The study of biodiversity spatial patterns along ecological gradients can serve to elucidate factors shaping biological community structure and predict ecosystem responses to global change. Ant assemblages are particularly interesting as study cases, because ant species play a key role in many ecosystem processes and have frequently been identified as useful bioindicators.Here we analyzed the response of ant species richness and assemblage composition across elevational gradients in Mediterranean grasslands and subsequently tested whether these responses were stable spatially and temporally. We sampled ant assemblages in two years  in two mountain ranges  in Central Spain, along an elevational gradient ranging from 685 to 2390 m a.s.l.Jackknife estimates of ant species richness ranged from three to 18.5 species and exhibited a hump-shaped relationship with elevation that peaked at mid-range values (1100–1400 m). This pattern was transferable temporally and spatially. Elevation was related to ant assemblage composition and facilitated separation of higher elevation assemblages (> 1700 m) from the remaining lower elevation species groups. Ant assemblages were nested; therefore species assemblages with a decreased number of species were a subset of the richer assemblages, although species turnover was more important than pure nestedness in all surveys. The degree of nestedness changed non-linearly as a cubic polynomial with elevation. These assembly patterns coincided more clearly over time than between the two study regions.We suggest double environmental stressors typical of Mediterranean mountains explained species richness patterns: drought at low elevations and cold temperatures at high elevations likely constrained richness at both extremes of elevational gradients. The fact that species turnover showed a dominant role over pure nestedness suggested current ant assemblages were context-dependent and highly vulnerable to global change, which threatens the conservation of present day native ant communities, particularly at high elevations. Predicting the response of biodiversity to the main drivers of global change has become a primary goal of modern ecology –3. ConseAnts are diverse and ubiquitous, exhibit strong interspecific interactions, and perform many functional processes in ecosystems ; therefoet al. [Previous research on ant diversity documented variable responses to altitudinal gradients, which showed a certain dependency on macroclimate. For instance, studies reporting monotonic decreases in species richness with elevation were frequent in temperate mountains –21, whilet al.  indicateet al. , 27, altet al. , 29. Theet al. , 31 or tet al. . Howeveret al. –21 and wet al. , 34 as det al. , 35–37.et al. [Understanding how ant richness responds to altitudinal gradients is not sufficient to predict how environmental changes (e.g. rising temperatures) will affect species geographic ranges, because knowledge on species composition patterns is also required. Species assemblages and their distributions along ecological and geographical gradients result from species tolerances to climatic (abiotic) factors and biotic interactions, which interplay with niche evolution to determine community composition –40. Kodret al.  acknowleet al. –50.In the present study, we explored patterns of ant species diversity and community composition in Mediterranean mountain grasslands, integrating spatio-temporal transferability. Specifically, we addressed the following questions: (1) what is the relationship between ant species richness and elevation; (2) how does elevation affect ant species composition; (3) what are the relative contributions of spatial turnover and nestedness in ant community composition; and (4) are our findings transferable spatially and temporally?2 = 0.80, F1,16 = 62.23, P < 0.0001 for maximum temperatures and R2 = 0.96, F1,16 = 371.2, P < 0.0001 for minimum temperatures; own data based on temperature loggers buried at ground level during the sampling period in 2014, We studied grassland ant communities in two mountains in the larger Sistema Central range, Sierra de Guadarrama  and Sierra de la Serrota . The two mountains are ~ 100 km apart with similar climatic, geologic, and biotic characteristics. Mean annual temperatures range from ~ 14 ºC at 600 m to ~ 4 ºC at the summits, and mean annual rainfall ranges from 550 mm to 1500 mm, with severe summer drought . Soil teSample sites were interspersed along the elevational gradient trying to keep a difference in elevation of 100 m between consecutive sites. The closer sampling sites were at least 1 km apart. Sites faced southeast to southwest, had gentle slopes (< 5%), and were not heavily grazed nor affected by anthropogenic disturbance . With this sampling restrictions, we strived to control the heterogeneity of sampling units by considering similar annual-dominated dry grasslands. We sampled 18 grasslands in 2014 (in Guadarrama range) and 12 in 2015  to the hemi-matrices of binary Bray-Curtis distances among grasslands derived from species occurrences in pitfall traps.To assess assemblage nestedness among grasslands surveyed on each mountain and year we calculated the matrix temperature (T)  and the The significance of nestedness indices was estimated by comparison with suitable binary null models, which were simulated by randomizing the original matrix transformed to presence/absence binary data. Two null models were built to encompass the range from maximally liberal (equitable) to maximally conservative (constrained) null models . The firThe relative degree of nestedness, i.e. how much a grassland was nested within the set of sampled grasslands in the same geographic area and time period, was evaluated by a nestedness rank, according to T. Poorer grasslands, i.e. those having lower species diversity which were a subset of richer grasslands, were assigned higher ranks. These ranks were estimated as the ordinate in nestedness plots built with the T index, which were calculated as (k—0.5)/n for k = 1…, n rows /18 = 0.97).The shape of the relationship between nestedness and elevation during the 2014 sampling in Guadarrama (n = 18) was explored using GAM with Gaussian errors and a thin plate regression spline for elevation. This model was validated using Guadarrama 2015 data . A low mean absolute error of predicted ranks would suggest the nestedness rank of plots did not vary between years. Similarly, the model built with Guadarrama 2014 sampling data was validated using the Serrota 2015 data . A low mean absolute error of predicted ranks would suggest the nestedness rank of plots did not vary between regions.SOR), Simpson-based multiple-site dissimilarity (βSIM), and nestedness-resultant multiple-site dissimilarity (βNES). βSIM accounts for spatial turnover and βNES integrates dissimilarity due to nestedness, while βSOR expresses the total dissimilarity between communities and equals the sum of βSIM and βNES [P-values were estimated through the equiprobable (‘r00’) null model .The contributions of spatial turnover and nestedness to the distribution pattern in Guadarrama 2014 data were calculated using three beta diversity indices: Sørensen-based multiple-site dissimilarity  and specialized packages vegan (v. 2.5–1) , and betWe detected 37 species, all recorded in Guadarrama  and 20 species in Serrota . A total of 15 species were common among the three surveys .P < 0.0001, D2 = 83.8%, Jackknife estimates of ant species richness in Guadarrama 2014 ranged from three (SE = 0) species in most of the highest grasslands to 18.5 (SE = 2.6) at intermediate elevations . Estimat2 between Bray-Curtis and NMDS distances = 0.95). This scaling separated grasslands roughly according to elevation, but not regions or years and suggested an elevation threshold of 1700 m a.s.l. was a suitable elevation to distinguish the two groups . This relationship among beta diversity components did not vary among surveys , transferred adequately to the 2015 Guadarrama dataset, and also to the spatial validation dataset in the 2015 Serrota range. Despite geometric constraints as mid-domain effects were described to explain these curve types , more reet al. [Most studies on ant communities reported species richness positively responded to temperature , 69, 70,et al.  reportedet al. , 35–37. et al. , 74. In et al. , so maxiAs anticipated, elevation also affected the taxonomic composition of ant communities. We observed a ca. 1700 m threshold separating high mountain ant species communities from those along the lower elevational gradient. This threshold seemed consistent among regions and between years. Furthermore, although nestedness was a significant component of beta diversity, most elevational shifts among communities were attributable to species turnover. This result suggested the communities with lowest ant species diversity (high mountain communities) were also the most unique in a regional context. The singularity of high elevation ant communities was previously observed in different biomes , 34, 35,http://www.madrid.org/nomecalles/Inicio.icm). Therefore, we assume all of them are equally accessible from the regional species pool. However, we cannot disregard other time-related mechanisms, which might explain community composition, such as priority effects favoring one or more species, depending on their relative order of arrival to a site [The examination of community nestedness suggested current ant assemblages are context-dependent and likely highly vulnerable to global warming and other anthropogenic changes. This is because, first, species turnover exhibited a dominant role over pure nestedness, which indicated different and characteristic groups of ant species occurred along an elevational gradient, and at the very least, ant species community composition was distinct at high elevations (> 1700 m) . We wanto a site , or metao a site .Overall, species taxonomic composition varied non-linearly with elevation. The insufficiency of the sampling mechanism emphasizes that ant assemblages are highly vulnerable to global changes on climate and anthropogenic impacts, which might affect species at the habitat and/or community level. It is unlikely species’ range shifts that track environmental change will maintain current ant species community composition.S1 Tablehttp://opengis.uab.es/wms/iberia/), and maximum and minimum soil) temperatures during sampling periods measured with data loggers buried at ground level . Occurrence of the species in the plots is given in the following columns.IDPlot: plot identification. Survey: G2014 for Guadarrama range in 2014, G2015 for Guadarrama range in 2015 and S2015 for Serrota range in 2015. For each sampling plot we give the elevation (in m), latitude and longitude , mean annual rainfall  and air temperature  according to the Atlas Climático Digital de la Península Ibérica ((XLSX)Click here for additional data file.S2 TableP-values estimated by equiprobable (‘r00’) binary null models and proportional resampling (‘quasiswap’). Significant P-values (P < 0.05) are in bold face. Fill: matrix fill (sum of 1s/sum of cells).Guadarrama range  and Serrota range . Indices are Nestedness metric based on Overlap and Decreasing Fill (NODF), NODFr for rows  and NODFc for columns  and matrix temperature (T). Numbers under headings for indices provide index values followed by (PDF)Click here for additional data file.S3 TableSIM) and the nestedness components (βNES) of beta diversity, and sum of both values (βSOR) for dry grassland ant assemblages in all study areas in central Spain. G2014: Guadarrama range 2014; G2015: Guadarrama range 2015; S2015: Serrota range 2015. P-values estimated by equiprobable binary null models (‘r00’) are given between parentheses. Significant P-values (P < 0.05) are in bold face.Multiple-site dissimilarities accounting for the spatial turnover (β(PDF)Click here for additional data file.S1 FigGeneralized additive model (GAM) of nestedness ranks (based on nestedness temperature index) on elevation in ant assemblages from Guadarrama range  fit to Guadarrama 2014 data and validated with Guadarrama 2015 data.(TIF)Click here for additional data file.S2 FigGeneralized additive model (GAM) of nestedness ranks (based on nestedness temperature index) on elevation in ant assemblages from Guadarrama range  fit to Guadarrama 2014 data and validated with Serrota 2015 data.(TIF)Click here for additional data file.S3 Fig2 = 94%), maximum (soil) temperature , minimum (soil) temperature , and mean annual rainfall . Regressions built for Guadarrama 2014 data . Data for Guadarrama 2015 (dark grey circles) and Serrota 2015 (white circles) are depicted as a reference. Temperatures measured in Celsius degrees and elevation in meters. Annual climatic data from the Digital Climatic Atlas of the Iberian Peninsula (http://opengis.uab.es/wms/iberia/). Soil temperatures measured for the sampling period (a week) with data loggers buried at ground level. Note that 2014 was cooler than 2015, in spite of which models for richness and nestedness built in 2014 validated well with data from 2015.Linear regressions between elevation and mean annual (air) temperature Click here for additional data file.


