The life of every organism is dependent on the fine-tuned mechanisms of protein synthesis and breakdown. The degradation of most intracellular proteins is performed by the ubiquitin proteasome system (UPS). Proteasomes are central elements of the UPS and represent large multisubunit protein complexes directly responsible for the protein degradation. Accumulating data indicate that there is an intriguing diversity of cellular proteasomes. Different proteasome forms, containing different subunits and attached regulators have been described. In addition, proteasomes specific for a particular tissue were identified. Cancer cells are highly dependent on the proper functioning of the UPS in general, and proteasomes in particular. At the same time, the information regarding the role of different proteasome forms in cancer is limited. This review describes the functional and structural heterogeneity of proteasomes, their association with cancer as well as several established and novel proteasome-directed therapeutic strategies. The proteasomes degrade the majority of intracellular proteins and represent a crucial element of the ubiquitin-proteasome system (UPS), which is involved in almost all cellular metabolic processes , 2. Flex1−7β1−7β1−7α1−7 arrangement , is a 150 Å to 115 Å, 700 kDa complex, assembled from 28 proteins arranged in four heptameric rings . The rinangement  19, 34), 341−7β1angement . The thrIt was assumed that the closed conformation of the alpha-subunit gates causes the majority of 20S proteasomes to be latent and incapable of degrading proteins without being activated by activators (described below). However, it was demonstrated that 25% of yeast 20S proteasomes have open gates , 37. Mor“Free” 20S complexes (proteasomes with no attached activators) is the most abundant form of proteasome, comprising 47–74% of all cellular proteasomes  5, 11–1–111–13; PSMB6), β2 (Z) (PSMB7), and β5 (X) (PSMB5) catalytic subunits, which mediate cleavage after acidic (β1), basic (β2), or hydrophobic (β5) amino acids, thus demonstrating caspase-like, trypsin-like and chymotrypsin-like activities, respectively  20S proteasome (cPs) contains the β1(Y)  contain the so-called immune catalytic subunits: β1i (LMP2), β2i (MECL-1), and β5i (LMP7) in place of β1, β2, and β5, correspondingly –69. The “immuno” –66, 70. “immuno” . Neverth“immuno” , type I “immuno” , 75, nit“immuno” , and gly“immuno” . Due to “immuno” , 78, 79.“immuno” . However“immuno” . Immunop“immuno” , 82–84. “immuno” , 42, 85.“immuno” , visual “immuno” , in the “immuno” , muscle “immuno” , and the“immuno” , 91. Fur“immuno” . Compare“immuno” . Convers“immuno” , 22–24. “immuno”  and high“immuno” –97; thus“immuno” .The overexpression of immunoproteasome genes is frequently observed in different cancer types and indicates a better prognosis for breast cancer patients , 100. Th26−35 epitope in tumor cells. It was shown that it is impaired in melanoma and HeLa cells following the upregulation of immunoproteasome subunits . This reduces the efficacy of epitope presentation and can facilitate tumor escape from immune pressure  intPs have β1/β2/β5i architecture. These proteasomes have slightly different activity than constitutive proteasomes or iPs  intPs contain β1i/β2/β5i catalytic subunits  (PSMB11 gene expression is regulated by the transcription factor Foxn1 (−/− mice have decreased levels of CD8+ single positive lymphocytes (The unique form of 20S proteasomes known as thymoproteasomes (tPs) is present in cortical thymic epithelial cells  113). T. T113). or Foxn1 . Comparior Foxn1 –119 withphocytes . Hence, phocytes .The expression of β5t has been reported only in thymomas . ImmunohPSMA8), they have a temporal expression profile and have been described solely in spermatocytes, spermatids and sperm (Spermatoproteasomes (sPs)  are testnd sperm , 125. Thnd sperm . Qian etnd sperm . In contnd sperm . Therefond sperm . The prend sperm . Spermatnd sperm .PSMA8 gene in large B-cell lymphoma, thymoma cells and testicular germ cell tumors  and Rpn . The lid subcomplex performs the deubiquitination of substrates and contains the Rpn subunits 3,5,6,7,8,9,11,12, and 15, where Rpn11 is a Zn2+-dependent metalloisopeptidase that cleaves ubiquitin chains from tagged proteins . It. It15). Many cancer cells of different origins are highly dependent on and have elevated levels of 26S proteasomes, indicating that their survival relies on the maximal utilization of these proteasome forms . Indeed,PSME1 and PSME2 genes, respectively . Hoectively , 186.2O2-treated cardiomyocytes PSME4 genin width , indicatin width . Interesin width . PA200 iin width , 240.https://www.proteinatlas.org/ENSG00000068878-PSME4/pathology]. The role of PA200 in cancer is mostly associated with DNA repair mechanisms, specifically the degradation of acetylated histones . T. T278). Generally, the specific function of hybrid proteasomes in cancer is unknown.Increased metabolic activity and the need to adapt to various stresses explain why many proteasome genes are upregulated in different tumor types. Thus, cancer cells are frequently more dependent on appropriate UPS function than normal cells, making the system an appealing target for cancer therapy. Accordingly, proteasome form diversity allows the development of new and the fine-tuning of known approaches for cancer management. For instance, the quantity and ratio of proteasome forms (constitutive proteasomes vs. immunoproteasomes) in tumor cells can predict the clinical effects of broad specificity proteasome inhibitors , 282, whOverall, the diversity of proteasome forms and the complexity of UPS provide exciting opportunities to optimize and fine-tune cancer therapy.AM: writing—original draft preparation. AM and VK: writing—review and editing and funding acquisition.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.


Cervical intraepithelial neoplasia (CIN) is a precancerous lesion of the uterine cervix that can regress or progress to cervical cancer; interestingly, it has been noted that young women generally seem to have higher rates of spontaneous regression and remission, suggesting a correlation between the patient's age and regression/progression rates of CIN. Even if the underlying mechanisms are still unclear, inflammation seems to play a pivotal role in CIN fate and inflammatory processes are often driven by mesenchymal stem cells (MSCs). This study was aimed at evaluating if age affects the behavior of MSCs from the cervix (C-MSCs) that in turn may modulate inflammation and, finally, regression rate. Fourteen samples of the human cervix were recovered from two groups of patients, “young” (mean age 28 ± 2) and “old” (mean age 45 ± 3), during treatment using the loop electrosurgical excision procedure (LEEP) technique. Progenitor cells were isolated, deeply characterized, and divided into young (yC-MSCs) and old cervixes (oC-MSCs); the senescence, expression/secretion of selected cytokines related to inflammation, and the effects of indirect cocultures with HeLa cells were analyzed. Our results show that isolated cells satisfy the fixed criteria for stemness and display age-related properties; yC-MSCs express a higher level of cytokines related to acute inflammation than oC-MSCs. Finally, in the crosstalk with HeLa cells, MSCs derived from the cervixes of young patients play a stronger antitumoral role than oC-MSCs. In conclusion, the immunobiology of MSCs derived from the cervix is affected by the age of donors and this can correlate with the regression rate of CIN by influencing their paracrine effect. In addition, MSCs from a young cervix drives an antitumoral effect by sustaining an acute inflammatory environment. High-grade cervical intraepithelial neoplasia CIN2–3) is a precancerous lesion of the uterine cervix with an incidence of 250,000–1 million annually in the USA. CIN can either resolve spontaneously or persist or progress to cervical cancer when not treated immediately –3 is a p, each paInterestingly, several researches , 6, 7 haInflammation is orchestrated by different cells of the immune system as well as by mesenchymal stem cells (MSCs) ; the exiFourteen women undergoing cervical excision for high-grade CIN, diagnosed after colposcopy-directed cervical punch biopsy following an abnormal cervical cytology, were enrolled in this study; seven patients were defined “young” (mean age 28 ± 2), and the others were defined “old” (mean age 45 ± 3). Patients provided their written informed consent to participate in the study, which was approved by the institutional ethics committees and was conducted in accordance with the Declaration of Helsinki.The cervical excisional procedures were performed with the loop electrosurgical excision procedure (LEEP) technique, in an outpatient setting under local anesthesia and strict colposcopic guidance, with 1.5–2.0 cm rounded loops, chosen according to the type of the transformation zone and the area of cervical tissue to remove.3), obtained from cone specimen, were placed into 6-well plates containing MSCGM medium , as previously described [Tissue fragments  conjugated antibodies (Becton Dickinson) against HLA-DR, CD14, CD19, CD34, CD45, CD73, CD90, and CD105. In addition, the expression of CD9 was analyzed as a distinguishing marker between MSCs and fibroblasts.For immunophenotyping, 2.5 × 10For the differentiation assay, cells were induced towards osteocytes, chondrocytes, and adipocytes using StemPro® Osteogenesis, Chondrogenesis, and Adipogenesis Kits , respectively . OsteogeCt−ΔΔ method [Ct = Ct(gene of interest) − Ct(control gene) and Δ(ΔCt) = ΔCt(yC − MSCs) − ΔCt(oC − MSCs). The values of the relative expression of the genes are mean ± standard deviation (SD) of three independent experiments. Primer sequences are reported in For RT-PCR, total RNA isolation, reverse transcription, and amplification were as previously described . mRNA ext method , where ΔTo assess the proliferation rate, the XTT Cell Proliferation Assay  was performed on cells after 72 hours of culture for the first 6 passages. The data about yC-MSCs are reported as percentages of the values measured in parallel in oC-MSCs (referred to as 100%) over three independent experiments.