to their maximum SWHC. When more than 50% of the plants in a replicate had reached R1, watering treatments were imposed and lasted through the R8 (full maturity) developmental stage. During this period (R1 to R8 stages), tubes were returned to either 100% (control) or 50% (drought stress) of the maximum SWHC by daily weighing and watering. There was no leaching of water from the drainage holes in the bottoms of the tubes at any time during the experiment, so total plant water use (WU) per day was calculated from the water additions at the top of the tubes. The whole-plant WU from planting to harvest was calculated as WU . The volumetric soil water content  was calculated from the TDR millisecond readings using a calibration curve developed for the specific soil mix used in this experiment  millisecond readings were recorded once per week for the duration of the study, from the planting date until the R7  stage. The TDR measurements were performed at five equally spaced points  periment .°C until a constant weight was attained. Then, the total aboveground (shoot) dry matter (SDM) per plant was determined, pods were threshed by hand, seed yield  was recorded, and the total number of seeds per plant was counted. Seeds per pod (SPP) was then calculated as the number of seeds divided by PN. Individual seed weight  was calculated by dividing the SY by the seed number. Harvest index (HI), the fraction of SDM allocated to the seed, was calculated as the SY divided by the SDM for each plant. After harvesting the aboveground plant parts, the soil and the intact root systems within each rooting column were carefully removed by pulling out the plastic liner after laying the tube down on its side. The root samples were washed, placed into labelled paper bags, and then oven-dried in a forced air drier at 80°C until a constant weight was attained  and then the final root dry matter (RDM) of each sample was recorded. Root-to-shoot ratio (R:S) was calculated as the ratio of RDM to SDM. Whole-plant water use efficiency  was calculated by dividing total plant dry matter  by total WU from planting to full maturity.About 5 to 10 days after the full maturity date when 95% of the pods were brown , all plants were cut at soil level and total aboveground plant fresh biomass was recorded. Immediately after harvest, the number of filled pods (pods with seeds) per plant  was counted. All aboveground samples were then dried in a forced air drier at 80× watering treatments), and the random effects of blocks, and block × cultivar interactions. We used the following statistical model:Analysis of variance was performed using the PROC GLIMMIX procedure of SAS Version 9.4 . A Type 1 error rate of 0.05 was used for all statistical tests. Since the dependent variables SY, PN, SPP, SW, SDM, RDM, R:S, TDM, HI, WU, WUE, and DTM were quantitative and continuous, a generalized linear mixed model (GLMM) was fitted with an identity link function and a Gaussian response distribution. The variances of the dependent variables were partitioned into the fixed effects of cultivar and watering treatments, and their interactions  of the jth watering treatment (control or drought stress) in the kth block, µ is the grand mean, ic is the cultivar treatment effect , jw is the watering treatment effect , iwjc is the interaction effect between cultivar and watering treatments, kB is the effect of the kth block (treated as a random effect), iBkc is the main-plot random error (treated as a random effect), and ijkϵ is the residual.where × water, cultivar × depth, water × depth, and cultivar × water × depth), and the random effects of blocks, and block × cultivar interactions. The random interaction term subject×depth was included in the model where the subjects (tubes) were assumed independent (identity covariance structure) and for depth three possible types of covariance structures  were compared. In each case, the most appropriate model was selected based on AICC and BIC fit statistics, no overdispersion based on the generalized Chi-square/df, and assessment of conditional studentized residual plots. Since the spacing interval between VSWC by depth measurements was equally spaced, the Kenward-Roger adjustment for bias correction for the denominator degrees of freedom was applied  error variance were tested by (1) plotting the studentized residuals against factor levels and predicted values; (2) generating a Q-Q plot and scatterplots of the residuals versus fitted values; and (3) performing a formal test of normality using a Shapiro-Wilk. Putative outliers, if any, were detected if the values of the studentized residuals were not within the range of -3.4 to 3.4 (4 to 3.4 .= SY per plant (drought stress)/SY per plant (control). The genetic variation for the various physiological traits was calculated using the cultivar least square means as the [/minimum value × 100]). The relationships among selected yield formation and related traits contributing to drought tolerance were investigated via correlation and regression analyses using the PROC CORR and PROC REG procedures in SAS Version 9.4 . The biplot display of principal component analysis (PCA) was also used to determine the relationship between multiple traits  using the Multivariate procedure of PAST Software Version 3.25 (Drought tolerance of each cultivar was quantified as the drought stress seed yield ratio (SYR), defined as the fraction of a cultivar’s seed yield per plant under control conditions (100% SWHC) that was maintained under drought stress conditions (50% SWHC), i.e., SYR ion 3.25 .Extreme temperatures occurred in the greenhouse during Replication 1 Figure 2p < 0.0001), PN by 51% (p < 0.0001), SPP by 4% (p < 0.01), SDM by 51% (p < 0.0001), RDM by 36% (p < 0.0001), TDM by 48% (p < 0.0001), WU by 51% (p < 0.0001), and DTM by 4 days (p < 0.0001). The drought stress treatment increased single SW by 6% (p < 0.0001), R:S by 35% (p < 0.0001), and WUE by 6% (p < 0.0001), as compared to the control watering treatment (p < 0.001) for every parameter measured. × treatment interaction effect: SY, PN, SDM, and WU. Traits for which there were significant cultivar effects but no interaction  are presented in The cultivars differed significantly /minimum value × 100]). The cultivars did statistically differ for their WU under control watering treatment conditions; Saska had the highest WU, 37.0 L plant-1, while OAC Lakeview had the lowest WU, 28.5 L plant-1, which amounted to a genetic WU variation of 30% .Using cultivar means averaged across watering treatments, genetic variation of 21% was recorded for SPP, 51% for SW, 93% for RDM, 34% for TDM, 92% for R:S, 33% for HI, 24% for WUE, and 14% for DTM Table 4p < 0.05) at several developmental stages,  for the drought stress to control ratios of SY, PN, SW, and WU, but not for the drought stress to control ratios of SPP and SDM. Among the 15 cultivars, two drought-sensitive cultivars  and three drought-tolerant cultivars  were identified. That is, the drought-tolerant cultivars had a significantly higher SYR  than the drought-sensitive cultivars (higher yield loss)  was associated with a smaller effect of drought stress on days to maturity, we regressed SYR on the drought stress to control ratio for DTM. However, there was no relationship between the two drought stress to control ratios , SDM , WU  and TDM  ; the drought stress to control ratio for WU also correlated with the ratio for TDM    and with high SYR   or SYR    had the longest SYR vectors, whereas the drought-sensitive cultivars (Saska and OAC Drayton) had the shortest ones, which were clustered on the opposite side to the drought-tolerant cultivars. Overall, the PCA indicates that the drought-tolerant cultivars were generally those that maintained high WU and SDM and, to a lesser extent, high PN, under drought stress conditions relative to control watering treatment conditions  and to characterize the physiological basis for any such differences in drought tolerance. Specifically, we wanted to see how a high SYR was related to drought effects on yield components , plant WU and dry matter (DM) production.While not producing symptoms of severe stress such as leaf wilting, which are also not common under Ontario field conditions, the drought stress treatment that was imposed through the entire reproductive period  resulted in a significant reduction in SY as well as every other trait that we measured, except for SW and HI. When averaged across the cultivars, the drought stress treatment reduced final plant DM and total plant WU by about 50%. SY was also reduced by about 50%, and almost all of this reduction could be attributed to a single yield component – PN. SPP decreased significantly under drought stress, and SW increased significantly, but these changes were very small compared to the change in PN. Effects of drought on DTM were also statistically significant, but small. These findings are consistent with our previous study and further indicate that PN is the most important yield component driving SY response to drought stress in this