In this study, we use vibrational optical coherence tomography (VOCT) to examine the morphology and stiffness of benign and cancerous lesions. Lesion images and 3D plots of weighted displacement versus frequency and depth were used to compare the cellular, dermal collagen, new blood vessels, and fibrotic composition of normal skin, actinic keratoses (AK), nodular and superficial basal cell carcinomas (BCCs), squamous cell carcinomas (SCCs), and melanomas. The results of this study suggest that benign and cancerous lesions differ based on the addition of new cells with increased resonant frequency and stiffness , new blood vessel peaks  that appear to be less stiff than normal blood vessels, and new fibrous tissue peaks  that are present in carcinomas but not in normal skin and only partially present (80 Hz and 130 Hz only) in AKs. Results obtained by creating images based on the location of the 80 Hz, 130 Hz, and 260 Hz peaks of cancerous skin lesions suggest that the fibrous tissue appears to surround the new cells and new lesion blood vessels. The results of this study suggest that the morphology and location of the fibrous tissues in relation to the new cancer-associated cells and lesion blood vessels may provide information on the invasiveness and metastatic potential of skin cancers. The invasiveness and metastatic potential of melanomas may be a result of the cancer-associated cells laying down fibrous tissue that is used as a pathway for migration. The new cancer-associated blood vessels in the vicinity of the new cancer-associated cells may promote this migration and eventual metastasis. The ratios of peak heights 50/130 Hz and 80/130 Hz of normal cells, new lesion cells, new lesion blood vessels, and fibrotic tissue may be used as a “fingerprint” for detecting melanoma and to differentiate it from other skin cancers non-invasively using VOCT. Organ fibrosis affects all major human tissues and can lead to the excessive accumulation of extracellular matrix (ECM) components, predominantly collagens. It can lead to compromised organ function and failure and is estimated to contribute to 45% of all deaths in the developed world . NumerouThe secretion of lysyl oxidase (LOX) by tumor cells initiates lysine-derived collagen cross-linking, thereby stiffening the tumor stroma. This creates mechanical forces that trigger the integrin-mediated formation of focal adhesions that initiate tumor cell invasion . LOX-medn) and collagen I (Col I), are known to interact with each other to regulate cellular behavior. ECM remodeling is primarily mediated by matrix metalloproteinases (MMPs) [n and Col I synergistically interplay in promoting a sustained altered signaling cascade to remodel the breast tumor stroma for invasive breast tumor growth [Two major extracellular matrix (ECM) components, fibronectin (Fs (MMPs)  and trans (MMPs) ,10,11,12r growth .n matrix; surrounding cells in the stroma of CAFs are reported to up-regulate Fn [n matrix comprising highly unfolded molecules [n matrix not only exhibits increased stiffness and viscosity, but also mediates enhanced pro-angiogenic signaling. The establishment of a fibrotic-like microenvironment via TGFβ-driven collagen I production may generate a fertile soil around dormant tumor cells that then drives the transition from dormancy to metastatic growth through a β1-integrin signaling mechanism [Cancer cells are unable to assemble their own Fulate Fn . These tolecules . This alechanism ,12.Both cancer cells and epithelial cell-derived cancerous tissue are stiffer than normal tissue ,14,15,16The purpose of this study was to provide non-invasive 3D maps of the location, stiffness, and morphology of the fibrous tissue and new cells deposited in cancerous skin lesions. This information is used to evaluate the relative relationship between the location of 80 Hz cells, 130 Hz blood vessels, and the location and orientation of the fibrotic tissue at 260 Hz that is deposited near each type of skin cancer. Preliminary results suggest that the geometric relationship, resonant frequency peak location, and relative peak heights of new cells, new lesion blood vessels, and fibrous tissue, as well as the ratio of the major resonant frequency peak heights, may provide information that can be used to “fingerprint” and predict the invasive and metastatic behavior of different skin cancers.Normal skin from 14 subjects  was studied in vivo using VOCT after informed consent was obtained, as reported previously ,19. Figu2. All subjects signed consent forms prior to enrolling in the study.Suspicious skin lesions identified by dermoscopy in the Dermatology Clinic at Summit Health  were biopsied and studied in vitro using VOCT on a sample microscope stand, as shown in OCT image collection was accomplished using a Lumedica Spectral Domain OQ 2.0 Labscope  operating in the scanning mode at a wavelength of 840 nm. The device generates a 512 × 512-pixel image with a transverse resolution of 15 μm and an A-scan rate of 13,000/sec. The beam size used was 0.25 mm in diameter, as discussed previously ,19. All Multiple cross-sectional OCT scans were collected using the volume scan software included in the Lumedica OQ Labscope and processed into 3D images using Image J software.The pixel intensities obtained from the grayscale images were plotted versus depth for each sample studied. The enhanced OCT images used darker-colored (blue and purple) regions to reflect lower pixel intensities, and the lighter (yellowish) regions reflected higher pixel intensity regions. Pixel intensities were processed using Image J software, analyzed with a MATLAB program, and plotted versus skin depth. Previous studies have shown that the images of normal skin and cancerous lesions seen by OCT correlate with the histological images seen in sections cut from tissue biopsies ,20,21.Maps of different skin anatomical structures were created by examining slices collected using the OCT volume scan app as well as by looking at changes in the pixel intensity vs. depth scans of the image. These data were combined with the measurement of the weighted displacement versus frequency data to create a 3D map of the components of each lesion ,19. ThesThe OQ Labscope 2.0 was modified by adding a 2 inch-diameter speaker placed about 2.5 inches from the tissue in the VOCT studies ,19. A siThe resonant frequency of a tissue component is defined as the frequency at which the maximum in-phase displacement is observed in the amplitude data for that component. The measured resonant frequencies are converted into elastic modulus values using a calibration equation (Equation (1)) based on in vitro uniaxial mechanical tensile testing and VOCT measurements made on the same tissue at the same time, as reported previously ,24. The 2 is the square of the resonant frequency, and E is the elastic modulus in MPa, as discussed previously [Calibration studies using in vitro uniaxial tensile testing and VOCT measurements were used to develop an Equation (1) for soft tissues. Since soft tissues have a density very close to 1.0, Equation (1) is valid for most tissues found in the body; where the thickness d is in m and is determined from OCT images, fneviously ,24. EquaNormal skin studies were conducted in vivo using a universal hand piece mount that was constructed to hold the OCT handpiece . Tissue Once VOCT studies were conducted, the biopsy samples were immersed in fixative and transported to the pathology lab for diagnosis. Histopathology on skin biopsies was conducted by a dermatopathologist after routine dehydration in alcoholic solutions, embedding in paraffin, thin sectioning, and staining with H&E. Mohs thin sections were processed after fixation by frozen sectioning and H&E staining. They were reviewed by a trained Mohs dermatopathologist who conducted the pathological analysis.Results of the VOCT experiments conducted on normal skin in vivo and excisions of AK, BCC, SCC, and melanoma illustrate that new resonant frequency peaks are seen in skin lesions. Note that the new cellular (80 Hz) and fibrotic peaks (260 Hz) seen in cancerous skin lesions are stiffer than those found in normal skin (50 Hz and 100 Hz). BCC, SCC, and melanoma all have peaks at 80, 130, and 260 Hz, indicating that there appears to be a relationship between the presence of new cells at (80 Hz), new lesion blood vessels (130 Hz), and fibrous tissue (260 Hz) in cancerous lesions. Previous published results indicate that the resonant frequency peaks seen in skin and skin lesions in vitro are like those observed in vivo ,22,23,24Like in the nodular BCC, the new cellular peak (80 Hz) and new blood vessel (130 Hz) peak of a superficial BCC  looks liThree-dimensional color-coded images of a melanoma are shown in Using a new non-invasive technique termed VOCT, we have shown that the morphology and stiffness of benign and cancerous lesions are different based on the addition of new cells with increased stiffness (80 Hz), new lesion blood vessels that appear to be less stiff (130 Hz), and fibrous