β-Gal activity was detected with a senescent cell staining kit  according to the manufacturer's instructions.SA-4 cells at passage 6th were seeded in a six-well plate and incubated overnight with the staining solution. β-Gal was microscopically revealed by the presence of a blue, insoluble precipitate within the cell, and the percentage of SA-β-Gal-positive cells was determined by counting at least 500 cells in each sample.Briefly, 7 × 10The expression of 86 genes related to senescence was analyzed by PCR array  in MSCs isolated from cervixes derived from young and old patients.2 First Strand Kits, following the manufacturer's instruction. mRNA expression was calculated by the 2Ct−ΔΔ method [Ct = Ct(gene of interest) − Ct(control gene) and Δ(ΔCt) = ΔCt(yC − MSCs) − ΔCt(oC − MSCs).Total RNA was isolated by using Masterscript RT-PCR System . cDNA synthesis was performed using SABiosciences RTt method , where ΔThe cell line HeLa (human cervix epithelioid carcinoma from Sigma-Aldrich) was cultured in EMEM (EBSS) + 2 mM glutamine + 1% nonessential amino acids (NEAA) + 10% foetal bovine serum (Sigma-Aldrich).HeLa cells were cocultured with MSCs to evaluate the paracrine effects exerted by stem cells isolated from cervixes of young and old patients.4 HeLa cells were seeded at the lower surface, and 5 × 104 of yC- or oC-MSCs were individually added the day after at the upper surface of a polycarbonate transmembrane filter in a Transwell filter system in a 6-well plate . Cells were cocultured for 72 hours. After cocultures, tumor cells were recovered and their number was assessed by an automated cell counter. HeLa cells cultured individually served as controls (mock). Data are reported as mean ± SD from three independent experiments.5 × 10β-actin, and RPE30 for data normalization. Of the three, GAPDH was chosen since it was the most stable one.RNA was isolated from HeLa cells, mock, and after individual cocultures with MSCs derived from young and old cervixes. The genes and the related primer sequences are summarized in Ct−ΔΔ method, where ΔCt = Ct(gene of interest) − Ct(control gene) and Δ(ΔCt) = ΔCt(HeLa cells cocultured with C − MSCs) − ΔCt. The values of the relative expression of genes of interest are referred to as mean ± SD over three independent experiments.Quantification of mRNA expression was calculated with the 2γ, TNF-α, IL2, IL4, IL5, IL13, IL10, TGF-β, IL17A, and G-CSF, were investigated by ELISA  as previously described [Selected cytokines related to acute and chronic inflammation, such as IL6, IL12, IFN-escribed . The anat-test using SPSS 17.0 software. p values ≤ 0.05 were considered significant.Statistical analysis of data from at least 3 independent experiments was performed using SPSS 19.0 software . All data are mean ± SD. For two-sample comparisons, significance was calculated by Student's MSCs were successfully isolated from the cervixes of all patients, subgrouped into “young” and “old.” No statistically relevant difference was found in each cellular group among the donors. Therefore, results are reported as mean ± SD for young and old patients in each analysis. For all the subsequent experiments, yC-MSCs and oC-MSCs were used at the same passage of culture.Cell cultures (from cervixes of young and old patients) showed a fibroblastoid morphology .Both cell types expressed all the stemness genes tested by RT-PCR, with a higher expression in cells derived from the cervixes of young patients. Considering as 1 the expression detected in MSCs from old cervixes, the same genes are from 1.74 ± 0.21 (OCT4) up to 12.05 ± 0.53 (KLF4) fold higher in MSCs from young patients . This trEvaluation of the stemness criteria identified by Dominici et al. demonstrated that cells were strongly positive for CD73, CD90, and CD105 and negative for HLA-DR, CD14, CD19, CD34, and CD45, as well as for the key marker CD9 .Cells were also capable of differentiating towards osteogenic, chondrogenic, and adipogenic lineages .XTT assay was performed after 72 hours of cultures of y- and oC-MSCs for the first six passages . yC-MSCsLooking at the data from the proliferation assay and with the aim of assessing potential differences between MSCs derived from cervixes of young and old patients, cellular senescence was tested.Senescence was evaluated in yC-MSCs and oC-MSCs at passages 6 and interestingly the number of blue cells (indicating senescence) was notably different between yC-MSCs and oC-MSCs  .X-fold in yC-MSCs. The expression of all genes revealed a dysregulation in yC-MSCs compared to oC-MSCs. A 3-fold cut-off was chosen for the significance; the expression of CCNA2, CCNB1, CDC25, CHEK1, E2F1, TERT, and PCNA was significantly higher in yC-MSCs than in oC-MSCs, while that of CDKN2A, CCNE1, NOX4, and SOD2 was downregulated in yC-MSCs compared to oC-MSCs  higher proliferation rate  higher levels of cMET, cFOS, cJUN, MMP11, CXCL12, and VEGF. These variations were significantly (p < 0.05) more evident in HeLa cocultured with oC-MSCs than with yC-MSCs  and after cocultures with yC-MSCs and oC-MSCs. Compared to mock HeLa , cocultured HeLa exhibited significantly  higher levels of IL2, IL6, IL10, IL12, IFN-γ, and TGF-β. The expression of IL4, IL5, IL13, IL17A, TNF-α, and G-CSF was not significantly different between oC-MSCs and yC-MSCs. Subsequently, C-MSCs were cocultured with HeLa cells and the secretion of the differently expressed cytokines was measured in HeLa mock (expressed as 100%) and in cocultured HeLa . Compared to the mock sample, HeLa cocultured with yC-MSCs showed a significantly (p < 0.05) increased secretion of IL6, IL10, and TGF-β, where the production of IL2, IL12, and IFN-γ did not change. Cocultures with oC-MSCs produce a significant increase only in the secretion of TGF-β , and the others were defined as “old” (mean age 45 ± 3). From all tissue biopsies, it was possible to isolate an undifferentiated cellular population able to satisfy all the minimal criteria to qualify as mesenchymal stem cells. Cells isolated from young cervixes were named yC-MSCs and those from old cervixes oC-MSCs. Both yC-MSCs and oC-MSCs were cultured as plastic adherent, were negative for CD14, CD19, CD34, CD45, and HLA-DR, and were positive for CD73, CD90, and CD105. Cells were also able to differentiate towards adipogenic, chondrogenic, and osteogenic lineages. These properties were equally displayed by yC-MSCs and oC-MSCs without evident differences. The subsequent analysis of the expression of genes related to stemness, such as OCT4, SOX2, NANOG, and KLF4, revealed the first discrepancy between young and old MSCs. yC-MSCs express a higher value of KLF4 than oC-MSCs. KLF4 directly binds to the promoter of NANOG to help OCT4 and SOX2 in regulating the expression of NANOG . This ob ± 2, andnescence . TERT plnescence –32.E2F1 and CHEK1 regulate, respectively, the expression of genes referred to cell cycle progression and the G2 DNA damage checkpoint , 34 and Among the downregulated genes in yC-MSCs, CCNE1  and CDKNγ, and TGF-β1) in MSCs from cervixes of young patients. Previous studies have suggested that decreased Th1 responses (strictly connected to acute inflammation) are associated with cervical carcinogenesis [To test if the involvement of inflammation in CIN progression could be age-related, the secretion of soluble factors related to acute and chronic inflammation was analyzed by ELISA in y- and oC-MSCs. The first evidence is the increased secretion of cytokines related to the acute inflammation . Firstly, it was assessed if factors secreted by MSCs may alter the proliferation of tumor cells, finding that cocultures with oC-MSCs produce an increase in HeLa proliferation rate. Subsequently, the expression of genes related to different mechanisms leading to tumor development was assayed in control and cocultured HeLa cells. PCR analysis revealed that, after cocultures with C-MSCs, HeLa cells expressed increased levels of all the selected genes and the observed increase was significantly higher in cocultures with MSCs derived from old than from young cervixes. In detail, the upregulation of cJUN, cFOS, and cMET after cocultures especially with oC-MSCs correlates with other works that underlined that these genes are implicated in cervical carcinogenesis –47. VEGFγ, and TGF-β (that were differently produced by MSCs from old and young cervixes) was tested after cocultures with HeLa.Finally, the secretion of IL2, IL6, IL10, IL12, IFN-γ compared to a mock sample, the level of IL6, IL10, and TGF-β were significantly increased after cocultures with “young” MSCs.While cocultures did not affect the secretion of IL2, IL12, and IFN-β displayed a significant increase in cocultures with oC-MSCs.Only TGF-Cytokines mostly secreted after cocultures with yC-MSCs belong to the Th1–Th17 pathways, indicating as the presence of neoplastic cells increase the mechanism of the host's immune surveillance.β decreased as tumor cells progressed from CIN to cervical carcinoma, indicating that the disruption of this signalling pathway might contribute to the malignant progression of cervical dysplasia [β in the presence of IL6 drives the differentiation of T helper 17 (Th17) cells, which can promote further inflammation and augment autoimmune conditions [A previous study demonstrated that the expression of TGF-ysplasia . In addinditions . The exanditions , there iFinally, IL10 plays a dual, controversial role in cervical carcinogenesis ; even ifIn conclusion, the present data suggest the following: (i) a mesenchymal stem cell (MSC) population persists inside the cervix; (ii) this cell population displays age-related properties; (iii) age may affect the regression rate of CIN by influencing the paracrine effect exerted by MSCs; and (iv) in the crosstalk with HeLa cells, MSCs derived from the cervixes of young patients play an antitumoral role. These results reinforce the evidence of the possibility of a conservative management of high-grade lesions  in young women with a desire for future pregnancy, avoiding unnecessary treatments with the preservation of reproductive potential.