culture system , which iOAC Bayfield grown under drought stress (about 16% higher than the control) in a previous study  under drought stress conditions. Similar increases in R:S and WUE under drought stress have been reported previously e.g., . In contus study , while HOverall, this experiment supports the following model of yield reduction due to drought stress: the drought stress treatment reduces whole-plant WU, which produces a nearly proportional decrease in whole-plant DM (since WUE increased only slightly) and in SY, with PN being the yield component most affected. This is consistent with the more general model that PN and thereby SN, primarily determines SY in soybean, with PN being strongly determined by plant growth rates during the “critical period” from about R2 to R5.5 e.g., . Also co× watering treatment interaction effects for SY, PN, SDM, and WU, indicating that the cultivars differed for their response to drought stress for those traits. The significant cultivar × treatment interaction effect for SY suggests that there is genetic variation for drought tolerance among these 15 cultivars. Similar to SY, the cultivars also differed for PN loss and SDM reduction under drought stress conditions. All four traits that showed significant cultivar × water interaction effects also showed a very uniform average 51% reduction due to drought stress, suggesting a quantitative, mechanistic relationship between them. Similar to our findings, × treatment interaction effects in soybean for SY, PN, SDM, and WU, while × treatment interaction effects for SY and PN but not for SDM (they did not measure WU in their work). × watering treatment interaction effect for SY among 10 soybean genotypes at one of their experimental locations.There were significant cultivar Similar genetic variation in susceptibility to yield loss under typical rainfed conditions in Ontario has been reported by Saska and OAC Drayton) and three drought-tolerant cultivars  were identified. We chose SYR a priori as an index of drought tolerance, but it must be recognized that SYR is increased when either the SY under drought stress is higher than expected, or the SY under control conditions is lower than expected. The latter case raises the possibility that cultivars identified as drought-sensitive according to SYR may just actually be unusually productive when water is plentiful. Indeed, both cultivars identified as being particularly drought-sensitive (OAC Drayton and Saska) had much higher than average yields under control conditions, while all three “drought-tolerant” cultivars had average (OAC Lakeview) or below average (OAC Champion and PRO 2715R) yields under control conditions. This may indicate that cultivars with high SYR are systematically those with poor yields under well-watered conditions, or it may just be a coincidence, unique to this particular set of cultivars. The latter possibility is unlikely given that cultivars such as OAC Lakeview and OAC Champion have been grown by Ontario farmers for close to two decades and still perform reasonably well for yield as compared to the more recent, modern ones. However, a broader selection of germplasm should be evaluated to determine if in fact high SYR is generally associated with average or low yield potential. Even if that is the case, a cultivar such as OAC Lakeview would still warrant further investigation, given that i) it produced the highest yield under drought stress while still producing average yield in the absence of drought stress, and ii) its mode of drought tolerance appeared to be unique, with PN being unusually insensitive to drought stress  under drought stress suffered more in terms of the loss to SDM and SY.OAC Champion and PRO 2715R are more closely associated with the SW ratio vector, and OAC Lakeview with the PN ratio vector.The PCA also revealed the expected relationships between changes in the various yield components. Among them, PN was the primary driver and predictor of SY, as compared to SW and SPP, but there was also evidence of the expected compensation between yield components. For example, those cultivars that saw the largest reductions in PN under stress (lower PN ratio) tended to be those that had higher stress:control ratios for SW and especially SPP, as also observed by × watering treatment interaction effect for DTM and the lack of association between SYR and the stress:control ratio for DTM indicate that cultivar differences in SY response to drought stress cannot be attributed to differences in the way drought stress affected maturity .In the current study, the lack of significant cultivar negatively auto-correlated with the stress:control ratio for WU, . Hence, on balance, it appears that the correlation between WUE under stress and SYR is not simply a mathematical artefact. High WUE can arise from reduced transpiration  or from higher photosynthetic response to leaf internal CO2 concentration  and three drought-tolerant cultivars . Drought-tolerant cultivars were those that maintained higher WU, SDM, and PN under drought stress conditions. However, the different cultivars had slightly different modes of drought tolerance in terms of yield components . Our study helps to define the physiological basis of soybean cultivar differences in drought tolerance, and provides direction for soybean breeders wishing to target specific traits that could improve soybean yield when soil water is limiting.In conclusion, drought stress significantly affected every trait we measured, except HI. There exists substantial genetic variation among Ontario-adapted commercial soybean cultivars for their response to drought stress, and there was a broad range of genetic variation for SYR among the cultivars tested. Based on their SYR, we identified two drought-sensitive cultivars  and the Natural Sciences and Engineering Research Council of Canada (Grant number: CRDPJ 513541 – 17).Financial support from the Grain Farmers of Ontario (Project number: GF2 0122) and the Natural Sciences and Engineering Research Council of Canada (Grant number: CRDPJ 513541 – 17) is gratefully acknowledged. The authors are also grateful to Godfrey Chu (University of Guelph), Laxhman Ramsahoi (University of Guelph) and Austin Bruch (University of Guelph) for their technical assistance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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The preservation conditions of fresh osteochondral allografts for clinical applications are critical due their objective: to transplant mature hyaline cartilage containing viable chondrocytes, maintaining their metabolic activity and also preserving the structural and functional characteristics of the extracellular matrix. The aim of the present study was to compare fluorescence confocal microscopy and flow cytometry techniques to evaluate the viability of the chondrocytes present in the osteochondral tissue, in order to determine their effectiveness and thus ensure reproducibility and robustness of the analysis. To this end, osteochondral allografts from human cadaveric donors were preserved at 4 °C for 3 weeks in a preservation medium supplemented with antibiotic and antifungal agents. Cell viability of chondrocytes was determined by monitoring the cartilage for 3 weeks of preservation by confocal fluorescence microscopy and flow cytometry, obtaining cell viabilities of 83.7 ± 2.6% and 55.8 ± 7.8% for week three, respectively. The confocal fluorescence microscopy approach is more advantageous and accurate, as it correlates better with actual cell viability values for monitoring osteochondral graft preservation, detecting only the cells that died a natural death associated with the preservation method. Cell viability is the main parameter to be evaluated when fresh osteochondral grafts are preserved for clinical applications ,2,3. MajThere is currently no systematic test for assessing cell viability in osteochondral grafts, nor studies that compare the use of flow cytometry with traditional confocal fluorescent microscopy. In the present study, femoral condyles were preserved, cell viability was monitored, and the percentage of viable chondrocytes was quantified using semi-quantitative confocal fluorescence microscopy and quantitative flow cytometry techniques. Establishing a reference method to assess viability would allow standard assessment of the quality of osteochondral allografts, facilitating the safe distribution of tissues for transplantation in young patients with large osteochondral defects who currently do not have effective alternative treatment to recover functionality of the affected joints.The aim of our study was to compare these two analytical techniques for quantitative determination of cell viability of cartilage chondrocytes present in osteochondral allografts for clinical use. We hypothesize that flow cytometry analysis will allow, using a simpler technology, a higher degree of system characterization for the detection of live, dead and early apoptotic cells compared with confocal fluorescence microscopy, which is unable to distinguish between the double staining that occurs between live and early apoptotic cells.Human samples were obtained, processed, and analyzed according to current guidelines in relation to the collection and preservation of human tissues for clinical use (EEC regulations 2004/23/CE and 2006/17/CE) and in accordance with the protocol and legal requirements for the use of biological samples and biomedical research in Spain (Law 14/2007 and RD 1716/2011). In addition, the acquisition, processing, and preservation of the tissues used in this study were carried out in accordance with Spanish law on the development and applications of organ transplants (RD 9/2014). All the information provided before donation, together with informed consent, guaranteed that the samples obtained were to be used for clinical applications and/or applied research. The use, protection, communication, and transfer of personal data complied with local regulations (Law 15/1999).