The aim of the study was to investigate the outcomes of temporal Zone II-sparing laser photocoagulation combined with intravitreal bevacizumab (IVB) in patients with Type 1 retinopathy of prematurity (ROP) in Zone I.  Medical records of 74 eyes of 37 infants were analysed. Only infants with Type 1 ROP in Zone I were included. Thirty-two eyes were treated with temporal-sparing laser + IVB. Both Zone I and temporal Zone II were spared to minimise potential visual field loss. Forty-two eyes were treated with laser alone conventionally. Early treatment outcomes, late complications, and refractive errors were analysed. p=0.011) and retreatment was required less  than in the laser-alone group. Retinal/preretinal haemorrhages occurred more often in the laser-alone group . Normal development of temporal retinal vessels was also observed in twelve eyes in the combined treatment group. No differences in late complications or refractive errors were observed between the groups.  The mean gestational age and birth weight of the enrolled patients were 25.7 ± 2.5 weeks and 798.8 ± 440.2 g, respectively. In the combined treatment group, plus sign regression was achieved faster (12.1 ± 6.2 days vs. 25.6 ± 21.3 days,  Temporal Zone II-sparing laser treatment combined with IVB showed good early treatment outcome and temporal retinal vessels development. Retinopathy of prematurity (ROP), a vasoproliferative disease associated with underdevelopment of the retina, is a leading cause of severe impairment of visual function in childhood . Mild caHowever, confluent laser scars on the retinal periphery may cause visual field restriction after the treatment. The area where laser photocoagulation was performed becomes atrophic, eventually restricting the patient's peripheral visual field. Several efforts have been made to reduce the area of laser photocoagulation using the recently highlighted intravitreal bevacizumab (IVB) treatment –8. But ITo minimise the visual field restriction caused by laser treatment, we saved a larger area in Type 1 ROP, including temporal Zone II, to maximise the combination effect compared with that in previously reported studies, which spared Zone I or the posterior pole. The functional and long-term structural outcomes were analysed.This study, performed retrospectively, followed the tenets of the Helsinki declaration and was approved by the Institutional Review Board of Asan Medical Center. Informed consent was obtained from all parents after providing an explanation of laser photocoagulation and off-label use of bevacizumab, if indicated. Potential risks and side effects of IVB were explained. After thorough consideration, all parents agreed to the treatment involving laser photocoagulation with/without IVB.The medical records of preterm infants who received laser photocoagulation alone or combined with IVB, and had been followed for at least 6 months in the period from Mar 2013 to Sep 2016 at Asan Medical Center, Seoul, Korea, were collected. Peripheral retinal laser photocoagulation was indicated when the fundus examination revealed Type 1 ROP in Zone I. Only infants with ROP in Zone I were included in the current study. Infants who received laser treatment due to Zone II ROP were excluded.Two distinctive patterns of treatment were performed . In one 2 therapy period were collected. A series of comorbid systemic diseases associated with ROP, such as necrotising enterocolitis, respiratory distress syndrome, ligated patent ductus arteriosus, bronchopulmonary dysplasia, sepsis, intrauterine growth restriction, poor weight gain, hydrocephalus, and intraventricular haemorrhage, were also documented.Before treatment and during follow-ups, the fundus was examined by indirect ophthalmoscopy and wide-field photography, if indicated. A RetCam  was employed to record the severity of ROP, extent of the laser scar, regression of new vessels, and development of peripheral retinal vessels after treatment. Patient baseline characteristics such as gestational age at birth, birth weight, multiplicity, APGAR score, ventilator care, and OEarly treatment outcomes/complications as well as late structural/functional complications were analysed based on fundus photographs and medical records. At 6–12 months after treatment, all treated infants underwent measurement of refractive errors by manual cycloplegic refraction performed by a skilled paediatric ophthalmologist. The results were recorded as measurements of spherical and cylinder astigmatism, and the spherical equivalent. These were also divided into the categories of emmetropia, mild (0–3 D), moderate (3–6 D), or high (>6 D) myopia, and hyperopia.t-, Fisher's exact, and Pearson's Chi-square tests were used. p values < 0.05 were considered to be statistically significant.Statistical analysis was performed using SPSS for Windows . The independent A total of 74 eyes of 37 infants were analysed. Thirty-two eyes of 16 infants were treated with temporal-sparing LIO + IVB, and 42 eyes of 21 infants were treated with LIO alone. The mean follow-up period was 19.7 ± 9.0 months. Out of the 32 eyes treated with temporal-sparing LIO + IVB, 10 (31.3%) showed AP-ROP. With the exception of the follow-up period being shorter in the temporal-sparing LIO + IVB group, there were no statistically significant differences in baseline characteristics and comorbid conditions between the two groups .p=0.011). Ten eyes in the LIO-alone group required retreatment after a mean 25 days from the initial treatment, while all eyes in the temporal-sparing LIO + IVB group were stabilised after the first treatment (p=0.004). Among the eyes requiring retreatment, 6 eyes were treated with LIO + IVB and 4 eyes were treated with additional LIO only. Retinal/preretinal haemorrhages were found more frequently in the LIO-alone group . No reports of endophthalmitis or adverse systemic safety issues related to IVB were found. Among late complications, structural complications like macular dragging or optic disc atrophy tended to occur frequently in patients treated with LIO alone. Functional complications such as strabismus or nystagmus showed no tendency between the groups. Refractive error measurements were performed 10.4 ± 5.0 months after treatment. No statistically significant differences were found between the two groups in the spherical equivalent (p=0.293) and refractive error (p=0.130) distributions.Treatment outcomes and complications are shown in Among the 32 eyes treated with temporal-sparing LIO + IVB, 24 eyes of 12 infants were able to analyse the retinal periphery with wide-field fundus photography after 1 month of treatment. The others were not able to do so because of poor peripheral visualisation. Normal development of temporal retinal vessels was observed in the 12 eyes (50%) of 6 infants. Vessel development progressed close to the laser scar, and the avascular area was barely visible . In the In the present study, the functional and structural outcomes in patients with Type 1 ROP were compared between those who underwent temporal Zone II-sparing laser photocoagulation combined with IVB and conventional laser photocoagulation alone. Wider preservation of the retina after regression of ROP was observed in eyes treated with temporal-sparing laser therapy combined with IVB compared with those treated with laser photocoagulation alone. Eyes undergoing the combined treatment required retreatment less frequently and showed faster regression of plus sign compared with eyes treated with LIO alone. Refractive changes and the incidence of long-term complications showed no differences between the two treatments. In 50% of eyes treated with the combined therapy, normal development of the peripheral retina was observed after complete regression of ROP.Temporal Zone II area was chosen to be spared in the current study. Saving the nasal area would be more beneficial in saving the temporal visual field in binocular vision. However, as shown in Because retinal atrophy and photoreceptor damage are inherent sequelae of laser photocoagulation, IVB monotherapy might be a good option to achieve ROP regression with a good anatomical outcome. However, several findings of delayed reactivation were reported in IVB monotherapy, sometimes as late as 3 years posttreatment , 12–14. p=0.065).As in previous reports , 7, 8, cIn spite of the advantages of IVB use in ROP treatment, delayed reactivation is a major concern. However, there was no reactivation in both groups followed up for 103.1 ± 43.6 weeks PMA in the current study. The follow-up period was shorter in the combined treatment group (84.4 ± 36.9 vs. 117.4 ± 43.6 weeks). Nonetheless, given that the mean time to recurrence was 19.2 ± 8.6 weeks after bevacizumab treatment in the BEAT-ROP study, our follow-up period was long enough to analyse long-term efficacy . TreatmeAfter treatment, there was no difference in mean spherical equivalent between the two groups at 6 months. Over 80% of the infants showed emmetropia or mild myopia. In a meta-analysis of 11 studies of a total of 378 eyes with intravitreal anti-VEGF treatment in ROP, the average spherical equivalent refractive error reported after anti-VEGF monotherapy ranged from +0.75 D to −3.75 D . In the As demonstrated in There have been reports that performing laser treatment in combination with bevacizumab injection increases the systemic levels of the drug, which decreases the levels of systemic VEGF and could possibly be associated with decreased neurodevelopmental outcomes , 27. TheOur study has several limitations. Besides the retrospective nature of the study design and small sample size, the major limitation is that multiple systemic and environmental factors influencing the treatment outcome, such as oxygen supply, nutrient concentration, intrauterine environment, and perinatal issues, may have been different between the treatment groups. We confirmed that major systemic risks were not significantly different between the two groups in Despite its limitations, the present study is valuable in that we demonstrated good structural and functional outcomes in minimally threatened visual fields and visualised them with wide-field fundus photography and fluorescein angiography. The previous studies had limitations in treatment standardisation or diagnosis because of a lack of images. We adopted a wide-field fundus-imaging system to determine the ROP stage, plus sign regression, retinal haemorrhages, extent of the avascular area, laser ablated area, and peripheral vessel advancement. This visualisation allowed us to standardise treatment and perform an appropriate comparison.Temporal-sparing laser treatment combined with intravitreal bevacizumab showed better early treatment outcomes compared with conventional laser treatment. There were no differences between the groups in long-term structural and functional complications, including delayed reactivation. Because of the preservation of the viable retina with peripheral vessel development and the low reactivation rate, temporal Zone II-sparing laser photocoagulation combined with intravitreal bevacizumab could be a good choice for Type 1 ROP patients to achieve complete regression while minimising peripheral field defects.


