Telocytes are CD34-positive cells with a fusiform cell body and long, thin cytoplasmic projections called telopodes. These cells were detected in the stroma of various organs, including the prostate. The prostate is a complex gland capable of undergoing involution due to low testosterone levels; and this condition can be reversed with testosterone replacement. Telocyte function in the mature prostate remains to be dermined, and it is not known whether telocytes can take place in tissue remodeling during prostate involution and regrowth. The present study employed structural, ultrastructural and immunohistochemical methods to investigate the telocyte’s phenotypes in the ventral prostate (VP) from control (CT), castrated (CS) and testosterone replacement (TR) groups of adult male Wistar rats. Telocytes were found in the subepithelial, perimuscular and interstitical regions around glandular acini. Telocytes from CT animals have condensed chromatin and long and thin telopodes. In CS group, telocytes appeared quiescent and exhibited layers of folded up telopodes. After TR, telocytes presented loose chromatin, abundant rough endoplasmic reticulum and enlarged telopodes, closely associated with bundles of collagen fibrils. We called these cells “telocytes with a synthetic phenotype”. As testosterone levels and glandular morphology returned toward to the CT group parameters, after 10 days of TR, these telocytes progressively switched to the normal phenotype. Our results demonstrate that telocytes exhibit phenotypic plasticity upon androgen manipulation and interact with fibroblast and smooth muscle cells to maintain glandular architecture in control animals and during tissue remodeling after hormonal manipulation. In addition to fibroblast population heterogeneity in normal tissues, demonstrated mainly in the dermis, fibroblasts exhibit great plasticity in pathological conditions, such as cancer-associated fibroblasts5 and as part of myofibroblasts7. This diversity adds complexity to the study of the stroma. Fibroblast morphology may also change in specific contexts such as hypoxia8, a finding that indicates that these cells exhibit a high level of phenotypic plasticity. The same alteration was verified in smooth muscle cells, which exhibit phenotypic plasticity with the alternation between contractile and synthetic phenotypes, the latter of which synthesises elements of the extracellular matrix during musculature development during certain physiological or pathological contexts10. Additionally, in pathological conditions, there may be a transition from a contractile to a migratory and phagocytic phenotype11.A few decades ago, researchers thought the stroma was a static environment that only act to support and nourish the epithelia. As such, the cellular milieu would be dominated by fibroblasts that would alternate between a more active state, the activated fibroblasts, whose function was restricted to the production of components of the extracellular matrix, and a less active state, the quiescent fibroblasts. However, there is now abundant knowledge that fibroblasts comprise a heterogeneous population of cells that may have different origins and functions in stromal maintenance12. Telocytes were detected in the myocardium14, skeletal muscle15, jejunum17, mammary gland18, joints19, sclera20, prostate22 and several other organs. The exact function of this cell type remains unclear, although there is evidence that telocytes exert organ-specific functions involved in homeostasis, remodelling, regeneration, repair, embryogenesis, angiogenesis and others23. The intimate association of telocytes with smooth muscle cells has been observed in several organs25. Indeed, it was proposed that telocytes play a supportive role in the prostate gland, including smooth muscle cell differentiation and contribution to tissue organisation during development26, but the function of telocytes in the mature prostate remains elusive.The stroma complexity has increased with the classification of a new cell type, the telocytes. Telocytes are CD34- or CD34- and c-Kit-positive cells that differ morphologically from fibroblasts, pericytes and smooth muscle cells since they have fine and long cytoplasmic extensions called telopodes. These structures are divided into dilated sections, podoms (with mitochondria), and fibrillar-like sections, the podomers that display a moniliform aspect28. In humans, the prostate responds to increases in testosterone (T) concentration, which can lead to pathological conditions such as hyperplasia30. Further, T deprivation or castration lead to prostate size reduction and hyperplasia reversion31. Antiandrogen therapies act to reduce the prostatic volume 15 to 25% through apoptosis and glandular epithelial compartment shrinkage32. In rodents, castration also reduced prostate volume and increased apoptosis of luminal epithelial cells33, while T replacement induced epithelial cell proliferation and restored the secretory epithelium activity35. In addition to changes in epithelial cells, androgen deprivation promotes smooth muscle cell transition from the contractile to the synthetic phenotype, without affecting its differentiation status37. Telocytes are closely associated with smooth muscle in the prostate22 and they play an important role in prostate tissue organisation during its initial development26, but little is known about telocyte behaviour in the context of androgen deprivation or T replacement. Thus, the present study uses histochemical, immunohistochemical and ultrastructural techniques to investigate whether telocytes are affected by castration and T replacement in the prostate and whether they play a role in prostate gland involution reversal due to T replacement.Prostate physiology depends on steroid hormones, concentrations of which change throughout life. In some species, the prostate gland undergoes drastic seasonal changesIn the CT group, light microscopy indicated possible telocytes or telocytes-like cells located in the sub-epithelial region and the perimuscular region of rat VP. These cells had spindle cell bodies, reduced cytoplasm and possible long and thin telopodes Fig. . Twenty-Telocyte characteristics are best observed using transmission electron microscopy. In the VP from CT rats, telocytes were found at three sites: subepithelial, close to the glandular epithelial basement membrane; perimuscular, involving the smooth muscle cell layer; and interstitial, distributed in the interstitium in close association with thickened collagen fibres and blood vessels. In the CT group, telocytes exhibited condensed chromatin and long and thin telopodes Fig. . We veriThere was marked activation of the subepithelial and perimuscular prostatic telocytes in the castrated animals after 3 days of testosterone replacement. Specifically, these telocytes demonstrated loose chromatin and a large cytoplasm filled with enlarged cisternae of rough endoplasmic reticulum and the Golgi apparatus, changes that indicated a synthetic phenotype. Smooth muscle cells, with a distended morphology, appeared at the transition from a synthetic to a contractile phenotype Fig. . CellulaAfter 10 days of TR, the subepithelial and perimuscular prostate telocytes exhibited loose chromatin and increased cisternae of rough endoplasmic reticulum and the Golgi apparatus. Subepithelial telocytes also made a sequence of several focal adhesions with the epithelial basement membrane and formed small spaces that contained fine bundles of collagen fibrils. Telocyte morphology progressively returned to the normal, non-synthetic phenotype, where fusiform cell bodies and less thick telopodes occur Fig. . ActivatTelocytes are CD34-positive cells. Thus, CD34 immunohistochemistry was performed in the VP to localize these cells. In CT animals, CD34 positive cells were localize in subepithelial region; the telopodes were immunolabelled Fig. . Three aIn order to evaluate whether castration and hormone replacement had effects on the presence of AR (androgen receptor) and Ki67 immunoassays were performed in ventral rat prostate of the control rats, 21 days after castration and 3 days of T replacement. In the control rats, strong immunolabeling for AR is verified in the prostatic epithelium and in stromal cells including some cells with fusiform nucleus in the perialveolar region, in which telocytes are normally detected Fig. . The immIn order to distinguish telocytes from other CD34 positive cells, as endothelial and hematopoietic cells, double immunofluorescence assays for CD34 and CD31, that labels endothelial and hematopoietic cells but not the telocytes, were performed. The CD34 positive cells were verified at the periphery of the prostatic alveoli in the control group, these cells have long cytoplasmic projections and are CD31 negative, which coincides with the profile of the telocytes Fig. . In the The serum testosterone was 7.1 ± 1.6 ng/mL in CT group. The T levels was below the detection limit (0.02 ng/mL) in CS21 group Fig. . TestostGlandular and cellular changes in epithelium, smooth muscle cells and telocytes due to castration and hormone replacement were presented as a schematic draw in the Fig. 38. Our data corroborate the