®® DyeCycleTM Ruby Stain , 0.9% NaCl , PBS , tobramycin , vancomycin , amphotericin A , cotrimoxazole , pronase , type II collagenase , DMEM , FBS , HEPES , Glutamine , and a penicillin/streptomycin/amphotericin cocktail .The following reagents were used in the study: Ringer’s lactate , calcein AM , 7-AAD , Annexin V , Vybrantwww.bancsang.net (accessed on 27 August 2022). Soft tissues adjacent to the joint area were removed and the bone area of the graft was washed with 0.9% NaCl to remove any remaining blood and fat residues. Condyles were stored for three weeks in physiological medium (Ringer’s lactate) supplemented with an antibiotic cocktail  and anti-fungal agent (amphotericin A).Osteochondral grafts were harvested from deceased donors within 24 h after death and stored at 4 °C. The study used 12 femoral condyles from three human cadaveric donors, obtained after consent to be used for scientific purposes. Femoral condyles were processed in class B cleanrooms under a class A laminar flow cabinet in our classified facilities (Barcelona Tissue Bank (BTB)—Banc de Sang i Teixits (BST); ®® DyeCycleTM Ruby Stain (5 µM for 15 min). The cell fluorescence signal (5 × 105 cells/mL and 5000 events per measurement) was acquired using a BD LSR Fortessa cytometer  using the following lasers and detectors: (i) Blu-ray/violet, 405 nm and 50 mW; (ii) Pacific blue, 488 nm and 50 mW; (iii) FITC, 640 nm and 40 mW; and (iv) Alexa Fluor, 647 nm and 50 mW. The data obtained were analyzed using FACS Diva software (Becton Dickinson).For the flow cytometry analysis, cartilage samples were obtained by sagittal cutting (400 mg) and chopped into small fragments. Pre-digestion was carried out with 1 mg/mL pronase dissolved in culture medium . Cartilage fragments were incubated for 1h at 37 °C with gentle agitation. A second digestion was subsequently performed by adding 3 mg/mL type II collagenase dissolved in complete culture medium , and incubated for 3 h at 37 °C with gentle agitation. After digestion, the cell suspension was filtered through a 70-µm pore membrane filter to remove undigested cartilage fragments. The cell suspension was then centrifuged at 2400 rpm for 5 min and the pellet was resuspended in PBS and divided equally into 6 aliquots. These were incubated at room temperature (RT) in darkness with the following probes: (i) calcein AM (4 µM for 30 min), (ii) 7-AAD (0.5 µg/mL for 10 min), (iii) Annexin V (0.5 µg/mL for 10 min), and (iv) Vybranthttps://imagej.net (accessed on 27 August 2022) through hand-made custom macros that quantified live and dead cells individually. Projection of an image of all z-stack planes together was split into 2 channels, to analyze the calcein and 7-AAD signals, respectively. A threshold value was selected in the histogram to define the value from which the signal would be counted as a particle. The segmentation tool was then applied to separate all sets of cells and differentiate them as a single unit. Finally, the particle analysis tool was applied to count the particles collected in the green and red channels, corresponding to the living and dead cells, respectively.For the confocal fluorescence microscopy assay, fragments of cartilage were obtained by sagittal cutting of the cartilage into 2 × 2 mm pieces. Samples were first incubated with 1 mL of 4 µM calcein AM dissolved in PBS for 30 min at RT in darkness with soft stirring. They were then washed twice with PBS and incubated with 1 mL of 0.5 µg/mL 7-AAD for 10 min at RT in darkness with soft stirring. They were then washed twice with PBS. The cell fluorescence signal was acquired by a Leica TCS SP5 confocal inverted microscope  on a DMI6000 stand, with the Leica Application Suite Advanced Fluorescence software , using a 10×/0.4 NA HC PL APO objective. Calcein images were taken with the 488 nm laser line and emission detection between 495 and 550 nm, taking two averages. Images of the 7-AAD were taken with the 561 nm laser line and emission detection between 574 and 666 nm, taking two averages. The transmitted light channel was used to set the limits of the tissue block in the tile scan area, but not for image acquisition. The channels of 3D stacks were taken sequentially in stack-by-stack acquisition mode. Scanner speed was set at 600 Hz in bidirectional mode. The cartilage fragment was scanned from the surface to the deepest area adjacent to the bone, and cartilage images of the superficial, middle, and deep cartilage area were acquired. The images were bidirectional, captured with a 10× magnification objective, at 1024 × 1024-pixel resolution (speed of 600 Hz) using digital capture software. Images were analyzed using ImageJ Fiji software .Experiments were performed with cartilage from femoral condyles of at least three different donors, and each assay was performed with three technical replicates. The percentage of cell viability is presented as the mean ± standard error of the mean (MD ± SEM) for: number of living cells, dead cells, and apoptotic cells, referring in all cases to the total cell numbers. ANOVA analysis followed by Dunnett’s post hoc test was used for comparisons; Cartilage digested and analyzed by flow cytometry showed cells of different sizes, each incorporating a different amount of calcein AM, as well as small artefacts that also incorporated it. To ensure that the detected calcein signal corresponded to a cell or artefact, cell characterization was carried out by flow cytometry. Calcein AM and 7-AAD were used to assess the percentage of viable and non-viable chondrocytes, respectively, following enzymatic digestion of cartilage .Four cell subpopulations were obtained A provide®® DyeCycleTM Ruby Stain (Ruby), without calcein, shows the DNA concentration, which allows the chondrocyte cell cycle pattern to be described, versus the FSC/SSC pattern, which is used to describe the cell morphology  the inability to discern apoptotic cells, including them as living cells and therefore overestimating counting them as living; and (2) the subjectivity of the post-analysis method used is a function of the hand-made macros used, which produces a deviation between the results found in different studies. The flow cytometry technique is superior in terms of accuracy, since it can distinguish between living, dead and apoptotic cells. However, it has more limitations than confocal fluorescence microscopy. The results obtained using this technique do not accurately reflect the real cell viability values, as it underestimates the number of living cells by counting cells affected by enzymatic digestion of the sample, which are not associated with the natural death due to the cartilage preservation method, as dead or apoptotic. In addition, it provides no information about cell distribution or morphology. It is less advantageous in terms of time and simplicity, since it requires a long protocol for digestion of the cartilage fragments, as well as incubation of the different fluorescence probes. Both stages require a total time of approximately 6 h until cytometry measurements can be performed. In contrast, the confocal fluorescence microscopy technique only requires about 45 min of sample preparation until the images can be acquired.Overall, although the post-analysis method used is complex, the confocal fluorescence microscopy approach is more advantageous and accurate than flow cytometry. It correlates better with real cell viability values because it does not require additional digestion steps that likely alter the osteochondral matrix tissue. Additionally, confocal fluorescence microscopy is methodologically simpler than flow cytometry in terms of sample preparation, despite the complexity of the post-analysis needed. Moreover, flow cytometry approach overestimates the number of dead cells as it is not possible to differentiate between cell death related to preservation method and the procedure itself. Regardless of the limitations of each technique, confocal fluorescence microscopy is an effective technique to evaluate cell viability and to assure sufficient quality of osteochondral grafts for clinical applications.