tissue (260 Hz) that is very stiff that are present in all carcinomas. These results suggest that the product of fibrosis that results from cell mutations appears similar for different types of skin cancers, including cancers that have invasive and metastatic potential. Whereas BBCs form primarily fibrotic tissue, other cancers that are invasive and can metastasize also appear to contain large amounts of normal cells (SCC) and large amounts of cancer-associated cells (melanoma). The 3D relationship between the new cells and new blood vessels (80 Hz and 130 Hz) and fibrous tissue (260 Hz) may provide a means for non-invasively assessing the invasive and metastatic potential of a skin cancer.The ability to noninvasively predict the invasive potential of skin cancers offers the potential for early evaluation of lesions that may be difficult to diagnose. Using VOCT, it is possible to capture and measure the stiffness of lesions as small as 0.05 mm, which is much smaller than can be seen by visual inspection and dermoscopy. Not only do skin cancers contain fibrotic tissue, but they also contain cancer-associated fibroblasts (CAFs) and new friable blood vessels.It has been reported that an epithelial–mesenchymal transition (EMT) results in a change of the cell phenotype of epidermal cells in epithelial-derived cancers. During EMT, epithelial cells lose cell–cell adhesion attachments and reorganize their cytoskeleton. Cell–cell connections loosen, and cells become motile and resistant to apoptosis . The depTransforming growth factor beta 1 (TGF-β1), tumor necrosis factor alpha (TNF-α), and hypoxia cooperate in the triggering of EMT both in cancer and fibrosis ,26, convFibroblasts found in carcinomas appear to exhibit an altered phenotype characterized by the increased expression of α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP), as well as increased secretion of extracellular matrix proteins (ECM), including fibronectin and fibrous type I collagen ,28. CAFsAs cancer progresses, tumor cells obtain the ability to break down the underlying basement membrane and invade into the surrounding tissue. Some cancer cells attain this invasive behavior through the acquisition of a mesenchymal phenotype via EMT . Recent Endothelial cells can be activated by cytokine signaling and express inflammatory markers, which can sustain or exacerbate the inflammatory process. Activated endothelium can recruit and activate leukocytes, thus perpetuating tissue inflammation, and the sustained stimulation of endothelial cells may lead to the endothelial-to-mesenchymal transition that also contributes to fibrosis . Though The products of inflammation may contribute to the formation of stiff fibrous tissue that is seen in the 3D reconstructions reported in this paper to provide a pathway along which cells to migrate. The invasive cancers, SCC, and melanoma, additionally exhibit large numbers of cells (50 Hz and 80 Hz) along with the fibrotic tissue, and melanoma, the most invasive cancer, exhibits large numbers of new cells (80 Hz) and new blood vessels (130 Hz) in addition to large amounts of fibrotic tissue. It is now possible, based on the ratios of the 50, 80, and 130 Hz peaks, to non-invasively determine the lesion composition of cells, new lesion blood vessels, and fibrous tissue to identify potentially metastatic lesions. The ability to classify lesions will prove useful in assisting the dermatologist to identify potentially metastatic epithelial cancers. The “fingerprint” of each cancerous tissue based on the normalized peak height ratios shown in Using a new non-invasive technique termed VOCT, the results reported in this pilot study have shown that the morphology and stiffness of benign and cancerous lesions are different based on the addition of new cells with increased stiffness (80 Hz), new lesion blood vessels that appear to be less stiff (130 Hz), and fibrous tissue (260 Hz) present in carcinomas. These results suggest that the product of fibrosis that results from cell mutation appears similar for different types of skin cancers. BCCs have extensive fibrotic tissue deposition, but the number of cells and the amount of new lesion blood vessels is limited. In contrast, cellular proliferation and fibrous tissue deposition are seen to increase in invasive cancers including SCC and melanoma.


Glioblastoma (GBM) is considered one of the most aggressive human cancers. Earlier, our group have demonstrated that alternative RNA splicing plays an important role in the regulation of the GBM phenotype. To continue this study, we analyzed the type of RNA splicing and the expression levels of the spliceosomal genes in a large number of tumor tissue samples and patient-derived GBM sphere lines. We demonstrated that the expression level of splicing factors allows dividing GBM patients into groups with different survival prognosis and also reflects the phenotype of the tumor. In addition, we identified the alternative splicing events that may regulate the GBM phenotype. Finally, we for the first time compared the expression profiles of the spliceosomal genes in different regions of the same tumor and identified splicing factors whose expression most significantly correlates with GBM patients’ survival. Aforementioned data emphasize the important role of pre-mRNA splicing in GBM progression. Glioblastoma is the most common primary brain tumor in adults. Despite the enormous efforts aimed at combating this disease, the existing methods of treating glioblastomas are mainly of a palliative nature and can increase the average patient survival from 3 months (without treatment) to 2 years  , 2. SuchPreviously, our and other groups obtained results indicating that the way of splicing of pre-mRNA molecules in tumor cells plays an important role in the formation of glioblastoma heterogeneity . For exaTo identify the splicing regulator proteins most strongly associated with the main properties of low-grade gliomas (WHO grades II–III) and glioblastomas (grade IV), we decided to conduct a bioinformatics analysis of available RNA sequencing data from the TCGA database (The Cancer Genome Atlas), as well as to perform sequencing of RNAs isolated from tumor tissue samples and primary cultures of glioblastoma cells collected by our laboratory over the past 5 years.We started our analysis by identifying the proteins involved in pre-mRNA splicing. Using the KEGG and NCBI databases, we created a list of 197 proteins that presumably affect splicing. It should be noted that this list is probably somewhat redundant, because, based on the analysis of protein structure and protein–protein interactions, only 50–150 proteins in the cell can directly regulate splicing , 12.After analyzing the expression levels of spliceosomal protein genes in more than 150 patients with glioblastomas, we showed that all glioblastomas can be divided into several groups according to the expression of splicing factors . The firMDM2, MDM4, FoxM1, hRAS, Notch1, ATM, TGM2, Drosha, etc. Next, we divided all detected splicing changes into eight 8 types: (1) altered 3' splicing site, (2) altered 5' splicing site, (3) alternative mRNA end, (4) alternative mRNA start , (5) single exon skip/add, (6) multiple exon skip/add, (7) mutually exclusive exons, and (8) intron retention (To understand how the changes in spliceosomal gene expression that we observed affect the process of pre-mRNA splicing, we compared alternative splicing events detected by transcriptome sequencing of proneuronal and more aggressive mesenchymal primary glioblastoma cell cultures. In total, we found 3370 alternative splicing events that were statistically significantly different between the two groups of samples. These events affected important tumor-associated genes such as etention . Analysietention .This result is in good agreement with the phenotypic differences between proneuronal and mesenchymal glioblastoma cells. For example, we have previously shown that mesenchymal cells are characterized by increased migration rate and invasiveness, greater resistance to DNA-damaging chemotherapeutic drugs, accelerated proliferation, and altered cell morphology . Thus, iAfter obtaining data on the expression of spliceosomal genes in tumors from different patients, we analyzed the levels of RNA splicing regulators within a single tumor. Previously, we demonstrated large phenotypic differences between cells from the center and periphery of glioblastoma . For thiFinally, we analyzed the TCGA database to identify the splicing factors whose expression is statistically significantly associated with survival in patients with grade II–III glioma and glioblastoma. Summarizing the above results, it can be postulated that splicing-regulatory proteins play an extremely important role in determining the phenotype of glioblastoma cells and, theoretically, can be used to predict the course of the disease in patients with glioblastoma. However, obviously, further studies are required to identify the functions of specific spliceosomal proteins.