effects of castration on the prostate and add complexity to this process by implicating a new player: telocytes. According to our ultrastructural and immunohistochemical results, these cells modify their phenotype after castration. Subepithelial telocytes show reduction or loss of their telopodes as well as nuclei dilation. These changes occur simultaneously with the progressive loss of focal adhesions with the disorganised secretory epithelium, indicanting a possible reciprocal interaction between telocytes and epithelial cells for maintenance the normal cellular phenotype.Castration has drastic effects on the prostate, leading to gland involution, as reflected in the loss of the secretory epithelium and the disorganisation of the supportive prostatic stroma39 and other organs25. These junctions are lost over the duration of castration, and smooth muscle cells undergo drastic changes as demonstrated by the acquisition of a synthetic phenotype marked by an irregular shape and associations with bundles of collagen fibres. Telocytes may play a broader role in adult prostate tissue organisation, not only by compartmentalising the stromal microenvironment26, but by also actively acting on the secretory epithelium and the smooth muscle layer. However, it should be noted that telocytes are also altered reciprocally by these other tissue components and prostate function maintenance depends on these multidimensional interactions.Smooth muscle cells in the prostate also establish adherens junctions with telocytes. Junctions between these cells were observed in the ileum35. Three days after T replacement, telocytes acquired a phenotype similar to the smooth muscle cell synthetic phenotype, with dilated cytoplasm, large nuclei and association with bundles of collagen fibrils, and focal adhesions with the epithelium basement membrane and adherens junctions with smooth muscle cells were restored. We term these cells “telocytes with a synthetic phenotype”. After 10 days T replacement, the telocytes progressively lost this phenotype and switched to the normal phenotype, and simultaneously the prostatic secretory epithelium and the smooth muscle cell layer were restored. Serum testosterone levels after 3 days of T replacement were higher than compared to 10 days of T replacement, and thus prostatic secretory epithelium restoration depends on the restoration of serum testosterone to that observed in the control group. As part of this restoration, the smooth muscle cells returned to the contractile phenotype as telocytes progressively reverted to their normal phenotypes. Our immunohistochemical data show that telocytes are AR positive and thus sensitive to testosterone, as previously observed by our group26, this possibly corroborates the role of this cell type in the reversion of prostate involution induced by castration. Previous data indicated that telocytes are also sensitive to estradiol26, and thus prostate telocytes respond to steroid hormones.Prostate involution reversal was observed by means of T replacementIn general, our data suggest that telocytes participate in both maintenance and reestablishment of the secretory epithelium and periductal/perialveolar musculature layer of the prostate after T replacement in castrated animals.Our data indicated the presence of telocytes and changes in their morphology during castration-induced prostate involution and prostate involution reversal due to T replacement. We observed morphological changes in telocytes during these processes, with the reduction or absence of telopodes and dilated nuclei during castration and the acquisition of a phenotype, which we termed synthetic, during the onset of T replacement. Finally, after 10 days of T replacement, the telocytes began to return to their characteristic phenotype, which occurred along with the progressive return of T serum levels to the control group level. Finally, our data demonstrated that prostatic telocytes are not static but rather change their phenotypes, and these cells are connected with both the epithelium and smooth muscle and are involved in a T-dependent mechanism that is important both in maintaining the functionality of the prostate and in reversing its involution.The animals were provided by the São Paulo State University . Adult male 3-months-old Wistar rats were housed in a temperature-controlled (25 °C) room on a 12 h light/dark cycle. All of the animals were housed in polyethylene cages, with ad libitum access to filtered water and rodent food. The experimental protocol followed the Ethical Principles in Animal Research and the Brazilian legislation established by the Brazilian Council of Control in Animal Experimentation and was approved by the Ethics Committee for Animal Use from the Institute of Biosciences of Botucatu/UNESP (CEUA protocol 492). The animals were divided into three groups: Control rats (CT), untreated castrated rats (CS), and castrated rats treated with testosterone replacement (TR).Castration was performed via orchiectomy at 90 days of age the anesthesy with 100 mL of ketamine (0.116 g mL) and 30 mL xylazine (0.02gmL) (1 mL applied per kg body weight). After 21 days of glandular involution, a group of castrated animals started to receive daily doses of testosterone propionate (4 mg/kg of body weight/day) (Sigma-CO) dissolved in corn oil vehicle, subcutaneous injected, for 10 days. CT and CS animals received only the vehicle.The animals from CS group were euthanised by lethal injection containing a mixture of an anaesthetic, ketamine , and a muscle relaxant, xylazine  after 3 and 21 days of castration, while TR group were killed after 3 and 10 days of the testosterone replacement . The ventral prostate (VP) were excised, weighted and immediately immersed in fixative.TM, New Brunswick, NJ, USA). The sensitivity of this assay was 0.02 ng/mL. The intra-assay and inter-assay variations for the T assay were 5.36% and 5.10%.Blood samples from each animal were collected at the time of death. The plasma was separated and stored at −80 °C. Plasma testosterone (T) concentrations were determined by means of automatic equipment  using specific reagents supplied by Johnson & Johnson Ortho-Clinical . Five-µm prostate sections were cut and mounted on silanized slides. Prior to staining, the sections were dewaxed, rehydrated and then unmasked in 0.01 M citrate buffer (pH 6.0). The anti-CD34 , anti-Ki67 , anti-androgen receptor  antibodies were applied to the sections and incubated overnight at 4 °C. A secondary antibody complexed with peroxidase was utilized and the detection with 3,3′-diaminobenzidine as substrate was done. Counterstaining was performed with Harris hematoxylin. Analyzes were carried out using a DMLB Leica Microscope and the images were obtained by a Leica DFC300FX digital camera connected to the microscope.Immunohistochemical assays of paraffin-embedded tissues sections were performed using the protocol described in Justulin et al.26. The prostate glands of rats of the control group, the group of 21 after castration and the castrated group submitted to 3 days of testosterone replacement were fixed by immersion in 4% paraformaldehyde  for 24 h. After fixation, the tissues were washed in water, dehydrated in ethanol series, paraffin embedded  and sectioned at 5 μm on a Leica microtome . Such sections of prostate gland were subjected to immunofluorescence assays to evaluate the presence of telocytes throughout the castration induced tissue remodelling and to distinguish these cells from other CD34 positive cells. Therefore, double-immunofluorescence assays for CD34  and CD31, a marker of endothelial and hematopoietic cells, were performed; these antibodies were incubated at a dilution of 1:100 overnight. On the following morning, the sections were incubated with goat anti-mouse antibody  and goat anti-rabbit  secondary antibodies diluted 1:200 in 1% BSA for 2 h at room temperature and then stained with DAPI . Histological sections were analysed with a ZeissImager M2 fluorescence microscope  coupled with AxioVision software .Immunofluorescences of paraffin-embedded tissues sections were performed using the protocol described in in Sanches 26. Tannic acid was chosen for better preservation and visualization of the fibrilar components. After washing with the same buffer, the material was post-fixed with 1% osmium tetroxide, washed again, dehydrated in graded acetone, and embedded in Epon 812. Ultrathin silver sections were cut using a diamond knife on a LKB ultramicrotome and contrasted with alcoholic uranyl acetate and lead citrate. Grids were examined in a Phillips CM 100 transmission electron microscope operating at 80 kV.Fragments of VP from different experimental groups were processed for TEM. Tissue fragments (1 mm3) were fixed with 3% glutaraldehyde plus 0.25% tannic acid in Millonig’s buffer containing 0.54% glucoseThe values are expressed as mean ± SD. One-way analysis of variance was performed to determine whether differences existed among all groups and then the Tukey Kramer post-test was employed to determine the significance of the differences. A p-value of < 0.05 was considered significant. Statistical analyses were performed using Instat version 3.0 software .