Background and Objectives: This study aims to describe the earliest renal lesions in patients with von Hippel-Lindau (VHL) disease, especially the multicellular microscopic pathologic events, to get information into the genesis of renal neoplasms in this condition. Materials and Methods: Multicellular events were identified, and 3dimensional reconstruction was performed in grossly normal kidney parenchyma from VHL disease patients by using H&E-stained slides previously prepared. Results: The lesions were measured and the volume of clusters was calculated. Immunohistochemistry was performed for downstream HIF-target protein carbonic anhydrase 9 (CAIX) as well as CD34 for assessment of angiogenesis. We divided lesions into four types according to lesion height/size. The number of lesions was markedly decreased from lesion 1  to lesion 2, then from lesions 2 to 3, and again from lesion 3 to 4. Distribution was highly consistent in the four cases, and the same decrement pattern was seen in all blocks studied. The volumes of clusters were measured and divided into three categories according to their volume. The most frequent pathologic event in VHL kidneys was category 1 , then category 2, and then category 3. Conclusion: We demonstrate that tracking histologic and morphologic changes in 3 dimensions of multicellular microscopic pathologic events enabled us to confirm a protracted sequence of events from smaller to larger cellular amplification events in VHL kidney. Von Hippel-Lindau (VHL) disease was first described in 1926 . The VHLPatients affected by VHL disease develop specific types of heavily vascularized tumors in a highly selective subset of organs. Multiple tumors occur frequently. These include retinal, cerebellar, brainstem and spinal cord hemangioblastoma, microcystic cystadenoma, epididymal cystadenoma, pheochromocytoma and paraganglioma, endolymphatic sac tumor, as well as renal clear cell carcinoma ,12,13,14Renal clear cell carcinoma (RCCC) shares histopathologic features with hemangioblastic central nervous system tumors and epididymal tumors. It is characterized by the proliferation of clear cells with abundant reactive vascularization ,15. MandAlthough clinical, radiological, and pathological data suggested that the precursors of RCCC in patients with VHL disease could be a benign or atypical renal lesion ,21,22, tLubensky et al. showed that 25 of 26 clear-cell renal lesions lost the wild-type allele, and that was the first molecular evidence that LOH of the normal VHL disease gene occurs in benign and atypical clear-cell cysts. This may represent an early event in the development and progression of RCCC in VHL .Characterization of precursor structures has provided more precise insight into the anatomic and cytologic origin of neoplastic processes associated with VHL disease. Precursor structures are far more numerous than frank tumors. VHL inactivation after the “second hit” is necessary but insufficient to develop mass-forming tumors ,23. CompThis study characterizes the multicellular microscopic pathologic events using a novel 3-dimensional algorithm consisting of methodological sampling and histological tracking. We tracked lesions in 3 dimensions to confirm their relationship to each other. This algorithm allows us to observe that microscopic clear cell proliferations follow a predictable pattern covering a morphological spectrum of histologic lesions from an isolated clear cell to potentially tumorigenic clear cell proliferation.Tissue has been obtained from seven kidneys from seven different patients obtained during post-mortem examination and used for this study. All tissues underwent fixation in 10% buffered formalin. Tissue collection included samples from four kidneys from patients with confirmed germline mutations of the VHL gene and established clinical diagnosis of VHL disease (designated as “VHL kidneys”). All VHL patients had additional VHL disease-associated tumors including hemangioblastomas, epididymal cystadenomas, endolymphatic sac tumors, and microcystic adenomas of the pancreas. Three patients were male and one female. The causes of death were pneumonia in two cases, metastatic renal cell carcinoma, and intracerebral hemorrhage.The VHL patients’ ages at the time of demise were between 50 and 65 years (average 58.5 years). Three control kidneys from patients with sporadic renal cell carcinoma, aged between 55 to 70 years (average 63.33 year), were also used for this study designated as “sporadic kidneys”.n = 7). Cuboids were then sliced into 0.3 cm × 1.5 cm × 1.0 cm blocks and a maximum of 30 blocks per cuboid was created. All tissue blocks without grossly visible pathologic change were paraffin embedded. An initial section was taken from each block and stained with hematoxylin and eosin (H&E) for histologic evaluation and identification for 3D reconstruction.All kidneys were sectioned randomly into maximally 10 cm × 1.5 cm × 1.0 cm columnar cuboids. The obtained cuboids of each kidney were inspected, and cuboids showing any grossly visible tumor were rejected. Of the remaining cuboids, the cuboid with the least grossly visible pathologic changes was identified and selected. For each kidney under study, one cuboid was selected  underwent antigen retrieval in a high pH buffer followed by 20 min incubation with the primary mouse monoclonal anti-human CAIX antibody. CD34, a mouse monoclonal from Agilent/Dako IR632, underwent antigen retrieval in low pH, then the antibody was incubated for 15 min, the linker 10 min, and the Flex-hrp for 10 min. HIF is a rabbit polyclonal antibody from Novus Biological (NB100-122). The Antigen retrieval was low pH. The antibody was diluted 1:50 and incubated 30 min. All immunohistochemical studies were developed with DAB for 10 min and counterstained with hematoxylin. All H&E and immunohistochemical slides were digitized and analyzed using ImageScope, version 12.1.0.5029. Vewport 12.1.3. Copyright © Aperio Technologies, Inc. 2003–2013.We recently described an algorithm for tissue procurement, processing, and 3D analysis of tissue blocks of interest . The his1-Chain: multiple adherent clear cells that fill more than 50% of a tubular circumference.2-Cluster: Collection of clear cells expanding and filling a renal tubule.3-Complex cluster: Multiple profiles of intratubular aggregates of clear cells next to each other, with possible growth outside the tubules.The quantification was confined to chains, clusters, and complex clusters. First, we annotated all lesions with the letter C with subsequent numbers (x) in each investigated block . Subsequently, we followed those lesions into the following H&E-stained slides which are 50 microns deeper than the previous slide. If the lesion (C1) still showed in that second slide, the lesion retained the same annotation of (C1), but we annotated that lesion as (C1-2), and if the other lesion (C2) still showed in first, second and third H&E-stained slide, it was annotated as (C2-3) and so on (where the number after the dash designates the number of H&E-stained slides in which the lesion was detectable).Knowing that the distance between H&E-stained slides was 50 microns we could calculate the lesion’s height within the lumen of the renal tubule and divide them as follows:Lesion 1 is equivalent to Cx-1 and is less than 50 microns in height in the tubular lumen.Lesion 2 is equivalent to Cx-2 and is less than 100 microns in the tubular lumen.Lesion 3 is equivalent to Cx-3 and is less than 150 microns in the tubular lumen.Lesion 4 is equivalent to all lesions Cx-4 and exceeds 150 microns in height in the tubular lumen. 2 + r22 + (r1 × r2)], and we did add to the calculated frustum volumes two pyramid volumes, one on the top of the frustum and another on the bottom, with the assumption that all our lesions extend above and down by 25 μm, using the formula of pyramid volume (V = (1/3) × π × h × r2). The lesion 2 volume (Cx-2) = one frustum volume + 2 pyramids volumes. Lesion 3 (Cx-3) volume= 2 frustum volumes + 2 pyramids volumes. We applied the following equations only to cluster lesions, and it represents a measurement of the volumes of lesions in cubic mm. The volume was calculated using a conical frustum as a model. The studied lesions were presumed to have a frustum shape. We measured the lesions by drawing a circle around the center of the lesion in a way that it approximated the area of the lesion. This circle annotation surface area was used as an equivalent to the lesion’s surface area, and then we measured the radius of that circle. Subsequently, we measured the radius of the same lesion in the 50 microns deeper from the previous slide The previous study  showed tAfter inspecting and annotating all the lesions, the total number of lesions visualized and annotated in the H&E-slides from 10 blocks of 4 VHL patients studied were 1194 chains, clusters, and complex clusters. The studied consecutive sections had a distance of 50 micrometer from each other. Those are subsequently subclassified as:Lesion 1 (Cx-1): 958 chains or clusters .Lesion 2 (Cx-2): 154 chains or clusters (lesion present in two consecutive sections only).Lesion 3 (Cx-3): 53 chains or clusters (lesion present in three consecutive sections only).Lesion 4 (Cx-4): 29 chains or clusters (lesion present in four consecutive sections only).The total number of lesions studied in each block of all VHL patients is demonstrated in Regarding the immunohistochemical studies, sixteen slides from seven blocks of four patients with VHL disease were stained with CAIX antibody. CAIX-positive cells were observed in all clear cell changes in all stained slides regardless of the size . 