A cluster randomised trial of mHealth and participatory learning and action (PLA) community mobilisation interventions showed that PLA significantly reduced the prevalence of intermediate hyperglycaemia and type 2 diabetes mellitus (T2DM) and the incidence of T2DM among adults in rural Bangladesh; mHealth improved knowledge but showed no effect on glycaemic outcomes. We explore the equity of intervention reach and impact.Intervention reach and primary outcomes of intermediate hyperglycaemia and T2DM were assessed through interview surveys and blood fasting glucose and 2-hour oral glucose tolerance tests among population-based samples of adults aged ≥30 years. Age-stratified, gender-stratified and wealth-stratified intervention effects were estimated using random effects logistic regression.PLA participants were similar to non-participants, though female participants were younger and more likely to be married than female non-participants. Differences including age, education, wealth and marital status were observed between individuals exposed and those not exposed to the mHealth intervention.PLA reduced the prevalence of T2DM and intermediate hyperglycaemia in all age, gender and wealth strata. Reductions in 2-year incidence of T2DM of at least 51%  were observed in all strata except among the oldest and least poor groups. mHealth impact on glycaemic outcomes was observed only among the youngest group, where a 47% reduction in the 2-year incidence of T2DM was observed .Large impacts of PLA across all strata indicate a highly effective and equitable intervention. mHealth may be more suitable for targeting higher risk, younger populations.ISRCTN41083256. The DMagic trial in rural areas of Bangladesh reported the first large-scale, population-level evidence concerning the effectiveness and cost-effectiveness of participatory learning and action (PLA) community mobilisation and mHealth interventions for reducing the prevalence of intermediate hyperglycaemia and type 2 diabetes in the general population and 2-year incidence of type 2 diabetes among individuals identified with intermediate hyperglycaemia. The PLA intervention achieved absolute reductions in prevalence and incidence of 21% and 9%, respectively. The mHealth intervention increased population-level knowledge and awareness of diabetes, but no changes in disease occurrence were observed.This study investigates the equity of the exposure to and effects of the PLA and mHealth interventions. Results show PLA community mobilisation to be an equitable population-level intervention that can benefit entire communities, including those often excluded from interventions, such as women, older adults and the poorest. Ongoing research is exploring models of national scale-up and adaptation to urban contexts in Bangladesh and elsewhere. mHealth health-promoting interventions may have a role to play in improving health outcomes in certain high-risk groups and should be considered as part of multicomponent, multisectorial responses to diabetes risk.The prevalence of type 2 diabetes mellitus (T2DM) is growing in South Asia due to a mixture of complex genetic predispositions and increases in obesity and overweight, sedentary behaviour, energy-dense diets and an ageing population.Health equity is defined as the absence of systematic disparities in health, or its social determinants, between more and less advantaged social groups.Being poor may cause one to avoid seeking care for diabetes due to high financial costs, resulting in worse health outcomes and possible inability to work, further leading to financial vulnerability.Equity in health and healthcare is central to the Sustainable Development Goals agenda,The DMagic cluster randomised controlled trial (cRCT) evaluated a participatory learning and action (PLA) community mobilisation intervention and mHealth phone messaging to prevent and control T2DM and intermediate hyperglycaemia in rural communities in Bangladesh.10.1136/jech-2021-217293.supp1Supplementary dataThe DMagic trial took place in 96 villages (approximate population of 125 000) across four purposefully selected subdistricts (‘upazillas’) in Faridpur District, Bangladesh, from June 2015 to June 2018. Villages, which acted as trial clusters, were randomly allocated to either the mHealth, PLA or control arm (32 in each). Men and non-pregnant women aged 30 years and older were the units of analysis.2 and has mainly an agricultural economy. The population is primarily Bengali and 90% are Muslim, with the remaining population being Hindu. Faridpur District is divided into nine upazillas (subdistricts), which in turn are divided into unions of approximately 25 000 population.Situated on the banks of the Padma River, Faridpur District has a population of over 1.7 million in an area of 2050 kmThe PLA intervention was implemented through 18 monthly group meetings focused on T2DM prevention and control. Groups worked through a cycle of problem identification, planning and implementing strategies to address these problems with the wider community, and evaluation. Groups were gender specific, and all members of the community were welcome to attend and participate in the intervention. Communities developed a range of context-specific strategies, including increased opportunities for exercise and access to fresh fruit and vegetables.The mHealth intervention involved voice messages promoting behaviour change to reduce diabetes risk.The 96 villages were randomly allocated (1:1:1) to the community mobilisation (PLA) intervention, mHealth intervention or control, with each upazilla constituting one stratum. The name of each village was written on pieces of paper which were drawn at random by community leaders and representatives at a public orientation and consent meeting in Faridpur town. The first eight villages per upazilla drawn from the bottle were allocated to arm A, the next eight villages to arm B and the final eight villages to arm C. After all 96 villages had been allocated , each of the three arms were randomly assigned to either the community mobilisation (PLA) intervention, mHealth intervention or the control group by simultaneous drawing of arm letter and intervention allocation from two separate bottles. Because of the nature of the interventions being tested, the intervention team could not be masked to allocation. The data collection team was masked to allocation at the cluster and individual levels during the baseline survey.Primary outcomes were (1) the combined prevalence of T2DM and intermediate hyperglycaemia among a random sample of adults aged 30 years and above and residing in study clusters, and (2) 2-year cumulative incidence of T2DM in a cohort of individuals identified with intermediate hyperglycaemia at baseline .Secondary outcomes included mean diastolic and systolic blood pressures; prevalence of hypertension; hypertension control (among those with known hypertension); mean body mass index; prevalence of overweight and obesity and abdominal obesity (waist to hip ratio >0.9 for men and >0.85 for women); health-related quality of life (using EuroQol-5 Dimensions-3 Level (EQ-5D-3L) score)A pre-intervention (baseline) survey to collect data on sociodemographic characteristics and primary and secondary outcomes was carried out among a random sample of 13 684 eligible individuals (permanent resident aged 30 years and older) between January 23 and May 30 2016. Sample size was based on overall trial objectives and is described in detail elsewhere.Sampling and survey methods were repeated among a new random sample of 13 687 eligible individuals for an end-of-study (postintervention) cross-sectional survey conducted between 16 January and 30 April 2018 to assess intervention effects on the prevalence of intermediate hyperglycaemia and T2DM and secondary outcomes in the general population.The 2470 individuals identified with intermediate hyperglycaemia in the baseline survey were also followed up in the end-of-study survey to measure incidence of T2DM among this cohort.The cross-sectional end-of-study survey was used to describe the reach of the PLA and mHealth interventions by comparing participants who received the intervention to those who did not, within each arm. Exposure to PLA groups was defined as those who self-reported participating in meetings monthly or every 2 months; we reasoned that monthly or bimonthly meeting attendance would give participants enough exposure to benefit directly. Participants who reported attending meetings less frequently were considered as non-participants.Mobile phones within this context are often a shared resource within households and the mHealth intervention encouraged sharing of message content. We therefore defined exposure to mHealth messages as those who self-reported ever having received a message or knowing someone who had received a message. A sensitivity analysis was also performed where we restricted the definition of exposure to include only those who directly