LMNA gene encoding lamin A affect the differentiation capacity of stem cells, but the mechanisms of this influence remain largely unclear. We and others have reported recently an interaction of lamin A with Notch pathway, which is among the main developmental regulators of cellular identity. The aim of this study was to explore the influence of LMNA mutations on the proosteogenic response of human cells of mesenchymal origin and to further explore the interaction of LMNA with Notch pathway. Mutations R527C and R471C in LMNA are associated with mandibuloacral dysplasia type A, a highly penetrant disease with a variety of abnormalities involving bone development. We used lentiviral constructs bearing mutations R527C and R471C and explored its influence on proosteogenic phenotype expression and Notch pathway activity in four types of human cells: umbilical vein endothelial cells (HUVEC), cardiac mesenchymal cells (HCMC), aortic smooth muscle cells (HASMC), and aortic valve interstitial cells (HAVIC). The proosteogenic response of the cells was induced by the addition of either LPS or specific effectors of osteogenic differentiation to the culture medium; phenotype was estimated by the expression of osteogenic markers by qPCR; activation of Notch was assessed by expression of Notch-related and Notch-responsive genes by qPCR and by activation of a luciferase CSL-reporter construct. Overall, we observed different reactivity of all four cell lineages to the stimulation with either LPS or osteogenic factors. R527C had a stronger influence on the proosteogenic phenotype. We observed the inhibiting action of LMNA R527C on osteogenic differentiation in HCMC in the presence of activated Notch signaling, while LMNA R527C caused the activation of osteogenic differentiation in HAVIC in the presence of activated Notch signaling. Our results suggest that the effect of a LMNA mutation is strongly dependent not only on a specific mutation itself, but also might be influenced by the intrinsic molecular context of a cell lineage.Lamin A is involved in many cellular functions due to its ability to bind chromatin and transcription factors and affect their properties. Mutations of  LMNA gene is associated with a disease called laminopathy. The most known pathological form of lamin A—progerin—causes a rare premature aging syndrome, or progeria. At the same time, point mutations of the LMNA gene encoding lamin A are more frequent and lead to diseases, in which various tissues of mesenchymal origin are damaged. Mutations are often tissue-specific, that is, certain mutations lead to the appearance of a single disease phenotype with a muscle, skeletal or adipose tissue being primarily involved. Dysfunction of the cardiovascular system is a common sign for many laminopathies and is considered as the leading clinical sign in lamin-associated cardiomyopathies and myodystrophies [Nuclear lamins are the main proteins of the nuclear envelope and provide the strength to the nuclear membrane, as well as the interaction of extra-nuclear structures with components of the nuclear matrix. Recently, it became clear that lamins play not only a structural, but also a regulatory role in a cell. Disruption or mutation of the trophies ,2,3,4,5.LMNA are not clear. It has been shown that changes in the processing of lamina may be involved in atherosclerotic processes during aging. Pre-lamin A could accumulate in the arterial wall and colocalize with degenerating smooth muscle cells in atherosclerotic plaques [The studies focusing on the cells of cardiovascular origin are still rare and mechanisms of cardiac pathologies associated with mutations in  plaques ,7. Proge plaques . Lamin A plaques . The acc plaques . In smoo plaques . How lamins regulate gene expression and cell differentiation remains unclear. Lamins directly bind to DNA, chromatin, nucleosomes and histones, but the physiological relevance of these interactions is still not certain . Long heLMNA could affect this interaction [One of the proposed mechanisms for the realization of the lamin pathological effect is a specific alteration of a particular cellular signaling system. We have recently shown that lamin A interacts with Notch signaling, influencing cellular fate and differentiation, and point mutation in eraction . Lamin Aeraction ,18,19,20eraction . Proosteogenic phenotype is one of the “default” cellular phenotypes that cells of mesenchymal origin could easily acquire at pathological state such as vascular and valvular calcification, atherogenic transformation, aging, and others. Notch is an important regulator of the osteogenic state of cells and is implicated in various stages of osteogenesis . LMNA gene. MADA is characterized by dysmorphic craniofacial and skeletal features, lipodystrophy, and metabolic complications as a consequence of severe alteration of cellular osteogenic differentiation and calcification processes [Mandibuloacral dysplasia type A (MADA) is an extremely rare autosomal recessive genetic disorder caused by the mutation of the rocesses ,26,27,28rocesses . LMNA mutations on the proosteogenic response of human cells of mesenchymal/cardiovascular origin and to further explore the interaction of LMNA with Notch pathway. To address this aim, we have chosen two LMNA mutations associated with MADA and compared LMNA-Notch interaction in four cell types of mesenchymal/cardiovascular origin—endothelial, smooth muscle, cardiac and valvular interstitial cells. We show that the effect of a LMNA mutation is strongly dependent on the cell type and thus is tissue-specific and might be reversed depending on cell type even inside mesenchymal lineages. The aim of this study was to explore the influence of The study was performed according to the Declaration of Helsinki and approval was obtained from the local Ethics Committee of Almazov Federal Medical Research Centre. Written informed consent was obtained from all subjects prior to tissue biopsy. g for 5 min and cells were put in growth medium , 67.5% M199, 10% fetal bovine serum (Hyclone), 1× nonessential amino, 50 units/mL penicillin and 50 µg/mL streptomycin , seeded on 0.1% gelatine-coated 96-well plate, and clonally expanded. After this, the cells were expanded for 1–2 weeks in Petri dishes  in growth medium, at 37 °C and 5% CO2. CMC immunophenotype was verified with flow cytometer GuavaEasyCyte6  using CD33, CD45, CD117, CD90, CD105, CD73, CD146, CD56 monoclonal antibodies  as previously described [Human and rat cardiac mesenchymal cells  from myocardial tissue were isolated and put in in vitro culture and induced to differentiation as previously described . Brieflyescribed .To obtain human aortic smooth muscle cells (HASMC), cultures the cells were isolated from the aortic wall by collagenase digestion as described previously . HASMC wHuman aortic valve interstitial cells (HAVIC) were isolated from aortic valves harvested during aortic valve surgery at the National Almazov Research Centre by collagenase treatment as described previously . Patientg for 5 min. The cell pellet was suspended and seeded on a 35-mm Petri dish covered with 0.2% gelatin in ECM . Primary cells between passages two and five were used for all experiments. All cultures were maintained in humidified 5% CO2 at 37 °C.Human umbilical vein endothelial cells (HUVEC) were isolated from the umbilical vein by collagenase dissociation . The veiLMNA WT, LMNA R527C and LMNA R471C bearing lentiviral constructs were described in a previous work [ous work  as well ous work . LentiviLentiviral production was performed as previously described . In shorLMNA-bearing viruses, RCMC were transduced with viruses bearing human LMNA and then immunocytochemical staining with antibodies against human lamin A was performed. The effective rate of RCMC transduction was 40–60%.Transgene expression from lentiviruses was verified. For NICD-bearing, virus transduction of cells was verified by Western blotting for active Notch domain (NICD). For The cells were grown on cover slides, then fixed for 10 min in 4% paraformaldehyde and treated by 0.1% Triton X-100/PBS for 3 min, then incubated in 1% BSA/PBS for 1 h. Then cells were incubated for 1 h with primary antibodies against lamin A . Secondary antibodies conjugated with Alexa488  were used. DAPI was used to visualize nuclei. Microphotographs were taken using AxioObserver Microscope  at ×20 and ×40 magnification with AxioVision software.LMNA WT or mutant LMNA. LMNA R527C and LMNA R471C were used in experiments. Osteogenic differentiation was induced in control and transduced cells by the addition of 50 mg/m ascorbic acid, 0.1 mM dexamethasone and 100 mM beta–glycerophosphate to the osteogenic medium  (ScienCell); for HCMC: DMEM/F12 was supplemented with 2% horse serum, 1× nonessential amino, 1× ITS, 2 mM L-glutamine, and 100 units/mL penicillin/streptomycin; for HASMC: DMEM was supplemented with 5% FBS (HyClone), 2 mM L-glutamine, and 100 units/mL penicillin/streptomycin; for HAVIC: DMEM was supplemented with 15% FBS (HyClone), 2 mM L-glutamine, and 100 units/mL penicillin/streptomycin). All experiments were performed with three biological replications, i.e., three independent experimental procedures. The expression of osteogenic markers were estimated after 3 days of differentiation using qPCR.Intact cells and cells were transduced with virus carrying wild type NOTCH1, NOTCH3, NOTCH4, HEY1, HES1, RUNX2, OPN, and COL1A1 are presented in RNA from cultured cells was isolated using ExtractRNA . Total RNA (1 μg) was reverse-transcribed with MMLV RT kit . Real-time PCR was performed with 1 μL cDNA and SYBRGreen PCRMastermix  or TaqMan in the Light Cycler system. The thermocycling conditions were as follows: 95 °C for 5 min, followed by 45 cycles at 95 °C for 15 s and 60 °C for 1 min. A final heating step of 65 °C to 95 °C was performed to obtain melting curves of the final PCR products. The corresponding gene expression level was normalized to GAPDH from the same samples. Changes in the target genes expression levels were