3. We subsequently subcategorized them into three groups from smallest volume to largest volume. In category one, there were 105 lesions (volume range 0.000034–0.00096 mm3). Category two, there were 10 lesions (volume range 0.00131–0.00956 mm3). Category three had four lesions (volume range 0.0102–0.0671 mm3).We calculated the volumes of all clusters examined, which are 119 in total. We found that they range in volume from 0.000034–0.0671 mmOur study demonstrates that careful microscopic examination of grossly normal kidney tissue in VHL patients revealed numerous clear cell chains and clusters in comparison to grossly normal kidney tissue in patients with sporadic clear cell renal cell carcinoma. We investigated the multicellular foci in the grossly normal cortex of VHL kidneys and counted them in 10 blocks of 4 different VHL cases. We identified that the count of these pathological foci was sequentially decreased from lesion 1 to lesion 2, then from lesion 2 to 3, and finally, the lowest was lesion 4, in each case, and each block studied. Distribution was highly consistent in the four cases. The same sequential decrement was noticed in clusters volumes, too: the most frequent category 1 (105 clusters), category 2 (10 clusters), and the least was category 3 (4 clusters).Given that we have cut through  the tested blocks, H&E stained them, and performed a 3dimensional analysis of these structures by which approach we establish certainty that clear cell proliferations do not represent extensions of larger pathologic processes that were invisible on the original section. We primarily analyzed all H&E-stained sections for structures of interest, followed by examining expression of CAIX and CD34.CAIX gene is regulated by the VHL/HIF system via a HIF responsive element in the promoter, and CAIX constitutes an attractive marker for HIF activation because of the inducible response and the stability of the protein [All samples from different multicellular clear cell foci examined were positive for CAIX, and it is well-established to detect activation of the HIF pathway. Expression of the  protein ,25.Mandriota et al.  confirmeOne observation we had in this study after performing CD34 immunohistochemical stain is the proportional increase in the density of vasculature with the increment of the size of clear cell lesions from chains to clusters, to invasive foci. This observation supports the fact that these clear cell foci promote angiogenesis, which allows tumor growth and possible angiogenic invasion with continuous growth.Given the findings in our studies, and since the microscopic clear cell proliferations follow a predictable pattern, we hypothesize that the renal neoplastic pathological events  evolve from a variety of microscopic precursors .Mubarak et al. showed that the most frequently observed pathologic events in VHL kidney sections were either isolated clear cells within a renal tubule or two or more clear cells adherent to each other, likely representing changes secondary to an amplification event. More significant accumulations of clear cells along the tubular lining, as well as clustering of clear cells within renal tubules, appeared to be in the continuity of smaller preceding events .Clear cell changes in renal tubules are not always neoplastic and can be seen secondary to metabolic or toxic etiologies  ,29,30. IIn other studies in patients with VHL disease, it has been shown that careful microscopic examination of grossly normal tissue of the spinal cord and epididymis revealed precursor structures that are numerically more frequent than tumors ,32. SpinShively et al. showed that in surgically resected nervous system hemangioblastomas of patients with VHL disease, HIF-1 alpha activation was associated with epithelioid structures, which were mainly seen in larger tumors. However, small tumors were composed mostly of poorly differentiated mesenchymal structures and did not show HIF-1 alpha activation. This suggests that the growth of nervous system tumors in VHL disease has a sequence of structural and molecular events .The fact that the microscopic renal cystic and solid neoplasms containing only clear cell cytological features were found in patients with VHL disease  gave us Of particular interest in VHL disease has been the relationship between cysts and tumors. Additional studies may better clarify whether cysts and tumors develop via independent mechanisms or whether they are merely variations of an essentially uniform pathogenetic process. While consequences of dysregulation of VHL and HIF pathways provide numerous potential clues , our appOur morphological data showed different types of lesions which represent intriguing candidates for progression into cystic lesions and tumors in the kidney. The genetic and epigenetic mechanisms for these progressive events remain to be clarified. Walther et al. investigated normal-appearing renal tissues from VHL patients . They deWhile previous studies by Mubarak et al. had been concerned with lesion quantification only, this study focuses on the larger simple and complex clear cell clusters, with more detailed information on their volumetric extent. Here we were able to not only show inverse correlation between lesion complexity and frequency, but—through 3D approach—between lesion size and frequency which we believe to represent the histomorphologic equivalent of linear “evolutionary tumorigenesis”, recently postulated for VHL disease-associated clear cell carcinoma after whole genome analysis of a series of renal carcinomas .Our findings demonstrate more than 1000 multicellular amplification events in less than one cubic cm of “normal” kidney tissue, the largest one measuring less than one thousandth of a cubic mm, undetectable by naked eye or radiologically. In the patients under study, VHL disease in human kidney therefore presents as wide-spread substitution of tubular cells with clear cells, only a small subset of which will undergo amplification into the next level of complexity. As we show here, this principle of protraction directly applies to lesion size.Previously, this principle of protraction has been radiologically documented in far more than 1000 times larger, radiographically detectable lesions . In conjOur findings may encourage diagnostic and academic pathologists to not only pay attention to primary tumor pathology in resection specimens, but also to adjacent normal kidney tissue; the quantity of our described precursor structures may give information on the “activity” of VHL pathology in these tissues, and may have prognostic implications for the likelihood of new tumor formation. Of particular current therapeutic interest is molecular targeting through immunotherapy , and resFinally, microdissection of precursor structures combined with targeted genetic analysis may give insight into earliest genetic changes leading to progression of precursors from single cell to complex cluster; 3D reconstruction could also be applied to serially sectioned frozen tissue blocks of normal VHL kidney allowing for mass spectroscopic analysis for earliest proteomic change after microdissection, and applicability of proteomics for diagnosis of primary VHL tumors has been previously demonstrated .The main limitation of our study is the small number of cases tested. However, this study has examined sequential levels cut at 50 microns . Each slide was digitized and annotated accordingly. Each annotation was followed in each cross -section to accurately assess the frequency, sizes, and volumes of these clear cell chains and clusters occurring in the grossly normal kidneys of patients with VHL. Similar studies in the literature are lacking.The study of morphological changes and histogenesis in VHL kidneys leads to a better understanding of the sequence of tumorigenesis. Additionally, this study has shown us the prevalence of these clear cell changes and sublocalization within the kidney parenchyma to be in close proximity to a certain anatomic/histologic structure in the kidney, which is leading us to a conclusion regarding the cell of origin in clear cell renal cell carcinoma . Further