According to the “developmental origins of health and disease” (DOHaD) concept, maternal obesity predisposes the offspring to non-communicable diseases in adulthood. While a preconceptional weight loss (WL) is recommended for obese women, its benefits on the offspring have been poorly addressed. We evaluated whether preconceptional WL was able to reverse the adverse effects of maternal obesity in a mouse model, exhibiting a modification of foetal growth and of the expression of genes encoding epigenetic modifiers in liver and placenta. We tracked metabolic and olfactory behavioural trajectories of offspring born to control, obese or WL mothers. After weaning, the offspring were either put on a control diet (CD) or a high-fat (HFD). After only few weeks of HFD, the offspring developed obesity, metabolic alterations and olfactory impairments, independently of maternal context. However, male offspring born to obese mother gained even more weight under HFD than their counterparts born to lean mothers. Preconceptional WL normalized the offspring metabolic phenotypes but had unexpected effects on olfactory performance: a reduction in olfactory sensitivity, along with a lack of fasting-induced, olfactory-based motivation. Our results confirm the benefits of maternal preconceptional WL for male offspring metabolic health but highlight some possible adverse outcomes on olfactory-based behaviours. In adisorders ,9,10.The “developmental origins of health and disease” (DOHaD) concept states that obese women give their children an increased risk of developing non-communicable diseases (NCDs) in adulthood. Early exposure to various exogenous or endogenous changes during preconceptional period, gestation, lactation or childhood and adolescence affect long-term health ,12,13. IObesity is also associated with reproductive issues such as infertility, obstetrical complications, birth defects and stillbirth . When liFurther work is required to determine the effect of maternal preconceptional BMI and WL on metabolic outcomes and olfactory behaviour related to food intake in the offspring. To our knowledge, no study has investigated the contribution of either maternal obesity linked to high fat consumption or preconceptional WL on the preservation, improvement or impairment of olfactory performance in the offspring at different ages, and with a focus on sex differences. Here, we evaluated whether a preconceptional WL may be used as a therapeutic tool to reverse the adverse effects caused by a gestational obesity (OB) on offspring’s phenotype, using a previously described animal model developed in our laboratory . MetabolThe COMETHEA ethical committee , registered with the Comité National de Réflexion Ethique sur l’Expérimentation Animale under the n°45, approved this protocol (visa 12/062), in accordance with European Union  legislation and the National charter on the ethics of animal experimentation.Four-week-old female and 7-week-old male C57Bl/6J mice were purchased from Harlan Laboratory  and housed in IERP animal holding facilities  at controlled temperature (22 ± 2 °C) with a 12 h light/12 h dark cycle. Mice had ad libitum access to water and food and paper towel was provided for nest building. After one week of adaptation, mice were placed in individual cages and randomly assigned to either the high-fat diet (HFD) or the matched control diet CD, . Food wan = 40; n = 98). At 14 weeks, after 2 months of HFD, mice with a weight above the threshold of mean CTRL weight +2 standard deviations, were considered to be obese [n = 26). The remaining mice stayed on HFD to develop a chronic obesity . Measurements of fasting cholesterolemia, glycaemia, insulinemia, leptinemia and oral glucose tolerance test (OGTT) were performed at the age of 13 and 22 weeks. From 23 weeks of age, females were mated with chow-fed C57BL/6J males  for one night. If no vaginal plug was observed, females were mated with another male according to their estrus cycle. The reproduction period lasted for 6 weeks. Females remained on their experimental diets throughout pregnancy and lactation. At E16.5, litter was last changed in the cage and nestlets were provided. All female mice gave birth by spontaneous vaginal delivery.We used two different season-matched F0 cohorts. From 5 weeks of age, mice of the control group received CD for the 4 months in preconception period  1, litters were reduced to six pups when necessary. Litter was changed in the cage at PND7 and F1 were weighed every 2 days until PND21. Then, male and female offspring were randomly fed a CD or HFD to obtain 6 F1 groups : CTRL-CDAfter 6 h fasting (8:00 a.m. to 2:00 p.m.), a bolus of glucose (2 g/kg body weight) was delivered into the stomach of conscious mice using a gavage probe. Glycaemia was measured prior glucose delivery (T0) and at 20, 40, 60, 90 and 120 min. Insulinemia was measured at 0, 20 and 60 min, except for the F1 mice at weaning, for whom only at fasting (T0) insulinemia was measured. The trapezoidal method was used to calculate the area under the curve (AUC) (auc function of the flux package in R) considering only values above the fasting level. Glycaemia from tail vein blood was measured in duplicate using an Accu-Chek Performa blood glucose meter . Tail vein blood (30µL) was collected in microvette tubes , centrifuged for 10 min at 2000 g at 4 °C. Insulinemia was measured with Mouse Ultrasensitive Insulin ELISA , according to the manufacturer’s instruction. Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was calculated as: fasting glycaemia (mol/L) × insulinemia (mol/L)/22.4.g at 20 °C, plasma collected and stored at −20 °C. For F0, total cholesterol level was measured by colorimetric dosage on Ortho Vitros Clinical Chemistry System instrument in Ambroise Paré Hospital . For F1, cholesterolemia was measured by enzymatic CHOD-PAP (cholesterol oxidase: p-aminophenazone) method . Leptinemia was measured with Leptin Mouse/Rat ELISA  according to the manufacturer’s guidelines.At 2:00 p.m., after 6 h fasting, submandibular vein blood (300 µL) was collected from conscious mice in tubes containing 5 U.I. of heparin . Samples were centrifuged for 10 min at 1500× At 6 months of age, F1 mice were weighed and sacrificed by cervical dislocation after 5–8 h fasting (from 8:00 a.m. to 13:00–16:00 p.m.). The weight of following tissues was recorded: subcutaneous (Sc), perirenal (Pr) and perigonadal (Pg) white adipose tissue (WAT), brown adipose tissue (BAT), liver, kidney and heart. Organ weights are presented relative to body weight (%).All behavioural tests were performed during the light phase, at 2:00 p.m., with water and food available, except when mentioned. Data were collected by experimenters blind to diet groups.It was performed as described previously in rats with slight modifications . Mice wev/v; four times, habituation), followed by decanal . The time spent sniffing the hole (in seconds) was manually recorded. Between each presentation, animals returned in their cage for 5 min. Animals that did not display any sniffing during the test were removed from the statistical analysis .The capacity of mice to discriminate two chemically distinct odorants was measured as described , in a laEOG recordings were performed from the centre of turbinates IIb and III of the olfactory epithelium in an opened nasal cavity configuration on male mice during light phase (2:00–5:00 p.m.) as already described . Heptaldp-value of p < 0.05 was considered to be significant.The data were analysed using the R software. Male and female offspring were tested separately as there was sexual difference for most phenotypes. For all tests, an adjusted For metabolic data, as variances were unequal, data were Boxcox transformed, using the powerTransform and bcPower functions of the car package and analysed with a likelihood-ratio test, using the lrtest function of the lmtest package, and adjusted with the Benjamini and Hochberg (BH) correction for multiple comparisons using the p.adjust function. Linear mixed models were used to model the evolution of metabolic phenotypes with time, using the lmer function of the lme4 package. Estimates of the slopes for each phenotype were reported as “β” and coded as Phenotype = Group + Time + Group × Time+random(ID × Time) and Phenotype = Group + Time + Diet + Diet × Time + Group × Time + Group × Diet × Time + random(ID × Time) for F0 and F1 respectively, where “Group” is the maternal group , “Diet” is the F1 post-weaning diet (CD or HFD), “Time” is the timepoint and “random(ID × Time)” is a random intercept and slope component for repeated measures. We used ANOVA to test for differences at each timepoint, using the aov function followed with Tukey’s post hoc test, coded as Phenotype = Group or Phenotype = Group + Diet + Group × Diet for F0 and F1 respectively. Effect size was reported as the proportion R2 of the variance in the phenotype of interest. The PCA function of the FactoMineR package was used in F0 dams.For olfactory scores, the distribution of the variance was heterogeneous, so nonparametric statistics were performed. N-ways ANOVAs were done using the aovp function of the Permutation Tests for Linear Models (lmperm) package, with factors: Groups × Diet in Figure 4a and Group × Diet × fasting in Figure 4c, Group × Diet × Odorant in Figure 5 (discrimination) and Group × Diet × Odorant dilution in Figure 6; or the nonparametric analysis longitudinal data (nparLD) package  with Gro2 and a v.test, which is a normalized difference between the average of the individuals of a class and the general average, corroborated the graphical results. Significance is reached when χ2p < 0.05 and v.test > 1.96 in absolute value. In the variables factor map, for a given dimension, the variables most correlated to the dimension are close to the circle. Significance is reached when 2r > 0.6 in absolute value and p < 0.05.A multiple factor analysis (MFA) was performed in F1 generation on a set of 18 variables regrouped in 3 variable categories , using the FactoMineR package . “Biochep < 0.05) . Nevertheless, WL dams remained +5% heavier than CTRL in the preconception period and at mating  (p > 0.05), indicating a normal regulation of feeding behaviour of HFD-fed dams , while all WL dams normalize these metabolic parameters except leptinemia  b. Dams f < 0.05) b. Diet efed dams c. After p = 0.8) and the percentage of gestation loss  were similar for the three groups of dams. Gestation time was similar between groups . We observed that the maternal group had a major effect on mortality of pups during the first postnatal week: CTRL and WL dams had more litters where at least one pup was weaned, compared with OB dams . Moreover, OB dams weaned fewer pups per delivered litter than CTRL or WL females . At sacrifice, one week after weaning, OB dams had an increased Sc, Pg, Pr and total WAT’s relative weight (p < 0.001), and a decreased kidney’s, heart’s and liver’s relative weight than CTRL dams . Maternal group influenced the male and female offspring weight, with OB differing from both CTRL and WL, except for PND7 males, where OB only differed from WL  and increased the proportion of SGA foetuses (+28%) at term of gestation. This effect was corrected in WL dams . As the  from WL a,b.p = 2.9 × 10−14, p = 2.0 × 10−12) and 3.5 . OB males were heavier than CTRL males at week 3 : OB-HFD males were 10.5% heavier than