calculated as fold differences using the comparative ΔΔCT method. Primer sequences for human Cells were transduced with lentivirus containing the 12xCSL-luc reporter described above. In the construct, the expression of the firefly luciferase gene is regulated by 12 CSL binding sites upstream of a minimal TK promoter and the level of CSL promoter activity indicates the transcriptional activation of a Notch pathway. Cells were lysed using Luciferase Assay System  according to the manufacturer’s recommendations 48 h after transduction. Luciferase activity was measured with Synergy2 . Samples were normalized by protein content using Pierce BCA Protein Assay Kit . t-test. A value of p ≤ 0.05 was considered significant. Statistical analysis was performed by using R software .Values are expressed as mean ± SD of triplicate experiments. Groups were compared using Student’s LMNA. For this, we transduced rat cardiac mesenchymal cells (RCMC) with lentiviruses containing human sequences of LMNA WT, LMNA R527C and LMNA R471C, and then stained the cells with an antibody specifically recognizing human lamin A and not recognizing rat lamin A  transduced with lentiviruses bearing wild type and mutant  lamin A . That alLMNA WT or mutant LMNA (LMNA R527C/LMNA R471C) and added LPS to culture medium. We used four types of human cells of mesenchymal/cardiovascular origin: human umbilical vein endothelial cells (HUVEC), human cardiac mesenchymal cells (HCMC), human aortic smooth muscle cells (HASMC) and human aortic valve interstitial cells (HAVIC). We analyzed the expression of osteogenic markers RUNX2, ATF4, OPN, and COL1A1 3 days after transductions (RUNX2 and COL1A1 in LPS-treated HAVIC compared to HAVIC without LPS (Control) in non-transduced cells HAVICs only. Next, we analyzed the effect of LMNA R527C and LMNA R471C compared to LMNA WT in LPS-treated cells. We detected a statistically significant increase of OPN and COL1A1 expression levels caused by LMNA R527C mutation in HASMC and HAVIC, correspondingly. LMNA R471C caused a significant increase of ATF4 and COL1A1 in HAVIC when compared to LMNA WT. Thus, four types of mesenchymal cells had individual early responses to the LPS treatment.Lipopolysaccharide (LPS) is widely used for its ability to mimic anti-inflammatory cellular response and has been shown to influence the proliferation and differentiation of mesenchymal stem cells . To asseductions . We obseRUNX2, ATF4, OPN and COL1A1 3 days after the induction of differentiation  and induced osteogenic differentiation. Transduction with LMNA R471C caused a significant change in the expression level of osteogenic markers: increase of RUNX2 and ATF4 in HCMC; increase of ATF4 in HASMC; increase of RUNX2 and decrease of ATF4 and COL1A1 in HAVIC. Transduction with LMNA R527C caused a significant increase in the expression level of RUNX2 in HASMC and HAVIC and a decrease in the expression level of COL1A1 in HAVIC. In addition, we found a very low level of OPN and COL1A1 genes expression in HUVEC and OPN gene expression in HCMC. These results show individual early response of each cell type on osteogenic induction and LMNA mutation. To explore proosteogenic capacity, we induced osteogenic differentiation in the cells by the addition of specific factors to growth medium. Osteogenic induction was analyzed by the early expression of specific osteogenic markers ntiation . We obseLMNA WT or mutant LMNA (LMNA R527C/LMNA R471C) expression in each cell type. Induction of osteogenic differentiation caused a statistically significant increase in the expression of NOTCH1 and NOTCH3 in HCMC compared to undifferentiated cells. In contrast, the induction of osteogenic differentiation in HASMC and HAVIC caused a statistically significant decrease in the expression of HES1 or NOTCH1, NOTCH3, NOTCH4, HEY1, and HES1, correspondingly. Next, we evaluated the effect of LMNA R527C and LMNA R471C mutations on Notch-related genes compared to LMNA WT. We detected a significant increase of the NOTCH3 expression level in HCMC caused by LMNA R527C mutation when compared to LMNA WT. HASMC with LMNA R471C were marked by a significant decrease of NOTCH3 expression level compared to LMNA WT. In addition, we demonstrated statistically significant changes of gene expression levels in HAVIC: decreasing NOTCH1 and NOTCH3 for cells with LMNA R471C, and increasing NOTCH4 and HES1 for cells witch LMNA R471C and LMNA R527C, correspondingly.To estimate the interaction of lamin A and Notch pathway, we analyzed the effect of mutant LMNA on the expression of Notch target genes in the four types of human mesenchymal cells: HUVEC, HCMC, HASMC and HAVIC . We tranLMNA are dependent on the cellular type and tissue.These data suggest that mutant lamin A counteracts with Notch signaling and confirms our earlier observation that the effect of mutations in LMNA R527C mutations.HCMC and HAVIC were selected for further analysis as they showed more pronounced effects of HEY1, indicating the activation of Notch signaling in both cell types at both time points . Osteogenic induction was confirmed by the expression of a specific osteogenic marker RUNX2 3 and 12 days after the induction of differentiation. RUNX2 expression level. LMNA R527C caused a significant decrease in the expression level of RUNX2 in HCMC 12 days after the induction of osteogenic differentiation. In contrast, there was an increase of the expression level of RUNX2 in HAVIC 3 days after the induction of osteogenic differentiation.To study the effect of Thus, we observed inhibiting action of LMNA R527C on osteogenic differentiation in HCMC in the presence of activated Notch signaling, while LMNA R527C caused the activation of osteogenic differentiation in HAVIC in the presence of activated Notch signaling.LMNA mutation.We summarized the data on gene expression in four cell lines in LMNA mutation in a given cell line.We asked what could be the reason for why the cells of the similar mesenchymal origin respond differently to the same condition. We analyzed the expression of Notch-related genes and proosteogenic genes and compared four mesenchymal lines used in the study by the level of expression of these genes . The expLMNA geneHow lamins influence cellular fate remains one of the important questions of cellular biology. Understanding the regulating mechanisms, which involve lamins, is also important for the search for therapeutically relevant approaches to laminopathies caused by point mutations in the LMNA mutations associated with bone tissue malformations. We show here that even the cells of close developmental origin, such as endothelial cells, cardiac mesenchymal cells, aortic smooth muscle cells, and aortic valve interstitial cells could have different consequences for the same conditions, such as specific proosteogenic treatment or specific LMNA mutation, in terms of their differentiation commitments. In this study, we have analyzed the proosteogenic response of four human primary cell lineages in the presence/absence of specific LMNA mutations associated with various disease phenotypes bring about their effect in a mutation specific manner: Each mutation caused a specific influence on the differentiation and proliferation capacity of MSC [Previously, using human mesenchymal stem cells of adipose tissue (MSC), we have shown that y of MSC . This exy of MSC ,37,38. LMNA mutations and, by extension, in all nuclear envelop disorders, there is an inappropriate association of heterochromatin with nuclear lamina upon differentiation [Direct binding of lamins to different regions of chromatin in the course of cellular differentiation led to the concept that differentiation might be dependent on the repression/activation of genes involved in this binding , howeverntiation ,41.LMNA R482L mutation associated with Dunnigan-type familial partial lipodystrophy contributes to the down regulation of Notch activation in undifferentiated and differentiated mesenchymal stem cells and decreases adipogenic differentiation when Notch is activated, thus supporting the hypothesis that lamin A interacts with Notch pathway [In our previous work, we showed that  pathway . In the  pathway . Notch i pathway . An impl pathway . In a re pathway .LMNA mutations, either leading to a lipodystrophy or associated with signs of premature ageing, also triggered vascular smooth muscle cell senescence with osteoblastic transdifferentiation and calcification [Lamin A has been shown to influence osteoblast differentiation  and coulfication ,45.LMNA R527C or LMNA R471C mutations. A recent study used three cell lines: normal human dermal fibroblast, HeLa and HEK 293 and suggested that less differentiated embryonic cells are very sensitive to lamin A imbalance, and its upregulation disturbs lamin C, which may influence gene expression and many regulatory pathways [We observed various responses of different cell lineages to similar treatments stimulating proosteogenic phenotype and to similar alterations of genetic background such as the introduction of pathways .Evidences exist that the expression of disease-causing lamin A mutations can drastically alter interactions with chromatin . For exaThe main conclusion that we draw from our study is that the action of lamin A is strongly dependent on the intrinsic molecular context. We observed a broad range in expression level of Notch-related and proosteogenic genes between the different cellular types used in the study. We suggest that this variability in gene expression level and balance among different signaling pathways could also contribute to spacial regulation of lamin interaction with chromatin and define its regulatory role in cellular fate. A comparison of four cellular models gives clues to understanding the variable and the still cryptic role of lamin A in driving cellular commitments to differentiation into a specific lineage.Outcomes of this cross-talk deserve further research for a more detailed understanding of the role of A-type lamins in cellular differentiation as well as in pathology.