Mentha spicata has been investigated as a source of essential oil (EO) and pharmaceuticals. The quantity and composition of EO from M. spicata cultivated in Palestine were analyzed seasonally over a three-year period. A significantly higher EO content was produced in summer and fall months (2.54–2.79%). Chemical analysis of EO revealed 31 compounds with oxygenated monoterpenes (90%) as the most abundant components followed by sesquiterpene and monoterpene hydrocarbons . M spicata can be characterized as a carvone chemotype (65%). EO and carvone have shown strong inhibitory activities against the principal enzymes associated with Alzheimer's disease (AD) and overweight diseases (cholinesterase and porcine pancreatic lipase) and also shown strong antidermatophytic activity against Microsporum canis, Trichophyton rubrum, T. mentagrophytes, and Epidermophyton floccosum. The pancreatic lipase inhibition and the synergism showed the potential activity of M. spicata EO and carvone and that their combinations with standard drugs can be useful for the treatment of obesity and overweight. The results also demonstrated that, in addition to their significant inhibitory activity against biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA), M. spicata EO and carvone had a strong inhibitory effect on metabolic activity and biomass of the preformed biofilm. The current study supports the utilization of M. spicata EO as a traditional medicine and opens perceptions to find more potent substances in the EO for the management of obesity, AD, and dermatophytosis and for combating drug-resistant bacterial infections.In the present study, the medicinal aromatic plant  Mentha spicata L.) is a cultivated perennial, rhizomatous, and glabrous plant of the Lamiaceae family and is one of the most common herbs grown commercially worldwide including Palestine. It has been considered one of the most important essential oil crops [Spearmint -carvone, which offers spearmint its unique smooth characteristic scent [M. spicata oil also contains noteworthy concentrations of limonene, dihydrocarvone, and 1,8-cineole [The most dominant constituent found in spearmint oil worldwide is R-, the fresh and dried spearmint EOs have been used for the treatment of obesity, dementia, hypertension, abdominal pain, digestive disorders, muscle spasm, flatulence, headache, fever, menstrual pain, asthma, cough, cold, depression, and others , 28–31. M. spicata EO as a folk medicine in TAPHM and open perspectives to find more effective substances from plant origin for the treatment of important human diseases.The objectives of this study were to (1) study the effect of seasons on the EO content and composition of spearmint cultivated in Palestine; (2) investigate the biological activities of the EOs focusing on their potential benefits in the treatment of obesity, Alzheimer's disease (AD), dermatophytosis, and antibiotic-resistant infections; and (3) verify the use of Samples of aerial parts were collected from a spearmint field in the BERC Experimental Station, Til, Nablus, Palestine, during three consecutive years (2015–2017). EOs were separated from the fresh aerial parts by hydrodistillation using a modified Clevenger apparatus following the conditions reported in .Determination of EO composition was performed using gas chromatography-mass spectrometry (GC-MS) following the conditions reported in , 27. TheMicrosporum canis, Trichophyton mentagrophytes, T. rubrum, and Epidermophyton floccosum, using the poisoned food technique as described by Gakuubi et al. [50) was assessed using Excel.Essential oil and its main component, carvone, were tested for their antidermatophytic activity against four dermatophyte species: i et al.  and Mohai et al. . EO and Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined following previously reported assays , 37.MRSA-BERC #01, was used to assess the potential of M. spicata EO and carvone to prevent biofilm formation. Aliquots of twofold serial dilutions (100 μL) of EO and carvone were prepared in tryptic soy peptone supplemented with the 0.2% glucose (TSBGlc) medium and added to the 96-well flat-bottom microtiter plate, with final concentrations ranging from 0.078 to 5 μL/mL. Bacterial suspensions  were then added to the plate. The TSBGlc medium was employed as a negative control. TSBGlc without essential oil was used as a blank [MRSA-BERC #01 to form the biofilm in the presence of carvone or EO was then determined using the 2,3,5-triphenyl-tetrazolium chloride (TTC) reduction and crystal violet (CV) assays as mentioned below.A biofilm-producing bacterial strain,  a blank , 39. AftM. spicata EO and carvone on established biofilms was tested following Sabaeifard et al. [μL of the bacterial suspension (1 × 106 CFU/mL) was added to each well in the microtiter plate and incubated for 24 h at 37°C to allow biofilm formation. After incubation, 100 μL of different concentrations of the EO was added to each well to give final concentrations ranging between 0.078 and 5 μL/mL. TSBGlc was used as the negative control and the blank. The plates were incubated for 24 h after incubation, the planktonic cells were removed by gently aspirating the cell suspension, and the plate was washed twice with PBS. Extra moisture was removed by tapping the plate on a sterile tissue paper. The microplates were left to dry in a laminar flow desk for 15 min in the upside-down position. The activity of EOs or carvone on the preformed biofilm was subsequently determined using the TTC reduction assay and CV assay [The effect of d et al. . In brieCV assay –45 as meμL of TSBGlc were transferred to each well, and the plates were then kept in the dark at 37°C for 4 h. Following incubation, the TTC solution was aspirated, and the plate was air-dried. Attached red formazan was dissolved in a mixture of acetone (20%) and ethanol (80%). The dissolved red formazan was moved to a new flat-bottom microplate, and absorbance was measured at 500 nm using Epoch Microplate Spectrophotometer (BioTek) [Biofilm metabolic activity was measured using the TTC assay following Sabaeifard et al. . In brie(BioTek) –45.The effect of EOs on the total biofilm biomass was measured by the CV staining protocol as described by Peeters et al. . In brieAnticholinesterase activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were measured spectrophotometrically using the NA-FB method following Ali-Shtayeh et al. . NeostigM. spicata EO to inhibit PPL was determined based on a spectrophotometric analysis as described previously [The ability of the eviously . OrlistaM. spicata EO/orlistat and carvone/orlistat was carried out. The evaluated concentrations were in the range of 7 dilutions with final concentrations of 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 μL/mL for EO and carvone and 0.063, 0.031, 0.016, 0.008, 0.004, 0.002, and 0.001 μg/mL for orlistat.The interactions of EO or carvone with orlistat were determined using a checkerboard method as described by Nikkhah et al. . A two-dA) = MIC50 (A) in combination/MIC50 (A) alone and FIC of compound B (FICB) = MIC50 (B) in combination/IC50 (B) alone, where A is EO or carvone and B is orlistat.The fractional inhibitory concentration (FIC) was calculated for the first well in each row of the microtiter plate containing the combination with lipase percentage of inhibition more than 50%, given as follows: FIC of compound A (FICThe FIC index (FICI) was calculated as follows:50 is defined in relation to the independent MIC50. A concave curve represents synergy, whereas convex curve and straight line indicate antagonistic and additive effects, respectively [The obtained results were inferred as follows: synergistic effect (FICI ≤ 0.5), additive effect (0.5 < FICI ≤ 1), no interactive effect (1 < FICI ≤ 4), and antagonistic effect (FICI > 4) , 48. Theectively .Averages and standard deviations for three simultaneous assays were tested using standard statistical methods. The relationships between the studied phytochemical compositions were expressed by Pearson's correlation coefficient (SPSS software 21).M. spicata varied quantitatively according to seasons. M. spicata demonstrated a significantly higher (p ≤ 0.05) EO content in summer and fall months when the plants were in full bloom  than that in winter and spring months when the plants have reached the end of their growing cycle (0.87–1.94%)  . The resn et al.  who alsoll bloom , 27.M. spicata EOs. Eleven of the components  show mean percentages more than 1%, which represent 90% of the total oils  can be considered carvone chemotypes. Our results are in consistent with those of other scientists who also found the EO of M. spicata to be of the carvone chemotype [The phytochemical analysis results of the EOs are detailed in tal oils . Three ohemotype , 50, 51.M. spicata plants (The percentage of carvone in the EOs of a plants  increaser = −0.88), pulegone (r = −0.83), and dihydrocarveol (r = −0.79), indicating very high reverse correlation between carvone and these components. Lower percentages of carvone in EOs were shown during winter and spring months accompanied by an increased concentration of limonene, dihydrocarveol, pulegone, and cis-carveol.The Pearson correlation exhibited significant negative correlation between carvone and other phytochemical components (not presented). Carvone was negatively correlated with cis-carveol ) comparable to that of EO yield with the highest OM percentages (94.7–95.3%) concurring with warmer seasons  characterized by higher plant growth rates when the plants were in full bloom and with the lowest OM percentages (84.8–86.4%) coinciding with cooler weather conditions in winter and early spring characterized by lower plant growth rates when the plants reached the end of their growing season  showed a dose-dependent activity against the test dermatophytes , the oil and carvone were generally more active on the mycelia growth of T. mentagrophytes than other tested dermatophytes; at 0.5 μL/mL, PI = 94.1% and 100% for oil and carvone, respectively  to inhibit or destroy biofilm on a polystyrene surface formed by MRSA.In the current study, we evaluated the ability of several doses of M. spicata EO exhibited a significant inhibitory activity on biomass and metabolic activity of MRSA biofilm formation in a dose-dependent manner (M. spicata EO reduced the number of adherent bacteria (biomass) by 71.3% at 5 μL/mL, and the observed biofilm inhibitory concentration (BIC50) was 0.37 μL/mL  evaluated by the CV assay, M. spicata and carvone showed significant effects on the biomass and oxidative metabolic activity of the preformed biofilm in a dose-dependent manner  and BuChE (IC50 = 35.0 μL/mL) inhibitory activities (50 = 19.01 μL/mL) and BuChE (IC50 = 32.33 μL/mL). It is therefore speculated that the anticholinesterase activity of the major monoterpenoid component (carvone) is accountable for the observed inhibitory effect, of the M. spicata aromatic plant [Several plant species from the Lamiaceae family are used in TAPHM to enhance memory , 31. Thetivities , matchinic plant , 71.M. spicata be further explored for possible useful effects on neurodegenerative disorders, such as AD, utilizing its cholinesterase inhibition activities.According to