received mHealth messages. Socioeconomic and demographic characteristics between exposed populations and non-exposed populations in the PLA and mHealth arm were described and compared with χ² and Fisher’s exact tests as appropriate.All individuals who provided blood glucose measurements at the end-of-study survey  were included in the analysis of intervention effect on the combined prevalence of T2DM and intermediate hyperglycaemia. In the intermediate hyperglycaemia cohort, all individuals for whom a baseline blood glucose measurement revealed intermediate hyperglycaemia and for whom an end-of-study blood glucose measurement was assessed were included in the analysis.Community representatives were involved from the early stages of the research and engaged through a community orientation meeting. We subsequently established community advisory groups, comprising community leaders and representatives, who were consulted on and influenced intervention and survey activities, considering the burden of the intervention and time required to participate in the research. Community leaders promoted participation in the interventions and surveys, and PLA intervention activities were largely determined by community participants themselves. Trial findings were shared with community members who contributed to the interpretation of the data through a process of participatory analysis described elsewhere.At the end-of-study cross-sectional survey, data were collected from 11 454 (83.7%) of 13 687 individuals. In the villages assigned to the control arm, physical measurements  and interview survey data were gathered from 3785 (83%) individuals; 44 (1%) provided only physical measurements, and 1 (<1%) completed only the interview survey. In the mHealth arm, 3797 (83%) completed the physical measurements and interview survey; 15 (<1%) completed physical measurements only; and 5 (<1%) completed the survey only. In the PLA arm, 3786 (83%) completed the physical measurements and interview survey; 12 (<1%) completed the physical measurements only; and 9 (<1%) completed the survey only.From the intermediate hyperglycaemia cohort identified during the baseline cross-sectional survey (n=2470), 704 (85%) in the control arm, 666 (84%) in the PLA arm and 714 (85%) in the mHealth arm were followed up.There were more male non-responders than female non-responders (1712 (23%) of 7520 men vs 721 (9%) of 7854 women, p<0·001). Male non-responders were younger than male responders , whereas female non-responders were slightly older than female responders . Reasons for non-response and loss to follow-up included death, pregnancy, migration and refusal.Reach of the interventions was assessed among all individuals who participated in the end-of-study survey from the cross-sectional sample.Applying a more restricted definition of exposure to include only individuals who directly received messages showed similar patterns of exposure across sociodemographic groups overall, though a greater proportion of women were classified as unexposed, indicating that women were more likely to be exposed indirectly .PLA resulted in similar large reductions in the combined prevalence of T2DM and intermediate hyperglycaemia among men  and women , across all wealth tertiles  and across all age groups . ResultsPLA resulted in reductions in the 2-year cumulative incidence of T2DM by 51% among men  and by 60% among women  . A consiNo significant impacts of the mHealth intervention on cumulative incidence of T2DM were observed. However, a potential intervention effect may be apparent in the youngest (30–39) age group .PLA and mHealth interventions had large positive impacts across all gender, age and wealth groups in relation to knowledge and awareness about the causes, symptoms, complications, prevention and control of diabetes, and, among individuals with diabetes, diabetes control and self-awareness of diabetic status . There wOur analysis of the impact of a PLA intervention across age, gender and wealth groups shows that despite socioeconomic differences in participation in PLA groups, the intervention achieved large, significant reductions in occurrence of intermediate hyperglycaemia and T2DM in all gender and wealth groups and in all but the oldest age group in communities where PLA was implemented. Exposure to the mHealth intervention was greater among younger, better educated individuals. Although mHealth intervention effects on primary outcomes were not observed in most groups, indications of a potential effect on 2-year incidence of T2DM among 30–39 year olds with intermediate hyperglycaemia may indicate a role for targeted mHealth interventions in diabetes prevention in high-risk individuals.While we saw some differences in who participated in PLA groups, impacts were relatively consistent. An explanation for this could be in the way PLA works at the community rather than individual level. There is evidence that non-participants might have been motivated to control and prevent diabetes through interacting with group participants and through the creation of an enabling environment for behaviour change. This helps explain how the benefits of the intervention spread across different groups.The PLA intervention had an equitable impact in men and women. Meetings held in groups segregated by gender may have made it easier for women and men to participate, with groups organised at times and locations convenient to participants and aligned with cultural norms. Group discussions and larger community meetings enabled men and women to come together to plan actions and may have made household conversations about dietary changes easier. For example, women could cook vegetables and less oily food without being criticised by their husbands, and families grew their own vegetables. Women also felt more able to ask for support to seek healthcare.Although participation in PLA meetings was higher in the younger age groups of women, the impact of PLA on combined prevalence of T2DM and intermediate hyperglycaemia was observed in all age groups. However, an impact on cumulative incidence of T2DM among the cohort with intermediate hyperglycaemia was not observed in people above 60 years of age. It has been shown that there can be significant reversal of intermediate hyperglycaemia to normoglycaemia in middle-aged people who undergo behavioural interventions such as increased exercise and dietary restrictions,Although participation in groups was greatest among people in the second wealth tertile (poor), positive impacts of PLA were observed across all socioeconomic strata. This ‘diffusion effect’ of PLA impact across socioeconomic strata was also reported by Houweling and colleagues, although in relation to neonatal health outcomes.Overall, high exposure to the mHealth intervention was observed, with over 80% of individuals in mHealth clusters receiving or knowing someone who received a message. Widespread accessibility and usage of mobile phones throughout the community explains this. Men and women were equally likely overall to be exposed to mHealth, and there was a higher likelihood for both male and female recipients to be younger and more educated. Marital status did not change the likelihood of receiving messages among men but did among women, where married women were significantly more likely to receive messages. This may be explained by mobile phone ownership in Bangladesh being much higher among men, and thus, married women more readily accessing a mobile phone.While overall the mHealth intervention did not impact intermediate hyperglycaemia or T2DM prevalence or T2DM incidence, the potential impact on the 2-year cumulative incidence of T2DM among the youngest group with intermediate hyperglycaemia in the mHealth arm is intriguing. One study assessing a medication adherence promotion intervention through mHealth for low-income adults with T2DM showed that older people were less likely to engage in the intervention.A potential limitation in our study is that of multiple hypothesis testing, increasing the risk of type I errors in relation to p value cut-offs of ‘statistical significance’. We have therefore applied the Bonferroni adjustment, which applies more stringent criteria of statistical significance. However, we also acknowledge that Bonferroni is widely regarded as a conservative approach to handling multiple testing and it increases the likelihood of type II errors.PLA community mobilisation for diabetes prevention and control is an effective and equitable population-level intervention. Further research should be conducted to evaluate the effect of PLA in rural areas of other LMICs with a similar high burden of T2DM. PLA should also be adapted and piloted in urban areas in Bangladesh to inform possible country-wide scale-up of the intervention. mHealth health-promoting interventions may have a role to play in improving health outcomes in certain high-risk groups and as part of multicomponent, multisectorial responses to diabetes risk.