MBTPS2).The ichthyosis follicularis, alopecia, and photophobia (IFAP) syndrome is a rare X‐linked genodermatosis characterized by noninflammatory spiny follicular hyperkeratosis, severe photophobia, and non‐scarring alopecia with variable severities. IFAP syndrome results from mutations in the gene encoding the membrane‐bound transcription factor peptidase, site 2  was identified in our patient. The heterozygous MBTPS2 mutation was identified in his mother but not his father.A novel missense mutation  in the membrane‐bound transcription factor peptidase, site 2 gene (MBTPS2 mutation in a patient with IFAP syndrome and thus expands the known MBPTS2 molecular repertoire.This study demonstrated a novel  Visual acuity was 0.25 in the left eye and 0.15 in the right eye. Ophthalmologic examination revealed conjunctival congestion and photophobia while cornea and ocular fundus were normal. He had slightly prominent ears. Dermatological examination revealed complete absence of hair, eyelashes, and eyebrows as well as chronic angular cheilitis around the mouth  according to the manufacturer's instruction. Genetic investigations for their daughters and grandmother were not available. Genomic DNA sample was fragmented to construct DNA library. Then the DNA library was amplified and purified by PCR in accordance with the manufacturer's instructions. Single‐read sequencing was performed by NextSeq500 (illumina). Then Align analysis, SNP analysis, and DIP analysis were conducted to obtain information of mutation sites from targeted region. At last, protein damage analysis was conducted to qualitatively predict the probability of the results by PolyPhen‐2.2.2. In addition, variants were confirmed by Sanger sequencing in final.MBTPS2 in exon 10 of the X phenotype of IFAP chromosome. The heterozygous MBTPS2 mutation was identified in his mother but not his father  of 3P of MBT5The IFAP syndrome is a rare disease characterized by typical presence of ichthyosis follicularis, alopecia and photophobia. Ichthyosis follicularis is the most common cutaneous manifestation presenting as noninflammatory follicular keratotic papules, which mainly involve the scalp and extensor extremities and give the sandpaper texture. Other cutaneous findings include hyperkeratotic psoriasiform plaques, lamellar scaling, angular cheilitis, periungual inflammation, and dystrophic nails. Congenital non‐cicatricial alopecia involving the scalp, eyebrows, and eyelashes is another distinct finding. Most of the cases have complete body hair loss, while some cases have thin and sparse hair  span the endoplasmic reticulum membrane. Mutations in MBTPS2 were also identified as genetic basis for IFAP syndrome with BRESHECK syndrome, keratosis follicularis spinulosa decalvans syndrome  and an X‐linked form of Olmsted syndrome (OMIM# 300918) , and the keratosis, ichthyosis, and deafness syndrome . IFAP syndrome is not treatable. Oral acitretin at a dose of 0.3 to 1 mg/kg/day had been used with improvement in cutaneous features but no changes for photophobia  in MBTPS2 gene was detected, which added new genetic information to this condition.The authors report no conflict of interest.


Three hundred and eight healthy Lebanese adults were included in this study. Data collection included a questionnaire for demographics and lifestyle in addition to a detailed dietary assessment using a culture-specific 80-item semi-quantitative food frequency questionnaire. This was coupled with anthropometric measurements and peripheral blood withdrawal for DNA and genotyping using Taqman allele discrimination assays. The two FTO candidate SNPs were not associated with risk of obesity in this population sample, yet there was a trend, though not a significant one, towards lower muscle mass among carriers of the risk allele of either FTO SNPs. To our knowledge, these results have not been previously reported. As for the TCF7L2rs7903146 variant, results were congruent with the literature, given that individuals who were homozygous for the risk allele had significantly higher body mass index (BMI) and body fat despite lower intakes of saturated fat. Similar interactions, though not significant, were shown with muscle mass, whereby individuals who were homozygous for the risk allele had lower muscle mass with higher intakes of saturated fat, a result that, to our knowledge, has not been previously reported.In contrast to the large number of genetic studies on obesity, there has been significantly less nutrigenetics investigation of the interaction between diet and single nucleotide polymorphisms (SNPs) in obesity, especially within Eastern Mediterranean populations. The aim of this study was to evaluate the potential interactions between three candidate SNPs, namely,  This exceeds BMI increases reported from high-income countries such as the USA (1.1–1.2 kg/m2 per decade) [2 per decade). These findings carry important public health implications given that the association between body mass index (BMI) and chronic diseases is continuous and entails a dose-response relationship [Obesity is increasingly recognized as a public health concern with its prevalence reaching alarming levels in several parts of the world, plaguing both high- and low-income countries and jeopardizing their ability to cope with the increasing cost of treating obesity-associated diseases ,3,4,5,6. decade)  and excetionship .The causes of weight gain and obesity are multifactorial, and efforts to identify these causes are crucial for the development of effective preventive strategies. In addition to high dietary energy intake and low energy expenditure, obesity is influenced by factors such as genetic predisposition. Although hereditability seems substantial, with at least a 40% contribution based on twin and family studies, genetic mechanisms predisposing people to obesity are still not very well understood and are at many times contradictory ,11. Recers1558902 and rs9939609 variants in the fat mass and obesity (FTO) associated gene, with the A allele being consistently linked to increasing BMI [AA genotypes have been seen to produce greater reductions in weight, increases in muscle mass and changes in fat distribution in response to a two-year high protein diet, with opposite effects having been observed in those who consumed low protein diets [AA individuals are also prone to significantly enhanced weight loss in response to consuming a diet that is low in saturated fat and high in polyunsaturated fat [AA genotype have an enhanced weight loss response with higher physical activity when compared to TT [Transcription factor 7 like 2 (TCF7L2) gene with fat interactions. For example, in a 10-week randomized hypoenergetic diet, TCF7L2 rs7903146 homozygous TT participants were seen to significantly lose 44% more weight in response to a moderate- to low-fat diet [In contrast to the large number of genetic studies on obesity  there hasing BMI  as well sing BMI . This riated fat , and to ated fat . Interesed to TT . It is ifat diet . A two-yfat diet .Melanocortin 4 Receptor (MC4R) and the Apolipoprotein gene family in Iranian populations [rs1558902 and rs9939609 in the FTO gene and the rs7903146 variant of the TCF7L2 gene, and macronutrient intake with regard to obesity, body fat and muscle composition.To our knowledge, there have been no diet-gene interaction evaluations for the Eastern Mediterranean population except for a couple of studies on dietary interaction with candidate SNPs in ulations ,22,23. TThis cross-sectional study builds on a previously recruited and described community cohort of unrelated Lebanese individuals . All subAfter signing their informed consent, 501 Lebanese subjects older than 18 years old were recruited from the Greater Beirut area between February and June 2014. A multi-component questionnaire was administered in an interview settin, to obtain information on demographic and socioeconomic characteristics, lifestyle habits, physical activity using the short version of the International Physical Activity Questionnaire (IPAC) , and medn = 501) was therefore performed based on the following criteria: Firstly, having complete anthropometric and dietary data, and secondly, having no known diagnosis of hypertension, dyslipidemia, diabetes, or hyperglycemia. The final sample size was 308.For the present study, participants who reported the diagnosis of a chronic disease or metabolic abnormality that could have affected their food consumption habits were excluded. The selection of subjects from the original survey  . Partici® DNA kit  and stored at −20 °C until analysis. Genotyping for the three candidate SNPs was performed using Taqman allele discrimination assays on the CFX384 Touch real-time PCR detection system from BioRad . The reaction mix contained 2× universal PCR Master Mix No AmpErase UNG  and 40× Taqman SNP genotyping assay  diluted to 1×, DNA, and Nuclease-free water to make up a total volume of 10 µL/well. The PCR protocol was as described by the manufacturer: 95 °C for 10 min, then 40 cycles of 92 °C for 15 s followed by 60 °C for 1 min. Ten percent of the samples were run in duplicate and showed 100% reproducibility.DNA was isolated from 300 µL peripheral whole blood using the Qiagen Flexigenep-value considered in this study and the statistical significance level was set at 0.016 (being the ratio of 0.05 over three). No power analysis was performed because the study aims were multifactorial and included interactions with a number of dietary factors on three outcomes with potential confounders. Moreover, there is little nutrigenetics literature on body fat and muscle mass, in addition to the fact that this study builds on an already available sample. Thus, this work is considered exploratory and hypothesis generating.Data were analyzed using Statistical Package for Social Sciences (SPSS) version 24.0 for Windows . Since there were three groups of analyses , Bonferroni’s correction was used for the FTO genotypes for assessment of LD. Note that all of the participants were Caucasian Arabs.Allele frequencies were calculated and tested for Hardy Weinberg Equilibrium (HWE) using the chi square test. A Kappa statistic for agreement was calculated for the two Data were described in terms of frequencies and percentages for categorical variables, and mean ± standard deviation (SD) for continuous ones. Means with associated 95% confidence intervals (CI) of continuous variables were also estimated where appropriate. Associations between genotypes and participant characteristics, anthropometric measurements, lifestyle factors, and diet were performed using chi square or one-way analysis of variance (ANOVA) as applicable.Dietary intakes of macronutrients, expressed