the results of the current study, it is highly recommended that the EO extracted from the aerial parts of M. spicata EO and its main component (carvone) were assessed for lipid-lowering activity through inhibition of porcine pancreatic lipase (PPL) (M. spicata EO and carvone had a lipase inhibitory concentration (MIC50) of 12.5 μL/mL. The fractional inhibitory concentrations (FICs) of the dual combinations of EO or carvone with orlistat are presented in In the current study, se (PPL) . M. spicM. spicata signifies a rich source of antilipase constituents. Jamous et al. [M. spicata exhibit a strong PPL inhibitory property with an IC50 of 1.19 mg/ml. However, to the best of our knowledge, EO from this plant or its phytochemicals have not been previously studied for their lipase inhibitory activity.In this regard, the PPL inhibitory activities may in part be elucidated by the high content of carvone and other monoterpenes. The results support the view that s et al.  have preMentha spicata may be used for the development of new formulations of nutraceutical products for the management of chronic diseases such as Alzheimer's disease and overweight. M. spicata EO can fulfill the growing demand of industries for constant and good raw natural sources of antidermatophytic, anticholinesterase, and antiobesity agents that could be safer than synthetic drugs. To attain the optimum oil yield with a high quantity and content of OMs (particularly carvone), M. spicata should be extracted when the plants are in full bloom. The strong inhibitory activity of M. spicata EOs on AChE and PPL and antidermatophytic properties may be due to their high concentrations of carvone monoterpenes. The results also showed that, in addition to their significant inhibitory activity against biofilm formation of MRSA, M. spicata EO and carvone had a strong inhibitory effect on metabolic activity and biomass of the preformed biofilm. Besides the biological activities described here, the results support the use of M. spicata EO as a folk medicine. The results also provide further evidence for the utilization of M. spicata EO and its principal constituents as an important source of anticholinesterase, antiobesity, antidermatophytic, and anti-MRSA agents.Because of its high biological activities, EO from


Ganoderma, commonly called reishi (in Japan) or lingzhi (in China), have been used in traditional medicine for thousands of years, and their use has gained interest from pharmaceutical industries in recent years. Globally, the taxonomy of Ganoderma species is chaotic, and the taxon name Ganoderma lucidum has been used for most laccate (shiny) Ganoderma species. However, it is now known that G. lucidum sensu stricto has a limited native distribution in Europe and some parts of China. It is likely that differences in the quality and quantity of medicinally relevant chemicals occur among Ganoderma species. To determine what species are being sold in commercially available products, twenty manufactured products  and seventeen grow your own (GYO) kits labeled as containing G. lucidum were analyzed. DNA was extracted, and the internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (tef1α) were sequenced with specific fungal primers. The majority (93%) of the manufactured reishi products and almost half of the GYO kits were identified as Ganoderma lingzhi. G. lingzhi is native to Asia and is the most widely cultivated and studied taxon for medicinal use. Illumina MiSeq sequencing of the ITS1 region was performed to determine if multiple Ganoderma species were present. None of the manufactured products tested contained G. lucidum sensu stricto, and it was detected in only one GYO kit. G. lingzhi was detected in most products, but other Ganoderma species were also present, including G. applanatum, G. australe, G. gibbosum, G. sessile, and G. sinense. Our results indicate that the content of these products vary and that better labeling is needed to inform consumers before these products are ingested or marketed as medicine. Of the 17 GYO kits tested, 11 kits contained Ganoderma taxa that are not native to the United States. If fruiting bodies of exotic Ganoderma taxa are cultivated, these GYO kits will likely end up in the environment. The effects of these exotic species to natural ecosystems needs investigation.Species of  Ganoderma is a large and diverse, globally distributed genus of wood decay fungi that includes species that cause white rot on a variety of tree species. In addition, practitioners of Eastern traditional medicine have prescribed the use of laccate (shiny) Ganoderma species, commonly referred to as “reishi” or “lingzhi,” as a preventative anti-inflammatory treatment or to enhance immunity  Karst has a native geographic distribution in Europe and some parts of China, while Ganoderma lingzhi Sheng H. Wu, Y. Cao, and Y.C. Dai is native from East Asia  does not regulate the marketing of fungal medicinal products. As with other herbal supplements, the lack of regulation can create a “buyer beware” market, because the integrity of the product lies with the manufacturer . With thtropicum . Surveyslucidum” . Further stricto . The goan = 8), sawdust inoculum (n = 7), or mycelial suspensions in syringes (n = 2). All of the kits were labeled as containing G. lucidum, except one that was labeled as containing Ganoderma curtisii. However, one GYO kit was labeled with G. lucidum sensu lato, acknowledging the ambiguity of this species. Furthermore, two manufactured reishi products were labeled as containing a combination of fungal species: (1) G. lucidum and Lentinula edodes, or shiitake (product AL-R4) and (2) Ganoderma tsugai (presumably the improper spelling of G. tsuage), G. lucidum, and G. applanatum (product AL-R11). In addition, twenty commercially available, manufactured reishi products were purchased based on the following criteria: (i) readily available for purchase online, (ii) labeled as including G. lucidum, and (iii) marketed with medicinal benefits. Manufactured reishi products were sold as capsules (n = 13), powders (n = 3), tablets (n = 1), coffee (n = 1) or tea (n = 1). With the exception of two products labeled as G. lucidum sensu lato, acknowledging the ambiguity of the species G. lucidum, all products were labeled as “G. lucidum” with health-promoting marketing statements such as “supports longevity,” “supports immune system,” “detoxifier,” and “botanical immune support.” In addition, all manufactured products contained the following label: “These statements have not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, cure or prevent any diseases.”Seventeen GYO kits that were marketed for medicinal use were purchased from mushroom cultivation companies in the United States. These kits were sold as colonized wooden dowels  of spawn inoculum with a sterile scalpel and placing them onto 2% malt extract agar (MEA)  prepared according to the manufacturer’s instructions with the addition of streptomycin (100 mg/l), 95% benomyl (4 mg/l), and 85% lactic acid (2 ml/l), which help to limit bacterial and the growth of fungi in the Ascomycota. Cultures were maintained on MEA slants as working stocks, and colonized agar disks were submerged in sterile water for long term storage  to determine the presence/absence of chlamydospores, which are diagnostic features for some of the laccate  species . Two sli′ CTTGGTCATTTAGAGGAAGTAA) and ITS4b (5′ CAGGAGACTTGTACACGGTCCAG)  was sequenced for all of the GYO kits using primers EF-Gano23F (5′ GGTGTCAGGCAGCTCATYGT) and EF-Gano887R (5′ CGAACTTGCARGCGATGTG), which were developed specifically for amplification of laccate Ganoderma species. For each PCR reaction, the following reagents were used: 12.5 μl of Immomix Red Master Mix , 8.5 μl of PCR-grade H2O, 1 μl BSA , 1 μl of each 10 mM primer, and 1 ng/μl of DNA template. Reactions were performed on a MJ Mini thermocycler  with the following thermocycler conditions: cycle of 95°C for 10 min and followed by 35°cycles of 94°C for 30 s, variable annealing temperatures of 55°C (ITS) or 62°C (tef1α) for 30 s, and 72°C for 1 min, followed by a final extension step of 72°C for 5 min, and then 4°C. PCR products were visually assessed on a 1% agarose gel that was stained with Gel Red  to confirm successful amplification. Amplicons were purified with Exo-SAP-IT  according to the manufacturer’s recommendations. Sanger sequencing was performed using the same primers through the Genewiz1 sequencing lab. Forward and reverse sequences for each sample were aligned and visually edited using GENEIOUS 10  according to the manufacturer’s instructions. The internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) was amplified by PCR with primers ITS1F (5GGTCCAG)  to identmitation . In addiEIOUS 10 . All San species . Identifn = 5, AL-R4, AL-R9, AL-R10, AL-R12, and AL-R19), samples were prepared as the other samples, except PCR products were not diluted. The library was purified with the Agencourt AMPure XP kit  to remove primer dimers before sequencing with a MiSeq 300 bp paired-end protocol  at the Interdisciplinary Center for Biotechnology of University of Florida. Raw data are available at NCBI’s SRA BioProject Accession SRP149732.If multiple taxa were present in an individual sample, Sanger sequencing would either yield a clean DNA sequence only for the dominant taxon or would yield a mixed sequence that would not be readable due to multiple overlapping sequence peaks. Accordingly, we performed Illumina metabarcoding sequencing in addition to Sanger sequencing for all of the manufactured reishi products because these are the products that are most likely to contain multiple species based on the recommendation of 2. Briefly, the overlapping 2 × 300 Illumina MiSeq reads were merged using USEARCH  were aligned using the MAFFT  evolutionary model with rapid bootstrapping and 1000 bootstrap replications, and the Bayesian analysis used a GTR evolutionary model with a gamma rate variation using four gamma categories, for one million generations with 4 heated chains and a burn-in length of 100,000. These unrooted trees were produced to place ITS sequences and ITS1 OTUs generated from Sanger and Illumina sequencing into lineages identified based on reliable reference sequences . The alignments and trees have been deposited to Treebase under submission #22867.Illumina sequence data were processed using the AMPtk pipeline (version 1.1.0)he MAFFT  plugin iin RAxML  and a Bain RAxML  plugins in vitro colony morphology of Ganoderma (n = 7) of the seventeen GYO kits constitutively produced smooth, intercalary to terminal, double-walled, hyaline, ovate to obpyriform chlamydospores that are consistent with species in the Ganoderma resinaceum clade (n = 7) of the manufactured reishi products were putatively made with mature fruiting bodies, based on the identification of generative and skeletal hyphae and basidiospores. Twenty-five percent (n = 5) were putatively made with immature fruiting bodies , twenty percent (n = 4) were putatively made with cultures (generative hyphae only), and ten percent (n = 2) were putatively made with only basidiospores. Five percent  of the manufactured products had no definitive Ganoderma tissues based on our slide mounts (Table 2).Of the GYO