To assess parents' experiences and satisfaction with care during very preterm birth and to identify domains associated with positive and negative experiences of care.Qualitative study using semi-structured interviews.Three neonatal units in tertiary care hospitals in South-East England.Thirty-two mothers and seven fathers who had a baby born before 32 weeks of gestation and spoke English well.Semi-structured interviews were conducted. Results were analysed using thematic analysis.Participants' experiences and satisfaction with care during the birth of their preterm baby.Overall, 80% of participants were extremely satisfied with the care during the birth of their preterm baby, seven were generally satisfied but felt some things could be improved and one was dissatisfied. Four key determinants of experiences of care were identified: staff professionalism, which included information and explanation, being calm in a crisis, appearing confident and in control, and conversely not listening to the woman; staff empathy, which included caring and emotional support, and encouragement and reassurance; involvement of the father; and birth environment.Although the determinants of experiences of care are generally consistent with previous research on term births, unique factors to preterm birth were identified. These were the importance of the staff appearing calm during the birth, and the staff portraying confidence and taking control during the birth. Women valued being listened to, and both they and their partners valued staff helping fathers to feel involved during the birth. Preterm birth is the most important single determinant of adverse outcome in terms of survival, quality of life, psychosocial and emotional impact on the family, and costs for health services. The highest mortality and morbidity occurs in babies born very preterm, defined as delivery before 32 weeks of gestation..Women's views and experiences of maternity services, especially care during labour and childbirth, are increasingly important to healthcare providers, administrators, and policy makers, and can influence decisions about the organisation and provision of services.13There has been little research into parents' experiences and satisfaction with care during very preterm birth. The aims of this study were to explore parents' experiences and satisfaction with their care during very preterm birth, and to identify the domains associated with positive and negative experiences of care.This qualitative study used semi-structured interviews. Recruitment took place between June 2011 and November 2011 at three tertiary care centres in South-East England. Ethics approval was obtained from the Kent NHS Research Ethics Committee.Parents were eligible if they had a baby born before 32 weeks of gestation in the previous 6 months and spoke English well. They were also eligible if only one member of the couple wanted to take part or if they were single. Parents of babies who died were also included. Women were not approached if the attending clinicians considered that they were too unwell to take part.Information about the study was available to parents in the neonatal units. Eligible parents were then invited to participate by responding to a letter of invitation. This was either posted or given to parents if they had been on the neonatal unit for longer than 2 weeks. Parents returned a card indicating their willingness to participate, and the study researcher then contacted them to discuss the study and arrange the interview. Before the start of the interview, participants were given the opportunity to ask questions, and gave written informed consent. All interviews were carried out by the first author (A.S.), who is a psychologist with experience of interviewing women in the perinatal period. Parents were informed that the interviewer was not associated with the hospital so as to encourage open and honest responses. Parents were offered the choice of an interview in a quiet room in the hospital or at their home. Interviews lasted approximately 45 minutes. They were recorded and then transcribed with all identifying information removed. Data collection ended when no new information emerged from the interviews and data saturation had been achieved.The interview schedule consisted of 10 open-ended questions that were used as a guide to explore parents' experiences and satisfaction with care during the birth (Box Can you describe what led up to your baby's birth?Can you describe what happened during the birth of your baby?Can you describe what happened immediately after the birth of your baby?Can you tell me about your experience of the care you received at birth and immediately after your baby was born?Was there anything particular about the care you received (at birth and immediately after your baby was born) that you were happy with?Was there anything particular about the care you received (at birth and immediately after your baby was born) that you were unhappy with?What aspects of your care (at birth and immediately after your baby was born) were the most important in terms of your level of satisfaction?Overall, how satisfied would you say you were with the initial care that you received?Do you have any suggestions about how immediate care at birth could be improved?Are there any other issues I haven't brought up that you feel are important and you want to talk through?nvivo 9 was used to organise the codes and themes. For this report, direct quotes are referred to by participant codes to ensure anonymity .Qualitative analysis of the transcripts used inductive thematic analysis to identify, describe, and analyse themes and patterns within the data.n = 5) or at the participant's home (n = 34). Participants were between 25 and 44 years old, most were white European, and they were either married or co-habiting (see n = 24) and hospital 2 (n = 15), but not from hospital 3 (n = 0).Of the 123 couples or single parents sent a letter of invitation, 39 returned cards indicating an interest in the research (32 mothers and seven fathers). All fathers who took part were married to, or cohabiting with, a mother who also took part in the research. Hence, when ‘couples’ are referred to this indicates 14 participants (seven men and seven women). Men and women in couples were interviewed separately, with the exception of two couples that asked to be interviewed together. Interviews were either in a quiet room in the hospital  indicated that they were extremely satisfied with their care, and said that there was nothing that could be improved.Seven participants (18%) indicated that although they were generally satisfied with the care certain things could have been improved, such as the provision of information.In addition, one woman (2%) was clearly dissatisfied with her care.Four main themes emerged as important determinants of either positive or negative experiences of care during preterm birth. This refers to the professional competencies that were important in determining parents' experiences of care. Positive experiences of care were associated with information and explanation, staff being calm in a crisis, and staff appearing confident and in control. In contrast, negative experiences of care were associated with staff being perceived as not listening to the woman.Provision of information was really important and was mentioned by 33 participants (85%). They wanted to be told what would happen during the birth (particularly if they were having a caesarean section), what type of anaesthetic would be administered, and what was going to happen to their baby when he or she was born. The anaesthetist was someone who stood out in participants' minds in terms of providing detailed information and explanations  described feeling frightened of what was going to happen during the birth and for the outcome of their baby. However, the calm attitude of the staff helped them feel more comfortable and at ease. For one woman this was the most important factor in her birth experience  expressed disappointment that the staff did not always listen to what they had to say. These women described telling staff that they felt they were in labour and close to giving birth, and often the staff did not believe or trust what they were saying, which left women feeling ignored and frustrated. One woman described how she tried to tell the midwife that she was about to have her baby, but was not listened to, and as a result no staff were present at the birth  spoke about the ‘warm and friendly’ attitude of the staff. In terms of satisfaction with their experience it was important that they were treated in a pleasant manner. The two very different quotes shown in Mothers spoke about the importance of a member of staff always being with them, and this generally referred to the presence of a midwife  mentioned wanting encouragement and reassurance from the staff. They understood that staff have to be realistic about the situation and the prognosis for their baby  described how the staff tried to delay the caesarean section so the father could get there for the birth. Three women (8%) also discussed that they had planned their partner's involvement in the birth, and therefore appreciated any effort the staff made to make them feel more involved  described that the radio was playing during the birth, which made the environment seem less frightening (1, Mother, C/S; The aim of this study was to explore parents' experiences and satisfaction with care during very preterm births. Overall, parents' experiences of care were positive, with only one mother expressing dissatisfaction with the care she received during the birth. This is reassuring, and typical of studies of satisfaction with maternity services generally.Information provision and emotional support from the staff during the birth are clearly important to parents. Parents were highly satisfied with the information they were given during the birth, which is in contrast to previous studies where mothers of preterm babies have perceived the staff as not having the time to give information before or during the delivery.23Overall, the determinants of parents' experiences with care were generally consistent with previous research on term births; however, two factors unique to preterm birth were identified. These were the importance of the staff appearing calm during the birth and staff taking control during the birth. A calm response shown by the staff has not been previously identified as associated with satisfaction with care during childbirth, although it has been identified in other areas of patient satisfaction with care, particularly relating to emergency situations.Although parents largely reported positive experiences of care, two areas emerged where parents felt things could have been improved. First, some women described being in labour or very close to giving birth, and the staff either not believing them or not appearing to listen to what they said. Similar experiences were reported in a national study of women's experiences of maternity care.should be like, and therefore just evaluate the status quo.In common with other studies of satisfaction with care, one possible limitation of this study is that parents may have been reluctant to criticise the professionals who have taken care of them and their preterm baby. This ‘halo effect’ may be even more pronounced in the parents of preterm babies as the staff have looked after their baby for many weeks.One-quarter of parents accepted the invitation to be interviewed, which is a good response for this type of study. However, the experiences reported in this study may not be applicable to all parents who have a very premature baby. For example, the sample mainly consisted of white, married women, and further research is needed to see how these experiences relate to parents from different backgrounds. The implications and conclusions of this study should be considered with this in mind.In spite of these limitations, this study provides a rich and detailed insight into parents' experiences with care during preterm birth, and the findings have important implications for practice. Provision of information and explanations to parents, and offering caring and emotional support, is extremely important to parents. The parents in this study were very positive about these aspects of care during the birth; however, the findings also suggest that women should be listened to more, especially when telling staff they were in labour or felt close to giving birth, and that fathers should be helped to feel involved in the birth. The experience of very preterm birth is different to a term birth, and existing questionnaire measures of satisfaction with care in parents of term babies may not be appropriate for this population of parents. Satisfaction with care is a multidimensional construct consisting of different components of care provision that affect satisfaction. Therefore, it is important that future studies include multidimensional measures of satisfaction with care, as single-item measures only provide a limited insight into parents' experiences.In summary, this study contributes to the limited literature on satisfaction with care during very preterm birth. Giving birth to a preterm baby is a distressing and traumatic time for most parents. In spite of this, the present study suggests that parents are very satisfied with the care provided during the birth, but that listening to women and involving fathers during the birth could increase this further.