as percent energy intakes, were categorized into tertiles based on statistical grounds. A within-tertile trend analysis was performed using general linear models. The results were adjusted for potentially confounding factors, namely, age, sex and physical activity, as applicable. To account for any effect modification of dietary intake on the associations between the genotypes and each of the three outcomes, a multivariate analysis was carried out while including an interaction term of the genotypes and the different dietary intake tertiles. The same approach was carried out for the effect modification of physical activity on the various associations.FTO rs1558902 A allele (46%), FTO rs9939609 A allele (45%), and TCF7L2 rs7903146 T allele (35%). These were all in HWE and the frequencies were similar to those for reported MAFs [FTO SNPs were in Linkage Disequilibrium (LD) (Kappa (95% CI) = 0.83(0.78–0.89)), and therefore, data for FTO rs9939609 are shown in supplementary tables only. The minor allele frequencies (MAFs) of the three SNPs were: ted MAFs . As expeFTO rs1558902 and TCF7L2 rs7903146. The majority of the included participants were female (62.7%); they were relatively young with a mean ± SD age of 39.79 ± 14.00 years. The majority performed some type of exercise (85.7%). There was a relatively high proportion of current smokers (43.8% for cigarette and 32.8% for narghileh), and approximately one fifth of the study population currently consumed alcohol (21.4%).Mean daily dietary energy intake was estimated at 3600.55 ± 2029.45 Kcal/day with carbohydrates making up the highest contribution (50.07%), followed by fat (39.00%), and protein (12.95%). Sugar was estimated to contribute 14.8% of energy intake, while the percent contributions of the various types of fat were as follows: 10.38% for saturated fat, 13.8% for monounsaturated fat (MUFA), and 10% for polyunsaturated fat (PUFA).FTO genotypes only  and 31.2% being obese (>30 kg/m2). Mean body fat was estimated at 26.24 ± 11.42 kg and muscle mass at 26.43 ± 6.37 kg. No significant differences were noted among the different genotypes  and categorized in the second tertile of % of energy fat intake were significantly heavier (mean (95%CI): 31.03 (26.64–35.43)) even after adjustment for age, sex, and physical activity. More importantly, there was a significant interaction between % of energy saturated fat and the TCF7L2 genotypes, in which it appears that those homozygous for the risk allele had a higher BMI despite lower intakes of saturated fat (tertiles 1 and 2)  a. This sTCF7L2 SNP, whereby subjects who were homozygous for the risk allele (TT) and categorized in the second tertile of % of energy fat intake were heavier (mean (95% CI): 31.34 (22.69–39.98)) . More importantly, there was, again and similarly to BMI, a significant interaction between % of energy saturated fat and the TCF7L2 genotypes whereby those homozygous for the risk allele had higher body fat despite lower intakes of saturated fat (tertile 1)  b. This sp = 0.04) with the FTO rs1558902 SNP, whereby participants who were homozygous for the risk allele (AA) had lower muscle mass when compared to those who were heterozygous or homozygous for the non-risk allele. This was true for the whole cohort and after categorization of dietary intake into tertiles   and categorized in the second tertile of % of energy polyunsaturated fat intake had higher muscle mass (mean (95%CI): 30.77 (26.34–35.20)) after adjustment    . Similars9939609 . There wd 0.016) . Interesrtile 3) c. No sigOf note is that no significant interactions were revealed between physical activity and the three SNPs concerning either BMI or body composition .FTO candidate SNPs are not associated with risk of obesity in this population sample, there was a trend, though not significant, towards lower muscle mass among carriers of the risk allele of both FTO SNPs. To our knowledge, these results have not been reported previously. As for the TCF7L2 rs7903146 variant, we have shown results that are congruent with the literature [This is the first nutrigenetics study performed on a Lebanese population. We have shown that although the two terature ,20 whereMC4R, loci in the FTO gene have been consistently linked with the risk of obesity [FTO being highly expressed in the hypothalamus and playing a role in the ghrelin, leptin, and melanocortin pathways [FTO SNPs that were revealed in Caucasians had no or weak evidence of association in non-Caucasian groups [FTO risk allele carriers reinforce the potential for uniqueness in differences in genetic variations in various populations and their differential role in obesity risk, although the current study may be insufficiently able to detect a significant association between FTO SNPs and body mass and composition. As for the potential interaction between FTO SNPs and diet with regard to body mass and composition, a recent systematic review and meta-analysis of 14 studies with various intervention types and durations has shown that individuals who carry the FTO T risk allele may benefit more from diet and lifestyle interventions when compared to non-carriers. Nevertheless, it is important to note that the benefit is relatively small . In addition, a meta-analysis of only randomized controlled trials  has shown that the FTO risk allele was not associated with any differential change in adiposity after dietary weight loss interventions [FTO variants in the current study is due to the study design being cross-sectional with a relatively small sample size when compared to, for example, Sonestedt and colleagues’ [FTO rs1558902 risk allele on body weight in East Asians but not in Caucasians or South Asians.In addition to  obesity ,35,36,37pathways ,39,40. Tpathways ,34,35,36n groups . These fventions . It is pleagues’  evaluatileagues’  who showTCF7L2 gene, which is known to play an important role in the transcription of the proglucagon gene and hence the synthesis of glucagon-like-peptide 1 (GLP-1), which is implicated in insulin regulation, appetite and food intake [TCF7L2 gene interaction with regard to obesity and weight loss [TCF7L2 rs7903146 T-risk allele are more sensitive to low fat weight-loss diets [T risk allele carriers are more prone to weight loss when they consume a Mediterranean diet that is rich in sources of unsaturated fat [T-allele confers a higher risk for obesity and adiposity in spite of lower saturated fat intakes. Of note is that it is unclear to us why only those who were categorized in the second tertile of percent energy from total fat and were homozygous for the risk allele (TT) had significantly higher BMI values. This is a finding that necessitates further investigation and may have appeared in the two other tertiles with a larger sample size. More importantly, this study is the first to show a potential link between lower muscle mass in individuals who are homozygous for the T-risk allele and who consume higher levels of saturated fat. It is unclear to us why subjects who were homozygous for the risk allele (TT) and were categorized in the second tertile of percent energy from polyunsaturated fat had significantly higher muscle mass. Increasing evidence, however, points towards differential effects of various types of fatty acids on metabolic, regulatory, and biological processes, with the concept of fatty acid sensing gaining more recognition [T-risk allele. The consumption of saturated fat has been on the rise in Lebanon as the nutrition transition unfolds [The d intake ,45, has d intake ,47. To oght loss ,20,48,49ss diets ,20 and oognition . It has ognition . One supognition . Similarognition . In our ognition . This hi unfolds , is in f unfolds .This study has several strengths. It was conducted on a random sample of the adult population living in Greater Beirut and followed a well-planned design, protocol, and methodology. Rather than estimating total fat alone, the dietary assessment performed in our study allowed us to differentiate between the intakes of various types of fatty acids and to contribute to the growing body of evidence on the potential differential effects of these fatty acids. In addition, we did not rely on BMI as the sole indicator of adiposity but rather performed a comprehensive body composition assessment where both components of fat mass and muscle mass were evaluated.The results of this study should, however, be considered in light of the following limitations, on top of the relatively small sample size. First, the dietary assessment performed in this study was based on an FFQ, which may be limited by measurement errors, reliance on memory, and the number of food items included in the food list. FFQs may also overestimate energy intake, which, coupled with the high prevalence of being overweight and obesity in our sample, may explain the observed high energy intake in the study population . DespiteFTO gene do not appear to interact with various macronutrients with regard to body mass and composition, altering dietary fat composition with respect to the TCF7L2 rs7903146 variant may differentially affect body parameters. These results should, however, be confirmed with a larger population sample and preferably a prospective interventional cohort design should they be translated into a personalized nutrition practice.In conclusion, the current results add to the growing literature on gene-nutrient interaction with obesity ,11. Alth