Bacteroides and an enrichment of opportunistic pathogenic species of the ESKAPE group, starting from the second week. Besides revealing the intestinal microbiota of Tunisian newborns, this study provides novel insights into the microbiota perturbations caused by ECS.Microbiota colonization is a dynamic process that impacts the health status during an individual's lifetime. The composition of the gut microbiota of newborns is conditioned by multiple factors, including the delivery mode (DM). Nonetheless, the DM's influence remains uncertain and is still the subject of debate. In this context, the medical indication and the emergency of a cesarean delivery might have led to confounding conclusions regarding the composition and diversity of the neonatal microbiome. Herein, we used high-resolution shotgun sequencing to decipher the composition and dynamics of the gut microbiota composition of Tunisian newborns. Stool samples were collected from 5 elective cesarean section (ECS) and 5 vaginally delivered (VD) newborns at the following time points: Day 0, Day 15, and Day 30. The ECS and VD newborns showed the same level of bacterial richness and diversity. In addition, our data pointed to a shift in microbiota community composition during the first 2 weeks, regardless of the DM. Both ECS and VD showed a profile dominated by Proteobacteria, Actinobacteria, and Firmicutes. However, ECS showed an underrepresentation of  First, ECS is undertaken without rupture of the fetal membranes, and newborns are delivered without passing through the birth canal. They are thereby not exposed to the maternal vaginal flora , and did not receive any antibiotic treatment. Only newborns delivered by elective and unlabored cesarean were included in this study. No complications during the delivery were reported. Anonymized mother's health records with their corresponding metadata were compiled. Clinical metadata concerning the newborns were also collected including birth weight, gestational age, gender, weight, and feeding mode .A total of 10 participants were retained, and newborns' stool samples were collected at three time points. The neonatal meconium from VD and ECS newborns was collected during the first 24 h (Day 0) at the regional hospital of Zaghouan and at the Aziza Othmana hospital, located in Zaghouan and Tunis, respectively. The collection was renewed for 2 weeks (Day 15) and 1 month (Day 30) after birth. The samples were transported in ice bags to the laboratory and then stored at −80°C until DNA extraction.Subsamples from each newborn stool (~200 mg) were used for DNA extraction. The solubilization and liquefaction of the samples were carried out using 1 ml of Tween 20 (10%). The solution was then vortexed horizontally for 20 min and centrifuged at 14,000 rpm for 10 min at 4°C. The obtained pellets were resuspended in a lysis buffer  for chemical and enzymatic lysis. Then, bead-beating steps were proceeded using BeadBug and PowerBead Tubes, Garnet 0.70 mm (QIAGEN), with a setting of 3 × 20 s pulses, followed by incubation for 7 min at 95°C. After a centrifugation step, the supernatant was transferred into 2 ml tubes containing 30 μl proteinase K and 400 μl AL buffer (QIAamp Fast DNA Stool Mini Kit) and incubated at 70°C for 10 min. DNA was recovered using phenol-chloroform extraction and purified using the QIAamp DNA mini kit (QIAGEN). In brief, protein precipitation was performed using the phenol/chloroform method, followed by DNA precipitation using absolute ethanol. The supernatant was transferred to the QIAamp spin column, and DNA purification was performed according to the QIAamp Fast DNA Stool Mini Kit manufacturer's instructions. DNA samples were quantified using the Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher™), and samples with >100 ng/μl DNA material proceeded to the paired-end (2 × 150 bp) metagenomic sequencing. Overall, we sequenced 33 samples: 9 participants at 3 time points, 1 participant at 2 time points (Day 0 and Day 30), and 4 technical replicates .Quality control and preprocessing were undertaken on the raw data as follows: Trimmomatic was run to trim low-quality bases and short reads   as well as viruses (212 OTUs) and Archaea (103 OTUs). The statistical analysis started with filtering ambiguous and rare bacterial taxa . First, Alpha diversity metrics (Shannon and Simpson indices and OTU richness) were analyzed using the microbiome R package (McMurdie and Holmes 2013).https://github.com/vmikk/metagMisc). The Friedman test was used to compare differences in alpha diversity between collection days (paired samples), followed by the Nemenyi post-hoc test using the PMCMRplus R package (available at https://cran.r-project.org/web/packages/PMCMRplus/index.html). When comparing alpha diversity between two unpaired groups, the non-parametric Wilcoxon was employed. Kruskal–Wallis test was used to assess statistical differences between more than two groups, followed by post-hoc Dunn's test (with Bonferroni correction). The clustering and heatmap were performed using the microbiomeutilities R package  using the function “phyloseq_phylo_div” from the R package PhyloMeasures   were delivered at a mean gestational age of 38.5 weeks (SD ± 1.22). Samples were collected on the birthday (Day 0), Day 15, and Day 30 after birth. The characteristics of the mothers and newborns are listed in p = 0.001, R2 = 0.210). Although not impacting the diversity, feeding mode was the second most significant factor explaining variation in neonatal gut microbiota . This suggests that the functions potentially brought by bacteria at Day 0 are distinct from those ensured at Day 15 and Day 30. At the phylum level, a pattern of increasing abundance composed of Firmicutes, Proteobacteria, and Actinobacteria is observed  .To address the impact of DM on newborns' gut microbiota, we carried out various taxonomic and functional diversity analyzes. Alpha and beta-diversity showed no significant difference neither in terms of diversity nor in bacterial community structure between ECS and VD groups (= 0.034) . SimilarActinomyces, Pseudopropionibacterium, and Citrobacter genera were overrepresented compared with the VD group. Interestingly, the microbiota of ECS newborns was enriched in the genus Clostridium. Among these genera, the Clostridium perfringens, an opportunistic pathogenic species, was detected as overrepresented. In addition, the ECS showed an overrepresentation of Enterobacter species, a highly virulent and antibiotic-resistant member of the ESKAPE bacterial group.Although the ECS and VD microbiota shares a similar community structure, marginal taxonomic differences do exist. To further characterize these differences, LEfSe analysis was performed to detect specific differentially abundant features at various taxonomic ranks in ECS and VD . In the Bacteroides genus , and Parabacteroides were identified as overrepresented in the VD group  and VD  in newborns delivered by cesarean . Financial support was also afforded by the foundation Mérieux. We would like to acknowledge the European project PHINDaccess: Strengthening Omics data analysis capacities in pathogen-host interaction (Grant Agreement ID: 811034).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Previous studies have demonstrated that exposed to the initial suboptimal intrauterine environment of gestational diabetes mellitus (GDM) may increase risk of cardiovascular disease in adulthood.In order to investigate the underlying mechanisms involved in the increased risk of cardiovascular diseases (CVDs) in the offspring of GDM, we applied a high-throughput proteomics approach to compare the proteomic expression profile of human umbilical vessels of normal and GDM offspring.A total of significantly different 100 proteins were identified in umbilical vessels from GDM group compared with normal controls, among which 31 proteins were up-regulated, while 69 proteins were down-regulated. Differentially expressed proteins (DEPs) are validated using Western blotting analysis. The analysis of these differently expressed proteins (DEPs) related diseases and functions results, performed by Ingenuity Pathway Analysis (IPA) software. Based on “Diseases and Disorders” analysis, 17 proteins  were described to be associated with CVD, especially in Anemia, Thrombus and Myocardial infarction. Functional analysis indicated that DEPs involved in many cardiovascular functions, especially in “vasoconstriction of blood vessel” . Upstream regulator analyses of DEPs identifies STAT3 as inhibitor of ACTA2, FGA, FGB, and FGG.The results of this study indicate that intrauterine hyperglycemia is associated with an elevated risk of cardiovascular risk in the offspring. A growing number of epidemiological and experimental studies suggest that exposure to adverse intrauterine environment during fetal development can be associated with chronic disease in later life, such as CVD, obesity, type 2 diabetes and cognitive disorder –6. The tEmerging evidence suggests that the vasculature of women with a prior case of GDM is permanently altered, predisposing them to CVD. GDM also increases the offspring’s risk of developing hypertension and CVD. Elevated systolic blood pressure (SBP) and diastolic blood pressure (DBP) , 8–11, iIn the present study, we aimed to investigate the cardiovascular risk proteins in offspring exposed to GDM. A proteomics analysis was conducted in umbilical vessels from newborns of mothers with GDM and normal controls using the isobaric tag for relative and absolute quantitation (iTRAQ)-labeling technique to compare the proteomic expression profile. We analyzed the related diseases and functions using ingenuity pathway analysis (IPA) software. The results of the present study may provide valuable information for further investigation of the mechanisms underlying the cardiovascular dysfunction induced by intrauterine hyperglycemia.°Cuntil use. The clinical characteristics of the proteomic participants included in this study are outlined in Umbilical cords from newborns of 25 mothers with mild GDM and 25 controls were collected by obstetricians in Shaoxing Maternity and Child Health Care Hospital, China. The Ethics Committee of Shaoxing Maternity and Child Health Care Hospital approved the study. All the participants enrolled in this study were with singleton pregnancy and ceased pregnancies with Cesarean section at full term. Tissue samples were stored snap frozen at −80GDM were diagnosed between gestational weeks 24 and 28 after overnight fasting (for 8–12 h) by an oral glucose tolerance test (OGTT). According to the IADPSG diagnostic criteria, GDM was defined as fasting venous plasma glucose concentration ≥ 5.1 mM and ≥ 10.0 mM at 1-hand/or ≥ 8.5 mM at 2-h after drinking a solution with 75 g glucose. All women with GDM enrolled in the present study experienced dietary management without insulin treatment. Participants with maternal obesity factor  and macrosomia were excluded. The work described in the present study has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). The study protocols were reviewed and approved by the Research and Ethics Committee of the Women’s Hospital, School of Medicine, Zhejiang University, China, and informed consents were provided by all participants.