An attractive alternative treatment for malignant tumors that are refractive to conventional therapies, such as surgery, radiation, and chemotherapy, is electrical-pulse-mediated drug delivery. Electric field distribution of tissue/tumor is important for effective treatment of tissues. This paper deals with the electric field distribution study of a tissue model using MAXWELL 3D Simulator. Our results indicate that tumor tissue had lower electric field strength compared to normal cells, which makes them susceptible to electrical-pulse-mediated drug delivery. This difference could be due to the altered properties of tumor cells compared to normal cells, and our results corroborate this. Vm is given as [ Electroporation, a nonthermal phenomenon, is used to enhance the permeability of biological cells and tissues –4. Here,given as R is the cell radius and δ is the angle between the electric field E and the radius vector. A detailed study of electric field distribution is necessary for an effective understanding of the tissue's behavior when subjected to electric field. In this study, a 3D modeling of tumor and normal tissues are simulated using ANSOFT. The behavior of tissue and the effect of electric field on them are noted in each case. Literature has indicated that changes in the electrical properties of abnormal breast are more significant compared to the breast normal tissues [ tissues . The sur tissues . The resVm=1.5ERc tissues .For fast and accurate simulation results, we chose the Maxwell's FEM Software from ANSOFT Corporation, USA. For this simulation study, a slightly modified version of the more detailed electrical model of a tumor tissue reported by Surowiec et al.  was used Three-dimensional models of breast lobe with single tumor and normal cell, with two tumor cells and two normal cells, are simulated for both needle and plate electrode configurations. The model developed using Maxwell 3D Simulator is shown in The electric field across the tissue model for plate electrodes configuration is shown in Figures From this electric field distribution values, the transmembrane potential is calculated for the applied voltage. Also, from the 3D simulated model of different tissue model the capacitance value is noted and this value is applied to the electrical model using MATLAB.For this simulation study, a slightly modified version of the more detailed electrical model of a cell reported by Schoenbach's team , 13 is uμsec and for a time period of one second.In the above electrical model, the capacitance of the normal cell is replaced by the capacitance value obtained from the ANSOFT model of the cancer cell developed in this study. To this electrical model, a pulsed electric field of the order of 1200 V/cm is applied with a pulse width of 100 y-axis) across each element with respect to time (x-axis) is shown in Figures The variation in voltage (Cancer cells have different electrical and metabolic properties due to abnormalities in structures . A healt In our research, the electric field distribution in normal and tumor tissues was investigated using 3D finite element analysis for plane and needle electrode configurations. The tumor tissues shows lower intensities of electric field compared to the normal tissues, possibly due to the altered characteristics of membrane potential, its composition and minerals such as potassium, magnesium, sodium, and calcium . These r The electrical characteristics of the membrane and the cytoplasm, such as the conductivity and permittivity of the membrane and the cytoplasm, as well as membrane thickness also govern the response due to electroporation in addition to the intensity and distribution of the electric field applied. Our results indicate that electric field analyses could be used for selecting suitable parameters for effective treatment of tumor tissues, since these parameters need to be optimized for various tumors/tissues and cells.


As downregulation of Ocln is also seen in SCC derived from other tissues, as well as in other carcinomas, we suggest this as a common principle in tumor pathogenesis, which may be used as a target for therapeutic intervention.Tight junction (TJ) proteins are involved in a number of cellular functions, including paracellular barrier formation, cell polarization, differentiation, and proliferation. Altered expression of TJ proteins was reported in various epithelial tumors. Here, we used tissue samples of human cutaneous squamous cell carcinoma (SCC), its precursor tumors, as well as sun-exposed and non-sun-exposed skin as a model system to investigate TJ protein alteration at various stages of tumorigenesis. We identified that a broader localization of zonula occludens protein (ZO)-1 and claudin-4 (Cldn-4) as well as downregulation of Cldn-1 in deeper epidermal layers is a frequent event in all the tumor entities as well as in sun-exposed skin, suggesting that these changes result from chronic UV irradiation. In contrast, SCC could be distinguished from the precursor tumors and sun-exposed skin by a frequent complete loss of occludin (Ocln). To elucidate the impact of down-regulation of Ocln, we performed Ocln siRNA experiments in human keratinocytes and uncovered that Ocln downregulation results in decreased epithelial cell-cell adhesion and reduced susceptibility to apoptosis induction by UVB or TNF-related apoptosis-inducing ligand (TRAIL), cellular characteristics for tumorigenesis. Furthermore, an influence on epidermal differentiation was observed, while there was no change of E-cadherin and vimentin, markers for epithelial-mesenchymal transition. Ocln knock-down altered Ca Tight Junctions (TJs) are multiprotein complexes formed by transmembrane proteins, e.g. occludin (Ocln), claudins (Cldns), and junctional adhesion molecules (JAMs), which are associated with intracellular plaque proteins, e.g. ZO-1, 2 and 3 and MUPP-1. From simple epithelia it is known that TJs seal neighbouring cells and control the paracellular pathway for solutes, water, and cells (barrier function). In addition, they restrict the diffusion of apical and basolateral membrane components (fence function), coordinate signalling molecules and play a role in cell differentiation and proliferation There are frequent changes in TJ protein localization and/or expression in the course of carcinogenesis. For Cldns, up- or downregulation, as well as altered localization were described, dependent on the tumor entity. For instance, in breast cancer Cldn-1, -2, and -7 are downregulated, while Cldn-4 is upregulated and in colorectal- and pancreatic-cancer Cldn-1, -2, and -7 are upregulated arcinomata in situ) of SCC and their presence is a marker for increased risk for the occurrence of SCC Squamous cell carcinoma (SCC) is the second most common skin malignancy accounting for the majority of non-melanoma skin cancer-related metastatic disease and death There is limited data about TJ protein alterations in human cutaneous SCC. Langbein et al. 2+-homeostasis in knock-down studies.These interesting but somewhat discrepant findings in only a limited number of SCC and BD cases and the putative impact of TJ protein alterations on tumor progression prompted us to investigate larger numbers of cutaneous SCC and their precursors including  sun-exposed skin, as well as healthy, non-sun-exposed skin with the aim to identify similarities and differences for TJ proteins. Because we observed a striking loss of Ocln in many malignant SCC in contrast to its precursors we hypothesized that this molecule might promote tumorigenic features in keratinocytes which was addressed by investigating the involvement of Ocln in cell-cell adhesion, apoptosis, proliferation, differentiation and CaThe samples for this study were obtained from our clinical department. They were used after diagnostic procedures had been completed. The local medical ethics committee (Aerztekammer Hamburg) approved this study (060900 and OB-008/04). All patients gave their written informed consent.Samples of human SCC , BD , KA , AK  sun-exposed skin  and non-sun-exposed skin  were obtained from our clinical department. Grading of the SCC was performed according to the world health organisation classification Immunofluorescence microscopy was performed as previously described Pre-confluent human primary keratinocytes were transfected with the respective siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Keratinocytes were then incubated for 24 h before seeding onto a fibroblast collagen gel as described below.5 fibroblasts was poured into cell-culture inserts  and allowed to gel for 2 h at 37°C without CO2. The gels were then equilibrated with KGM medium  at 37°C in a CO2 incubator for 2 h, and 1.5×106 keratinocytes, transfected with the respective siRNAs, were seeded onto each collagen gel. After overnight incubation at 37°C the medium was removed from both the inserts and external wells, and 10 ml serum-free keratinocyte defined medium (SKDM), consisting of KGM without bovine pituitary extract and supplemented with 1.3 mM calcium , 10 µg/ml transferrin (Sigma), 50 µg/ml ascorbic acid (Sigma), and 0.1% bovine serum albumin (Sigma), was added to each external well. The organotypic skin models were cultured for 7 days and medium was changed every second day.Organotypic skin models were prepared as described previously 2+ conditions by using HiPerFect Transfection reagent  according to the manufacturer’s instructions. Briefly, cells were transfected by a fast forward protocol. 