kits, 100% of the pure cultures had generative hyphae with clamp connections and were consistent with anoderma . Forty-oum clade . No othen = 17) of the GYO kit samples and 70% (n = 14 of 20) of the manufactured reishi products. Sequences of tef1α were successfully generated for 100% (n = 17) of the GYO kit samples and amplification of the manufactured reishi products was not attempted. The 31 ITS sequences (MH160056-MH160086) and 17 tef1α sequences (MH168053-MH168069) were deposited in Genbank. Of the GYO kits, two products were correctly labeled as G. curtisii (AL-M17) and G. lucidum (AL-M16). The other 15 GYO kits were mislabeled as G. lucidum, and were actually identified as G. lingzhi (n = 7), G. sessile (n = 4), G. resinaceum sensu lato (n = 3), or G. curtisii (n = 1). All of the manufactured reishi products for which we generated sequences were mislabeled as G. lucidum, and subsequently identified with Sanger sequencing as G. lingzhi, except one sample (AL-R6) that was identified as G. applanatum. These results are presented in Table 1.ITS sequences were successfully amplified for 100% (n = 19) contained at least one Ganoderma species (Table 3). However, G. lucidum sensu stricto was not detected in any of the products. Three products each contained only a single Ganoderma species detected by Illumina meta-barcoding: G. applanatum in AL-R6 and G. lingzhi in AL-R7 and AL-R14 . We also detected multiple Ganoderma species in 16 of the 19 manufactured products. Because of PCR biases linked to next-generation sequencing methods, the quantification of species abundance in a community based on the abundance of sequences in a sample is not generally considered reliable (Ganoderma spp. in each sample (Table 2). The majority of products  contained a high abundance of G. lingzhi sequences, with only negligible numbers of sequences (< 5% of the reads) from other species of Ganoderma or other relevant taxa. Five out of the 19 successfully sequenced products had > 5% sequence abundance from other Ganoderma or relevant fungal species other than G. lingzhi .Amplification and sequencing of the genomic library was successful for 19 of the 20 manufactured reishi products tested (AL-R12 failed). All successfully sequenced manufactured reishi products . The ITS sequences generated in this study clustered with significant statistical support with the reference sequences.The maximum likelihood and Bayesian phylogenies constructed with sequences from Sanger and Illumina sequencing gave identical results, so only the unrooted RAxML phylogeny is presented . The majority of the GYO kits and manufactured reishi products indicated that they were made with “Ganoderma lucidum.” However, no manufactured reishi products and only one GYO kit (AL-M16) actually contained G. lucidum sensu stricto. The incorrect identification of Ganoderma species in these products is presumed to be non-intentional and likely due to the complicated taxonomic problems within the laccate Ganoderma as well as the nomenclatural changes that have occurred in recent years  or geographically separated regions within North America   . Based ois genus ,b. It alG. lucidum in our survey, we found that 86% of them included a product substitution. Most of the time G. lingzhi was substituted for G. lucidum. As a medicinal product, this reishi “substitution” is probably more appropriate than the G. lucidum indicated on the label. G. lingzhi, which is native to East Asia, was recently circumscribed as one of the species previously mislabeled as G. lucidum sensu lato in Asia  pilei and belong to the subgenus Elfvingia (G. curtisii and G. resinaceum produce laccate (shiny) pilei as in G. luccidum but belong to two distinct subclades within the subgenus Ganoderma , and nearly half of the products from the GYO kits sold in the United States contained the Asian species G. lingzhi despite being labeled as G. lucidum.G. lucidum and G. lingzhi were not only genetically distinct based on the beta-tubulin gene, but were also chemically different. Extracts of G. lingzhi produced significantly more triterpenic acids than extracts made with G. lucidum sensu stricto , and biochemical properties associated with each one of these unique isolate of G. lucidum. The “antler form” showed significantly more production of phenolics, flavonoids, polysaccharides and ganodermin, compared to the fruiting bodies that formed a true pileus (kidney-shaped cap form) . Just as there is a dearth of research exploring biochemical differences among the newly elucidated charides . Furthercharides . Lastly,charides . Similarap form) . As pharGanoderma species, there are many ecological implications in regard to cultivating non-native Ganoderma taxa outside of their native range. Of the 17 GYO kits purchased by companies in the United States, 11 purchased kits were of Ganoderma taxa that are not native to the United States. These taxa included G. lingzhi (native to Asia), G. lucidum (native to Europe and Asia), and G. resinaceum sensu lato (native to Europe). Previously, it was shown that monokaryotic isolates of the North American native G. sessile (previously considered in publications as G. lucidum) were compatible with monokaryotic European isolates of G. resinaceum, a species native to Europe  of a single genotype could create a genetic bottleneck in the wild populations. This would ultimately lead to a reduction of genetic diversity in wild populations. This phenomenon has been previously shown with the common button mushroom Agaricus bisporus in California, where cultivated genotypes have escaped cultivation and displaced native genotypes  of 1994, the FDA is not responsible for analyzing the contents of dietary supplements . Accordid others , the UniG. lucidum contain multiple Ganoderma species. The fact that these products are inaccurately labeled and/or contain a mix of species, but are nonetheless sold for medicinal uses, raises questions regarding the authenticity of the fungal products used by this industry. Important questions are also raised such as: Do all Ganoderma species produce similar quality and quantities of medicinally relevant compounds? Can phylogenetic placement of a Ganoderma species predict the presence or effectiveness of medicinally relevant compounds? Are all Ganoderma preparations  equally useful for medicinal purposes? Does growing non-native Ganoderma species present risks that they may displace native decay fungi or cause root rot or detrimental decay on native trees? These questions should be addressed in subsequent research focusing on the cultivation and manufacturing of reishi products.Our research shows that GYO kits and manufactured products  marketed as The conception of the paper was a collaborative idea with AL, BR, MS, and JS. The work was funded by a grant received by AL, JS, and RB. The microscopy and molecular lab work was conducted as a collaborative effort by AL, CT, and MJ. The manuscript was written by AL. The manuscript was edited and reviewed by AL, BR, MJ, CT, MS, RB, and JS.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.


Smoking is a known predisposing factor to exacerbations in CF patients. But the effects of second-hand tobacco smoking are not yet clear. Hence, this study determined the clinical and spirometric presentations and urinary cotinine levels among cystic fibrosis patients over seven years of age in relation to their parent’s smoking history.In this cross-sectional comparative study, 58 consecutive cystic fibrosis patients older than seven years of age were enrolled. These patients were divided into two equal groups: those with second-hand tobacco smoking and those without. Pulmonary function tests and hospital admission rates were compared across the groups.The mean hospital admission times were 5.1±2.4 in the group with passive smoking, and 2.6±1.3 times in the group without (P<0.001). The cotinine level was reversely correlated to time interval passed from previous admissions  in passive smokers and  in non-passive smokers. In the analysis of FEV1 with urine, there was a significant but negative relation between FEV1 and cotinine (P= 0.002). Besides, in the analysis of FE 25–75 and urine cotinine, there was also a significant and negative relationship (P=0.001).From our findings, we conclude that pulmonary function tests and hospital admission rate in patients with cystic fibrosis are associated with urinary cotinine level and household second-hand tobacco smoking. Cystic fibrosis (CF) is the most common hereditary respiratory disease of childhood with autosomal–dominant pattern of transition and increasing trend of incidence –3. The sThe available therapeutic approaches are generally conservative and complete cure for these patients is not accessible –15. AccoIn this cross-sectional comparative study, 58 consecutive cystic fibrosis patients older than seven years of age were enrolled. These patients were divided into two equal groups: those with second-hand smoking and those without.The age, sex, body mass index (BMI), family history of respiratory diseases, symptoms in patients, chief complaints, computed-tomography (CT) scan findings, clubbing severity, spirometric parameters, the times passed from last admission, relative status of parents, smoking by parents and cigarette packs they used per year (according to IUATLD Questionnaire) were assessed in the two groups. Two main variables — annual hospital admission times and pulmonary function test — were evaluated and compared across the groups. Also the association of different factors was compared with these two variables.t-tests were used; P values less than 0.05 were considered statistically significant.Data analysis was performed among 58 subjects: 29 patients with second-hand smoking and 29 patients without. Cotinine level was evaluated with ELISA test. Data were analyzed using SPSS (version 20.0) software . Chi-square, Kendall, and independent-sample The age, sex, BMI, family history of pulmonary disease, and relative status of parents were the same across the groups . Only faThe mean hospital admission times were 5.1±2.4 times in group with passive smoking and 2.6±1.3 in group without (P<0.001). The cotinine level was reversely correlated to time interval passed from previous admission  in passive smokers; and  in non-passive smokers. In the analysis of FEV1 with urine, there was a significant but negative relation between FEV1 and cotinine (P=0.002) . In the Patients with CF may show more respiratory response to air pollution in and out of home and in other closed environments. Smoking is a cause for exacerbation of disease leading to decreased quality of life in patients with CF , 20. HowIn the study by Raju et al. , it was The study by Campbell et al.  demonstrSmyth et al.  reportedFrom our findings, it may be concluded that pulmonary function tests and hospital admission rates in patients with cystic fibrosis are associated with urinary cotinine levels and household second-hand smoking. However, further multi-center studies with larger sample sizes are required to attain more definitive results. Also from our findings, it is recommended that tobacco cessation and prevention programs be integrated as an important component for parents with CF children.