°C for 5 min. After each sample was cooled to room temperature, the sample was loaded onto an ultrafiltration filter . We added 200 μL UT buffer  to the filter and centrifuged it at 14,000 g at 20°C for 30 min. Subsequently, 100 μL of iodoacetamide solution (50 mM iodoacetamide in UT buffer) was added for blocking reduced cysteines, and, the samples were incubated for 20 min in darkness. Then the filters were centrifuged at 14,000 g at 20°C for 20 min. The filters were washed with 100 μL UT buffer at 14,000 g for 20 min. This step was repeated 2 times. Then, 100 μL dissolution buffer  was added to the filter, and it was centrifuged at 14,000 g at 20°C for 30 min, and, this step was repeated twice. Finally, 40 μL of trypsin  buffer (2 μg trypsin in 40 μL dissolution buffer) were added, and, the samples were digested overnight at 37°C. Each filter unit was transferred to a new tube and centrifuged at 14,000 g at 20°C for 30 min. The concentration of resulting peptides was determined by UV light spectral density at OD280  labeling kit . Three umbilical arteries from the control group (C) were labeled with mass 114, 115 and 116 isobaric iTRAQ tags, the other three umbilical arteries from the GDM group were labeled with mass 117, 118, and 119 isobaric iTRAQ tags. Identical quantities of peptide mixtures from the 6 peptides mentioned above were labeled with reagent 113 and served as sample IS . According to the manufacturers protocol, The labeling reactions were incubated for 2 h at room temperature before further analysis.http://www.uniprot.org/) and the concatenated target-decoy database. The parameters were set as follows: Trypsin as digestion enzyme, cysteine carbamidomethylation as a fixed modification, Oxidation (M), Gln→Pyro-Glu (N-term Q), iTRAQ 8 plex (K), iTRAQ 8 plex (Y), and iTRAQ 8 plex (N-term) as the variable modification.After iTRAQ-labeling the peptide samples were combined and subsequently purified using a strong cation exchange (SCX)-cartridge: Polysulfoethyl 4.6 × 100 mm column . For LC-MS/MS analysis of the resulting peptides, we followed a previously described method , ACTA2 , IDH3A , GAPDH . Subsequently, membranes were washed 10 min for three times with TBST, then each membrane was incubated for 1 h at room temperature with the appropriate secondary antibody . The protein intensities were determined and analyzed using Odyssey® Imager .Human umbilical arteries were lysed in 1 × RIPA buffer with 1μg/mL leupeptin and 1μg/mL phenylmethylsulfonyl fluoride. Aliquots containing 30 μg of protein samples were separated by 12% SDS-PAGE and transferred electrophoretically to a nitrocellulose transfer membrane . After blocked with 5% BSA in TBS containing 0.01% Tween 20 (TBST) for 1 h at room temperature, the membrane was incubated overnight at 41 and Java Tree view software2 were further used to evaluate the capability of the resulting feature proteins in differentiating the two groups of samples. IPA software  was employed for functional analysis of the identified DEPs between the two groups. In the functional network analysis, the DEPs were represented as nodes, and the biological relationship between two proteins was represented as an edge (line), which was supported by the published articles or the canonical information embedded in the IPA database. Downstream biological processes analysis, which was used to predict the downstream effects of the DEPs based on the observed gene expression changes, was also on the strength of the canonical information embedded in the IPA database. The calculated z-score can be used to infer the activation states (“increased” or “decreased”) of implicated biological processes. Fisher’s exact test was used to calculate a p-value to determine the probability that the association between proteins in the dataset, and the biological process could be explained by chance alone.Proteins exhibiting at least a ± 20% fold change in expression were determined as significantly different. The Cluster 3.0t-test, and P < 0.05 was considered statistically significant.All analysis was performed with the software Statistical Analysis Software . Data are displayed as means ± SEM. Statistical evaluation was conducted using an unpaired Student’s P > 0.05). The serum glucose concentration at 0,1, and 2 h of OGTT in the GDM group were significantly higher than that in the normal pregnancies . Three pairs of the collected umbilical vessels were selected to compare the proteome profiles between GDM and controls by iTRAQ-LC-MS/MS.Twenty five pregnancies with GDM and 25 normal controls were enrolled in the present study, and the specific clinical characteristics of the participants and delivery data were shown in http://proteomecentral.proteomexchange.org) via the iProX partner repository  in the umbilical arteries between GDM and controls in genome-wide level, iTRAQ-LC-MS/MS was performed in 3 GDM samples and 3 control samples. In the present study, a total of 1,653 proteins were quantified in the umbilical arteries from both GDM and the controls . The maspository  with thepository . Hierarcpository , indicatTo validate the results carried out by iTRAQ-LC-MS/MS, Western blotting was conducted in additional three umbilical vessels from GDM and three normal umbilical vessels. Based on the significant expression difference and the biological function in the cardiovascular system, three proteins, including FGA, ACTA2, and IDH3A, were selected for further investigation. As shown in ®, QIAGEN)3 for pathway, disease and function, and network analysis. Three cardiovascular signaling pathways identified by IPA software  were described to be associated with CVD, especially in Anemia, Thrombus and Myocardial infarction . Based oThe term “upstream regulator” as used in IPA refers to any molecule that can affect the expression of another molecule. In upstream regulator analysis, STAT3 was predicted in inhibited state (z-score = −2.196) and regulated ACTA2, FBN1, FGA, FGB, and FGG .Cardiovascular and metabolic disorders often present in adult life, but may have their origins in changes to the intrauterine environment during fetal development. Previous studies including epidemiological investigations and experimental projects have demonstrated the alteration of vascular function and cardiovascular system in the offspring of GDM , 24. TheSeveral studies implicated that the association between maternal hyperglycemia and childhood metabolic outcomes was attenuated after adjusting for covariates including maternal BMI and weight gain during pregnancy , overallThe physiological and pathological regulation of cardiovascular function derives mainly from the collaboration between vascular endothelial cells and VSMCs (Vascular smooth muscle cells). Previous studies stated that characteristics and alterations of umbilical vessels could offer valuable information since umbilical vessel cells experienced the effect of same intrauterine environment , 25–27. Based on the identified DEPs in umbilical arteries between GDM and normal controls, our results suggested that the “Cardiovascular System Development and Function” was impaired exposure to intrauterine hyperglycemia. In this content, the top related function was “Vasoconstriction of blood vessel,” this is consistent with a large number of epidemiological results. These suppressed physiological processes in the umbilical vessels from GDM might lead to impaired vascular repair under stress or diabetic vessel pathological damage.In this study, we found 20 DEPs  related with Cardiovascular system. ACTA2 gene mutations in adults associated with thoracic aortic aneurysm and dissection (TAAD) . TransgeIn summary, in the present study, we discovered that the expression profile of proteins in umbilical vessels of newborns from GDM group was different from that in normal controls. The bio-informatics analysis suggested that some DEPs might play important roles in cardiovascular dysfunction of GDM children. Our findings would contribute to the exploration of the potential mechanism underlying the dysregulated balance of apoptosis and autophagy in vessels, angiogenesis and endothelial cell dysfunction in the offspring of GDM pregnancies. However, deeper analyses will still mostly need to be performed to explore the key factors and potential preventive and therapeutic strategies of cardiovascular dysfunction in GDM offspring.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of Shaoxing Maternity and Child Health Care Hospital. The patients/participants provided their written informed consent to participate in this study.H-TP and Y-MX: writing—original draft. H-DZ: methodology. X-LS: formal analysis. BY: visualization. H-GD: validation. R-JX and J-LD: data curation. JZ and TZ: project administration. All authors contributed to the article and approved the submitted version.