CTRL-HFD  compared with CTRL F1  and post-weaning diet (p = 0.0005) influenced retrieving time, with an interaction between factors . Furthermore, CD mice born to OB dams displayed faster retrieving times (CTRL-CD vs. OB-CD p = 0.03). This effect was not observed in WL group . In males, only post-weaning diet affected retrieving time  and post-weaning diet , with an interaction between these factors . CD-fed mice displayed shorter retrieving time and improved their performance over time (p = 2.7 × 10−4), while HFD-fed mice did not (p = 0.47). In females, there was also an effect of maternal group  and interaction between maternal group and post-weaning diet . Concerning CD-fed mice, OB females were more performant than CTRL (p = 4 × 10−4), but there were no WL vs. OB and CTRL vs. OB differences . Post-weaning HFD thus impaired olfactory learning/memory performance between D1 and D3 and, in females, OB mice fed a CD were the best-performing.The evolution of olfactory performance was evaluated by repeating the test three days in a row b. For bop = 3.5 × 10−8), fasting (p = 3 × 10−9), as well as an interaction between maternal group and fasting (p = 4 × 10−3) and between post-weaning diet and fasting , but not WL-CD (p = 0.09). In males, a post-weaning diet and fasting effects were observed (p = 4 × 10−16 and p = 6 × 10−7), as well as an interaction between maternal group and post-weaning diet , but not in OB-HFD and WL-CD (p = 0.25 and p = 0.1). In both sexes, fasted mice on HFD still displayed the worse performance regardless of maternal group . In females, there was a global effect of odorant repetition (p = 3 × 10−14) and an interaction between odorant presentation and post-weaning diet , but CD-fed mice showed a continuous diminution of sniffing time between P1 and P4, whereas HFD-fed mice showed a slight increase at P3 (p = 2 × 10−30) and a post-weaning diet effect  nor post-weaning diet .As all animals displayed odorant habituation whatever their sex, maternal group or post-weaning diet, we tested the mice’s ability to discriminate two odorants by presenting a new odorant  as fifth presentation. Comparative analysis of sniffing time between the fourth pentanol and the decanal presentation showed that mice globally discriminated between these two odorants . Not the post-weaning diet (p = 0.74), but the maternal group had an impact on the responses to odorants (p = 0.012), with no interaction , but not compared with offspring born to OB . There was no difference between OB and CTRL. Therefore, there was a decrease in the olfactory sensitivity for a non-food odorant of offspring born to WL dams, whatever the post-weaning diet.At 6 months of age, we performed EOG recordings on male’s olfactory epithelium (OE) to analyse the amplitude of electrophysiological responses to the application of increasing heptaldehyde (HEP) concentrations from the 6 groups . Amplitueraction . Offspri2 test, p = 0.0234), with an anti-correlation of the OB group . Separation between OB and WL offspring occurred on the two first dimensions (42.5% of the variability) with an association  of these maternal groups to the second dimension  and the second dimension was represented by “Biometry”  and contributed to the differences in the first dimension (r² > 0.6). On the first dimension of the graph of variables (28.1% of the variability), OB offspring were associated to variables leptinemia, liver relative weight, cholesterolemia, body weight and Sc WAT (p < 1 × 10−5), while WL offspring were associated with variables food intake and Pg WAT (p < 1 × 10−5) (p < 7 × 10−10) and WL offspring with food odour preference (p < 6 × 10−6)  on all the data previously described. Variables were separated into three classes: “Biochemistry”, “Biometry” and “Behaviour”. In line with the previous results, there was a major effect of sex and post-weaning diet a. We the × 10−5) g. On the × 10−6) g.Therefore, in both sexes, post-weaning diet had a major effect. More interestingly, the maternal weight trajectories influenced the offspring’s phenotype in HFD-fed males.The main effects on metabolic and olfactory phenotypes are linked to the post-weaning diet.There is a noticeable influence of the maternal group: OB-HFD are more susceptible to develop obesity than CTRL-HFD males, OB-CD females are the best performing in the cookie test, fasting does not improve the performance of WL-CD. In HFD-fed offspring, MFA reveals differences between OB and WL mice.While maternal WL has positive effects on offspring metabolism, there is a programming effect of the preconceptional intervention on olfactory sensitivity.Sex differences are observed: maternal obesity impairs male metabolism, and improves olfactory performances in female fed a CD. Odorant habituation is sensitive to the postweaning diet in female only.We investigated the effects of a maternal obese environment and preconceptional WL on the evolution of selected metabolic measures over time, along with obesity-induced olfactory phenotypes in offspring. We found that:To our knowledge, this is the first study addressing the respective role of preconceptional/perinatal and postweaning nutrients modulation on combined metabolic and olfactory outcomes, both known to contribute to food intake regulation, with a special focus on sex differences.As expected, HFD post weaning was a major contributor of both metabolic and olfactory disturbances. It indeed explains more than 45% of the variance in MFA, and was in line with other studies on metabolic outcomes of HFD . InteresThis suggests that disturbance of olfactory sensitivity or cognitive function, or both, could develop along with metabolic imbalance in juvenile age in a maternal lipid-rich context, thus possibly fueling the vicious circle of appetite dysregulation and unhealthy food or nutrients choices . Our finWe observed only a weak effect of maternal obesity on both metabolic and behavioural phenotypes. We previously reported that maternal obesity leads to an increased SGA phenotype at term of gestation . Here, wFrom our previous results of increased SGA phenotype in this model  and fromWe previously showed that preconceptional WL was beneficial to foetal growth, with a partial normalization in placental and hepatic gene expression patterns . This suThe decrease in olfactory sensitivity in WL males and the resistance of WL-CD to fasting could stem from the preconceptional caloric depletion associated with the transition to CD in mothers or to a maternal stress linked to the procedure of food transition per se, through the rapid lost weight. Indeed growing evidence suggests that preconceptional adversity may affect the progeny even in adulthood . InteresOur data pointed out a noticeable male-preferential impact in the body weight conditioning by maternal obesity, as it was previously reported . AccordiIn conclusion, we demonstrated that maternal preconceptional weight loss roughly normalized the survival and metabolic profile of the offspring, but induced a reduction in the sensitivity of the olfactory mucosa. Maternal weight loss was not associated to any other deleterious phenotype. It remains unclear whether the programming effect of maternal weight loss is due to maternal stress or nutrition per se.In humans, preconceptional weight loss is obviously recommended to obese women, yet few studies have been conducted on the consequences for the offspring. We believe that more animal experiments and human cohorts are needed to clarify the beneficial effect of maternal preconceptional weight loss on the offspring’s health.


Bailey’s molecular subtypes were differentially distributed within the two groups, with the immunogenic subtype being underrepresented in the “B/T” group (p = 0.005). Uni- and multivariate analyses indicated that PDAC anatomic location was an independent prognostic factor. Finally, the supervised analysis identified 334 genes differentially expressed. Genes upregulated in the “head” group suggested lymphocyte activation and pancreas exocrine functions. Genes upregulated in the “B/T” group were related to keratinocyte differentiation, in line with the enrichment for squamous phenotype. We identified a robust gene expression signature (GES) associated with B/T PDAC location, suggesting that head and B/T PDAC are different. This GES could serve as an indicator for differential therapeutic management based on PDAC location.The association between pancreatic ductal adenocarcinoma (PDAC) location (head vs. Body/Tail (B/T)) and clinical outcome remains controversial. We collected clinicopathological and gene expression data from 249 resected PDAC samples from public data sets, and we compared data between 208 head and 41 B/T samples. The 2-year overall survival (OS) was better for the head than for the B/T PDACs (44 vs. 27%,  Pancreatic ductal adenocarcinoma (PDAC) is a major public health problem worldwide with 260,000 deaths annually , and itsMany studies have demonstrated that cancers of the right and left colons have different molecular characteristics ,12,13,14Through in-depth molecular characterization, large-scale genomics provides the opportunity to address such a “long-lasting question”. Here, to determine whether head and B/T PDACs are similar or different diseases, we have studied clinicopathological and gene expression data of 249 resected pancreatic carcinoma samples, including 208 head and 41 B/T tumors.We collected clinicopathological and gene expression data of 264 clinical pancreatic carcinoma samples from four public data sets, including 249 operated primary cancer samples with informed cancer localization .Before analysis, gene expression data were processed. First, each data set was normalized separately: quantile normalization for the available processed data from Illumina set and Robust Multichip Average (RMA)  with thet-test analysis was applied to expression levels of each gene using linear models with empirical Bayes statistic included in the limma R package [p-value ≤ 5%, and a q-value ≤ 25%. The classification model was then computed using a metagene-based approach with the significant genes and statistics for ponderation. Using a cut-off of 0, samples were classified as “head-like” or “body/tail-like”. Robustness of the resulting classification model was assessed in the independent validation set , i.e., the three remaining sets Analyses were done in R and associated packages. Ontology analyses were performed using DAVID Bioinformatics Resources 6.8 [We first defined the molecular subtypes of pancreatic cancer samples in each data set separately as defined in the original publications: the three Collisson’s subtypes  were cla package . Genes wrces 6.8 .Overall survival (OS) was calculated from the date of diagnosis to the date of death from pancreatic cancer. Follow-up was measured from the date of diagnosis to the date of last news for living patients. Survivals were calculated using the Kaplan–Meier method and were compared with the log-rank test. Uni- and multivariate prognostic analyses were done using Cox regression analysis . All statistical tests were two-sided at the 5% level of significance. We followed the reporting REcommendations for tumor MARKer prognostic studies (REMARK criteria) .n = 219, 88%) and were classified as AJCC stage II tumors . Following the TNM classification, most samples were pT3 tumors , most had at least one lymph