Iron deficiency (ID) is common in overweight and obese individuals (OW/OB) but the mechanism is uncertain. Greater blood volume (BV) in OW/OB may increase hemoglobin (Hb) mass and iron requirements, and confound iron biomarkers by hemodilution. Quantification of BV/PV changes in OW/OB is challenging and a formula to estimate BV/PV based on anthropometric indices would be valuable. In normal weight (NW) and OW/OB women, we aimed at: (1) measure BV and assess whether differences in BV affect concentrations and total circulating mass of Hb and iron biomarkers; (2) develop an algorithm describing BV in OW/OB.n = 62) by using the carbon monoxide-rebreathing method, body composition by dual energy X-ray absorptiometry, and iron and inflammatory status.In a cross-sectional study, we measured BV in NW, OW, and OB non-anemic women (P < 0.05), respectively. In OW/OB compared to NW, total circulating masses of IL-6, hepcidin, Hb, and sTfR were higher, while total mass of serum iron was lower . An equation including height, body mass and lean mass to estimate BV in all BMI groups (R2 = 0.76).OW and OB women had 11 and 16% higher mean BV and PV compared to NW (An equation based on anthropometric indices provides a good estimate of increased BV in OW/OB women. In OW/OB women, there is an increase in Hb mass that likely increases iron requirements for erythropoiesis and circulating TfR mass. At the same time, higher hepcidin concentrations may lower serum iron mass. Both these mechanisms may increase risk for ID in OW/OB women. Body mass index (BMI) is negatively correlated with serum iron and positively correlated with serum transferrin receptor (sTfR) in children and adults . CompareSeveral simple equations exist for the estimation of BV in individuals but they are mainly based on data from NW subjects –20. AlthTherefore, our study aims were: 1) to quantitatively measure BV and PV, and iron and inflammatory status biomarkers in OW/OB women and NW women; (2) use the measures in [ to quantThis study was part of a larger study that investigated the influence of body mass and fat on iron absorption in NW, OW, and OB women where the sampling methodology and response rates are published in detail . Briefly2); 20 with overweight ; and 20 with obesity (OB: BMI 30–39.9 kg/m2). At screening, we informed women about the study aims, procedures, and associated risks. Inclusion criteria for the study were: [2; [The subjects in this study were 64 women: 24 with NW  on two separate occasions. The typical error was 1.2% with an R2 = 0.98 [At screening, we obtained a fasting blood sample via forearm venipuncture for analysis of hemoglobin, and iron and inflammation status. We determined body composition and BV by using dual energy X-ray absorptiometry (DXA) and CO-rebreathing, respectively. We scanned all participants on a DXA GE Healthcare Lunar according to the manufacturer’s specifications . We performed the scan analysis using the GE encore software version 11.40.004. To minimize fluctuations in BV, we performed all measurements, if possible, between 7 and 14 days after the beginning of the last menstrual cycle. The optimized CO rebreathing method  is a rou2 = 0.98 .We measured Hb concentration by using a Coulter counter  with 3-level quality-control material  on the day of blood collection. We measured serum iron and total-iron binding capacity (TIBC) by using a colorimetric method as described previously . We measU test, as appropriate. To study associations between continuous variables, we used bivariate Pearson’s or Spearman correlations and multiple linear regression models with adjustments for confounding variables. Differences were considered significant at P-values < 0.05.We performed statistical analyses using IBM SPSS Version 20 . We checked data for normality by visual observation and by using the Kolmogorov–Smirnov and Levene’s normality tests. Non-normally distributed data were logarithmically transformed for statistical analysis. The mean (±s.d.) for normally distributed data, geometric mean  for data with normal distribution after log-transformation or median (interquartile range) for non-normally distributed data even after log-transformation values for each parameter were determined. We applied nonparametric tests for data that remained non-normally distributed after logarithmic transformation. We assessed differences between NW, OW, and OB using analysis of variance (ANOVA) with post hoc Bonferroni correction and Kruskal–Wallis test, followed by Mann–Whitney R2 was used to determine the accuracy of this approximation.To create a new equation for the calculation of BV, we included height, body mass, body surface area (BSA), BMI, total lean, and fat mass in a regression analysis, and screened for those that best estimated measured BV. P < 0.05); OB had significantly greater lean body mass than NW (P < 0.05). Total BV and RBC volume (mL) were greater in OB and OW compared to NW  while blood and RBC volume in mL/kg were significantly lower in OB and OW compared to NW . Mean PV was higher by 11% in the OW (P = 0.07) and 16% in the OB (P = 0.010) compared to the NW women. PV in mL/kg was lower in OB and OW compared to NW (P < 0.001). Although Hb concentration (g/L) was not significantly different among the 3 groups, total Hb mass (g) was 11% greater in OW (P = 0.015) and 16% greater in OB (P = 0.005) compared to NW, while Hb mass in g/kg was 14% lower in OW and 22% lower in OB  compared to NW. Serum concentrations and circulating masses of CRP, IL-6, AGP and hepcidin were greater in OB versus NW (P < 0.05). There were no significant differences in concentrations of sTfR or SF among the four groups. However, total masses of sTfR and SF were greater in OW/OB compared to NW . In OW/OB compared to NW, concentration of serum iron was lower (P = 0.025) and total serum iron mass was lower (P = 0.014). In OW/OB compared to NW, concentrations of serum TIBC was lower (P = 0.04) but total mass of TIBC was not. Transferrin saturation was lower in OW/OB, but this was not statistically significant  dropped out of the study; 62 women completed the study and were included in the analyses. Age, anthropometry, body composition, and inflammatory biomarkers are shown in Table R = 0.74, P < 0.001) and total lean mass , than with fat mass , waist circumference , height  or BMI .Correlations between body mass, lean and fat mass, and absolute BV are presented in Fig. R = 0.32, P = 0.01), body iron stores , SF , and negatively associated with BMI , body mass , fat mass , AGP , BV , and PV . Mass of serum iron was positively associated with circulating mass of SF  and hepcidin mass . Moreover, mass of SF was positively associated with hemoglobin mass , mass of inflammatory markers , BMI , body mass , and fat mass  but not with total lean mass . In multiple linear regression analysis, total body fat  but not lean mass  was a significant negative predictor of serum iron when corrected for body iron stores. Moreover, BV was a significant negative predictor for serum iron  when corrected for body iron stores. Baseline BMI was a significant predictor for total serum iron mass  and TfR mass  but not for total serum TIBC mass  when corrected for body iron stores. PV was a significant predictor for total TIBC mass  and TfR mass  but not for total serum iron mass , when corrected for body iron stores.Correlations between relative and absolute iron status indicators with anthropometry, body composition, BV and inflammation biomarkers are shown in Table Two equations for the estimation of measured BV were created using stepwise regression with a forward selection of covariates. They are shown below and their performance is shown in Table R2 = 0.69, P < 0.01)(1) Blood volume (mL) = 4698.8 × height (m) + 32.5 × body mass (kg)−5342.1 (2) Blood volume (mL) = 2686.7 × height (m) + 18.9 × body mass (kg) + 66 × lean mass (kg)−3932.3 , using fixed ratios for normal values, with different ratios for men and women. Estimated BV calculations are usually performed with the formula: body mass (kg)×average blood volume (mL/kg). In the clinical practice BV is estimated to be 65–75 mL/kg . However similar to our results different studies have shown that total BV and its components, plasma and red blood cells, decreases per unit body mass with increasing degree of obesity –43. In aEstimation of BV in OW/OB is challenging since BV is not a constant fraction of body mass. Currently existing formulas for the calculation of BV , 45–49 iThe strengths of the present study include: (1) the assessment of BV using the CO-rebreathing method in women with a wide range of BMI, including an assessment of the reproducibility of the CO-rebreathing method; (2) co-measurement of lean and fat mass using DXA; and (3) measurement of a number of different biomarkers of iron and inflammation status including serum iron, sTfR, TIBC, transferrin saturation, hepcidin, and IL-6 and individual calculation of their circulating masses. A limitation of the study is that we have only studied Swiss women; whether the findings and the derived equations apply to men and to other ethnic groups is uncertain. Also, the steps involved in BV measurement using the CO-rebreathing method present potential opportunities for error. However, parameters that could influence the BV measurements were standardized to minimize measurement errors, and the reproducibility of the method was verified by a test–retest correlation analysis and the CV was low (1.2%). Due to the small sample size and a cross-sectional design, our results should be interpreted carefully and no conclusion regarding cause and effect can be made.In conclusion, BV and PV is significantly increased in OW/OB compared to NW and this affects interpretation of iron biomarkers and provides insights into comparative iron metabolism in these groups. Our new proposed algorithm provides a good estimate of BV in OW/OB and could be used in further studies that require estimates of BV changes in OW/OB.


N = 443) varied in age, country and clinical seniority but comprised mainly genetics health professionals (82%) and BWA users (93%). Oncology professionals perceived reproductive, hormonal (exogenous) and lifestyle BC risk factors as more important in BC risk assessment compared to genetics professionals . BWA was used more frequently by respondents who gave high weight to breast tumour pathology and low weight to personal BC history as BC risk factors. BWA use was positively related to the weight given to hormonal BC risk factors. The importance attributed to lifestyle and BMI BC risk factors was not associated with the use of BWA or any of the other tools. Next version of the BWA encompassing additional BC risk factors will facilitate more comprehensive BC risk assessment in genetics and oncology practice.The BOADICEA breast cancer (BC) risk assessment model and its associated Web Application v3 (BWA) tool are being extended to incorporate additional genetic and non-genetic BC risk factors. From an online survey through the BOADICEA website and UK, Dutch, French and Swedish national genetic societies, we explored the relationships between the usage frequencies of the BWA and six other common BC risk assessment tools and respondents’ perceived importance of BC risk factors. Respondents (The online version of this article (10.1007/s12687-018-0362-8) contains supplementary material, which is available to authorized users. Breast cancer (BC) is a major public health problem for women with almost 1.7 million new BC diagnoses and 521, 900 BC deaths estimated worldwide in 2012  that aims to inform the development and implementation of a comprehensive genetic test in BC risk assessment and to combine this with additional non-genetic BC risk factors. The latter will be achieved through further development of the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) model and BOADICEA Web Application (BWA)   to invite them to complete the survey . A total of 225, 170, 37 and 32 individuals were contacted from the British, French, Dutch and Swedish NGS, respectively.The survey questions addressed the following: (1) practice in genetic counselling and testing for cancer predisposition; (2) importance attributed to BC familial, hormonal, reproductive and lifestyle BC risk factors; (3) knowledge/usage frequency of the Gail  and body mass index (BMI) BC risk factors. The BOADICEA model allows for extensive family history; tumour pathology such as oestrogen and progesterone receptors and HER2, CK5/6 and CK14 status of BC in family member(s) to be taken into account , use frequency on a five-point Likert scale ranging from never to always (see Table n = 21).A preliminary version of the overall survey was designed following survey method recommendations  