Among the compared technologies, MTR results in the highest yield, profit, labor productivity, nitrogen-, phosphorus-use efficiency, and water productivity (at par), and is positive for children’s welfare and the overall energy productivity, indicating better sustainability and has the potential to replace PTR. Direct seeded rice has the highest yield gap  but has the lowest production cost (can reduce the cost of production by $130 ha−1), and the highest greenhouse gas (GHG) reduction potential. SRP PIs are capable for assessing the sustainability of rice establishment technologies except for a few indicators, for example food safety and workers health and safety, which are more applicable to watershed and household level indicators, respectively. The SRP PIs provide scientific evidence and practical impetus for the selection and promotion of sustainable rice production technologies.Sustainability of rice production systems is a prime concern for Asia to maintain food security and to support economic growth. This gain in productivity not only depends on agricultural inputs but also depends on social and environmental factors. To address these emerging issues, new resource- and capital-efficient and profitable technologies have been introduced. The conventional method of rice production  is considered as highly input intensive. As an alternative, dry direct seeded rice (DSR) using seed drill has been promoted to save labor and production costs compared with PTR. Similarly, machine transplanted rice (MTR) has been also considered and promoted in many rice growing countries of South and East Asia. Economic, environmental, and social performances of DSR and MTR  were compared to the PTR using Sustainable Rice Platform (SRP) defined 12 Performance Indicators (PIs) (version 1.0) as a gauge to measure their sustainability. For that, a household survey was conducted on 652 households in Odisha India during 2016. The gaps, i.e., the target to achieve better sustainability, were computed for most of the indicators from the difference between top 10th percentile and the population mean value of the indicator. The results indicated a yield gap of 1.35 t ha  •Significant yield, profit, labor productivity, nutrient-use-efficiency gaps exist with current practice of rice production.•Machine transplanted rice had relatively minimal trade-off than manual transplanting & dry direct seeded rice.•Optimization of fertilizer, water, pesticide, labor, and production costs are necessary for improving sustainability.•Direct seeded rice has the highest greenhouse gas reduction potential than conventional and machine transplanted.•A significant scope exists to further improve the sustainability indicators to well define sustainability of rice production. It is grown in more than one-fifth of the total gross cropped area and contributes more than one-fourth of the total calorie intake . In the Sustainability is “meeting society’s present needs without compromising the ability of future generations to meet their own needs” . Mainten−1 season−1)  Laik et aeason−1) , and redeason−1) . For theeason−1) . A simileason−1) . In the machines .http://www.sustainablerice.org), is such a holistic multi-stakeholder platform co-convened by United Nations Environment (UN-Environment) and the International Rice Research Institute (IRRI). Its aim is to promote sustainability in rice production and profitability with reduced environmental footprint and with better social values, and sustainability in rice consumption, supply and trade in global rice sector  of these alternate crop establishment technologies being promoted as the drivers for sustainable intensification of rice in India and elsewhere. The Sustainable Rice Platform  optimization . Similar22.1th indicator) was used. The survey questionnaires were derived from the set of SRP indicators  was computed by dividing the amount of grain yield by total amount of irrigation and rainfall water (L). For each farmer,the total amount of irrigation water applied and the amoount of rainfall received during the crop growing period was computed. The amount of irrigaiton water computed from the number of irrigation and the approximate depth of irrigation applied , phosphorus-use efficiency (PUE), pesticide-use efficiency, GHG emission, health and safety, child labor, and women empowerment were calculated as defined by SRP . A detai applied  from the applied . Bird co applied  and Stua applied . Three s applied  and for 2.43 ha−1 during the crop growing period computed), number and type  of manual labor (person-hour) starting from nursery and land preparation to harvesting and threshing, the amount of seed, amount and type  of fertilizers, and amount and type of pesticides  used, and amount of compost applied were recorded through recall during survey and computed  among three rice establishment methods. For all three establishment methods, four energy use efficiency indicators, i.e., agronomic energy input (AEI), energy output/grain yield energy (GYE), net energy balance (NEY), and net energy yield productivity (NEYp) were computed . Questiocomputed .2.5All indicators were computed using the Excel Version of the Field Calculator. The attainable (exploitable) yield gap was computed from the difference between the mean of top 10th percentile and the population mean, and the percent yield gap was calculated by dividing this difference by the mean value of the top 10th percentile. Using the same approach, attainable gaps in other indicators, i.e., profitability, labor productivity, nitrogen- and phosphorus-use efficiency, water productivity, and greenhouse gas emission also were computed and displayed in frequency distribution indicating top and bottom percentile values. To compare the sustainability and trade-offs among performance indicators (including energy) and input use among three establishment methods and three yield gap category farmers , radar/spider diagrams  were used. Descriptive statistics (mean and standard deviation) are presented wherever it is applicable. For the comparison of establishment methods across the PIs, analysis of variance (ANOVA) was computed considering the number of farmers as replication and establishment method as treatment using the unbalanced generalized linear model (GLM) function of R version 3.5.33.1The application of the production inputs  varied and was highest in manual PTR . Farmers−1) was significantly lower than MTR (51 kg ha−1) and PTR (54 kg ha−1). Seeding window of DSR was similar to nursery establishment for MTR or PTR. Due to early seeding, the water input as rainfall was maximal in DSR followed by MTR and PTR. The N fertilizer rate used in DSR and MTR were lower than in PTR, and in overall the N rate applied in all establishment methods was lower than the recommended N rate for rice in Odisha. Farmers apply only 50–60% of the recommended rate of fertilizer in irrigated rice and 25–30% of the recommended fertilizer rate in favorable rainfed rice in Puri district. Around 97% of farmers applied all N, P and K fertilizers, 29% of farmers applied urea fertilizer also as basal, 47% of farmers applied muriate of potash fertilizer at topdressing, and 4% of farmers applied diammonium phosphate as well in topdressing. The farmers generally applied 1.8 ± 0.6 fertilizer types at basal, and 3.0 ± 1.1 types of fertilizers at topdressing. Most of the farmers (99%) applied fertilizers in splits (62% one split and 37% in two split applications at top-dressing). The MTR farmers applied the highest number and amount of irrigations followed by PTR. The lowest water use was by the DSR farmers. On average, there was more than one pesticide application for all establishment methods and the highest number of applications in MTR.Interestingly, the seed rate with DSR (41 kg ha3.2−1) followed by PTR (4.64 ± 0.77 t ha−1) and the lowest in DSR (4.12 ± 1.01 t ha−1). The overall mean yield gap of rice in Odisha was 1.35 t ha−1. Among different technologies, the highest yield gap of 1.6 t ha−1 (38%) was in DSR, followed by MTR at 1.5 t ha−1 (30%), and the lowest at 1.1 t ha−1 (23%) for manual PTR  had the lowest production cost; $100 ha−1 (17%) lower than MTR ($602 + 127 ha−1) and $130 ha−1 (21%) lower than PTR ($632 + 132 ha−1) (−1 grain followed by DSR ($0.13 kg−1 grain), and PTR ($0.14 kg−1 grain). Most of the cost saving in DSR came from avoiding nursery establishment ($44 ha−1) and reduced labor in crop establishment ($56 ha−1). Crop establishment (nursery + transplanting/sowing costs) under DSR required only 9% of the total cost, while it required 19% with MTR and 23% in manual PTR (Dry direct seeded rice ($502 + 120 ha32 ha−1) . In termnual PTR .Fig. 3Co−1), and labor productivity (108 kg grain day−1) were with MTR followed by DSR  and PTR  (−1), was higher by 34% and 26% than PTR ($350 ha−1) and DSR ($371 ha−1), respectively. The coefficient of variation of net profit was the highest with DSR (67%) followed by PTR (57%) and the MTR (42%). Compared to PTR, MTR and DSR both decreased the number of labor use by 42%, and increased the labor productivity by 45 and 28 kg grain day1, respectively  . With MTectively . The freectively .Fig. 4Le3.4−1 grain) and similar for MTR and PTR (2400  kg kg−1 grain). The highest GHG emission was from the PTR (17% higher than DSR) followed by MTR (5% higher than DSR) (Both N- and P-use efficiencies were highest in MTR followed by at par in DSR and PTR . Water phan DSR) . The frehan DSR) , while fhan DSR) .Fig. 5Le3.5Based on the SRP PIs, the majority of the farmers fall under the category of gold and acceptable pesticide applicator in Odisha . A higheOverall, 21% of farmers did not apply any pesticide, 16% of farmers applied pesticide one time, 25% of farmers applied pesticides twice, 30% applied them three times, and 9% applied them more than three times. When farmers applied pesticides, 67% of farmers applied herbicides, 69% insecticides, 42% of farmers applied fungicides, and 4% of farmers applied rodenticides SI . Less th3.63.6.1From the computation of food safety indicator as defined by SRP, only 3% of the farmers responded that they are aware of risks in food safety from cadmium, chromium, mercury, and lead, and none of them could specify the name of the heavy metal which is problematic in their location. This response was similar for crop establishment methods (varied between 2 and 5%).3.6.2Fifty percent of the farmers practice poor to fair health safety mostly due to the poor management of pesticides . Only 403.7Rice farmers in Odisha have poor to fair child support, and child labor is considerable in farm activities . Total sWomen empowerment was loosely defined in the questionnaire. It was difficult to attribute differences in the scores based on the specific technologies, as the influence is not direct and the causal relationships are not clear. The dimensions used for assessing empowerment are driven by social and cultural norms and economic status of households. So, it is hard to judge whether some technologies are more or less sustainable with regard empowerment. This needs more in-depth research including qualitative methods.Using the current questionnaire, 80% of the women who responded indicated that empowerment was poor . A disagHowever, it was also observed that women’s control over decisions regarding use of household and personal incomes increased with land holding. This is reflected in the overall higher empowerment scores generally observed in households with larger landholdings.However, we also observed some trade-offs, where better women empowerment occurred with DSR and MTR  as manua3.82 equivalent and lowest pesticide use efficiency  than PTR . Machine transplanted rice had the highest GYE, NEY, and NEYp . In termThe frequency distribution of AEI and NEYp  showed D44.1Machine transplanted rice showed the better performance with highest yield, profit, labor productivity, nitrogen-, phosphorus-use efficiency, and water productivity (at par), and the overall