100000 cells/ml were transfected with a 1∶1 mixture of HiPerFect reagent (1∶200) and 100 nM of Ocln-siRNA or control-siRNA, respectively. Efficiency of knock-down was controlled by Western-blot and qRT-PCR analysis after 2, 3 or 5 days (depending on the experiment). Silencing of Ocln in a human organotypic skin model was performed as described Primary human keratinocytes were transfected under low Ca2+ conditions were performed 48 h after seeding of 8000 cells/well and siRNA-treatment. Subsequently, Ca2+ levels were elevated to 2 mM to induce differentiation and 48 h later proliferation experiments were performed under high Ca2+ conditions. The BrdU-ELISA kit from Roche  was used following manufacturer’s instructions.Proliferation experiments under low Ca2) were evaluated per model (n = 4). In each visual field the number of Ki67 positive cells was normalized to the total number of basal cells which were evaluated by DAPI staining.Proliferative (Ki67-positive) cells were stained by MIB-1 and at least 5 visual fields . The numbers of particles in the squares were determined automatically by utilizing FIJI software Intercellular adhesion under high Ca2+ conditions and transfected for 48 h with the indicated siRNAs. Then cells were transferred to siRNA-free medium for another 24 h and subsequently exposed to TRAIL  or irradiated with 150 mJ/cm2 to induce apoptosis. Irradiation was performed in PBS, and then the solution was replaced by fresh medium.For TRAIL treatment and UVB-irradiation, keratinocytes were plated in 96-well plates (8000 cells/well) under low CaPLUS kit (Roche) according to the manufacturer’s instructions. This assay is defined as release of mono- and oligonucleosomes due to DNA fragmentation in the cytoplasm of apoptotic cells. Cytotoxicity was determined after 3 h and 20 h of TRAIL treatment as well as 4 h and 16 h after UVB irradiation by a lactate dehydrogenase (LDH) release assay . Relative apoptosis was calculated by dividing the absorbance of treated cells by the absorbance of non-transfected non TRAIL-or UV-treated control cells. The percentages of cytotoxicity were calculated according to a control with completely lysed cells due to lysis buffer treatment.Apoptosis was evaluated at 3 h and 20 h of TRAIL treatment and at 16 h after UVB-irradiation by using the Cell Death Detection ELISA2+ permeability was performed in Ussing chambers specially designed for the investigation of cell monolayers grown on culture plate inserts 2; Millipore), cultured under high Ca2+-conditions for 48 h, and investigated in the Ussing chambers. Ca2+ permeability was determined from biionic potentials and the Goldman–Hodgkin–Katz equation as reported in detail previously Determination of Ca2+-barrier-formation and apoptosis was determined by Student’s t-test. TJ protein expressions were cross-tabulated with tumor grading, differentiation markers, and sun-exposure and the significance of associations were tested using Fisher’s Exact Test. P-values <0.05 were considered as statistically significant .Statistical significance of proliferation, adhesion, CaIn normal, non-sun-exposed skin Cldn-1 and JAM-A were predominantly found at the cell-cell borders of all living epidermal layers, while localization of Cldn-4, Ocln and ZO-1 was mainly restricted to cell-cell borders of the stratum granulosum , 3. In aIn contrast, in SCC samples there was a complete loss of staining for ZO-1 in 25% of the tumors, for Ocln in 80% , for CldIn 81% of the positive cases for Cldn-1, there was a downregulation of the protein in the lowermost and in 60% in the uppermost layers of epidermal tumor parts , Table 2In the invasive parts of the tumors , Ocln anThis indicates that complete loss of Ocln and downregulation of Cldn-1 in the lowermost and JAM-A and Cldn-1 in the uppermost layers is a frequent feature in SCC. This also applies for the broader localization of Cldn-4 and ZO-1.We wondered whether the alterations found in SCC may also be seen in in-situ carcinomas  or in tumors of lower malignancy (KA).Complete loss of Ocln was found in only 5% of AK, 11% of BD, and 29% of KA, demonstrating that its absence is much more characteristic for SCC (80%). Contrariwise, broader localization of Ocln was found more frequently in the precursor tumors . Frequency of broader localization was similar between the tumor entities. Furthermore, there was no clear-cut difference between SCC and the other tumors as concerning Cldn-1, Cldn-4, and JAM-A . SimilarThe data indicate that alterations found for Cldn-1, Cldn-4, ZO-1, and JAM-A are common for UV-induced skin tumors while the frequent loss of Ocln appears as specific for SCC.Because the alterations for Cldn-1, Cldn-4, and ZO-1 were observed in all UV- promoted skin tumors and a broader localization of Ocln was frequent in all tumor entities except for SCC, we wondered whether these alterations could also be identified in chronically sun-exposed skin. Indeed, a broader localization of Ocln, ZO-1, and Cldn-4 was seen more frequent in the epidermis of sun-exposed as compared to non-sun-exposed skin with high statistical significance  . We founAltered differentiation is a common feature in tumor progression. Accordingly, irregularity of epidermal differentiation markers involucrin and TG1 was described in SCC Because Ocln was also described to be involved in epithelial-mesenchymal transition Further, we looked for correlations between involucrin, and TG1, on one hand and Ocln expression on the other hand in the SCC samples as well as for correlations of Ocln with the SCC grade. We found that Ocln-positive tumors were to a higher percentage differentiated tumors  than Ocln-negative tumors (25.9%) . CharactWe asked whether the alteration of Ocln might also influence keratinocyte adhesion, because loss of adhesion is also a prerequisite for tumor invasion and metastasis. Indeed, significantly reduced keratinocyte-keratinocyte adhesion was seen in response to Ocln knock-down, as shown by a cell adhesion assay in a time kinetic analysis .2+-homeostasis. Therefore, we investigated whether decreased Ocln levels may also alter Ca2+ permeability of keratinocyte-sheets. Indeed, increased paracellular Ca2+ permeability (126% ±5%) was observed in Ocln knock-down cells as compared to control-siRNA-treated cells  and 20 h  of TRAILThus, Ocln knock-down selectively prevented apoptosis induction by TRAIL and UVB, which may suggest a role of Ocln loss in tumor progression of SCC, based on apoptosis resistance to different stimuli.Of note, the effects of Ocln knock-down were not seen in all keratinocyte cultures. The effects on TRAIL-induced apoptosis were seen in 50% of the primary keratinocyte cultures (n = 14) that originated from different donors, and the suppressive effects on UVB-induced apoptosis were seen in 40% of cultures (n = 10). This was independent from the level of Ocln knock-down. All primary cultures that showed an effect of Ocln knock-down on UVB-induced apoptosis showed a similar effect when using TRAIL.No primary cytotoxicity (necrotic cell death) was seen in response to TRAIL at early time points (3 h). It was only enhanced in the TRAIL-treated keratinocyte cultures after 20 h, interestingly both in the Ocln-siRNA treated cells and in control cells , arguing2+ conditions when treated with Ocln-siRNA, whereas at high Ca2+ conditions cell proliferation was slightly reduced. Also in 3D skin models no significant influence of Ocln knock-down on keratinocyte proliferation was evident (data not shown).Increased proliferation represents a characteristic feature of in-situ and malignant tumors. Therefore, we asked whether Ocln might also contribute to the regulation of keratinocyte proliferation. We did, however, not observe increased proliferation of human keratinocytes in 2D cultures at low Ca2+ homeostasis. This hints for a significance of the Ocln loss proven here in a large panel of cutaneous SCC as compared to SCC precursors and to normal skin. In addition, we demonstrate that changes of Cldn-1, Cldn-4, JAM-A, and ZO-1 can not only be found in SCC but also in precursor lesions and sun-exposed skin.Here, we demonstrate that downregulation of Ocln in keratinocytes results in decreased cell-cell adhesion, reduced susceptibility to induction of apoptosis, altered epidermal differentiation, and altered CaWe identified a frequent loss of Ocln in cutaneous SCC which was not found in its precursors or sun-exposed and non-sun-exposed skin. In line with our results loss or downregulation of Ocln has also been described in most samples of lingual and bronchial SCC Previous reports of cutaneous SCC could not identify such an Ocln loss though the protein was partly found to be restricted to cells with keratinization such as cancer pearls 2+-permeability, apoptosis, and proliferation.Addressing the question of consequences of reduced Ocln levels in keratinocytes, we investigated epidermal differentiation, markers for EMT, cell-cell adhesion, CaWe demonstrate that downregulation of Ocln results in downregulation of involucrin and increase of TG1 protein levels in 3D skin models, indicating an influence of Ocln on epidermal differentiation. Several studies described a downregulation of involucrin in poorly differentiated SCC, while it was strongly expressed in highly differentiated SCC 2+ permeability in keratinocytes and therefore altered Ca2+-homeostasis. Increased permeability might result in an altered tissue Ca2+-gradient which is known to influence keratinocyte differentiation 2+-homeostasis is also likely to be involved in reduced cell-cell adhesion observed in our Ocln knock-down keratinocytes, because Ca2+ is known to be essential for the formation of functional adherence junctions and desmosomes 2+-permeability in Ocln knock-down cells could also be a secondary effect due to the alteration of other TJ proteins, namely Cldn-2 and 12 which are critical for Ca2+-permeability in the intestine 2+-permeability, its downregulation counteracts the increased permeability and might be a compensation mechanism.As a putative underlying mechanism of altered epidermal differentiation we identified an increased paracellular CaDownregulation of Ocln has been described to be involved in EMT. Especially in the context of TGFβ induced EMT recent studies in simple epithelial cells indicate high significance of Ocln as a key regulatory component mediating complex formation of PAR6 together with type I TGFβ receptors. Upon exposure to TGFβ, Par6 is phosphorylated and binds to Smurf1, an E3 ubiquitin ligase, which in turn mediates ubiquitination of RhoA. Loss of Rho is important for the dissolution of TJs and for EMT 2+ conditions. Under these conditions only a low proportion of Ocln is present at the cell-cell borders 2O2-induced cell death We observed a role of Ocln in apoptosis sensitivity of keratinocytes. Thus, reduced apoptotic responses of keratinocytes were seen in response to TRAIL and UVB after Ocln knock-down. TRAIL activates the extrinsic apoptotic pathway, while UVB may induce both, extrinsic and intrinsic apoptotic pathways For Cldn-1, we found downregulation in uppermost and lowermost layers of cutaneous SCC and predominantly absence in the invasive parts, but a complete loss was only found in 9%. This is in agreement with Ouban et al Looking for explanations for the common alterations of Cldn-1, Cldn-4 and ZO-1 identified in the various skin tumors, we hypothesized that chronic UV exposure might induce some of these changes. Therefore, we investigated sun-exposed versus non-sun-exposed skin. Indeed, we observed a significant downregulation of Cldn-1 in lower layers and broader localization of Ocln, ZO-1, and Cldn-4 in a high proportion of chronically sun-exposed skin. Our data fit nicely to results reported in an UVB-irradiated murine skin model In conclusion, we demonstrate a frequent loss of Ocln in cutaneous SCC but not in its precursors. We confirm our hypothesis that Ocln knock-down in keratinocytes is involved in the promotion of tumorigenic features and show reduced susceptibility to TRAIL- and UV-induced apoptosis as well as reduced cell-cell adhesion. This may play a role in SCC tumorigenesis, as well as in other carcinomas, as loss of Ocln is a common feature in tumors. Other changes of TJ proteins were identified in cutaneous SCC as well as in precursor lesions and sun-exposed skin, and may therefore characterize initial steps in tumorigenesis induced by UV-irradiation.