The aim of this study was to evaluate the effects of being overweight on the ability to conceive, fertilization rate, and in vivo development of embryos in regularly cycling, spontaneously ovulating, and naturally mated female mice. The study was based on statistical analysis of data collected during 14 experiments with identical design, performed on 319 control and 327 obese mice, developed in an intergenerational model of obesity induction which eliminates the impact of aging and high-fat feeding. Six-week-old mice with a vaginal sperm plug were slaughtered on embryonic days 2, 3, or 4, and the flushed contents of the oviducts and uteri were assessed by stereomicroscopy. The results showed no association between being overweight and the proportion of ovulating or fertilized females. On the other hand, a strong association was found between being overweight and ovulation yield. On embryonic day 2, significantly higher numbers of eggs were recovered from the oviducts of fertilized obese mice. Maternal overweight status was also associated with higher developmental capacities of preimplantation embryos. In conclusion, contrary to studies based on the high-fat-diet model, in female mice fed regular chow, being overweight was associated with an increased ovulation quota and higher developmental rate of fertilized oocytes. Being overweight did not impact ability to conceive. On the other hand, as documented in our previous studies, the quality of oocytes and blastocysts recovered from overweight mice developed in an intergenerational model of obesity was low. Assessment of the impact of being overweight on the ability to conceive and early development of conceptus in women has been carried out in numerous systematic studies. However, many of these have produced conflicting outcomes, documenting no or a negative effect of being overweight on particular reproductive parameters . To respect specifics of reproductive research, an intergenerational model of diet-induced obesity was established for the production of outbred mice that are overweight . Mice deSo far, the intergenerational model of diet-induced obesity has been used to evaluate the effect of female overweight status on various qualitative parameters of ovarian cells, oocytes, and early embryos, such us lipid deposition, metabolic activity, hormonal activity, apoptosis incidence, gene expression, and DNA methylation ,20,21,22In general, quantitative reproductive parameters are characterized by high individual variability in females. Furthermore, they might be influenced by a number of external factors, such us season, health, stress, libido, or quality of sperm. Not all of these factors can be eliminated when planning experiments on living animals; however, their impact on results can be minimized through the use of a large sample size.The aim of the current study was to evaluate the effects of female overweight status on the ability to conceive, ovulation quota, fertilization rate, and in vivo development of preimplantation embryos by means of statistical analysis of data collected during 14 experiments with identical design performed between the years 2011 and 2020 on control and obese mice using an intergenerational obesity model.Mus musculus) of the outbred ICR (CD-1 IGS) strain . Overweight females were produced using a breeding protocol based on over-nutrition of animals during intrauterine and early postnatal development  and allocated into four groups: females delivered from control dams were classified as normal controls with physiological body weight (±20 g) and body fat (7–8%) or lean controls spontaneously displaying decreased body weight and body fat (<7%); females delivered from experimental dams were classified as obese mice with significantly elevated body weight (±24 g) and fat (>11%), or obesity-induction resistant experimental mice with physiological body weight and slightly elevated body fat (8–11%). In the following reproductive experiments, only control mice with physiological body weights and body fat and obese experimental mice with significantly elevated body weight and body fat were used.At day 35, spontaneously ovulating control and obese female mice were mated with males of the same strain  for one or more nights. In each experiment, a cohort of 50 males with inbreeding-excluding origin and previously proven fertility was used for mating purposes. To minimize the impact of paternal obesity or stress, males were fed the standard pellet diet only and housed individually. Successful mating was confirmed by the identification of a vaginal sperm plug every morning.Mated dams from both groups were sacrificed using cervical dislocation and were subjected to ova/embryo isolation on embryonic day 2 of pregnancy , on Female mice with a vaginal sperm plug were classified as “mated females”. Female mice with developing corpora lutea and at least one ova or embryo in the reproductive tract were classified as “ovulated females”. Female mice with at least one ≥2 cell embryo in the reproductive tract were classified as “fertilized females”.Successful fertilization was confirmed by the presence of ≥2 cell embryos in the maternal reproductive tract. Flushed ova with normal morphology were classified as unfertilized oocytes. Ova showing abnormal morphology  were classified as unfertilized and degenerated oocytes. Embryos displaying 2 to 16 cells were classified as cleaving-stage embryos, embryos displaying >16 cells were classified as morulas, and embryos displaying a blastocoele cavity were classified as blastocysts. Embryos with abnormal morphology  were classified as degenerates.Statistical analysis was performed using StatsDirect 3.2.8 software .Categorical variables are presented as counts and relative frequencies. Data showing departures from normality are presented as medians with the interquartile range (IQR). Descriptive and univariate analyses were performed in order to identify important reproductive characteristics associated with maternal obesity.Fisher’s exact test was conducted to test for an association between maternal overweight status and proportion of ovulating and fertilized females on day 2, 3, and 4 after mating. Variables are quoted with the corresponding odds ratio (OR) along with the respective 95% confidence interval (CI).Poisson regression models were fitted for the outcomes of the number of recovered ova and embryos on embryonic day 2, 3, and 4; and for the outcomes of the number of recovered fertilized ova (≥2 cell embryos) and unfertilized ova (oocytes and degenerates) on ED 2, the number of quickly developing embryos (≥8 cell) and slowly developing embryos (<8 cell) and ova on ED 3, and the number of blastocysts and slowly developing embryos (morulas and <16 cell embryos). The outcomes were regressed against the presence of maternal overweight status in donor mouse females. The selected model variables are quoted with the corresponding regression coefficient (B), along with the respective 95% confidence intervals (CI).p < 0.05.The significance level was set at This study was performed on 319 control and 327 obese female mice showing the presence of a vaginal sperm plug after spontaneous ovulation and overnight mating with the males .proportion of ovulating females or between maternal overweight status and the proportion of fertilized females on any day of early pregnancy (p = 0.153).The results of Fisher’s exact test showed no association between maternal overweight status and the regnancy . Indepenregnancy . On embrtotal number of ova recovered from fertilized mice (p = 0.0008). Further analysis of ovulated ova showed that maternal obesity was also associated with a significant increase in the number of fertilized ova  and a non-significant increase in the number of unfertilized ova   . On embrdevelopmental capacities of embryos recovered from fertilized mice (p = 0.0007) and a significantly higher number of blastocysts on ED4 . In counterpart, maternal obesity was associated with a non-significantly decreased number of slowly developing embryos on ED3  and a significantly decreased number of slowly developing embryos on ED4 .The results of the Poisson regression model indicated an association between maternal overweight status and zed mice : OviductIn fertilized female mice, the majority of oocytes recovered on ED2 and ED3 showed normal morphology. The occurrence of degenerated oocytes was very rare . On ED4, the proportion of degenerated oocytes/embryos reached 0.047% in the control group and 0.042% in the overweight group. No statistical difference between the groups was detected on any embryonic day.Analysis of data collected from 14 independent experiments showed that being overweight, in spontaneously ovulating six-week-old female mice, might have a positive effect on their ovulation rate .Similarly, significantly increased numbers of ovulated oocytes have been previously documented in female mice fed a high-fat diet and displaying elevated body weight, higher