energy productivity, indicating it has a high potential to replace manual PTR . Whereas−1; and increased labor productivity by more than 20–60 kg day−1. However, there were trade-offs of a large yield gap (41%) and having a large percentage of farmers in the bottom decile compared to the other two crop establishment methods  and P fertilizer rates (19 kg P ha−1) are below the state recommended rate of 80–100:40:40 kg N:P2O5:K2O ha−1  yield gap of 1.52 t haountries . If we cK2O ha−1 . This isK2O ha−1 . In OdisK2O ha−1 .Overall the performance indicators worked quite well in assessing the trade-offs with different technologies; however, there is significant room for improvement in these indicators versus scale of assessment: while most of the indicators worked well to evaluate the technologies at plot scale, clearly some of the indicators e.g. food and health safety, child labor and women empowerment are not always technology or field specific. Some of these are more appropriate to be measured at the regional level while others may work at the household level. Sustainable Rice Platform performance indicators (version 1.0) are not structured well at this stage to dissect the difference of technological intervention in terms of food safety. As food safety is more a regional/household indicator, most of the farmers responded generically irrespective of technology. This indicator could be computed in a better way via more explanatory questioning related to food safety; avoiding laboratory analysis could make the indicator computation simple. Similar to food security, performance indicators for health and safety are more relevant at the household level. Most of the respondents were sharing information based on their practices on their farm rather than for a specific technology.The computed energy input in different operations  and the 4.31.The household in this study were farmers. It was relatively easy for the farmers to respond to questions on their current practices, but they found it difficult to respond to indicators which are more at the landscape level .2.The application of rodenticide, molluscicide and avicide are sporadic and minimal. Further, the ingredients could be many fold more toxic than the molecules of herbicides and fungicides. So, while computing pesticide use efficiencies, consideration of the LD50 rate of the pesticide could make the computed value more convincing. Computation of the pesticide indicator may focus only on the applied pesticides  or use computations that consider how much active ingredient is applied from the most commonly applied pesticides: herbicides, insecticides, and fungicides.3.Computing water productivity in terms of L of water per kg of grain would provide an easier index for practitioners and extensionists to understand, than an indicator expressed as kg of grain per L of water.4.Similarly, some expressions such as high GHG score are misleading to assess the sustainability performance of rice production practices.5.Women’s empowerment in households is driven by prevailing social and cultural norms and other intersectional factors. They are not technology-specific. In fact, technology use by women and their ability to make decisions regarding them is influenced by their empowerment status. While reduction in labor use and associated drudgery is welcome, some poor women who depend on wage labor for transplanting might also lose employment and incomes due to mechanization. This might open up opportunities for some entrepreneurial women to engage in service provision role. The current indicators do not allow capturing such tradeoffs in a technology-specific context. While the use of structured questionnaire and indicators is helpful, much insights would be gained from using mixed methods for such assessments.6.Indicators of energy use can be expressed using different indicators, and the outputs can be confusing for extension specialists and farmers. For example, GEY, AEI, NEY, NEYp, differ with different technologies via the input use, machines use, time and intensity of field operations, and the final outputs (grain and straw). We contend that because NEYp is more representative, then the inclusion of NEYp as the 13th performance indicator of sustainable rice cultivation could add value through providing a clearer message than the other measures, especially for technology comparisons and impact evaluation.We suggest the following considerations and refinements to the measurement of the performance indicators for sustainable rice cultivation:5The differential trade-off among different SRP PIs with different rice establishment methods in Odisha indicated MTR is the best performed has advantage over other establishment methods considering its economic, environmental and social indicators performmance as defined by SRP. Further, a relatively high gap (target to achieve) in yield (41%) and similar gaps in profit, labor productivity, NUE, PUE, and water productivity indicated sustainability can further be improved. Opportunities were identified to both decrease the yield gap and increase profitability by optimizing N and P fertilizer use, irrigation, pesticides and labor. Such optimization is crucial to reduce the nutrient mining and improve the land productivity. Our findings clearly show the benefits and positive potential (reduce sustainability trade-off) of MTR in Odisha as an alternative to PTR. Machine transplanted rice provided higher yield and net profit, and better performance of most of the SRP indicators and measures of energy use efficiency. There is scope for reducing the environmental footprint by reducing GHG emissions through the adoption of DSR. MTR-technology appeared to be the best in terms of increasing water productivity, NUE and PUE. The highest labor productivity in MTR and DSR suggests the opportunity of mechanization in rice production using alternative establishment methods and thus improving labor productivity. Machine transplanted rice also reduces drudgery and child labor, and therefore opens potential new income generation opportunities through women and youth engagement as entrepreneurial service providers. Except for food safety and workers health and safety, which turned out to be more watershed and household level indicators, SRP defined PIs (version 1.0) and the gap approach provide an effective set to measure the sustainability  of different technologies, and provided the target value to achieve for establishing sustainable rice production and sustainable intensification.


We describe the outcomes of 16 cases of imported loiasis in Italy. Patients had microfilaremia <20,000/mL and were treated with high-dose albendazole for 28 days and a single dose of ivermectin. This combination might be an effective treatment option in nonendemic areas, when diethylcarbamazine, the drug of choice, is not available. Loa loa is a filarial nematode transmitted by tabanid flies of the genus Chrysops, which inhabits forested areas of West and Central Africa  eyeworm observed in the eye conjunctiva within the previous 2 months; 2) detection of L. loa microfilariae in the peripheral blood, determined by the leukoconcentration method ; or 3) Calabar swellings associated with eosinophilia (>500 eosinophils/µL) within the previous 2 months. In addition, only patients with >12 months of follow-up were included. The reference Ethics Committee (Comitato Etico per la Sperimentazione Clinica delle Province di Verona e Rovigo) approved this study in July 2016 (study protocol no. 33908). We reviewed the medical records of patients with loiasis admitted to IRCCS Sacro Cuore Don Calabria Hospital  during 1993–2016 who had been treated with albendazole (400 mg 2×/d for 28 days), followed by ivermectin, . For study purposes, we defined a case of loiasis by having previously stayed in an endemic country plus meeting L. loa microfilariae in their blood; 5 had Calabar swellings and eosinophilia; none had an observed eyeworm. Of note, all 6 migrants had eosinophil counts of 500–1,500/µL, whereas all expatriates had eosinophil counts >1,500/µL, in line with previous observations  in the analysis . Eleven Acanthocheilonema vitae as source of antigens , returned positive results for all patients. We also evaluated serologic results retrospectively for patients diagnosed before 2014. Liver function  had been checked after ≈2 weeks from the onset of treatment with albendazole; no changes were observed in any patient. We recommended that blood tests be repeated every 2 months until patients were free of microfilaremia and that patients make annual follow-up visits for 4–5 years. However, these recommendations were seldom followed, and follow-up visits had to be tailored to each patient’s characteristics and needs. One patient received a further course of ivermectin due to the persistence of low-level microfilaremia 4 months after treatment; testing at subsequent follow-up visits showed no microfilaremia. No severe adverse events were reported, although 2 patients reported itching. A commercial ELISA test, based on Posttreatment follow-up periods ranged from 14 months to 12 years. Within 6 months after the end of the treatment, 15 (93.8%) of 16 patients had recovered completely, as demonstrated by disappearance of symptoms, normalization of eosinophil counts, and negative microfilaremia. The remaining patient reported presence of Calabar swelling and microfilaremia 1 month after treatment, so he received a single course of diethylcarbamazine, after which symptoms disappeared within 2 months. Our retrospective study indicates that using the combination of albendazole and ivermectin resulted in a high rate of recovery  in patients with loiasis with microfilaremia <20,000/mL. By comparison, a study by Klion et al. found that only 38% of 32 patients treated with diethylcarbamazine were cured after 1 course of therapy, and some patients continued to be symptomatic despite >4 courses of treatment , highlighting the need for guidelines for treating loiasis in nonendemic areas, where there is no risk of reinfection. Because data are scant, additional information about the treatment and outcome of loiasis imported into nonendemic countries is useful for clinicians. A previous study pointed out wide heterogeneity of treatment strategies for imported loiasis  plus a single 200 µg/kg dose of ivermectin might be recommended for loiasis patients with microfilaremia <20,000/mL as an alternative to diethylcarbamazine, which is the treatment of choice for loiasis but is often unavailable. In case of clinical cure (absence of symptoms) but persistence of low levels of microfilaremia, an option might be a second course of ivermectin to achieve clearance of microfilariae (