Diffusion tensor imaging (DTI) based fiber tractography (FT) is the most popular approach for investigating white matter tracts in vivo, despite its inability to reconstruct fiber pathways in regions with “crossing fibers.” Recently, constrained spherical deconvolution (CSD) has been developed to mitigate the adverse effects of “crossing fibers” on DTI based FT. Notwithstanding the methodological benefit, the clinical relevance of CSD based FT for the assessment of white matter abnormalities remains unclear. In this work, we evaluated the applicability of a hybrid framework, in which CSD based FT is combined with conventional DTI metrics to assess white matter abnormalities in 25 patients with early Alzheimer’s disease. Both CSD and DTI based FT were used to reconstruct two white matter tracts: one with regions of “crossing fibers,” i.e., the superior longitudinal fasciculus (SLF) and one which contains only one fiber orientation, i.e. the midsagittal section of the corpus callosum (CC). The DTI metrics, fractional anisotropy (FA) and mean diffusivity (MD), obtained from these tracts were related to memory function. Our results show that in the tract with “crossing fibers” the relation between FA/MD and memory was stronger with CSD than with DTI based FT. By contrast, in the fiber bundle where one fiber population predominates, the relation between FA/MD and memory was comparable between both tractography methods. Importantly, these associations were most pronounced after adjustment for the planar diffusion coefficient, a measure reflecting the degree of fiber organization complexity. These findings indicate that compared to conventionally applied DTI based FT, CSD based FT combined with DTI metrics can increase the sensitivity to detect functionally significant white matter abnormalities in tracts with complex white matter architecture. Diffusion tensor imaging (DTI) based fiber tractography (FT) is currently the most widely used method to reconstruct fiber pathways in the brain, despite its well known limitations in regions with complex white matter architecture In the past decade, several advanced approaches for characterizing the intra-voxel diffusion profile have been developed to overcome the limitations of the second-rank diffusion tensor model We hypothesize that if CSD based FT is more accurate in reconstructing fiber bundle trajectories in regions with “crossing fibers”, it should be more sensitive to microstructural abnormalities underlying cognitive dysfunction than DTI based FT in these tracts. In tracts without “crossing fiber” regions, both methods should perform equally. To test this hypothesis we used a hybrid framework, in which CSD based FT is combined with conventional DTI metrics to assess white matter abnormalities in patients with early Alzheimer’s disease (AD). This allowed us to examine the microstructural properties of specific white matter pathways in relation to memory performance, while overcoming the well-known limitations of DTI based FT in regions with “crossing fibers”. We evaluated this CSD-DTI framework for two white matter tracts: one specifically selected because it contains many regions of “crossing fibers”, i.e. the superior longitudinal fasciculus (SLF) and one with only one fiber orientation, i.e. the midsagittal section of the corpus callosum (CC). Diffusion measures in these tracts have been previously shown to be altered in patients with AD compared to controls using tract based analyses In this paper, we examined whether CSD based FT combined with DTI metrics can increase the sensitivity to detect functionally significant white matter abnormalities in tracts with complex white matter architecture compared to conventionally applied DTI based FT.The medical ethics committee for research in humans of the University Medical Center Utrecht, the Netherlands has approved this research. Informed written consent was obtained from all participants. All clinical investigation has been conducted according to the principles expressed in the Declaration of Helsinki. Exclusion criteria were a history of stroke in the last 2 years, a history of stroke with subsequent cognitive deterioration, schizophrenia or other psychotic disorders, major depression, alcohol abuse, brain tumor, epilepsy or encephalitis. Incapacitated patients with a severe stage of AD, indicated by a clinical dementia rating score >1 Twenty five patients , 19 with early stage AD and 6 with amnestic mild cognitive impairment (a-MCI) were recruited via a memory clinic at the University Medical Center Utrecht. Probable or possible AD was diagnosed according to the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer Disease and Related Disorders Association (NINCDS-ADRDA) criteria 3, 2.5 mm slice thickness (without gap), 48 slices, repetition time 6638 ms, echo time 73 ms, flip angle 90 degree, acquisition matrix 88×88 (in plane resolution of 2.5 mm) and reconstructed at 128×128, 45 isotropically distributed diffusion-sensitizing gradients with a b-value of 1200 s/mm2, and one b = 0 s/mm2 image 2 image was on average 33.5 with a standard deviation of 11.7 MRI data were collected using a Philips 3.0 Tesla scanner . Diffusion MRI data were obtained using a single-shot spin echo EPI sequence with the following parameters: field of view = 220×220×120 mm2 image with ElastixThe DTI data sets were corrected for eddy current induced geometric distortions and subject motion by realigning the diffusion-weighted images (DWIs) to the b = 0 s/mmExploreDTI software package (www.exploredti.com). We reconstructed the SLF and the CC using both FT methods with a uniform seed point resolution of 2 mm3 and a maximum deflection angle of 30 degrees. For the DTI based FT an FA threshold of 0.2 was applied. Analogously, the applied termination threshold for CSD based FT was a fiber orientation distribution (FOD) value of 0.1 (the harmonic degree of the estimated FOD coefficients was limited to 6) Standard deterministic streamline DTI The SLF, including SLF II, III and the arcuate fasciculus The CC was reconstructed as described previously 2−λ3/λ1) Diffusion parameters: fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (DR), axial diffusivity (DA), and the normalized planar diffusion coefficient  Differences in configurational tract characteristics  after DTI and CSD based FT were analyzed with paired-samples T-test. The composite memory score and diffusion measures were all normally distributed. The relation between mean DTI metrics  and memory performance was evaluated using linear regression analysis adjusted for age, sex, and level of education. Differences in the relation between DTI metrics and memory obtained with DTI- versus CSD-based FT was calculated using Steiger’s Z-statistic for dependent correlations Because crossing fibers affect the tensor estimation To examine the possibility that the relation between diffusion measures and memory performance is affected by tract volume 3) CSD: 19.88±5.25; DTI: 10.10±2.78; p<0.001). In 75% of the patients the tract length was longer with CSD compared to DTI based FT (mean tract length ± SD (mm) CSD: 109.2±10.9; DTI: 98.3±13.6; p<0.001). The approximate tract volume of the CC segment was also larger for all patients with CSD compared to DTI based FT (mean tract volume ± SD (cm3) CSD: 9.59±0.98; DTI: 7.03±0.94; p<0.001).For the SLF, lower FA values of the SLF were associated with worse memory performance for both FT methods, but this association was only statistically significant for CSD based FT (standardized regression coefficient (95% CI) DTI: 0.39 ; p = 0.054, CSD: 0.41 ; p = 0.042) , model 1We also assessed the relation between diffusion parameters and cognitive performance in a tract without “crossing fibers”: the midsagittal segment of the CC. The FA of the CC was not significantly associated with memory performance with either tractography method, whereas a trend was observed for an association between memory and mean MD ; p = 0.051, CSD: −0.37 ; p = 0.074) , model 1This is the first report on the application of CSD based FT to detect white matter abnormalities in patients with (early) AD. Our results indicate that 1) CSD based FT in combination with DTI metrics significantly increased the sensitivity to detect a relation between white matter abnormalities and memory performance in a tract with “crossing fibers” (SLF); and 2) the relation between DTI metrics and memory was comparable between both FT methods in a tract without “crossing fibers” .In line with our expectations, fibers of the SLF were more likely to terminate in regions with “crossing fibers” with DTI-based FT. By contrast, with CSD based FT the SLF continued beyond these crossings to more temporal and dorsal frontal regions, which is in line with descriptions from autopsy studies increased mode of anisotropy in patients with MCI compared to controls only in areas where the SLF intersects the projection pathways The adverse effects of “crossing fibers” on the interpretation of diffusion measures such as MD and FA have been previously demonstrated e.g. , but theVoxels with “crossing fibers” are more likely to be included with CSD based FT. We therefore used the planar diffusion coefficient as a covariate to overcome the confounding effects of these “crossing fibers” on the diffusion metrics in relation to cognition. If two fiber populations within a voxel “cross” or “kiss”, the shape of the diffusion tensor becomes more planar (disc-like). As a result, a voxel with intact crossing fibers can have a similar FA value compared to a voxel with a degenerating non-crossing fiber population. However, the planar diffusion coefficient between these voxels will be different The effects of DTI metrics on cognitive performance were more prominent for DR than for DA, suggesting that the observed association is more likely driven by myelodegeneration than by a loss of axonal integrity Our study has a number of limitations. One is the modest sample size, which may have decreased our sensitivity to detect a relation between structure and function. Still, we were able to replicate previously observed associations between diffusion measures and AD severity Finally, the use of the planar diffusion coefficient as a quantitative measure to characterize “crossing fibers” may be valid in cases were two fiber bundles intersect or overlap, but may not be directly applicable in regions where three or more fiber bundles intersect. Although previous work reported that no more than two fiber populations could be observed in the SLF Since DTI based FT fails in regions with “crossing fibers”, more accurate methods to characterize the microstructural properties of fiber pathways are in need. Here we showed that CSD based FT combined with standard DTI metrics increases the sensitivity to detect functionally significant white matter abnormalities in a tract with “crossing fibers” in patients with early AD compared to DTI based FT. The use of a hybrid CSD-DTI framework is therefore a promising tool to detect functionally significant white matter changes in regions with complex white matter architecture.Figure S1Adjusted correlations between diffusion parameters of the SLF and memory performance. Top row: the relation between FA/MD and memory with DTI based fiber tractography. Bottom row: the relation between FA/MD and memory with CSD based fiber tractography. FA/MD and memory are expressed as standardized residuals after adjusting for age, sex, level of education, and the planar diffusion coefficient Click here for additional data file.Figure S2Unadjusted correlations between diffusion parameters and memory performance. Top row: the raw FA values of the SLF (left) and medial segment of the CC (right) with DTI based fiber tractography. Bottom row: the FA of the SLF (left) and medial segment of the CC (right) with CSD based fiber tractography.