node involved , but only 10 had detectable metastases at diagnosis (4%). Within the whole population, 26 tumors (10%) were located in the tail, 15 (6%) in the body, and 208 (84%) in the head of the pancreas. The 2-year OS of the whole population was 41% (95% CI: 0.33–0.49), with a median follow-up of 11 months (range: 0–156.4).A total of 249 PDAC samples were included in the analysis. Their clinicopathological and molecular data are summarized in Samples were then characterized at the molecular level. The Bailey’s classification identified the four subtypes: ADEX (21%), immunogenic (18%), pancreatic progenitor (22%), and squamous (39%). Regarding the Colisson’s classification, 47%, 35%, and 18% of PDACs were classified as classical, exocrine-like, and quasi-mesenchymal, respectively. According to the Moffitt’s “Tumor” classification, 45% of samples were basal-like and 5% were classical. The Moffitt’s “Stroma” classification was distributed as activated (63%) and normal (37%).p = 0.039) than patients from the “head group”, had more pT1 tumors (p = 0.049), less pathological lymph node involvement (p = 0.001), less tumors from ductal type (p = 0.015), and less stage 2 tumors (p = 0.04). No difference between the two groups was observed concerning the tumor grade.Patients were divided in two groups based on the tumor anatomic location: “head” or “B/T” of the pancreas. A total of 208 patients (84%) had a “head” PDAC and 41 (16%) had a “B/T” PDAC. As shown in p = 0.0058). Accordingly, the immunogenic subtype was more frequent in the “head group” than in the “B/T group” (p = 0.0058). No significant difference was observed between the two location groups regarding the Collison’s subtypes and the Moffit’s tumor subtypes. By contrast, there was more Moffit’s activated stroma subtype in the “B/T group” than in the “head group” (p = 0.006).In molecular term, all Bailey’s molecular subtypes were represented in both “head” and “B/T” tumors. However, the distribution was significantly different, notably the immunogenic subtype was almost absent from the “B/T group”  in the “head group” and 14.1 months (range: 1–60) in the “B/T group”, and the respective 2-year OS were 44% (95% CI: 0.36–0.54) with a median follow-up of 11 months, versus 27% (95% CI: 0.14–0.49) with a median follow-up of 9.6 months, revealing longer survival in the “head group” than in the “B/T group” (p = 0.0078), the PDAC location (p = 0.0453), the Bailey (p = 0.006), and Moffitt (“Tumor” p = 0.00076 and “Stroma” p = 0.0002) classifiers were associated with OS . The hazard ratio (HR) for death was 0.63 (95% CI: 0.41–0.99) in the head vs. B/T classes. In multivariate analysis, the PDAC location (p = 0.0047), the pN status (p = 0.0028), Moffitt tumor (p = 0.0497), and stroma classifiers (p = 0.0232) remained significant, suggesting independent prognostic value.We then compared the prognostic value of the head versus B/T anatomic location with that of other clinicopathological variables and molecular subtype classifiers . In univariate analysis , the pN p = 0.002; Since AJCC TNM staging is the major prognostic factor used in clinical practice, we analyzed OS in comparable situations, i.e., in groups matched according to the TNM staging. The “head group” conserved a better 2-year OS (67%) than the “B/T group” (17%) when looking at tumors classified as T3-4N0M0 (p < 0.0001)  . It was n = 137) and the other sets pooled as a validation set (n = 112). A total of 334 genes were differentially expressed, including 278 (83%) genes upregulated and 56 (17%) genes downregulated in the “head group” compared to the “B/T group” (p < 0.0001). Importantly, its robustness as a classifier for B/T samples was confirmed in the independent validation set  (To identify a gene expression signature (GES) of “head” versus “B/T” PDAC, we used the TCGA cohort as a learning set (T group” . A “locact test) .p < 0.0001), more specifically involving genes from inflammatory response (p < 0.0001), adaptive immune response (p < 0.0001), G-protein-coupled receptor signaling pathway (p < 0.0001), transmembrane receptor protein tyrosine kinase signaling (p < 0.0001), cell surface receptor signaling (p < 0.0001), and cellular defense response (p < 0.0001) pathways. Such activation of “Immune-related” pathways is coherent with above-mentioned enrichment of the immunogenic subtype in the “head group”. The immune signature showed a strong immune infiltrate, more specifically involving upregulation of mRNA related to unconventional γδ-T cells (p < 0.0001), to the presence of a cytotoxic activity potentially at work and B-cells-related markers (p < 0.003). Finally, numerous cytokines, chemokines, and related receptors were secreted in “head” tumors and involved in immune cell recruitment and function (p < 0.0001).Regarding the 278 genes identified as upregulated in the “head group” , two majp < 0.0001), proteolysis (p < 0.0001), and digestion (p < 0.0001) process. These genes were related to pancreatic secretion (p < 0.0001), fat, protein and vitamin digestion and absorption (p < 0.0001), and glycolysis/gluconeogenesis/drug metabolism (p < 0.01).The second major pathway upregulated in the “head” PDAC was related to pancreas function, notably triglyceride catabolic process (p < 0.0001), more specifically to squamous tumors differentiation. Some of the downregulated genes were also related to the nervous system, notably dopaminergic synaptic transmission (p < 0.0001) and positive regulation of calcium ion import (p < 0.0001) that function as regulators of pancreas endocrine function, such as glucagon synthesis and positive regulation of cAMP biosynthetic process (p < 0.0001). Finally, several genes were related to tumor aggressiveness in “B/T” tumors, including genes involved in cellular proliferation, survival, and epithelial to mesenchymal transition. Some of these genes are associated components of the EGFR and/or ERBB and/or TP53 pathways. Other genes are involved in the regulation of cell proliferation and local invasion and the acquisition of drug resistance mechanisms and/or TGF-β-induced EMT.Regarding the 56 genes identified as downregulated in the “head” compared to “B/T” PDAC , associaXCELL [XCELL gene signature-based data allow identifying immune and stromal cell types present within the tumor.Because tissues are complex mixtures consisting of numerous noncancerous cell types, we searched for correlation between “head” vs. “body/tail” transcriptomic profiles and a variety of innate and adaptive immune cells, stromal cells, and many other cell types that are found in the tumor. For this, we used the XCELL  and the XCELL  gene expp = 0.0005) and Immune_Score (p = 0.0006) were significantly correlated with the “head” PDAC (p = 0.0007), Epithelial_cells (p = 0.0017), Smooth_muscle (p = 0.0123), and TGFβ (p = 0.0299) gene signature-based profiles. As mentioned earlier, these signatures are suggestive of ongoing epithelial-to-mesenchymal transition (EMT) process. Altogether, these results confirmed the results of our “location” GES in term of differential pathways involved in the “head” versus “B/T” PDAC.Those signatures showed correlations between the “head” transcriptional PDAC profile and the T and B immune infiltrates  as well as some antigen-presenting cells . Accordingly, the Microenvironment_Score (ad” PDAC . By contn = 150 cases), there was no significant difference in survival between the Head and the B/T groups except for the Squamous subtype. If location and associated surgery only had an impact on survival, it would have been detectable for all molecular subtypes of PDAC, and not the Squamous subtype only. It is also possible that the molecular differences related to the head versus B/T locations are not tackled by the molecular subtypes.Currently, the AJCC TNM classification is the only prognostic factor used in clinical practice to assess survival of resected PDAC and guide treatments decision. However, recent data suggest that PDAC localized in the “head” might have a better survival than PDAC localized in the “B/T” zone of the pancreas ,27. So fOverall, we were interested in defining the differential gene expression signature related to PDAC location. A recent study used pre-established “genes programs” issued from the PDAC classification of Bailey  to conduTNS4, NTSR1, and PNCK are three genes involved in these pathways. Tensin-4 (or TNS4) functions as an oncogene and promotes cell proliferation and/or motility in numerous solid tumors, including pancreatic cancer [In line with the shorter survival of B/T tumors, our GES analysis between “head” and “B/T” PDAC revealed that B/T tumors were probably more aggressive tumors, being highly proliferative and more prone to EMT. The higher proliferation rate could explain why “B/T” tumors often display bigger size at diagnosis . Tumor rc cancer , gastricc cancer . NTSR1 cc cancer  and is ac cancer ,53,54. ABecause Langerhans islet concentration is much higher in the tail than in the head of the pancreas, theoretically, patients with pancreatic “B/T” cancer are more prone to have islet dysfunction and subsequent diabetes. Data comparing the onset of pancreatic cancer-induced diabetes between “head” and “B/T” PDAC are limited. Here, we have shown that the endocrine functions of the pancreas seem to be relatively more efficient in the “B/T” than in the “head” PDAC. Even in the presence of the growing tumor, the endocrine cells, notably insulin-secreting cells, seem to maintain their function. Insulin-positive endocrine cells seem to be “refractory” to malignant progression. This can explain why postoperative diabetes is higher in patients receiving distal (B/T) pancreatectomy than in those treated with pancreaticoduodenectomy ,55 and tPD1, BTLA, TNFSF14 (LIGHT), and TIGIT, which are the sign of immune exhaustion or inhibition of tumor infiltrating lymphocytes. Gamma-delta T cells are part of the unconventional T cells involved in the initiation and propagation of immune responses. It is thus plausible that the reactivation of this cytotoxic immune infiltrate, achievable with the help of immune checkpoint inhibitors and/or potent analogues with more or less selective stimulatory activity for γδ-T cells (such as bromohydrin diphosphate (BrHPP), anti-KIR, phosphoantigen, or ligation of NKG2D, for example), can benefit to “head” PDAC. As for many solid tumors, boosting the immune system, especially with neoadjuvant therapeutic agents might be highly beneficial for “head” PDAC.Another striking information related to our analysis was the absence of the immunological subtype in “B/T” PDAC. This highlights a profound defect in the recruitment of leukocytes within “B/T” PDAC and suggests that these tumors are probably noneligible for immune therapy-based treatment. The obvious defect in immune response was mainly noticeable due to the absence of CD8 γδ−T cells and B cells in “B/T” PDAC. Because this transcriptomic analysis results from the whole tumor piece, we cannot know whether these cells were infiltrating or surrounding the tumor. These cells however overexpressed immune checkpoint inhibitory molecules, such as In conclusion, we have identified a robust GES associated with B/T PDAC samples. The nature of genes differentially expressed suggests that different therapeutic managements might be applied in clinics depending on the “head” or the “B/T” location of tumor .