amounts of abdominal fat, and insulin resistance at the age of 21 weeks . On the Our observations contradict the results of population-based studies in humans. In women, the association between obesity and anovulatory infertility has been documented several times (reviewed in ). HoweveThe majority of other previous experimental studies showed no effect of female obesity on ovulation rate: 7- to 20-week-old mice with diet-induced obesity showed similar numbers of recovered oocytes ,26,27, zThe mechanism of the positive effect of being overweight on the ovulation rate was not evaluated in our study. However, based on previous findings, we can hypothesize that positive energetic balance accompanied by increased endocrine activity of pancreas and adipose tissue probably influences gonadotropin secretion in the pituitary gland and subsequently stimulates the development and ovulation of a higher number of follicles in the ovaries of obese mice. It has been shown that increased circulating insulin levels are associated with increased luteinizing hormone (LH) secretion through elevated expression and secretion of gonadotropin-releasing hormone (GnRH) in mice ,31. FurtOn the other hand, it has been proposed that due to LH hypersecretion and an increased LH:FSH ratio, the resumption of oocyte maturation in developing follicles is frequently impaired by obesity in women ,3. OocytSimilar findings have been documented in animal experimental studies: mice with diet-induced obesity showed increased occurrence of abnormal morphology of mitochondria, elevated levels of reactive oxygen species, and epigenetic modifications in GV oocytes ,12; a lop > 0.05, chi-square test). These results are in accordance with previously published findings recorded on day 1 of pregnancy: At 11 h after supposed spontaneous ovulation, successful fertilization was confirmed in 70.34% and 79.84% of oocytes recovered from obese and control females, respectively [Interestingly, the above stated facts did not affect the overall incidence of fertilization in overweight female mice in the current study: There was no difference in the average percentage of ≥2 cell embryos flushed from the oviducts of obese (93.36 ± 1.25%) and control dams (94.22 ± 1.23%) on embryonic day 2 .The results of the current study showed that the cleavage rate for preimplantation embryos developing in the reproductive tract of obese dams was significantly higher than that in controls . A shortBased on these observations, we might hypothesize that the oviducts of obese females probably provide a beneficial microenvironment for embryonic cell growth. Unfortunately, there are limited data on the composition of oviductal fluid in obese women or overweight animals to directly support this hypothesis ,45,46. HOn the other hand, the quality of such “quickly derived” embryos is disputable. As shown in our previous studies, blastocysts recovered from obese mice displayed significantly elevated incidence of apoptotic cell death , alteredLower quality of blastocysts may not necessarily lead to implantation failure; however, an altered genomic or metabolic profile would have a significant impact on the further development of the conceptus or growth of the offspring. The documented fetal growth retardation ,27 and dNevertheless, our finding of a positive effect of maternal overweight status on the cleavage rate of preimplantation embryos in vivo contradicts the outcome of two previous studies on 12- and 19-week-old mice showing no differences, or even a slight decrease, in developmental capacities of growing embryos recovered from dams with diet-induced obesity ,28. OncePublished data on the effect of being overweight on the developmental dynamics of recovered zygotes or 2 cell embryos in vitro are controversial as well: some authors have documented no effect ,26, wherThe results of this “population study” performed on pubertal mice developed in an intergenerational model of obesity showed no impact of female overweight status on the frequency of spontaneous ovulation or natural fertilization. On the other hand, female overweight status was strongly associated with elevated ovulation quota. Furthermore, data on the proportion of fertilized and unfertilized ova recovered from fertilized dams suggest that when negative effects of superovulation, aging, and high-fat consumption are eliminated, obesity-induced alterations in qualitative parameters of matured oocytes do not necessarily have to be linked to fertilization disorder. However, a higher ovulation yield did not result in higher embryo recovery on day 4 of pregnancy (current study) or in higher litter size on day of delivery (previous study ). This sLimitations of the study: Since reproductive physiology and the overweight scoring system (% of body fat vs. BMI) differ between rodents and human, inter-species extrapolations are limited. Since sperm quality analysis was not performed during selection of male mice, the impact of male subfertility on the results cannot be excluded.


Brain barriers are crucial sites for cerebral energy supply, waste removal, immune cell migration, and solute exchange, all of which maintain an appropriate environment for neuronal activity. At the capillary level, where the largest area of brain–vascular interface occurs, pericytes adjust cerebral blood flow (CBF) by regulating capillary diameter and maintain the blood–brain barrier (BBB) by suppressing endothelial cell (EC) transcytosis and inducing tight junction expression between ECs. Pericytes also limit the infiltration of circulating leukocytes into the brain where resident microglia confine brain injury and provide the first line of defence against invading pathogens. Brain “waste” is cleared across the BBB into the blood, phagocytosed by microglia and astrocytes, or removed by the flow of cerebrospinal fluid (CSF) through perivascular routes—a process driven by respiratory motion and the pulsation of the heart, arteriolar smooth muscle, and possibly pericytes. “Dirty” CSF exits the brain and is probably drained around olfactory nerve rootlets and via the dural meningeal lymphatic vessels and possibly the skull bone marrow. The brain is widely regarded as an immune-privileged organ because it is accessible to few antigen-primed leukocytes. Leukocytes enter the brain via the meninges, the BBB, and the blood-CSF barrier. Advances in genetic and imaging tools have revealed that neurological diseases significantly alter immune–brain barrier interactions in at least three ways: (1) the brain’s immune-privileged status is compromised when pericytes are lost or lymphatic vessels are dysregulated; (2) immune cells release vasoactive molecules to regulate CBF, modulate arteriole stiffness, and can plug and eliminate capillaries which impairs CBF and possibly waste clearance; and (3) immune–vascular interactions can make the BBB leaky via multiple mechanisms, thus aggravating the influx of undesirable substances and cells. Here, we review developments in these three areas and briefly discuss potential therapeutic avenues for restoring brain barrier functions. They allow communication between the brain and peripheral immune system, facilitate cerebral supply of oxygen, glucose, ions, and various regulatory molecules while promoting the efflux of metabolic waste and limiting the influx of neurotoxic molecules, pathogens, and leukocytes. Over the past 15 years, the proposed existence of the glymphatic system, (re)discovery of the meningeal lymphatics, and advances in our understanding of cerebral immunosurveillance, the blood–brain barrier (BBB), and blood supply have given important new insight into how the brain functions. The brain exhibits an immune-specialized status to minimize damage to its largely non-proliferating (and hence long-lived) cells, and therefore, leukocyte–vascular interactions and trafficking are tightly controlled. Neuroinflammation and microvascular dysfunction are hallmarks of various neurological diseases including, but not limited to, Alzheimer’s disease (AD), stroke, brain tumors, epilepsy, and multiple sclerosis (MS). Capillaries are the site of the greatest vascular resistance within the brainFollowing an overview of the brain vascular network and CSF transport routes, we will review recent advances in immune cell trafficking, immune modulation of cerebral blood flow (CBF), and BBB properties in various neurological diseases.2Blood enters the cerebral cortex from its surface through penetrating arterioles (PAs), goes through a vast network of capillaries for brain–blood and blood–brain solute exchange, and exits via ascending venules (AVs) .