(TIF)Click here for additional data file.


Super-macromolecular complexes play many important roles in eukaryotic cells. Classical structural biological studies focus on their complicated molecular structures, physical interactions and biochemical modifications. Recent advances concerning intracellular electric fields generated by cell organelles and super-macromolecular complexes shed new light on the mechanisms that govern the dynamics of mitosis and meiosis. In this review we synthesize this knowledge to provide an integrated theoretical model of these cellular events. We suggest that the electric fields generated by synchronized oscillation of microtubules, centrosomes, and chromatin fibers facilitate several events during mitosis and meiosis, including centrosome trafficking, chromosome congression in mitosis and synapsis between homologous chromosomes in meiosis. These intracellular electric fields are generated under energy excitation through the synchronized electric oscillations of the dipolar structures of microtubules, centrosomes and chromosomes, three of the super-macromolecular complexes within an animal cell. The choreography of microtubules, centrosomes and chromosomes during mitosis and meiosis is beautifully designed by nature. Finely regulated and synchronized movements of these super-macromolecular complexes against the entropic forces within a dividing cell ensure the fidelity of the genetic material in both daughter cells. Currently, several models exist for the mechanisms of chromosome congression and spindle body assembly during M phase such as the search and capture model, kinetochore-mediated k-fibre formation, kinetochore motors contributing to congression, and the polar wind model. The mechanisms evoked by these models probably overlap, so there is redundancy among them, since mutations in the genes involved have only mild effects on chromosome congression during mitosis . Many opCellular electric fields have been studied in various cell types, and several studies have reported the existence of dielectrophoretic forces around cells -8; electCoherent oscillations in microtubules can be explained by Fröhlich’s theory, which describes a system of oscillators with energy supply, linear and nonlinear coupling with a heat bath. If a sufficient energy supply is provided to this system, condensation of energy occurs in the lowest mode leading to its coherent excitation ,24. ElecIn this article, we integrate research from several disciplines to provide an ‘electric’ view of the dynamics of these super-macromolecular complexes in mitosis, meiosis and other relevant cellular events. From our theoretical point of view, many of the unidentified forces regulating major cellular dynamic events during mitosis are probably electric forces generated by the synchronized oscillation of the electric dipoles within these super-macro organelle structures. Chronic exposure to extremely low frequency electric fields could affect several key steps of mitosis and neuronal cell physiology, resulting in an increased risk for cancer.The electric field of the microtubule is generated by the synchronized oscillation of α and β tubulins. These tubulins form electric dipoles during microtubule polymerization; under intracellular energy excitation, synchronized oscillation of α and β tubulin subunits generates a longitudinal electric field around the microtubule -15  . As in mMechanisms of centrosome separation and bipolar spindle body assembly have been discussed in a recent review . The proAndrews et al. have studied the effects of high frequency (range 2 to 50 MHz) electric fields on mammalian (human and Chinese hamster) chromosomes in vitro. They showed that such chromosomes can be oriented, aligned and translated by an oscillating electrical force. They also observed that above certain threshold field strengths the chromosomes orient themselves with their long axes along the field direction. The dependence of this threshold on frequency was measured and was found to be much larger at low than at high frequencies . Using eHowever, the physical mechanisms that regulate higher order packaging of M phase chromosomes are still not well characterized. Here we present a hypothesis of chromosome compaction. We apply a pulse-coupled oscillation clustering model to the dynamic events of chromosome packaging and inter-/intra-chromosomal organization. During chromosomal packaging, differentially compacted regions form partially synchronized electric oscillators interacting with an elastic electromagnetic field. According to the physical pulse-coupled oscillator model, unsynchronized pulse-coupled oscillators with proximal natural frequencies form synchronized oscillation clusters at a given coupling strength. As the coupling strength increases, these synchronized oscillation clusters merge with each other -40.During M phase chromosome compaction, the 30 nm chromatin fiber is initially formed by the electrostatic forces between neighboring nucleosomes. Under intracellular stochastic energy excitation, electric dipolar oscillation would be generated between neighboring nucleosomes. After oscillation synchronization and coupling, regulated electric oscillation is generated along the 30 nm chromatin fiber, and the oscillation coupling process further compacts that fiber -14,41. TTheoretically, the oscillation clustering model explains the closely juxtaposed configuration of duplicated chromosomes during M phase, which is counter-intuitive from the perspective of electrostatic repulsion between duplicated chromosome arms. As the homologous chromosomal regions develop synchronized oscillation clusters with identical natural frequencies, they tend to cluster together. The same scenario could apply to synapsis during meiosis; the electric oscillations of homologous chromosomes couple with each other, preventing synapsis between non-homologous chromosomes.The intracellular electric fields described in the foregoing sections could facilitate several cellular events during mitosis and meiosis. First, the metaphase bipolar spindle microtubules are formed through microtubule nucleation from the γ tubulin ring complexes (γ-TuRCs) at the centrosomes and retrograde delivery of peripheral microtubules by motor proteins ,44-46. TAccording to the physical organization of the duplicated chromosome arms, the condensed electric chromosomal fields around the centromeric regions could attract microtubule fragments to the sister kinetochores through electric interaction, which is consistent with observations of kinetochore movement along uncaptured microtubules, forming K-fibers (kinetochore associated microtubules) . Thus, eIn meiosis, the kinetochores are positioned at one side of the duplicated chromosome dimers, so the two sister chromosomes do not separate. The electric oscillation clustering between homologous chromosomal regions results in synapsis and recombination between homologous chromosomes; the electric fields generated by two duplicated homologous chromosomes can be viewed as two identical partially-entrained electric oscillation clusters, constituted by sub-chromosomal clusters throughout the chromosome arms. Such clusters in homologous chromosomes share identical electric frequencies, so the close juxtaposition between homologous chromosomes at synapsis is achieved through electric clustering and coupling among them. Synapsis does not occur during mitosis probably because the chromosome configuration caused by the opposing outward-pulling forces of the kinetochores at the opposite sides of duplicated centromere disfavors inter-chromosomal electric attraction. In addition, this event may be regulated by synaptonemal complex proteins .Magidson et al. reported that chromosomes adopt a toroidal/ring shape organization of after NE breakdown, which facilitates spindle assembly during M phase . Their oNumerous reports indicate that extremely low frequency electric fields can increase the risks of certain types of cancer . MicronuTo characterize these intracellular electric fields and study their cellular functions further, biophysicists should develop more detailed mathematical and physical models for chromosome electric fields and their role in M phase chromosome compaction, to allow these fields to be described and calculated more precisely and to predict the dynamics of related cellular events. The dynamic of changes of the electric fields in a living cell during mitosis and other cellular processes could be visualized using live cell imaging technologies such as nano-sized voltmeters . It woulThe authors declare they have no competing interests.Yue Zhao conceived the general concepts and wrote the article; Qimin Zhan shared insights and gave advice. Both authors read and approved the final version of the article.