Euphorbia pulcherrima Willd. ‘Flame’, ‘Mable Bell’, ‘Green Star’, ‘Pink Bell’, and ‘Peach Bowl’ cultivars were investigated in this study. Stem cuttings in a propagation bench were drenched regularly with a solution containing either 0 (control) or 50 ppm of silicon (Si treatment) from potassium silicate (K2SiO3), with a 25 °C mean air temperature and 80% relative humidity (RH) under 70% shading. The results showed that the ‘Flame’ treated with Si had a significantly higher survival ratio as compared with that of the control (P ≤ 0.05) and that the Si treatment improved number of roots, length of longest root, fresh root weight, and dry root weight in all cultivars except ‘Mable Bell’. Supplementary Si increased the content of magnesium (Mg) and decreased the content of boron (B) and zinc (Zn) in the roots. The content of sulfur (S) in the shoots was increased by supplementary Si. The relative expression of Lsi1 and Lsi2 was higher in ‘Peach Bowl’, while it was lower in ‘Mable Bell’ and ‘Green Star’, which may be caused by the differing accumulation of Si in the shoot. Overall, supplementary Si had beneficial effects during cutting propagation of poinsettia cultivars, although these effects were cultivar-dependent.The effects of silicon (Si) on root development, mineral content, and expression of Si transporter genes in  Euphorbia pulcherrima Willd.) is one of the most famous ornamental plants for its colored bracts. The attractive bracts can remain fresh during the winter season, which makes it popular as a Christmas decoration as well as a general indoor decoration. In addition to its ornamental value, the latex from poinsettia can be used as a veterinary medicine for livestock to kill maggots [Poinsettia  and low silicon 2 (Lsi2) genes absorb Si from the substrate and transport it to the xylem [Lsi1 in plants will result in either reduced Si uptake or low Si accumulation [Silicon (Si) is largely present in the soil. However, only soluble silicon oxide in the form of monosilicic acid [Si(OH)he xylem . The supmulation ,10. Analmulation . Furthermulation . As infoLsi1 and Lsi2 was determined as they play essential roles in absorbing and transporting Si. Overall, we supplied five poinsettia cultivars  with Si during the cutting propagation stage, aiming to obtain information on how Si affects the cutting propagation of these cultivars.Our previous studies have revealed the beneficial effects of Si on rooting , adventiEuphorbia pulcherrima Willd. ‘Flame’, ‘Mable Bell’, ‘Green Star’, ‘Pink Bell’, and ‘Peach Bowl’ were collected from the Rural Development Administration (RDA) of the Republic of Korea. The cuttings were harvested and transported to the experimental site in June within a time span of one day. The cuttings were then grown in foam wedge substrate in trays  and kept on a fogged (fogged for 10 min every 15 min) propagation bench with a mean air temperature of 25 °C and relative humidity of 80% for four weeks in a glasshouse at Gyeongsang National University, Jinju, Korea. To avoid differences caused by original materials and location of trays on the bench, cuttings were randomly selected for each treatment, and the location of trays was separated by cultivar, treatment, and replicate.Terminal cuttings of 2SiO3) until the substrate was thoroughly soaked. The solutions were applied every three days for four weeks. The additional potassium introduced by K2SiO3 was balanced with reduced KNO3, and the resultant nitrate loss was compensated with nitric acid. In details, the composition of the nutrient solution for the control (the control solution) was as follows (in ppm): 708.00 Ca(NO3)2·4H2O, 246.00 MgSO4·7H2O, 505.00 KNO3, 230.00 NH4H2PO4, 1.24 H3BO3, 0.12 CuSO4·5H2O, 4.00 Fe-EDTA, 2.20 MnSO4·4H2O, 0.08 H2MoO4, and 1.15 ZnSO4·7H2O. The composition of the nutrient solution for the Si treatment (the Si solution) was as follows (in ppm): 50.00 K2SiO3, 708.00 Ca(NO3)2·4H2O, 246.00 MgSO4·7H2O, 439.51 KNO3, 230.00 NH4H2PO4, 1.24 H3BO3, 0.12 CuSO4·5H2O, 4.00 Fe-EDTA, 2.20 MnSO4·4H2O, 0.08 H2MoO4, 1.15 ZnSO4·7H2O, and 40.80 HNO3. The pH of final solutions (both the control and Si treatment) was adjusted to 6.0 using 1 mol·L−1 hydrochloric acid. Each treatment consisted of three replicates, and each replicate contained 15 cuttings.Cuttings in trays were drenched with a nutrient solution containing either 0 (control) or 50 ppm of Si (Si treatment) from potassium silicate , boron (B), copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), potassium (K), calcium (Ca), magnesium (Mg), phosphorus (P), and sulfur (S), dried root or shoot samples from each treatment were mixed together and ground with a stainless mill . The samples were ashed at 525 °C for 4 h in a Nabertherm muffle furnace . The ash was dissolved in 5 mL 25% HCl, followed by dilution with 20 mL of warm distilled water. The content was then measured three times for each treatment using an inductively coupled plasma (ICP) spectrometer .Lsi1: 5′-GGCTCTACGGTTCTCCTG-3′ (forward), 5′-CCTGCTTGTGCTCCTAAT-3′ (reverse). Lsi2: 5′-CCGTCCAGTAGGGTAGAGT-3′ (forward), 5′-ATGCCAATGGTTACAAGGT-3′ (reverse). 18S (reference gene): 5′-ATGATAACTCGACGGATCGC-3′ (forward), 5′-CTTGGATGTGGTAGCCGT-3′ (reverse). The relative gene expressions were calculated using the 2−ΔΔCt method.Roots were obtained from poinsettia plants cultivated for four weeks. Total RNA was extracted using the Easy-Spin Total RNA Extraction Kit  and then reverse transcribed to cDNA using the PrimeScript RT Reagent Kit . Three biological replicates and two technical replicates were adopted for each treatment. The reactions were carried out on the Rotor-Gene Q detection system  machine. The primers used in this study are as follows: t-test (P ≤ 0.05). Moreover, data from the quantitative RT-PCR results were analyzed by Duncan’s test (P ≤ 0.05).The data analysis was performed with the SAS statistical software Release 8.2 . The differences between the control and the Si treatment were tested by Student’s The cuttings went through stresses caused by the delay in transit and high temperatures. As a result, some of the cuttings rotted at the base of the stem after cultivating for several days. Without Si supplementation, the ‘Flame’ cuttings had a survival ratio of 66.7%, whereas the survival ratio of ‘Flame’ cuttings treated with Si was 93.3%. Supplementary Si did not significantly affect the survival ratio of the other cultivars. ‘Green Star’ treated with Si, ‘Mable Bell’ in the control, as well as the control and Si-treated ‘Pink Bell’ and ‘Peach Bowl’ had survival ratio of 100% .The roots were fully developed after four weeks of cultivation A. ExceptF-test results showed that Cu was not significantly affected by supplementary Si in the root and shoot, while levels of B, Fe, Mn, Zn, K, Mg, and S were significantly influenced by supplementary Si.Mineral analyses were conducted for both the shoots and roots of poinsettia cultivars ; Table 2Lsi1 and Lsi2 was investigated in the roots of the poinsettia cultivars. The results illustrated that the expression of Si transporter genes was greatly different among the poinsettia cultivars , and Ca . It can Lsi1 and Lsi2 results in greater Si uptake in rice [Lsi1 and Lsi2 and Si accumulation were inversely related in both the root and shoot. A similar result was found in rice. The expression of Si transporter genes was reduced by Si accumulation in the shoot [Lsi1 and Lsi2 (Lsi1 and Lsi2 are highly expressed in both ‘Flame’ and ‘Peach Bowl’, the increase of Si content in ‘Flame’ is greater than that in ‘Peach Bowl’. It is speculated that there may have allelic variations in the Lsi1 between ‘Flame’ and ‘Peach Bowl’, and as a result, the capacities of Si uptake of both cultivars are different. Thus, the dramatically increased Si content in ‘Flame’ and the reduced Si content in the roots of ‘Mable Bell’ and ‘Pink Bell’ and in the shoot of ‘Mable Bell’ may be explained by the different Si uptake capacities and/or the different expressions of Si transporter genes among the poinsettia cultivars. The reasons for how supplementary Si differently affects different poinsettia cultivars during cutting propagation requires further investigation to be clearly explained.Higher expression of  in rice . Intereshe shoot . These ahe shoot . As showand Lsi2 . AlthougLsi1 and Lsi2 expressions and Si accumulation were shown to be inversely related in the roots of poinsettia. In addition, Si accumulation significantly varied among the poinsettia cultivars in this study. Thus, more academic research is required to explain these results.Supplementary Si has the potential to improve the survival ratio of poinsettia during cutting propagation. Root development characteristics, including the number of roots, length of the longest root, fresh root weight, and dry root weight were enhanced by Si supplementation, possibly because of silicon’s role in reducing transpiration. Supplementary Si during cutting propagation also changed the mineral uptake in poinsettia. However, these changes vary among the different cultivars.


Scientific Reports 10.1038/s41598-019-42095-z, published online 10 April 2019Correction to: In the Supplementary Information file originally published with this Article, the University of Alaska Museum specimen IDs were omitted from Supplementary Datasets 1-4.In addition, the original version of Table 2 contained an error in the Parameters column for the Linear model where,b = 1.19 ”“now reads:a = 1.19 ”“These errors have now been corrected in PDF and HTML versions of the Article, and in the accompanying Supplementary Information file.


Olea europaea L.) growing has outstanding economic relevance in Spain, the main olive oil producer and exporter in the world. Fruit skin properties are very relevant for fruit and oil quality, water loss, and susceptibility to mechanical damage, rots, and infestations, but limited research focus has been placed on the cuticle of intact olive fruit. In this work, fruit samples from nine olive cultivars  were harvested from an experimental orchard at three different ripening stages , and cuticular membranes were enzymatically isolated from fruit skin. The total contents of cuticular wax and cutin significantly differed among cultivars both in absolute and in relative terms. The wax to cutin ratio generally decreased along fruit maturation, with the exception of “Marfil” and “Picual.” In contrast, increased water permeance values in ripe fruit were observed uniquely for “Argudell,” “Morrut,” and “Marfil” fruit. The toluidine blue test revealed surface discontinuities on green samples of “Argudell,” “Empeltre,” “Manzanilla,” “Marfil,” and “Sevillenca” fruit, but not on “Arbequina,” “Farga,” “Morrut,” or “Picual.” No apparent relationship was found between water permeability and total wax coverage or the results of the toluidine blue test. The composition of cuticular waxes and cutin monomers was analyzed in detail, and sections of fruit pericarp were stained in Sudan IV for microscopy observations. Skin surface topography was also studied by means of fringe projection, showing large differences in surface roughness among the cultivars, “Farga” and “Morrut” fruits displaying the most irregular surfaces. Cultivar-related differences in cuticle and surface features of fruit are presented and discussed.Olive ( Except the vine, there is no plant which bears a fruit of as great importance as the olive. Pliny the Elder (attributed)Olea europaea L.) tree is considered one of the oldest crops to have been domesticated by humans  autochthonous Spanish cultivars  were hand-collected at an experimental orchard located at IRTA-Mas Bové  from trees supplied with drip irrigation. Annual rainfall is 500 mm, and takes place mainly in April–May and September. Fertilization and cultural practices at the orchard are the usual in the producing area. Olives were picked at three different maturity stages  based on skin color during the usual harvest period (September to December) in 2016. Maturity index (0–7), fresh weight (g), flesh-to-stone ratio, and water content (% humidity) were determined on 50 fruits per cultivar and maturity stage. For the assessment of length and diameter (mm), 10 fruit were used  for the enzymatic isolation of cuticular membranes (CM). Disks were incubated at 37°C in cellulase/pectinase solution (0.2% (w/v) cellulase, 100 U ml−1 pectinase, and 1 mM NaN3 in 50 mM citrate buffer at pH 4.0 until no more material was released, and then washed in citrate buffer  until no material was left in suspension. After thoroughly rinsing in distilled water, CM disks were dried at 40°C, weighted, and then pooled and kept in hermetically capped vials until analysis. Cuticle yields were expressed per unit of fruit surface area (µg cm−2).Disks of fruit exocarp (two disks per fruit) were excised with a cork borer. Thirty to 75 olive s, depending on fruit size, were processed so to obtain around 100 cmlsewhere . Because−1) for 24 h at room temperature, with