–in vivo, have revealed that pericytes throughout the capillary bed are contractile and that those of at least the first to third capillary branching order from PAs (where first-order refers to the first branch) contribute to neuronal activity evoked increases in CBF (–14CBF is controlled by smooth muscle cells (SMCs) around arteries and arterioles and by pericytes with contractile processes wrapping circumferentially around capillaries.s in CBF .7–13 Per–,,–Respiratory motion, ciliary beating, vessel pulsation driven by the heart and spontaneous vasomotion of SMCs, and possibly pericytes of the first to third capillary branching order drive the flow of CSF, which facilitates brain buoyancy, waste clearance, and brain antigen presentation to systemic immune cells.33.1The brain is surveyed by microglia and astrocytes in the parenchyma, macrophages  olfactory nerve rootlets into lymph vessels or by entering the blood via arachnoid villi and granulations somehow connecting to venous sinuses.,Peripheral blood leukocytes  can also3.2,,–,Recruitment of leukocytes into the brain is a key hallmark of numerous CNS disorders.Since pericytes embedded in the basement membrane constitute the outermost layer of blood vessels, they are ideally situated to control multiple components of the immune response, as has been highlighted in the periphery. For example, during tumor necrosis factor (TNF) or interleukin ,,Intriguingly, while pericytes orchestrate and facilitate leukocyte entry in the periphery, in the CNS it was recently demonstrated that pericytes reduce the permissiveness of the vasculature to leukocyte entry. Mice lacking the ,Furthermore, pericytes are involved in neuronal control of the CNS immune response, by sensing and relaying signals to neurons early in systemic inflammation. Following systemic lipopolysaccharide (LPS) stimulation in mice, ,,Chemokines are presented to leukocytes to facilitate migration into inflamed tissues -a process facilitated in part by the binding of tissue-derived chemokines on the abluminal side of ECs to atypical chemokine receptor 1 (ACKR1), a receptor that facilitates the internalization, transportation, and presentation of chemokines to leukocytes on the luminal aspect of ECs.,In cancer, pericyte deficiency leads to increased leukocyte infiltration in murine experimentally induced tumors, IL-6 upregulation, and hypoxia.In summary, it is now clear that pericytes are key mediators in the sensing and propagation of the inflammatory response. However, the role of pericytes seems to be highly organ-specific and dependent on the inflammatory stimulus. Crucially, detailed analysis of pericyte-leukocyte interactions in the CNS is required to understand whether mechanisms are shared between the CNS and the periphery.4,–,,–,Over 100 years after their discovery, which highlighted their association with the vasculature  signal directly to mural cells to modulate CBF. This is facilitated by their close association with mural cells. Unlike microglia, however, PVMs lack ramified processes and ,––,–In the healthy murine brain, circulating leukocytes stall transiently in 0.4% of cerebral capillaries, possibly by interacting with the glycocalyx or selectins on ECs,,,Similar vascular blocks by leukocytes can be observed in patients with AD4.3,,––,There are numerous immune-related molecules with known effects on blood flow. They include adenosine, angiotensin II (Ang II), prostaglandin ne cells .There are established mechanisms by which these immune molecules may modulate the contractile tone of pericytes and SMCs . Molecul,–Depolarization evokes traction . In muraSignaling via GC or GPCRs coupled to 4.4,,–Microglia generate vasodilating adenosine by hydrolyzing ATP using the membrane-bound ectoenzymes CD39 and CD73.eceptors . In cont,,,–Microglia can also produce the prostanoid –,,Since microglial ,,,,,,Neurons also signal to microglia to alter blood flow via the chemokine fractalkine (FKN/CX3CL1). FKN is predominantly present in neurons (membrane-bound or released in a soluble form) and binds to CX3CR1 selectively expressed in microglia or ,,,,ROS generated by NADPH oxidase (NOX2) in immune cells can be released in the brain by microglia, PVMs or infiltrating leukocytes,,Superoxide  cell release of IL-17 into the circulation reduces the activity of eNOS, which reduces CBF by 25%.–,,Importantly, systemic infection evoked decreases in CBF may enhance cognitive decline in patients. For instance, 62% of hospitalized COVID-19 patients present with a reduction in brain energy supply, most commonly, in the form of an ischemic stroke5,,–The BBB is a highly specialized vascular barrier that limits the influx of serum proteins, leukocytes, and toxic substances such as glutamate and ATP into the brain parenchyma and pumps out waste—functions that are primarily mediated by ECs, pericytes, and astrocytes . The BBB,In the brain, ECs show a lower rate of transcytosis than ECs of any other organ.,,,,,,,Key to the development and maintenance of the BBB are pericytes, which are essential for the development of tight junctions between ECs,,–Ablation of microglia in the healthy murine CNS does not compromise BBB integrity.Recent data also suggest that microglia play dual roles in the regulation of the BBB, dependent on the timing of the inflammatory response. Initially, following LPS challenge in mice, microglial contact with cerebral blood vessels protects BBB integrity, by increasing expression of the tight junctional protein claudin-5,,,While microglia play key roles in the regulation of the BBB, peripheral leukocytes predominantly contribute to BBB breakdown during pathology. These actions can be mediated by ROS and MMP release, which damage EC junctions, for example by interfering with the ,In addition, prolonged stalling of neutrophils in vessels may impair the integrity of the BBB by enhancing endothelial actin depolymerization and the breakdown of adherens junctions between ECs, as occurs in the periphery.In summary, microglia can play protective and detrimental roles in the regulation of the BBB, but peripheral blood leukocytes are predominantly destructive in pathology.6,,,–,,Immune–vascular interactions are a therapeutic target for various neurological diseases. CBF decreases occurring after stroke or early in the progression of AD could, for instance, be therapeutically targeted by inhibiting FKN receptors or blocking ROS production, respectively.,Another clinical approach could involve recruiting immune cell subsets with protective properties to the diseased brain. Promising murine studies highlight that recruitment of regulatory T cells improves cognitive function in AD,In conclusion, immune–vascular interactions play both homeostatic and pathogenic roles in the CNS depending on the context and injury. This field is currently at an exciting stage, where future work will identify new therapeutic avenues to help combat CNS disease.


Besides, due to the good encapsulation ability of the hydrogel, the nanomaterials can be released in a controlled manner by changing laser parameter, irradiation time, etc. The experimental results show that the PB+Ce6@Hy system we developed can generate a large amount of reactive oxygen species (ROS), which can be combined with the photothermal effect to kill tumor cells, as a result, tumor proliferation has been adequately inhibited. This combined PTT/SDT dynamic strategy provides a new perspective for Ce6-induced cancer therapy, showing great potential for clinical application.Sonosensitizer-mediated sonodynamic therapy (SDT) has emerged as a promising anti-tumor strategy. However, this strategy of continuous oxygen consumption further exacerbates the hypoxic tumor microenvironment, which limits its therapeutic efficacy. In this study, we designed a multifunctional hydrogel (PB+Ce6@Hy) that simultaneously co-delivers nanozyme prussian blue (PB) and sonosensitizer chlorin e6 (Ce6) for the realization of photothermal therapy (PTT) and enhanced SDT. When the hydrogel reaches the tumor tissue through local injection, the 808 nm laser can induce the hydrogel to warm up and soften, thereby triggering the release of PB and Ce6. PB can interact with endogenous H Due to the limited penetration depth of light, phototherapy is not enough for deep tumors, which limits the development potential of photothermal therapy (PTT) and photodynamic therapy (PDT) –4. Based2O2) in tumors to generate oxygen, which is expected to alleviate the hypoxic microenvironment of tumors. Hu et al. designed a novel PMPT nanomaterial that not only alleviates hypoxia tolerance and enhances ROS generation during photodynamic processes, but also inhibits the MTH1-regulated DNA damage repair pathway, resulting in aggravated oxidative damage and cell death (Prussian blue nanoparticles (PB) are a class of inorganic substances assembled from transition metal ions or lanthanides through cyano bridging ligands . Becausell death . It has in vivo biological barriers such as liver and kidney clearance effects. Traditional drug delivery systems (i.e. intravenous injection) have a series of problems such as low efficiency of nanomaterials reaching tumor tissue, premature leakage of cargo, and profound toxicity caused by the long-term existence of the carrier in the body  and nanozyme PB into a lowmelting agarose hydrogel to prepare a multifunctional nanosystem (PB+Ce6@Hy) for enhanced SDT/PTT Scheme 1in vivo for tumor therapy. Simultaneously, rheological curves of agarose hydrogel were detected, as the temperature increases  could raise the tumor temperature to nearly 48 degrees. A large number of basic researches and clinical applications have proved that when tumor tissue is continuously heated for a certain period of time, the growth of tumor cells is blocked, disintegrated, and even leads to death (6 cells per mL) were subcutaneously injected into each mouse to establish the tumor models. Subsequently, tumor growth was monitored every 2 days to assess the primary effect of the treatment system. As shown in in situ and generate O2, which greatly strengthens Ce6-induced ROS production effect, tumor growth was greatly inhibited. in vivo model that both PB@Hy and PB+Ce6@Hy systems could alleviate the hypoxic microenvironment .