This study explores the effects of month of birth  on the chances of survival to age 100. Months of birth for 1,574 validated centenarians born in the United States in 1880–1895 were compared to the same information obtained for centenarians' 10,885 shorter-lived siblings and 1,083 spouses. Comparison was conducted using a within-family analysis by the method of conditional logistic regression, which allows researchers to control for unobserved shared childhood or adulthood environment and common genetic background. It was found that months of birth have significant long-lasting effect on survival to age 100: siblings born in September–November have higher odds to become centenarians compared to siblings born in March. A similar month-of-birth pattern was found for centenarian spouses. These results support the idea of early-life programming of human aging and longevity. Studies of centenarians (persons living to age 100 and over) are useful in identifying factors leading to long life and avoidance of fatal diseases. These studies may be a sensitive way to find genetic, familial, environmental, and life-course factors associated with lower mortality and better survival . SeveralMonth of birth often is used by epidemiologists as a proxy characteristic for environmental effects acting during in-utero and early infancy development. These early effects include temperature and sun exposure during in-utero and early postnatal period, nutritional status during early development, exposure to infectious agents, and other factors , 13, 14.Little information is available on the month of birth association with exceptional longevity. To our knowledge, there is only one study that examines the effects of month of birth on longevity . In thisIn this study, we analyze the effects of month of birth on survival to age 100 years using a large set of centenarians born in the United Sates in 1880–1895 and their shorter-lived siblings and spouses. Siblings share early childhood conditions including parental socioeconomic status, genetic background, and geographical location while spouses share common adulthood environment. It was shown that longevity has a significant familial component –32 suggehttp://wc.rootsweb.ancestry.com), which is one of the largest publicly available repositories of online genealogies. Search for centenarians in the Rootsweb database was conducted with assistance of the web-automation technique [This study compares centenarians to their shorter-lived siblings (who share common childhood conditions and genetic background) and spouses (who share common adulthood environment) using a large set of computerized family histories. Family histories  proved to be a useful source of information for studies in historical demography  and biodechnique , which aechnique , availabechnique , which iPrevious studies demonstrated that age misreporting and age exaggeration in particular are more common among long-lived individuals , 39. TheWe used only those records of centenarians whose age was successfully confirmed through the DMF (with matched birth and death years). We added only few cases where death year was different from that found in the DMF . Our previous work with centenarian data cleaning showed that incorrect death dates was the main source of errors in genealogical records of centenarians . At the As a result of data quality checks, we found 1,574 records of centenarians born in 1880–1895 with verified birth and death dates. Given the fact that longevity is often clustered in families, we found other centenarians in the studied families (born outside the 1880–1995 time window) so that the total number of centenarians increased to 1,945 persons. Distribution of centenarians according to their lifespan is presented in Birth dates were reconstructed for all centenarian siblings using information available in computerized genealogies and early censuses. The procedure of death date verification using DMF is not feasible for validating death dates of shorter-lived siblings or spouses (used as controls) because data completeness of DMF is not very high for deaths occurred before the 1970s . State dData for 10,885 siblings of 1,574 centenarians were used in this study. As a result, each case (centenarian) had about 7 control siblings on average. The sibship size (eight siblings on average) in the studied centenarian families is higher than the average number of children in American families reported by the 1900 USA Census: 5.12 ± 0.01; data obtained from the 5% sample of the US 1900 Census from the integrated public use microdata series (IPUMS) . Larger This study explored the effects of month of birth on the likelihood of survival to age 100. Centenarians (cases) were compared to their “normal” shorter-lived siblings (controls) or spouses using a within-family analysis. This approach allows investigators to study the within-family differences, not being confounded by the between-family variation. Long-lived persons born in 1880–1895 were used as cases. Siblings were born in a wider time window than centenarians but on average in the same year. Taking into account relatively high child mortality in the 19th century, we conducted analyses with different lifespan cut-offs in order to study late-life survival to advanced ages and evaluate the stability of results. The main approach used in this study is based on the comparison of children within rather than across families. A similar approach was applied for comparison of centenarians to their spouses.Differences in the month-of-birth distributions between centenarians or their siblings and the general population according to the 1900 US Census were assessed with the chi-square test. Standardized residuals were calculated in order to determine which months of birth may be major contributors to rejection of the null hypothesis (in the case it is rejected). When the absolute value of the residual is greater than 2.00, it indicates that it was a major influence on a significant chi-square test statistic. The chi-square test was also used to examine whether gender or longevity is related to the month of birth.P values  are lower than (i/m)Q, where i is a rank of P value, m is the total number of tests, and Q is the chosen FDR. The level of FDR in the Benjamini and Hochberg procedure was set to 0.10.Statistical analyses of the within-family effects for 1 : n matched study were performed using a conditional multiple logistic regression model (fixed-effect model) to investigate the relationship between an outcome of being a case (long-lived person) and a set of prognostic factors , 47. TheP < 0.001 for both centenarians and their siblings). df = 1; P = 0.025). As shown in df = 11, P = 0.045; siblings survived to age 20: chi-square test statistic 19.50, df = 11, P = 0.053). At the same time, we found no statistically significant association between month of birth and gender.Comparison of month-of-birth distributions for centenarians and their shorter-lived siblings with month-of-birth distribution for persons born in 1880–1890 and enumerated by the 1900 US Census showed statistically significant differences  found that life expectancy at age 80 depends on month of birth . In thisOur within-family study follows centenarians and their siblings from birth until the end of their life while previous studies analyzed a cross-sectional sample of the USA death certificates for persons belonging to multiple birth cohorts. For this reason, our results do not depend on the secular changes in seasonality of births and infant deaths. Another advantage of this study is its within-family design, which controls for unobserved characteristics of childhood conditions and parental genetic background. This study confirms the existence of month-of-birth effects on longevity and shows that these effects can be observed even after controlling for unobserved between-family variation. Some limitations of this study should be mentioned. Due to the data collection from computerized genealogies, we cannot be certain that centenarians (and controls) represent a random population sample. This limitation is not crucial for the analytical approach applied in this study, which tests specific hypothesis of seasonal birth effects on longevity, but may pose a question about generalizability of results. It is believed that the RootsWeb source of online family histories has more individuals with larger families and better offspring survival. Indeed our sample of centenarians has larger families compared to the general population. This deviation from the general population may potentially affect the results of univariate analyses when month-of-birth distributions for centenarians and siblings are compared to the general population. However, in the within-family analyses, we compare siblings with each other rather than with the general population, so the difference in family size does not affect the results of hypothesis testing about the month-of-birth effects on longevity . Also, cAnother problem is that some month-of-birth effects become not statistically significant after adjustment for multiple comparisons. For example, month-of-birth effects become nonsignificant when survival of siblings after age 70 is studied. However, the overall pattern of month-of-birth effects on longevity shows consistency across different age cut-offs suggesting a stability of the observed seasonal pattern. In addition to that, independent analyses on centenarian spouses demonstrated a similar pattern of month-of-birth effects on longevity. Finally, the conditional logistic regression analyses suggest that despite significant effects of months of birth on relative survival the effect sizes of month-of-birth effects on survival to 100 are small and explain about 2% of the variance of becoming a centenarian. This small percentage of explained variance is related to very high variability of individual lifespan, which has a substantial stochastic component . There are several possible explanations of why month of birth may affect mortality and health later in life. One explanation suggests that nutritional status of mother during pregnancy may affect fetal development in utero , 56. NutBirth weight often serves as an indicator of nutritional status during early development and was shown to be dependent on month of birth. For example, in Greece, infants born during the autumn and winter seasons of the year had significantly increased birth weight and gestation age . Recent Early seasonal impacts on subsequent adult lifespan may include not only seasonal vitamin deficiency, but also other seasonal impacts, such as infectious diseases. Seasonal peaks of disease occurrence are typical for many infections . The mosEffect of environmental temperature during the time of birth or conception may be another possible explanation for low proportion of centenarians among individuals born during the summer and spring months. For example, British women experiencing higher summer temperatures during their first year of life and hence suffering severe diarrhea and dehydration in infancy had higher blood pressure at older ages . High amIt was suggested that schools or other professional training organizations, which have a deadline for admission, may favor children who are somewhat older compared to their peers . This so-called deadline hypothesis  predictsAnalysis of the existing literature suggests that persons born in the fall months in the United States could avoid extremes of very high and very low ambient temperatures during their first month of life as well as high summer temperatures during conception. Persons born during the fall months also did not experience an early exposure to infectious diseases, which were common during summer, early winter, or spring months in the past. Seasonal pattern of the USA mortality for children below age one month in the past supports this suggestion. According to the USA statistics, mortality below age one month in 1940 was the lowest in September–November  suggestiThis is the first study of association between month of birth and exceptional longevity, which controls for early-life shared conditions and common genetic background. We developed a large sample of validated centenarians, their siblings, and spouses to study early-life seasonal effects on human longevity. We found significant associations between month of birth and longevity: individuals born in September–November have higher likelihood of becoming centenarians compared to March-born individuals. These results are consistent with the reports of higher life expectancy for persons born in the end of the year , 21, 22