constant shaking. Chloroform extraction was done three times, and the chloroform extracts were pooled, incubated 15 min in an ultrasonic bath and filtered. Dewaxed CM (DCM) were dried and kept in hermetically capped vials for subsequent analysis of cutin monomers. The chloroform extracts were concentrated at 40°C using a rotatory evaporator, and waxes then transferred to a pre-weighed vial, dried in a vacuum concentrator at 40°C until complete dryness, and weighed to calculate total wax yields (µg cm−2). Dotriacontane (C32) was then added as an internal standard, and samples were derivatized during 15 min at 100°C in N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine , in order to obtain trimethylsilyl (TMSi) ethers and esters from free hydroxyl and carboxyl groups, respectively.CM samples  were dewaxed in chloroform  was used for quantitative analysis of cuticular waxes, in the same chromatographic conditions as described above excepting that, at the last step, the oven was held at 310°C for 13 min and that a higher carrier gas flow (1.3 ml min−1) was used. Data are expressed as a relative percentage . Average chain length (ACL) of acyclic wax compounds was calculated as the weighted average number of carbon atoms, defined asWax samples (1 µl) were injected in on-column mode into a gas chromatography-mass spectrometry (GC-MS) system for compound identification and quantification. This GC equipment (Agilent 7890N) was coupled with a quadrupole mass selective detector (Agilent 5973N) and equipped with a capillary column . Compounds were identified by comparison with their retention times with those of standards, and through their electron ionization-mass spectra using a mass spectral library (NIST 11 MS). Chromatographic conditions were as follows: oven was set at 100°C for 1 min, then raised by 15°C minnC is the percentage of each acyclic wax compound with n carbon atoms.where −2). Heptadecanoate (C17) and tricosanoate (C23) were added as internal standards, and then samples were derivatized during 15 min at 100°C in BSTFA and pyridine . Derivatized samples (1 µl) were finally injected in on-column mode into a GC-MS and a GC-FID system for compound identification and quantification, respectively, under the same chromatographic conditions as described above for the analysis of cuticular waxes.DCM samples  were hydrolyzed for 2 h in 2 ml of 1 M HCl in 100% MeOH, esterified in the same solution during 2 h at 80°C, and added 2 ml saturated NaCl after cooling down. Cutin monomers were extracted three consecutive times in 2 ml hexane for 10 min using a mixer and centrifuged at 20°C. The collected supernatants were pooled and transferred into a pre-weighed vial, dried completely using a vacuum concentrator at 40°C, and then weighed to calculate total cutin yields (µg cmJ in g m−2 s−1) of the samples were calculated from changes in the fresh weight (ΔW in g) over time (Δt in s) and surface area (A in m2) as indicated below:Transpiration from the whole fruit was determined gravimetrically from measures of water loss over time as described elsewhere . Eight tP in m s−1) was calculated from the transpiration rate (J) divided by the driving force:The permeance .where Micro-topography of samples (25 olives per cultivar) was captured at two locations (180° apart) on the equatorial area of each individual fruit using fringe projection equipment . Topography data were collected with an x-y resolution of 26.83 µm and z  resolution of 2 µm. Subsequent calculations to extract surface roughness descriptive parameters  were conSa, Stm, Spm, Svm, Sk, and S , and the resin was dried at 45°C for 24 h.Pericarp fruit samples were chopped to little cubes (roughly 2 mm per side) and fixed in a formaldehyde-acetic acid (FAA) solution [5% (v/v) formaldehyde and 5% (v/v) glacial acetic acid in 1:1 (v/v) ethanol-distilled water] for 12 h. Samples were dehydrated in aqueous solutions containing increasing ethanol concentrations up to 100% (v/v). Dehydrated samples were transferred to Eppendorf tubes for infiltration and polymerization in Technovit 7100Resin-embedded samples were cut in 4-µm-thick sections using an ultramicrotome , and subsequently stained on a slide for 15 min in a Sudan IV lysochrome solution [5% (w/v) in 85% (v/v) ethanol] in order to visualize the lipidic constituents of fruit cuticles. Excess staining was removed by rinsing in 50% (v/v) ethanol, and samples allowed to dry at room temperature. Olive pericarp sections were observed and photographed using a microscope (Leica DM4000 B) with a coupled camera (Leica DFC300 FX). Cuticle thickness was determined from five images obtained from five different fruit per cultivar and maturity stage with the Fiji image processing software .® Pro 13 software. Results were calculated as means ± standard deviations. Multifactorial analysis of variance (ANOVA) procedures were applied, with cultivar and maturation stage as the factors, and means were compared with the Student’s t test (p ≤ 0.05). PCA was used to help the interpretation of the data set obtained, using the Unscrambler software, version 9.1.2 . Data were centered and weighed by the inverse of the standard deviation of each variable, and full cross-validation was run as a validation procedure.The statistical analyses were conducted with the JMPOlive cultivars assessed in this study included some preferentially used for oil production , for manufacturing of table olives , or for both purposes. The choice of genotypes comprised representatives of very early , early (“Sevillenca”), medium , and late  ripening patterns , as wellDifferences in surface characteristics were found among the nine olive cultivars assessed. Green fruits were stained with toluidine blue in order to visualize pores, cracks, or defects on the surface. Two groups of cultivars were revealed by the TB test: fruits of “Argudell,” “Empeltre,” “Manzanilla,” “Marfil,” and “Sevillenca” were stained, whereas those of “Arbequina,” “Farga,” “Morrut,” and “Picual” were not, even after leaving the fruit samples in the staining solution for several hours.Sa, Stm, Spm, Svm, and Sk). On the contrary, fruit of “Sevillenca,” ‘Empeltre,” “Arbequina,” and “Argudell” had smoother skin surface than other cultivars based on lower values of vertical parameters together with higher horizontal roughness as shown by S, representative of peak-to-peak spacing  did not change significantly along fruit maturation  out of the nine cultivars studied, while a significant decrease was observed for the rest of the genotypes   showing water permeance values above 11 × 10−5 m s−1  to 8.13  × 10−2) and as a percentage over total cuticle  in “Picual” mature fruit, consistent with thicker cuticles in these samples  and hexacosanol (C26) were the predominant alcohols  over total cutin monomers identified . A similar trend, with the exception of ‘Morrut,” was observed for 16-hydroxyhexadecanoic acid, “Manzanilla” and “Picual” fruits showing the highest amounts of this compound. The relative percentages of ω-hydroxy fatty acids with midchain hydroxyl groups remained steady throughout fruit maturation, “Farga” samples showing the highest values (29.4% at the green stage)  and monocarboxylic (3.3 to 21.5%) fatty acids were also quantitatively important. With the exception of “Morrut” fruits, the content of dicarboxylic fatty acids decreased along maturation, while that of monocarboxylic fatty acids was significantly augmented in all the cultivars analyzed, excepting “Marfil” (Bactrocera oleae): among the cultivars used in this work, “Empeltre,” “Farga,” “Manzanilla,” and “Sevillenca” are characterized by severe incidence of infestation, while “Arbequina,” “Argudell,” “Marfil,” “Morrut,” and “Picual” are less susceptible to this plague , and ACL of acyclic wax compounds. However, no consistent relationships were found among all these variables. The toluidine test as well as cuticle yields or thickness were also apparently unrelated to water permeance values. No apparent connection was observed either between the presence of discontinuities on fruit surface as revealed by the toluidine test and the susceptibility to infestation by olive fly  values, because adjacent peaks would be close to each other. Among the olive cultivars considered herein, the lowest values for horizontal roughness were observed for “Farga,” “Morrut,” and “Picual” samples, together with deeper valleys as shown by Svm . Higher percentage of infested fruits , cuticle thickness has been reported to increase during fruit development up to the mature green or breaker stage, and then to decrease thereafter until attaining full ripeness (S. lycopersicum L.), green and mature pepper (Capsicum annuum L.), and apple  fruit in comparison with olive (Mangifera indica L.) is reportedly thinner   and apple , ranging respectively from 0.9 to 4.9 × 10−5 m s−1 (−5 m s−1 (Prunus avium L.) (1.4 to 3.7 × 10−4 m s−1)  .−2) over fruit ripening of a model species such as tomato have been shown to be cultivar-dependent (Citrus sinensis Osbeck) (Hippophaë rhamnoides L.), cranberry (Vaccinium oxycoccos L.), or lingonberry (Vaccinium vitis-idaea L.), but much higher than those in black currant (Ribes nigrum L.) or bilberry (Vaccinium myrtillus L.)    fruit, f Osbeck) . Cutin yctively) . Stable ctively) . Both cuctively) .Vaccinium spp.) (Pyrus sinkiangenesis Yü and Pyrus bretschneideri Rehd)  in mature grapes (Vitis vinifera L.) . This may be relevant to understand resistance or tolerance to stress factors, as this physiological stage of the fruit coincides with environmental conditions which favor the development of pests and diseases  . Completum spp.) . Whereasri Rehd)  as well fera L.) , and of fera L.) . In olivfera L.) . An invefera L.) . Triterpfera L.) , and havfera L.) . In thisdiseases , and ind24 and C26 being the most abundant compounds within both wax types. However, the percentage of n-alkanes over total waxes was very low in comparison with other fruit species: for example, n-alkanes accounted for 29.4% of total waxes in “Jesca” peaches at harvest (S. lycopersicum L.)  .18-type cutin monomers were 2 to 3.7 times more abundant than the C16-type. Even so, 9/10,16-dihydroxyhexadecanoic was quantitatively the main ω-hydroxyacid with midchain hydroxyl groups identified in cutin samples, and an important compound in quantitative terms in cutin composition of all the olive cultivars considered herein. This compound has been reported to be prominent in cutin composition of mango (Capsicum sp.) (Diospyros kaki Thunb.) has been found to contain as much as 43.7% 9,10-epoxy-18-hydroxyoctadecanoic acid together with 17.4% 9/10,16-dihydroxyhexadecanoic acid (16 or C18) dominate cutin composition  . In contoic acid . In a grposition , with agThis comparative study provided new insights in genotype-related differences in surface and cuticle features of olive fruit. Information on the chemical composition of both cuticular waxes and cutin in fruit of nine olive cultivars is reported for the first time, as well as water permeability at different maturity stages. Data on fruit micro-topography were also obtained by means of fringe projections, revealing genotype-related diversity of surface microstructure. Although water permeance of olive fruit might be controlled or fine-tuned by different cuticle-related traits, none of those considered herein appeared to suffice by itself to determine this trait, suggesting that additional properties of waxes and cutin, such as their physical structure or biomechanical properties, significantly influence the barrier functions of plant cuticles. Even so, the bulk of results reported herein should establish the basis for a better comprehension of olive crop adaptations to the surrounding environment. Further research will be paramount to elucidate the role of cuticle properties in olive resistance to plagues, rots, and adverse environmental conditions. The comprehension of these relationships would be thus very relevant for improving orchard management.All datasets generated for this study are included in the article/supplementary material.CD, JG, AR, and IL collected the samples. CD carried out the biochemical analyses. HH and MR determined cuticular transpiration. P-HL and AE were in charge of skin topography determinations. AR was responsible of the experimental orchards and the physicochemical characterization of fruit samples. TC and FG contributed to sample processing. CD and VM did the microscopy observations. CD and IL conceptualized and wrote the manuscript. All the Authors revised and approved the manuscript.This work was funded by grant AGL2015-64235-R from the Plan Nacional de I+D, Ministry of Education and Science, Spain. CD is the recipient of a predoctoral scholarship granted by the Universitat de Lleida. PH-L was recipient of a PhD scholarship from Zespri™ International. HH was supported by an Oversea Study Program, Guangzhou Elite Project, China.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.


Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality and morbidity in low- and middle-income countries (LMICs) including Bangladesh. But no systematic review has been carried out in Bangladesh, which portraits the burden of COPD and its risk factors. Therefore, this systematic review was conducted to find out the prevalence and risk factors of COPD in Bangladesh. We searched PubMed, Google Scholar, Popline, and Banglajol from January 1, 1972 to April 30, 2017. We included studies that reported the prevalence and/or risk factors of COPD among Bangladeshi people. Two researchers independently searched and screened all the articles and extracted data from nine eligible studies. The whole process was verified by another researcher. Quality assessment was performed using a checklist adopted from published articles on quality assessment guidelines of observational studies. Discrepancies were resolved by consensus. Data analysis was done thematically. The pooled COPD prevalence among Bangladeshi adult was 12.5%  using the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria and 11.9%  using the lower limit of the normality (LLN) criteria. The prevalence was higher among males, low socio-economic group, rural residents, and biomass fuel users. Tobacco consumption, exposure to biomass fuel, old age, and history of asthma were identified as major risk factors of COPD. COPD prevalence is high in Bangladesh. It is a timely need for the policy-makers and public health professionals to take pertinent steps for prevention and control of COPD in Bangladesh. Chronic obstructive pulmonary disease (COPD) is defined by the American Thoracic Society (ATS) as a progressive and partially reversible disease of respiratory tract featured by the limitation of airflow that takes place as a result of chronic bronchitis or emphysema . There aCOPD is a global public health concern of the 21st century. In 2005, worldwide, 5% deaths were attributable to COPD . AccordiIn Bangladesh, a number of studies have been conducted focusing on two major NCDs, namely diabetes and hypertension -12. But Materials and methodsSearch StrategyWe systematically searched international  as well as national (Banglajol) databases to identify all potential publications on COPD in Bangladesh. We used both medical sub-headings and plain text for the following keywords: “epidemiology”, “prevalence”, “burden”, “risk factors”, “etiology”, “causality”, “aetiology”, “chronic obstructive pulmonary disease”, “COPD”, “pulmonary emphysema”, “emphysema”, “chronic bronchitis”, “bronchitis”, “obstructive lung disease”, “Bangladesh”. Using these keywords along with a Boolean operator, we prepared the global search term to identify the relevant publications. The team also manually searched the bibliography of initially selected articles  to identify more studies related to COPD. We used ‘Preferred Reporting Items for Systematic Review and Meta-Analysis’ (PRISMA) guideline for reporting this systematic review .Inclusion and Exclusion CriteriaThis systematic review included published articles based on the following criteria: a) study reported data from Bangladesh; b) study published between 1st January 1972  and 30th April 2017; c) study reported prevalence of COPD or emphysema or chronic bronchitis or obstructive lung disease; d) study reported single or multiple risk factors of COPD or emphysema or chronic bronchitis or obstructive lung disease; e) quantitative study; f) study published in the English language. However, studies were excluded based on the following criteria: a) study did not report data from Bangladesh; b) study published in other languages than English; c) qualitative study; and d) conference proceedings, book chapters, and editorials. At first, two researchers searched and screened all the articles individually. Another researcher critically reviewed the overall search and screening process to ensure the consistency. Finally, the full text of selected publications was assessed for eligibility by all three researchers. During the initial screening, it was not clear whether studies that focused on COPD or other respiratory diseases were included. However, while assessing the full text of these articles, any study that did not focus on COPD or obstructive lung disease was excluded. Any discrepancies during this process were resolved by group consensus.Quality AppraisalThree researchers independently determined the risk of bias of included studies. This study used a quality assessment checklist to assess the quality of the included studies. This checklist was prepared based on the criteria described in ‘Strengthening the Reporting of Observational studies in Epidemiology (STROBE)’ and other published methodological studies -16. The 1. Whether the study objective was clearly defined?2. Whether the sampling technique was random or non-random?3. Whether adequate sample size was used based on sample size calculation?4. Whether COPD was measured objectively?5. Whether COPD was measured based on any international criteria?If the study objective was clearly defined, we scored it as “1”, otherwise “0”. If the included study used random sampling, we scored it as “1”; however, if the included study used non-random sampling or sampling technique was not clearly mentioned, we scored it as “0”. If the sample size calculation was clearly described and required sample was obtained or, if at least 384 samples were obtained, we scored it as “1”, otherwise “0”. If the researchers measured COPD objectively (using spirometry), we scored it as “1”, but, if they measured COPD subjectively (self-reported or symptom-based) or did not clearly mention how COPD was diagnosed, we scored it as “0”. If they measured COPD based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria, we scored it as “2”; if they used any other international criteria, we scored it as “1”; and if it did not mention any criteria, we scored it as “0”. Later, the number for each study was added to obtain the final score. The maximum score was 6. If any study gets 6, we considered it as “high quality” study, scores 4 and 5 indicate “moderate quality”, and the lowest scores 0, 1, 2, and 3 indicate that the study was of "poor quality". All the discrepancies that arose during the quality assessment process were solved by the group consensus.Data ExtractionA data extraction form was created in excel to conduct this review. This form included (a) title, (b) journal name, (c) name of authors, (d) publication year, (e) year of data collection, (f) study objective, (g) study setting , (h) study design, (i) sampling strategy (random/non-random), (j) sample size, (k) study population, (l) outcome assessment (objective/subjective), (m), diagnostic criteria for COPD, (n) prevalence , (o) prevalence , (p) risk factors, (q) authors’ conclusion, and (r) researchers’ observation. Two researchers independently extracted data using the data extraction form. Another researcher cross-checked and critically reviewed both the files extracted by the individual researcher. Any forms of dispute arose during the data extraction process were resolved by the group consensus. Data analysis was done thematically.ResultsSearch ResultInitially, 1,639 articles were identified through a database search. Following duplicate removal, title and abstract screening, 24 articles remained for full-text assessment. The full text of these 24 studies was critically reviewed, of which nine studies met the eligibility criteria. Therefore, finally, nine studies were included in this systematic review. The overall process was done using the PRISMA flow diagram  ,24. ThesRisk FactorsFor this review, we considered only high- and moderate-quality studies to identify the risk factors -19,21-22Tobacco Consumptionp < 0.001) and former smokers  were at a higher risk to experience COPD after adjusting potential confounders [p < 0.001) [Tobacco consumption, in the form of smoking or chewing, was found associated with an increased prevalence of COPD in four studies ,21-22,24founders . Biswas Biomass Fuel Usep = 0.047) [A significant association was reported with the development of COPD and the use of biomass fuel for cooking purpose in three studies ,19,22. T= 0.047) .Old Agep < 0.001; 60 to 69 years, OR: 4.7, 95% CI, 3.5-6.4; p < 0.001) [Old age was reported as a risk factor for COPD by two studies ,24. Alam< 0.001) .History of Asthmap < 0.001) [A high-quality study reported that individuals with a history of asthma had seven times higher probability of developing COPD, even after adjusting for smoking history and other potential confounders  .OthersMale gender, low socio-economic status, low body mass index (BMI), and long-time exposure to the hazardous environment as a result of longer employment history (helpers of human haulers) were also identified as risk factors of COPD in some studies; however, evidence of statistical significance was not clearly reported for these factors.DiscussionTo the best of our knowledge, this is the first systematic review that reported the prevalence and risk factors of COPD in Bangladesh. Although several prevalence studies have been conducted on COPD in Bangladesh, there is a scarcity of large-scale studies like nationwide COPD survey. This systematic review reported that the overall prevalence of COPD among Bangladeshi adults was 12.5% using the GOLD criteria. This finding is in accordance with the finding of a recent systematic review on the global and regional prevalence of COPD where it was reported that globally 11.7% individuals are currently suffering from COPD -28. COPDWe were not able to find any study that could provide strong evidence on risk factors of COPD using longitudinal study design (cohort or case-control) or nationally representative sample. This result brings attention to the lack of availability of good-quality NCD-related data in developing countries as stated in a previous study . Along wHowever, it was revealed from the existing literature that there is a strong association between tobacco use and COPD. Tobacco consumption, especially smoking, is established as one of the most important risk factors of COPD in all over the world . StudiesOur study identified biomass fuel use as a risk factor for developing COPD specifically among rural women. This finding is consistent with the result of other studies, which reported that biomass smoke exposure significantly increases the risk of developing COPD, especially in women ,38. A reOlder age was identified as an important risk factor for developing COPD by some of the studies. It is well established that COPD prevalence, mortality, and morbidity increase with age . Like otCurrently, 12.5% of Bangladeshi adults are suffering from COPD in Bangladesh. This prevalence is much higher among tobacco users, biomass fuel users, and elderly people. As COPD is accountable for a substantial extent of mortality and morbidity, it is a timely need for policy makers and public health professionals of Bangladesh to prioritize COPD as an important health issue. Population awareness should be raised on health impact and economic burden of COPD by using mass media and including these issues in the national curriculum. Arranging policy dialogue is also imperative for promoting multi-sectoral response to combat COPD burden in Bangladesh.


More than a decade ago, the World Health Organization’s (WHO) age-friendly cities and communities program was established to guide communities in preparing for rapid population aging. WHO identified eight domains of livability that influence health and wellbeing of older adults—housing, one of the eight domains is the focus of this study. Partnering with our local Age Friendly initiative, we examined housing preferences and values for residents of a mid-sized urban community. A cross-sectional survey was administered online via Qualtrics and through face-to-face data collection across the community . Participants aged 30+ completed a researcher-devised survey about housing-related preferences and values. Data were analyzed using descriptive and inferential statistics. Of our 518 respondents, half were 60+ and most were women (73.2%). Younger respondents (age 30-59) were more likely to be married or living with a partner (73.7%). There was little difference in housing preferences between older and younger respondents or among those with varying financial means. Analysis revealed the top five housing considerations were: “safety” (78.8%), “affordability” (73.2%), “privacy” (51.9%), “proximity to services I frequently use” (50.6%), and “accessibility” (42.4%). An unexpected finding was that almost 50% of older respondents expressed a willingness to share a home with a roommate. Data suggests that city planners and property developers should prioritize these preferences when planning for the housing related needs of older residents. Alternative housing models, such as cohousing, should be explored further. Future research should examine interpretation of such housing preferences .