Fat storage-inducing transmembrane proteins (FITMs) were initially identified in 2007 as members of a conserved endoplasmic reticulum (ER) resident transmembrane protein gene family, and were found to be involved in lipid droplet (LD) formation. Recently, several studies have further demonstrated that the ability of FITMs to directly bind to triglyceride and diacylglycerol, and the diphosphatase activity of hydrolyzing fatty acyl-CoA, might enable FITMs to maintain the formation of lipid droplets, engage in lipid metabolism, and protect against cellular stress. Based on the distribution of FITMs in tissues and their important roles in lipid droplet biology and lipid metabolism, it was discovered that FITMs were closely related to muscle development, adipocyte differentiation, and energy metabolism. Accordingly, the abnormal expression of FITMs was not only associated with type 2 diabetes and lipodystrophy, but also with cardiac disease and several types of cancer. This study reviews the structure, distribution, expression regulation, and functionality of FITMs and their potential relationships with various metabolic diseases, hoping to provide inspiration for fruitful research directions and applications of FITM proteins. Moreover, this review will provide an important theoretical basis for the application of FITMs in the diagnosis and treatment of related diseases. Nicotiana benthamiana or suspension cells of Nicotiana tabacum, Cai et al. found that FITM2 was specifically localized in the ER and was often concentrated at ER-LD junction sites , were first identified by Kadereit and colleagues in late 2007, and are considered as two key genes involved in lipid droplet (LD) formation [/2, FITMson sites . FITM1 aon sites , 5. Concon sites . This reSaccharomyces cerevisiae [Caenorhabditis elegans. The sequences of FITM family orthologs in non-vertebrates exhibited a lower homology than those of vertebrates , which are also known as FITM1 and FITM2 (fat storage-inducing transmembrane proteins), confirming that they can induce the accumulation of lipid droplets [FITM1 gene is located at 14q12, while the mouse Fitm1 gene is located at 14C3. Meanwhile, they have similar gene structures, consisting of two exons and one intron, and encoding proteins of 292 amino acids. The human FITM2 gene is located at 20q13.12, while the mouse Fitm2 gene is located at 2H3. Both encode proteins of 262 amino acids. However, their genetic structures differ significantly as the human FITM2 gene comprises two exons and one intron, whereas the mouse Fitm2 gene consists of four exons, with the smallest fourth exon being only 7 bp in length. Concerning the spatial structures of these proteins, Gross et al. found that FITM proteins had six transmembrane domains, with both N- and C-termini localized to the cytosol. This is based on the predicted topological models for the FITM family using N-glycosylation site mapping and indirect immunofluorescence techniques [Both FITM1 and FITM2 genes encode proteins of over 200 amino acids and have 50% similarity to each other in terms of amino acid sequences. FITM1 and FITM2 do not have homology to known proteins or protein domains found in any species; thus, FITM genes comprise a unique gene family [chniques . FurtherThe distribution of FITM1 and FITM2 is significantly different in a variety of tissues. Mouse FITM1 mRNA was highly expressed in heart and skeletal muscle, but at lower levels in the liver, kidney, and testes, while FITM1 proteins were detected primarily in skeletal muscle, with lower levels in the heart [−/− mice further supported that the FITM family might represent the direct target genes of PPARα [In primary human skeletal muscle cells, the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) was able to stimulate the expression of the mRNA and protein levels of FITM1, and enhanced the formation of smaller lipid droplets, with only a modest increase in the triacylglycerol (TAG) content in oleate-incubated skeletal muscle cells . Furtherof PPARα . The aboIn addition, Fornes et al. found that mice injected with dihydrotestosterone, following 4–10 weeks of a high-glycemic diet, gained more liver weight on GD18.5 and had higher FITM1 mRNA expression compared with normal female mice , providiSeveral studies have shown that FITMs are a class of endoplasmic reticulum proteins that participate in the formation and assembly of lipid droplets. When Kadereit et al. first identified the functions of the FITM family, they applied radiolabeled glycerol as a precursor for triglyceride (TG) to label newly synthesized TG, finding that human HEK293 cells expressing FITM1 or FITM2 contained similar amounts of labeled synthesized TG at all indicated time points (before 6 h) in comparison with mock-transfected cells. Subsequently, HEK293 cells were transfected with FITM1 or FITM2 and treated for 3 h with radiolabeled glycerol, after which lipid droplets were isolated and radioactively TG-quantified. The results showed that the quantity of labeled, newly synthesized TG in lipid droplets was four- to sixfold higher than that in the control cells, suggesting that FITM1 and FITM2 did not affect TGs biosynthesis, but rather partitioned TGs into lipid droplets . AdditioLipid droplets (LDs) are organelles that store lipids consisting of a neutral lipid core (triacylglycerols and sterol esters) , where tWith further understanding of the mechanisms behind lipid droplet budding from the ER, FITM proteins are revealed to be essential for stimulating the budding of nascent LDs from the ER. The elimination or reduction of SCS3 and YFT2 negated the capacity of LDs to bud from the ER; thus, LDs remained within the ER membrane , 27. A rBriefly, FITMs might maintain normal LD formation primarily through two major steps of de novo LD biosynthesis including the nucleation step and the budding step Fig. . ClearlyArabidopsis thaliana, whereas enhanced the oil content by 13% in some FITM2-expressing Arabidopsis thaliana seeds [Since FITMs are intimately associated with the formation of LDs, which always play central roles in lipid and energy metabolism, FITMs may be involved in lipid metabolism. Therefore, numerous studies have also examined whether FITMs could be involved in the biosynthesis of TGs. For example, Kadereit et al. initially discovered that the knockdown of FITM2 expression in 3T3-L1 adipocytes generated a dramatic reduction in lipid droplets and a significant decrease in the total cellular TGs. Thus, they proposed that an inhibition in the ability to generate lipid droplets caused a partial feedback inhibition in TGs biosynthesis . In contna seeds . Moreovena seeds . Thus, iWith regard to the correlation between FITMs and phospholipids metabolism, Hosaka et al. first speculated that SCS3 was involved in regulating inositol synthesis, but they did not quantify levels of inositol phospholipids in SCS3 mutants or provide direct evidence that SCS3 regulated this pathway . HoweverThe distinct distribution of FITMs within tissues and their capacity to bind TGs raised the notion that FITM1 is vital for the generation of small LDs involved in the rapid mobilization of TAGs during increased ATP demand . By contProper ER functionality is essential for cells; however, various conditions can interfere with its operation, leading to a build up of proteins, referred to as ER stress . Unfolde2O2, amphotericin B, and azole [Concerning the potential relationship between FITMs and ER morphology, Becuwe et al. revealed that the absence of FITM2-mediated hydrolysis of acyl-CoAs in FITM2-knockout cells led to excess luminal acyl-CoA, which could disrupt ER membranes, possibly by acting as detergents to destabilize membranes or by altering phospholipid balance, indicating that the enzymatic activity of FITM2 could indirectly influence ER morphology and stress . Anothernd azole , which iIn summary, it is certain that FITM2 deletion results in abnormal lipid metabolism and equally leads to ER stress. Additionally, Han et al. found that ER stress induced the higher expression of lipid-related genes leading to abnormal lipid metabolism . As a reCaenorhabditis elegans, FITM2 deficiency led to a significant reduction in intestinal lipid droplets and dramatic defects in muscle development, leading to the death of almost all homozygous FITM2 animals as larvae [The process of lipid droplet formation often exerts an important role in both cellular physiology and disease. Given that FITMs play a vital role in lipid droplet formation, as well as lipid metabolism, downregulated FITMs might generate lipid homeostasis-associated disorders or diseases. In s larvae . Similars larvae . Consequs larvae . Converss larvae , 44. Rats larvae . The oves larvae . The obtAfter sequencing and exploring a consanguineous family from Pakistan, who were ascertained to have a novel deafness–dystonia syndrome with motor regression, ichthyosis-like features, and signs of sensory neuropathy, Seco et al. confirmed that the patient’s symptoms were caused by a mutation in the FITM2 gene. This provided the first independent evidence for the involvement of FITM2 in human disease . SubsequFITMs are typically highly expressed in heart muscle; thus, it is not surprising that the dysfunction of FITMs can lead to the occurrence of cardiac diseases. It was clear that the lipid content of myocardiocytes was increased in failing hearts , and theCurrently, the roles of the FITM family in cancer pathogenesis have received increased attention. Interferon-γ (IFN-γ) resistance is usually considered to be an intrinsic conserved cancer mechanism of cytotoxic T lymphocytes (CTLs) immune evasion for cancer cell. Lawson et al. found that FITM2 was required for maintaining the fitness of several types of cancer cells following exposure to CTL-produced IFN-γ . AdditioTaken together, this study reviews the structure, distribution, expression regulation, and main functions of FITM proteins and their association with a variety of metabolic dysregulations or diseases. FITMs are initially observed to be involved in lipid droplet formation. However, subsequently, their functions have been found to go far beyond the partitioning of TG into LDs. Current studies have demonstrated that FITMs not only affect lipid metabolism homeostasis, but also protect against cellular stress, even though the cause–effect relationships between FITM2, abnormal lipid metabolism, and ER stress remain uncertain. Accordingly, FITMs proteins are found to be involved in various metabolic diseases, including type 2 diabetes, lipodystrophy, cardiac diseases, and several types of cancers. Nevertheless, the mechanisms underlying the effects of FITMs in various diseases are not well understood. More studies on FITMs may provide new concepts and strategies for treating these diseases in the future. The liver is an important organ involved in lipid metabolism. However, there are few studies on the relationships between the FITM family and liver diseases including NAFLD, nonalcoholic steatohepatitis (NASH), liver cirrhosis, and HCC. In the past, NAFLD research has limited its focus on TG accumulation in hepatocytes . In fact