As assisted reproductive technology (ART) methods become the mainstream of infertility treatment, it has become even more critical to reassess its safety. Following the results of a study published by the Robinson Institute in the New England Journal of Medicine, the risk of ART, especially intracytoplasmic sperm injection (ICSI), has never been so closely scrutinized. This paper traces the origins and development of ICSI, assesses the risks documented in the literature, and finally interprets the implications of the study for couples contemplating therapy. We support the need for continued vigilance towards ICSI and the importance in investigating male-factor infertility as a prequel to its use. In 1992, the potential of intracytoplasmic sperm injection (ICSI) [New England Journal of Medicine. Davies et al. [ICSI involves the in vitro injection of preselected spermatozoa into the cytoplasm of a mature oocyte after ovarian superovulation and oocyte retrieval. ICSI quickly became the favoured technique for cases of male-factor infertility, as it was discovered that the basic semen parameters, such as having a low sperm count or less motile sperm , had lits et al.  concludeAs the number of children born from ICSI procedures has exponentially increased, greater attention has focused on the safety of the procedure. The vast majority of evidence demonstrates that there is no difference in the rates and types of congenital malformation when comparing ICSI and standard IVF pregnancies –15. This transmission of genetic anomalies  imprinting disorders congenital malformations developmental abnormalities. The potential concerns regarding ICSI offspring relate to four general areas of investigation: (1) Transmission of Genetic Anomalies. A considerable proportion of males requiring ICSI have very low sperm counts, which is associated with a greater risk of carrying chromosomal abnormalities [malities , 17. Spemalities . This mamalities , 20. Since pregnancy can be achieved in couples wherein the male partner harbors such abnormalities, the risk that male offspring might later manifest disorders including infertility is very real. In fact, there is limited evidence that paternal sex chromosomal disorders, including microdeletions, are rarely transmitted to male ART offspring , 22. Y c(2) Imprinting Disorders. There are suggestions of an increased incidence of epigenetic abnormalities after augmentation with ICSI. The developmental process of genetic imprinting involves the exclusive expression of specific genes from only one parent. However, imprinting disorders occur when this imprint is not set correctly. Gamete micromanipulation may lead to altered gene expression of imprinted genes as crucial epigenetic reprogramming events occur during germ cell development and early embryogenesis [ogenesis . Suboptiogenesis , 26. Somogenesis . As such(3) Congenital Abnormalities. The relationship between ART and congenital malformations was first reported by Hansen et al. [n et al.  who obsen et al. . The majn et al. –31. ASRMn et al. , 32. Pren et al. , 33. Hown et al. . These f(4) Developmental Abnormalities. Despite the genetic risks and congenital malformations that are reported, several studies found no significant differences in the long-term developmental outcome of ICSI offspring. Basatemur provided reassuring information regarding the physical growth of ICSI children up to the age of 12 years [12 years . Another12 years . It willGiven these conflicting results surrounding birth defects and ICSI, it has been impossible to know which infertility treatments differentially increase the rate of birth defects, or whether there is an underlying association with maternal and paternal factors that necessitate infertility treatment in the first place. The study “Reproductive Technologies and the Risk of Birth Defects,” conducted by the Robinson Institute in South Australia, attempts to answer some of these questions. Davies et al. present data from the largest registry to date, comparing 6163 births from ART, including those form IVF, ICSI, and ovulation induction, with some 308,000 unassisted conceptions (that included terminations and stillbirths) over 16 years. They look at rates of birth defects before the age of 5 years from births from each mode of ART, births from mothers with a history of infertility, and births from mothers with neither a history of infertility nor treatment. In analysis adjusted for confounders , the risk of any birth defect with ART was 8.3% compared with 5.8% in natural pregnancies . ART was associated with an increased rate of any defect and multiple defects, as well as with cardiovascular, urogenital, and gastrointestinal abnormalities, and cerebral palsy. When ART methods were analyzed independently, there was a 9.9% risk of birth defects using ICSI, compared to 7.2% using IVF. This finding will alleviate the concerns of couples that use IVF, as the risk of defect is not increased beyond their biological risk. Conversely, ICSI was 77% more likely than unassisted pregnancies to have a birth defect, compared to 26% for IVF. What was more striking was that when confounders were taken into consideration, this risk for IVF fell to 7%, whilst with ICSI it remained at 57%. After multivariate adjustment, the association between IVF and the risk of any birth defect was no longer significant, whereas the increased risk of any birth defect associated with ICSI remained significant.These results can be explained by one of two ways; either the ICSI procedure itself is dangerous or there may be some underlying factors associated with the couples that choose ICSI. This is reinforced as the study also found that pregnancies from mothers that have a history of infertility with natural births were also at an increased risk of conceiving a child with birth defects. So part of the chance of having a child with birth defects after assisted conception results from parental factors related to infertility—but it is not clear what those factors are.A limitation of the study rests in its use of data from 1986 to 2002. Since then, assisted reproduction has advanced from the methods used in embryo fertilization to the introduction of vitrification which allows a woman to freeze their eggs for more than two decades. So are the results from a generation ago still relevant? In addition to the maternal confounders that the study accounts for, many others remain. Davies et al. do not account for unknown paternal infertility factors . These may be important contributors to the increased chance of a birth defect after ICSI. Earlier studies have demonstrated that ICSI children carry a higher risk of chromosomal aberrations related to the severity of the male infertility ; howeverThere are several explanations for why the results of this study record high risks of birth defects in comparison to similar studies in the past. For their overall results, the authors did not separate twin pregnancies from singleton pregnancies. Twin pregnancies are known to be at higher risk of all birth defects, especially cerebral palsy. They also did not distinguish single embryo transfer from double embryo transfer. Even if it results in a singleton birth, previous research has shown double embryo transfer to be associated with a higher risk of birth abnormalities than single embryo transfer . In addiThe authors of this study do not discuss the results of a recent Swedish population-based study on 15 570 ART infants (9372 ICSI pregnancies)  which deThis study reminds us that the success of ICSI has been a double-edged sword. Inarguably, it has gone where IVF has not, offering men who were previously rendered infertile the possibility of becoming fathers. However, simultaneously it has hampered progress on the development/refinement of diagnostic tools (particularly related to spermatozoa-oocyte interaction), and perhaps of medical treatments for male infertility. Ironically, in an age of burgeoning knowledge surrounding reproductive function, the focus on the causes of infertility or subfertility in a couple has decreased. Unfortunately and frequently, patients are enrolled directly into ICSI programs with little clinical and diagnostic workups to avoid the costs of male-infertility testing. Such views have been labeled as “insidiously lazy”  and “a dThis study shows that it is still not known whether the risks associated with ICSI are related to the procedure or to inherent sperm abnormalities. What is known is that couples with infertility have modest excess risk for having children with birth defects, despite controlling for maternal factors, regardless of how or whether the infertility is managed. More importantly, it highlights the need for continued vigilance, especially with ICSI. There should be more vigorous evaluation and treatment of the male patient as part of the routine evaluation of the infertile couple and not the use of empirical therapy. If ICSI is deemed the most suitable method in the therapeutic armamentarium, then the associated risks of birth defects must be conveyed to the couple. It is imperative that the children being born through ICSI and other ART continue to be monitored. Through these methods, we will hopefully be able to balance improvements in conception rates and at the same time minimize the chance of successful pregnancies resulting in children with birth defects.