using a chi-squared test.Logistic multiple regression analyses .Overall, 443 respondents completed at least 50% of the survey, comprising 316 (71.3%) and 127 (28.7%) via the BOADICEA website and NGS survey sources respectively  ranged from 24 (5%) (BRCAPRO) to 61 (14%) (Myriad).The BWA was used at least occasionally by 413 survey participants (93%) (Table r = 0.51), Claus (r = 0.45), BRCAPRO (r = 0.44) and IBIS (r = 0.41) (data not shown).Regarding the number of tools that were used, the most common observation was the use of one tool at least regularly, by 36% of respondents. The usage frequency of the BWA and other tools was uncorrelated; only the usage frequency of Gail was correlated to the use other tools, i.e. Myriad  time in minutes to input clinical data ranged from 3.6 (3.6) (MSS) to 15.6 (10.8) (BWA) .To estimate BC risk, cancer family history was perceived as most important by most respondents (72%). Non-genetic BC risk factors were perceived as most important by between 1.3%  and 5.4%  of respondents (Table p ≤ 0.01). More genetic counsellors/nurses than other health professionals considered breast tumour pathology as most important (p ≤ 0.01) , alcohol consumption (p ≤ 0.0001), oral contraception (p ≤ 0.01), HRT (p ≤ 0.0001) and physical exercise (p ≤ 0.01).Oncology health professionals attributed higher importance to the following BC risk factors than genetics health professionals: breastfeeding  and IBIS . In addition, genetic counsellors/nurses used the MSS more frequently than clinical geneticists . Longer clinical experience was associated with a more frequent use of the Gail (OR [CI] = 2.97 [1.01–8.79]), Claus (OR [CI] = 3.06 [1.23–7.54]) and IBIS (OR [CI] = 2.61 [1.12–6.10]) tools. The level of clinical activity and specific cancer genetic counselling and testing training was not associated with the use of any of these tools.To estimate BC risk, respondents who attributed less importance to personal BC (OR [CI] = 0.49 [0.28–0.86]) and more importance to breast tumour pathology (OR [CI] = 3.90 [1.90–7.97]) used the BWA more frequently; the reverse was (unexpectedly) observed for the Gail  and Claus  tools. The use of the BWA was (unexpectedly) associated with the importance given to hormonal (exogenous) factors (OR [CI] = 1.77 [1.20–2.60]). Considering reproductive BC factors as important was associated with more frequent use of the Gail (OR [CI] = 3.06 [1.67–5.65]), Claus (OR [CI] = 1.88 [1.13–3.11]) and IBIS (OR [CI] = 4.81 [2.86–8.14]) tools and less frequent use of the MSS (OR [CI] = 0.57 [0.38–0.86]). IBIS was used less frequently by individuals who considered breastfeeding as important (OR [CI] = 0.53 [0.33–0.86]).Among non-genetic BC risk factors, the importance attributed to lifestyle and BMI risk factors to estimate BC risk was not associated with the use of any of these tools.Respondents to this survey were mostly BWA users (93%), and few were unaware of other BC risk assessment tools commonly used in cancer genetics clinics. Thus, results presented here describe factors that potentially influence the respondents’ choice of using the BWA or another tool.Uptake of a BC risk assessment tool may depend on its perceived validity, which is related to the type and number of BC risk factors incorporated within the underlying statistical model and any accompanying validation studies. So, in line with the characteristics of the BC risk assessment tools . Respondents’ professional background appeared related to the importance attributed to familial or hormonal, reproductive and lifestyle factors. This may explain the distinct uptake of some BC risk assessment tools according to the type of respondents’ profession. Indeed, oncology specialists attributed more importance to hormonal and lifestyle factors and also used the Gail and IBIS tools more frequently than clinical geneticists. However, genetic counsellors and nurses also used these tools more frequently as well as the MSS, which may be related to role sharing between medical and non-medical genetics clinicians. BWA use was not related to the type of health profession or the use of other tools, suggesting that this tool may respond to various clinical needs.In contrast, the Gail tool was moderately associated with the use of other tools such as Claus, Myriad, BRCAPRO and IBIS. In fact, our respondents were mostly genetics clinicians, and validation studies suggest that Gail is less appropriate to assess BC risk in hereditary high-risk populations [Evans and Howell Uptake of BC risk assessment tools may be related to usability and ease of data entry (e.g. depending on the amount of information) and accessibility . In this study, only the uptake of Claus and the MSS was related to the usability of the tool   was related to increased clinicians’ seniority.The survey design allows us to comment on the associations, but we cannot ascertain the directionality of the relationships, nor their specificity over time, along stages of the BC genetic counselling process.Our study has several limitations:Our study provides novel insights into the acceptability of the BWA by analysis of BC risk factors incorporated in this tool and other common BC risk assessment tools. These results suggest that while the BWA does fulfil various clinicians’ needs, it does not incorporate some BC risk factors considered important by its users. The results of our analyses will help to identify perceived deficiencies in the BWA and guide further software development , so that the next version of the BWA will facilitate more comprehensive BC risk assessment in genetics and oncology practice.ESM 1(DOC 109 kb)


Studies regarding blood pressure of Nepal have demonstrated a contrasting prevalence. We aimed at providing a generalized estimate of the prevalence of hypertension and prehypertension in urban, suburban, and rural areas of Nepal.This systematic review followed PRISMA guidelines. A thorough search of PubMed, EMBASE, and Web of Science was performed, and studies satisfying the eligibility criteria were reviewed. Pooled prevalence was calculated by random-effects model, and the sources of heterogeneity were explored with meta-regression and subgroup analysis.Twenty-three studies with 99,792 subjects were identified, and the estimated rate of hypertension and prehypertension were found to be 27.3% (95% CI: 23.8–30.9) and 35.4% (30.3–40.8). The prevalence of hypertension was 28.4% (22.4–34.7), 25.5% (21.4–29.8), and 24.4% (17.9–31.6) among urban, suburban, and rural populations, respectively. Moreover, rates of hypertension were found to be substantially higher in male  compared to female , and significantly higher among the middle-aged  than among younger adults . Further, prehypertension prevalence was found to be highest in rural areas  followed by urban areas  and lowest in suburban areas .Our study identified an alarming situation of hypertension among Nepalese males and middle-aged, and a situation of concern with prehypertension in rural areas affecting almost 40 % of the population.The online version of this article (10.1186/s41256-019-0102-6) contains supplementary material, which is available to authorized users. High blood pressure (HBP) is responsible for more than half of all strokes and coronary disease , and is One study analyzing HBP in developing countries pointed out that the prevalence of hypertension in Nepal was still at a low level (< 20%) , in contThence, we aimed at filling this gap using strong meta-analytic evidence by merging existing scientific literature to obtain a robust generalized estimate of the prevalence of both hypertension and prehypertension in the urban, suburban, and rural parts of Nepal.This systematic review was conducted following PRISMA  guidelines , and theAn initial search for studies by a combination of Medical Subject Headings (MeSH) terms consisting of ‘hypertension’ and its relevant synonyms was performed using PubMed, EMBASE, and Web of Science. The search was restricted to publications from January 2000 to August 2018 that were conducted on the human species and published in the English language. . The results were further narrowed by adding ‘Nepal’ as another key word. The search details of PubMed were as follows: ((“hypertension”[MeSH Terms] OR “hypertension”[All Fields]) OR “high blood pressure”[All Fields] OR “elevated blood pressure”[All Fields] OR “raised blood pressure”[All Fields]) AND  AND ((“2000/01/01”[PDAT]: “2018/08/31”[PDAT]) AND “humans”[MeSH Terms] AND English[lang]). A manual search for additional potential studies was performed using the references cited in the retrieved reviews and original research articles. The reason why excluding studies conducted before year 2000 was the definition of ‘hypertension’ we use being lastly revised and implemented in year 1999.Two authors  independently reviewed the titles and abstracts culled from the searches, and full texts of potentially eligible studies were downloaded and further screened for final inclusion in our study. The eligibility criteria for inclusion were: 1) original articles from non-hospitalized and population-based surveys reporting HBP prevalence ; (2) respondents without restriction to specific age groups or populations; (3) containing information on the study location ; (4) presenting HBP prevalence without other associated comorbidities; (5) using a non-convenience sampling method and with a sample size above 500 participants. For studies published in more than one edition, we considered the most comprehensive one. When there was uncertainty or disagreement between the two authors as to the eligibility of a study, another author (Yuantao Hao) was asked for guidance to reach a consensus. The study selection process is shown in Fig.  originalAll data were extracted independently by two authors  using a standardized protocol. The characteristics recorded for each eligible study included the first author’s name, year of publication, study design, sampling methods, age range of participants, net sample size , number of events (hypertensive and pre-hypertensive cases), corresponding prevalence, methods of BP measurement , diagnostic criteria of hypertension, response rate, survey year, survey site, and location of study .We then performed a quality assessment of the retained studies based on the completeness of the eligibility criteria, sampling strategy, age range, sample size, non-response rate, survey year, explanation of limitations of the study, and BP measurement techniques , 15. In χ2) test and quantified it with I2 index. I2 is defined as the proportion of total variation provided by between-study variation, and was denoted the values of 0, 25, 50, and 75% which corresponds to no, low, moderate, and high heterogeneity, respectively [p < 0.05 from Cochrane’s chi-square (χ2) test or large I2 demonstrate substantial heterogeneity. Random-effects model was constructed to address heterogeneity in the pooled prevalence. An univariate meta-regression was performed by setting possible confounders as moderator with function ‘rma.glmm’ in the ‘metafor’ package to explore for sources of heterogeneity , then we categorized studies into subgroups according to findings from the meta-regression and performed meta-analysis for each subgroup respectively to certify the results of the meta-regression. We applied the symmetry of funnel plots and did both Begg’s adjusted-rank correlation test and Egger’s regression asymmetry test to evaluate publication bias [p–value of less than 0.10 was considered indicative of statistically significant publication bias.Meta-analysis was performed using the packages ‘meta’ and ‘metafor’ in R software (version 3.4.2). To weaken the effect of studies with extremely small or large prevalence on the overall estimate, we transformed the data with the Freeman-Tukey double arcsine function before pooling the prevalence . We estiectively . A p < 0ion bias , 19; a pIn total, 953 studies were retrieved, of which 23 met the inclusion criteria in the primary review of hypertension prevalence , 20–36. n = 12) was preferred, nine others used a digital sphygmomanometer, and two did not provide any information about the measuring device used. The frequency for taking BP measurements varied from one to four and the intervals between each measurement ranged from half a minute to half an hour. Each study was scored and ranked accordingly; out of these only three studies were identified as low-qualified [As can be observed from Table ualified , 30, 32.The HBP situation in Nepal is illustrated by the Forest plot in Fig. The pooled regional estimates of hypertension were 28.4% (95% CI: 22.4–34.7%), 25.5% (95% CI: 21.4–29.8%), and 24.4% (95% CI: 17.9–31.6%) for the urban, suburban, and rural areas respectively. HBP was found to be substantially affecting male  as compared to female , and this tendency was found to be consistent in three residential areas .Figure I2 = 99% and P < 0.01) among retained studies. Meta-regression findings indicated that sample size (P = 0.928), gender ratio (P = 0.948), inception of survey (P = 0.721), definition of hypertension (P = 0.363), quality score of study (P = 0.347), and measurement device (P = 0.769) were not associated with the heterogeneity observed (Table P = 0.001) and quality grade of each study we ranked (P = 0.027) were identified as potential sources of heterogeneity.Previous results showed a considerable heterogeneity  in contrast to that below 40 years  and one of their included studies accounted for males only was not representative of Nepalese in general. Thus, a more comprehensive analysis was necessary to reveal the present Nepalese HBP situation.The pooled prevalence of HBP obtained from our study was found to be 27.3% (95% CI: 23.8–30.9%), higher than that reported in low-income countries (23.1%), but similar to the average rate in the SAARC regions (27.1%) and that reported in the Nepal nationwide STEPS survey (25.7%) , 38. A pPrevious literature on HBP prevalence often categorized residents by urban and rural regions, while we subdivided residents into three categories, namely urban, suburban, and rural, for an improved practical and factual analysis. Based on the regional analysis, although the HBP status appeared somewhat more severe in urban regions, we found that the prevalence of the three areas was comparable, and this was in accordance with an insignificant difference between urban and rural habitation presented in previous studies , 10. TheHigher HBP prevalence in male compared to female was found in our study, which was similar to that of two previous systematic reviews , 38. It The pooled prehypertension prevalence in our findings was similar to the estimated global figure (35.4% vs. 38.0% respectively) while higher than that of the SAARC region (29.6%) . FurtherThe reported HBP prevalence varied considerably across the included studies indicated a notable heterogeneity. The results of meta-regression and subgroup analyses suggested that heterogeneity was directly associated with the age of participants and the quality grade of the included studies. Further, we found that the prevalence was significantly higher in the middle-aged (≥40 years) compared to that in younger adults (< 40 years). Based on the wide and multi-database literature-search , we are the first to provide strong evidence for such an age boundary. A reasonable explanation was given by a previous study which suggested that age-related structural changes in blood vessels gradually lead to narrowing of the vascular lumen, and consequently could increase the risk of acquiring HBP . For stuIt is worth mentioning that Nepal has developed the national Multi-sectoral Action Plan for prevention and control of NCDs (2014–2020), including hypertension prevention and management for the prevention of cardiovascular disease . One of To our knowledge, this is the first comprehensive report targeted at evaluating the scientific literature on the prevalence of hypertension and prehypertension in the urban, suburban, and rural populations of Nepal. Despite such findings, there are some limitations of this study that should be mentioned. Studies from only three of the most commonly used platforms  were searched. Several included articles provided only a crude prevalence with no specified hypertensive events. Further, a noteworthy heterogeneity was observed among retained studies so that large-scale nationwide and more representative epidemiological studies are needed to confirm the results of this study.This systematic review identified an alarming situation of hypertension in Nepalese male and middle-aged (≥40 years), as well as a situation of concern regarding the prehypertension status which affects almost 40 % of the rural population. Improving the population-level awareness of HBP; facilitating routine screening aiming at those high-risk groups through integrating of NCDs with primary health care; and effective implementation of NCDs Multisectoral Action Plan (2014–2020) offer potential means for addressing the burden of increased blood pressure in Nepal.Additional file 1:Prisma Checklist. (DOC 64 kb)Additional file 2:Scoring table for quality assessment of the retained studies. (DOCX 14 kb)


N-acetylcysteine (NAC) supplementation and smoke cessationanalyses. Exposure to CS increased the production of ROS and induced molecularchanges in the autophagy pathway, including an increase in phosphorylated AMPKand ULK1, reduction in phosphorylated mTOR, and increases in SESN2, ATG12, andLC3B levels. NAC supplementation reduced ROS levels and reversed all molecularchanges observed upon CS treatment, suggesting the involvement of oxidativestress in inducing autophagy upon CS exposure. When exposure to CS was stopped,there were decreases in the levels of oxidative stress, AMPK and ULK1phosphorylation, and autophagy-initiating molecules and increase in mTORphosphorylation. In conclusion, these results suggest the involvement of ROS,SESN2, AMPK, and mTOR in the CS-induced autophagic process in the lung.Many pathological conditions linked to cigarette smoking are caused by theproduction of reactive oxygen species (ROS). The present study was conducted toanalyze the effect of ROS on the lungs of Swiss mice exposed to cigarettesmoking, focusing on autophagy-mediated mechanisms, and investigate theinvolvement of SESN2, AMPK, and mTOR signaling. Mice were exposed to cigarettesmoke (CS) for 7, 15, 30, 45, and 60 days; the control group was not exposed toCS. Only mice exposed to CS for 45 days were selected for subsequent The human airway system is continuously exposed to numerous chemical toxicants, whichlead to pulmonary exacerbations. Long-term exposure to cigarette smoke increases therisk of pulmonary-related diseases .In respCigarette smoke (CS) exposure induces the autophagy process to maintain cellularhomeostasis. Additionally, autophagy flux may contribute to increasing epithelialcell loss. Although numerous studies have evaluated CS-induced autophagy, theimportance of autophagy components in CS-linked oxidative stress conditions remainsunclear. Autophagy components including autophagy-related protein (ATG) familykinase, Unc-51-like autophagy activating kinase (ULK1), and ULK2 in mammals are keyregulators of autophagy initiation. The effect of the cigarette smoking on thesecomponents in the lungs is not well-understood. Additionally, the molecular basis ofthe inhibitory function of mammalian target of rapamycin (mTOR) in lung autophagyregulation is poorly understood. AMP-activated protein kinase (AMPK) is anotherstrong candidate for influencing autophagy function and acts as an energy sensor toregulate cellular homeostasis . AlthougSESN2 belongs to the family of highly conserved antioxidant proteins. Although itdoes not possess intrinsic catalytic antioxidant activity, SESN2 plays an importantrole in suppressing ROS . As a faad libitum. The animals were maintained at70% humidity and a temperature of 20 ± 2°C, witha 12 h light–dark cycle. The mice were periodically checked toverify their pathogen-free status. The study was divided into three stages.Two-month-old Swiss male mice were used for the experiments. All experimentalprocedures were performed in accordance with the Brazilian Guidelines for theCare and Use of Animals for scientific and didactic purposes and local ethicscommittee of the Extremo Sul Catarinense University (protocol number –016/2013). A standard diet  andwater were provided n = 10) and treated with CS for 7,15, 30, 45, and 60 days; non-exposed animals were used as a control and wereevaluated for 60 days. After the exposure period, fragments of the rightlung were collected for western blotting and dichlorofluorescein (DCFH)analysis. SESN2, AMPK, and mTOR expression levels were measured. Each timethe animals were exposed to CS, the control group  was placedin the laboratory without exposure to CS.Sixty animals were randomly divided into six groups(N-acetylcysteine (NAC)administration. The selection of 45 days of CS exposure for NACsupplementation was based on the levels of SESN2, AMPK, and mTOR obtainedfrom stage 1 results. We used NAC supplementation to counteract the ROSinvolvement in activating the autophagy process. Briefly, 40 mice wererandomly divided into four groups(n = 10): Cont (non-CS exposed);Cont + NAC (non-CS exposed + NACsupplementation); 45 (CS exposure for 45 days); and45 + NAC (45 days of CS exposure + NACsupplementation). The antioxidant NAC  was administered orally every day by oral gavage using acannula as previously described [To investigate the involvement of ROS in activating autophagy, mice exposedto CS for 45 days were selected for escribed . After tn = 10): Cont (non-CS-exposed);45 CS (45 CS exposure days); 45 CS exposure days + 7 daysof cessation; 45 CS exposure days + 15 days of cessation;45 CS exposure days + 30 days of cessation; and 45 CSexposure days + 45 days of cessation. After treatment,fragments of the right lung were collected for western blotting and DCFHanalysis.The involvement of ROS in initiating autophagy was evaluated by performingcessation of exposure to CS for different periods after 45 days of CSexposure. Briefly, 60 animals were randomly divided into six groups to remove the debris, and thesupernatant was used for protein quantification using the Lowry method [thr172, phosphomTORser2448, phospho ULK1ser317, Atg12  and Sestrin 2, LC3B . The membranes were then incubated with peroxidase-conjugatedsecondary antibody for 2 h at room temperature. Next, the membranes wereincubated for 2 min with enzymatic substrate and exposed to the RX filmon a radiographic development cassette. The intensity and area of the bands werecaptured using a scanner (HP G2710), quantified using the Scion Image program, and expressed as arbitrary units. Afterblotting, the membranes were probed again with α-tubulin antibody to serveas the protein loading control.Lung tissue was collected and immediately homogenized in lysis buffer  at 4°Cwith a Polytron MR 2100 . The lysate wascentrifuged at 12,851 × y method . A totaly method . Antibodp < 0.05).SPSS version 18.0 for Windows  was used for dataanalysis.The data were expressed as the mean and standard error of means(mean ± SEM) and analyzed statistically by one-way analysisof variance (ANOVA), followed by Tukey’s post-hoc test. The level ofsignificance was set to 95% . A significant reduction inDCF levels at 15, 30, and 45 days of cessation was observed as compared to CSexposure for 45 days group A.AdditiROS-induced autophagy in response to CS in COPD is not well-understood. CS inducespro-oxidants to increase oxidative stress in epithelial cells and other cells;autophagy is involved in this mechanism. The present study demonstrated the effectof CS on molecules involved in autophagy. We first established the time of CSexposure for ROS to reach high levels. Considering that the lungs are the primarytargets and are susceptible to oxidative damage caused by smoking, ROS levels wereevaluated in the lung tissue after different exposure times. The present studyrevealed a significant increase in DCFH oxidation at 30, 45, and 60 days of CSexposure. A study by Raza and colleagues  exposed To verify the changes in the cellular redox status and autophagy induced by anantioxidant, the mice were treated with NAC. DCF levels were significantly reducedat 45 days of CS exposure + NAC as compared to at 45 days CSexposure without NAC. This result was expected, as NAC is a powerful thiolicmolecule with antioxidant properties. NAC exerts antioxidant activity directlythrough its three sulfhydryl groups, which provide electrons to neutralize freeradicals, and indirectly by replenishing the intracellular levels of glutathione. In the After exposure to exacerbated stimuli, increased levels of SESN2 can activate AMPK,resulting in the negative regulation of mTOR ,9,12. InAutophagy occurs when AMPK and mTOR interact through coordinated phosphorylation ofULK1 ,26. In tIn conclusion, the present study revealed that exposure to CS led to an increase inROS in the lung tissue along with increased SESN2 protein levels and phosphorylationof AMPK and decreased mTOR and ULK1 phosphorylation. Exposure to CS also led toincreased levels of the autophagy marker molecules ATG12 and LC3B. These events weredecreased by NAC supplementation and the cessation of exposure to CS. In future,detailed studies of this crucial mechanism of SESN2-dependent autophagy responsewill increase the understanding of the pathogenic effect of CS-associated chronicairway diseases.