Autism spectrum disorders (ASD) are associated with complications of pregnancy that implicate fetal hypoxia (FH); the excess of ASD in male gender is poorly understood. We tested the hypothesis that risk of ASD is related to fetal hypoxia and investigated whether this effect is greater among males.Provincial delivery records (PDR) identified the cohort of all 218,890 singleton live births in the province of Alberta, Canada, between 01-01-98 and 12-31-04. These were followed-up for ASD via ICD-9 diagnostic codes assigned by physician billing until 03-31-08. Maternal and obstetric risk factors, including FH determined from blood tests of acidity (pH), were extracted from PDR. The binary FH status was missing in approximately half of subjects. Assuming that characteristics of mothers and pregnancies would be correlated with FH, we used an Estimation-Maximization algorithm to estimate HF-ASD association, allowing for both missing-at-random (MAR) and specific not-missing-at-random (NMAR) mechanisms.Data indicated that there was excess risk of ASD among males who were hypoxic at birth, not materially affected by adjustment for potential confounding due to birth year and socio-economic status: OR 1.13, 95%CI: 0.96, 1.33 (MAR assumption). Limiting analysis to full-term males, the adjusted OR under specific NMAR assumptions spanned 95%CI of 1.0 to 1.6.Our results are consistent with a weak effect of fetal hypoxia on risk of ASD among males. E-M algorithm is an efficient and flexible tool for modeling missing data in the studied setting. The autism spectrum disorders (ASD) comprise a group of neurodevelopmental conditions that are associated with impaired verbal and non-verbal communication and social interaction, and restricted and repetitive patterns of behavior, which typically manifest before age of 3 years. AlthougParallel line of research suggests that neonatal encephalopathy caused by perinatal asphyxia/hypoxia in infants born at term is associated with neurological impairment later in life. TherefoIn fact, one study of 239 children who survived neonatal encephalopathy reported that 4.2% were diagnosed with an ASD. This represents, after adjustment for confounders, about a 6-fold increase in ASD risk compared to ASD rates among 563 children who did not have neonatal encephalopathy (0.9%). The smaNone of the epidemiological studies conducted to date studied direct effects of hypoxia at birth on ASD. Therefore, we tested, in a population-based birth cohort, the hypothesis that risk of ASD is related to fetal hypoxia and investigated whether this effect is greater among males.th edition (ICD-9). Children matched in APHP file to AHW records were followed-up by the investigators until March 31, 2008 for (a) ICD-9 codes indicating ASD (299.0 or 299.8) listed with the physician billing record, (b) child's residency and mortality  and (c) mothers' socioeconomic status. Outcome was defined using most liberal definition from our previous work: at least one ASD claim by any physician because such case definition showed good sensitivity and specificity and did not affect associations between studied risk factors and ASD[This cohort and analysis of association of perinatal risk factors with risk of ASD are described elsewhere, without consideration of fetal hypoxia, defineds and ASD. Study pCausal model considered in this paper is shown in Figure r ≤ 1), the ratio of expected probabilities of having hypoxia in X missing group and non-missing group for a given set of Zs. Given information from Zs, setting = 1 means we assume MAR. Any case with < 1 amounts to postulating a special case of NMAR. There is no data on how much smaller than 1 r should be in our particular setting. Such values can arise from separate experiments (not possible in our situation), or sensible guesses of values of r can be made for the purposes of sensitivity analysis (route we followed); yet another approach is to follow fully Bayesian paradigm and place a prior on r. It is not possible to optimize r from our data using EM algorithm. We considered in sensitivity analyses a range of r spanning values consistent with excellent (r = 0.1) to moderate (r = 0.7) additional clinical judgment indicating the likelihood of positive test, over and beyond Zs. Note that r = 0.5 suggests that those not tested are only at 50% risk of hypoxia (given Zs) compared to those who were tested: this is consistent with the notion of predictive ability of the clinical judgment, beyond Zs, that the hypoxia test is positive. As we shall see below , was missing in approximately one half of our study subjects. Based on the notion that some variables (Zs) including characteristics of mothers and pregnancies would be correlated with the exposure X, we used an EM algorithm to tacklThere were 273,343 singleton live births in Alberta according to APHP records between 1998 and 2004, among whom 25,970 children could not be identified by AHW and 28,421 either died, or lost residence during follow-up; a further 62 had missing gender, leaving 218,890 children for analysis. There were no systematic differences on observable data among births that were included and excluded (details not shown). In 48%  of births, fetal hypoxia tests via blood pH were performed. In a minority of births multiple tests were performed . Given uncertain role that prematurity plays in ASD etiology[We will next examine ASD rate modeled using E-M algorithm among births of all gestational ages. Table  etiology, we coulr from 0.1 to 0.5), it appears that point estimate of the effect (OR) of hypoxia on risk of ASD among males who were born at full-term increases to 1.2-1.3 with 95%CIs that lie approximately between 1.0 and 1.6. Even if deviation from the MAR assumption was weak (r = 0.7), the resulting change in inference is dramatic: 95%CIs widen somewhat and shift from 0.91-1.29  towards the region conventionally thought of as "significant". Thus, the use of EM-algorithm for dealing with the special missing value problem in this project in conjunction with sensitivity analysis using the specified NMAR assumption altered statistical inference and was therefore useful.Our results are consistent with a weak effect of fetal hypoxia on risk of ASD that is confined to males. It may well be that some latent indication or an unknown correlated of testing for hypoxia at birth are responsible for the increased risk of ASD among males and therefore investigation of role of hypoxia at birth in ASD remains a fruitful area for research.The authors declare that they have no competing interests.All authors credited with this work made substantial intellectual contribution to the submitted manuscript. IB led overall project; the current analysis address the hypothesis he developed and attempted to test. IB, SF and LZ designed the study, oversaw data acquisition, contributed to analysis and interpretation of the data and co-wrote the manuscript. XW and YY devised and implemented data analysis via E-M algorithm, they led the preparation of technical appendices and made substantial contribution to writing the main body of the manuscript. All authors reviewed and approved the submitted version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/11/2/prepubEM Algorithm with either MAR or NMAR assumptions. Mathematical details of EM algorithm implemented in this article.Click here for fileImplementation of EM Algorithm for missing values in SAS software. SAS code that was used to implement EM algorithm presented in this article with annotation that allow adapting it to similar applications.Click here for file


Echinococcus granulosus. The highest prevalence of the parasite can be found in different parts of world like Africa, Australia, and South America. This infection can occurs in almost any part of the body. Here we present clinical, radiological, histological features and treatment of a multi vesicular osseous hydatid disease of the mandible in an Afghan 5 year old boy with a firm swelling in the right side of mandible.Hydatid disease is a common and major public health issue caused by parasite  Hydatid disease is a parasitic infection that has a worldwide geographic distribution and occurs in all continents including circumpolar, temperate, subtropical, and tropical zones , 2 and cEchinococcus granulosus. The adult parasite (0.5–1cm) resides in the intestine of animals such as dogs, foxes, wolfs and jackals , firm & measured about 10×10 cm. The over lying skin of the swelling was normal. The dentition and oral mucosa were normal . His genIn extra oral radiographic studies  a well-defined multi lacunar lesion in the right side of angle and body of the mandible was seen. According to the CT scan of the lesion, it seems that there were more than six lacunae in the cyst. No other abnormality findings were detected .No abnormalities were found on standard blood tests. The patient's serum was assessed for hydatid cyst antibody by ELISA and the results were negative.On excisional biopsy, we observed multi lacunar cyst with well-defined, firm and thickened wall. The cyst was located without any attachment to the peripheral bone. Several septums, separated this cyst .Hemovac tube was inserted to the site and soft tissues were sutured in three layers. Mebendazol (10 mg/ kg/day) was prescribed for 12 weeks. Patient visited a week after operation and was satisfied of the outcome. He was examined clinically and radiologically 18 months after surgery. No evidence of recurrence was seen and serologic test by ELIZA was negative .We excised the cyst and reshaped the body and angle of right side of mandible . Then weHydatid cysts can occur in any part of the body. Osseous hydatidosis is rare comparing to involvement of soft tissues. The prevalence of bone infection is 14% and 1.1% in endemic and non-endemic regions, respectively .The diagnosis of hydatid cyst should be considered in patients with a cystic mass, who lived or visited in/an endemic area . DiagnosE. granulosus and host defense had dictated the progress and structure of the cyst and maybe calcification in this case had been a protective mechanism to decrease the growth rate of the lesion.Osseous hydatidosis may manifest as bone pain and deformity, particularly among patients in 30–60 years old ages . HydatidThe differential diagnosis of skeletal hydatid cyst includes other infections disease (e.g. tuberculosis), fibroma, lymphoma, giant cell tumors, brown tumors, metastatic lesions, and other benign cystic lesions . The preCurettage of the lesion and drug therapy (Albendazol or mebendazol) have been reported to be sufficient treatment  but many