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewers MS and PQ declared a shared parent affiliation with the author MC at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Exportin-1 (XPO1) is a key player in the nuclear export pathway and is overexpressed in almost all cancers. This is especially relevant for non-Hodgkin lymphoma (NHL), where high XPO1 expression is associated with poor prognosis due to its oncogenic role in exporting proteins and RNA that are involved in cancer progression and treatment resistance. Here, we discuss the proteins and RNA transcripts that have been identified as XPO1 cargo in NHL lymphoma including tumour suppressors, immune modulators, and transcription factors, and their implications for oncogenesis. We then highlight the research to date on XPO1 inhibitors such as selinexor and other selective inhibitors of nuclear export (SINEs), which are used to treat some cases of non-Hodgkin lymphoma. In vitro, in vivo, and clinical studies investigating the anti-cancer effects of SINEs from bench to bedside, both as a single agent and in combination, are also reported. Finally, we discuss the limitations of the current research landscape and future directions to better understand and improve the clinical utility of SINE compounds in NHL.  Mammalian cells are segregated and organized into different compartments in part through the use of lipid membranes. The deployment of membranes in this manner creates functional hubs that separates processes such as energy production (mitochondria), from proteins/fat synthesis and calcium storage (endoplasmic reticulum) and containment of genetic material (nucleus). The correct spatiotemporal localization of molecules is controlled by a variety of mechanisms and a failure to compartmentalize molecules in time and space can lead to diseased states. The nuclear compartment is separated from the cytosol by the nuclear envelope and transport between these two locales is regulated by the nuclear pore complex (NPC). The NPC is a semi-selective barrier that allows the free passage of molecules between 40 and 60 kDa and requires active transport mechanisms for molecules exceeding this molecular weight limit . Given tThe molecules requiring active transport into and out of the nucleus rely on the activity of the karyopherins which constitutes a family of proteins that handle both nuclear import and export functions . ExportiXPO1 cargo has been examined broadly using yeast and HeLa cells , but litThe tumour suppressor proteins p53, p73, p21, p27, and FOXO1 inhibit cell cycle progression and are activated following DNA damage ,12. ThesOther studies have inhibited XPO1 using the novel small molecule KPT-330 and used immunofluorescence staining or fluorescent microscopy to observe changes in protein localization ,16. ThesThe NFkB pathway is central to many aspects of cancer progression and negatively regulated by IkB . While tSignal transducers and activators of transcription (STATs) belong to a family of transcription factors with a variety of functions. While some STAT proteins have anti-cancer activity, others act to promote cancer growth and progression by regulating genes involved in cell growth, inhibition of apoptosis and immune evasion . STAT3 aMore recently, RNA as XPO1 cargo has been gaining attention and interest. As nucleic acids to not contain canonical XPO1 NESs, RNA export is facilitated by associating with a wide range of adaptor proteins that contain nuclear export sequences (NESs) that allow them to be identified and exported by XPO1 . SeveralWhile most mRNA is exported via the TAP/NXKF1 pathway, some specific types of mRNA are exported via XPO1 . For exaFurthermore, XPO1 has been found to facilitate the nuclear export of unprocessed viral mRNA . TranscrSeveral bodies of work in the field have identified other potential RNA cargo. While the additional RNA cargos were not identified as XPO1 targets in lymphoma, they bear mentioning here as potentially important cargo in NHL despite the lack of definitive, supporting evidence Ribosomal RNA (rRNA), small nuclear RNA (snRNA), and micro RNA (miRNA) are exported into the cytoplasm by XPO1 with the help of adaptor proteins such as NMD3, the cap binding protein CBC, and the phosphorylated adaptor for RNA export (PHAX) . Gene siSimilarly, rRNA and snRNA has been identified as cargo in yeast , but resOur current understanding regarding selinexor’s mechanism of cellular cytotoxicity in a preclinical context is based on a collection of studies interrogating the activities of the various SINE compounds. KPT-185 is commonly used for in vitro experiments due to its high potency, however, its murine and human in vivo use is limited by its poor pharmacokinetics and oral bioavailability . For theIn vitro experiments with KPT-185 have demonstrated its cytotoxicity and potential mechanisms for its anti-cancer activity in NHL. Various NHL cell lines and patient tumour samples treated with KPT-185 demonstrated growth inhibition, reduced cell viability, and induced apoptosis after 72 h . AnalysiInterestingly, Western Blot and PCR analyses in these experiments also found that KPT-185 decreased XPO1 protein expression, but increased XPO1 mRNA expression . It was Xu et al. examined KPT-185 efficacy in the context of wild type and mutant p53. Mutations in this tumour suppressor protein are seen in about 30% of NHL cases, and are associated with poor prognosis due to treatment resistance . PreclinFurthermore, Western Blot and immunofluorescence staining determined that increasing KPT-185 concentrations resulted in an increase in nuclear localization of major tumour suppressor proteins such as p21, p27, p73, and FOXO3, indicating that a restoration of the nuclear pool of these proteins may play an important role in cytotoxicity, and may be of particular importance in the context of cells with mutant p53 [The significance of the tumour suppressor protein family in promoting cancer cell death was further observed in MCL, as KPT-185-mediated nuclear restoration of p53 activated transcription of its target genes such as FAS, PUMA, and DR5, which act to promote apoptosis . AdditioIn vivo, mouse models treated with KPT-276 demonstrated shrinkage of their NHL tumours without any visible signs of toxicity or excessive weight loss in these animals . The oraWhile most preclinical studies in lymphoma utilize KPT-185 in vitro and KPT-276 in vivo, studies using selinexor (KPT-330) provide the greatest insight on the efficacy of SINE molecules prior to initiating clinical trials as they are directly translatable to the compound that has been used in humans. To date, the preclinical studies for selinexor in lymphoma have mainly examined cytotoxicity and cell cycle arrest in various subtypes of NHL . Here, sA list of combination therapies with selinexor for NHL can be found in Preclinical studies have examined selinexor in combination with the Bruton’s Tyrosine Kinase (BTK) inhibitor zanubrutinib in DLBCL and mantle cell lymphoma (MCL) . BTK is In this light, zanubrutinib has found application as a compound used to treat B-cell lymphomas characterized by chronic BCR signaling and several studies using zanabrutinib in relapsed/refractory MCL can be found in the literature . TaranteIn vitro studies using three ABC-DLBCL and two MCL cell lines involved treatment with increasing concentrations of selinexor and zanubrutinib for 72 h and measuring its effects on cell growth. The combination was found to be synergistic at lowering cell count in both MCL lines and 1 DLBCL line, and additive in another DLBCL line. The one DLBCL cell line where the combination had no significant benefit was the zanubrutinib-resistant SUDHL-2 line, indicating that while selinexor is able to be effectively combined with zanubrutinib, it is not able to overcome resistance to the drug .To determine the mechanism by which this combination was effective, Tarantelli and colleagues examined the drugs’ effects on the cell cycle. Whereas individual treatments with selinexor or zanubrutinib mostly arrested cells in the G1 phase of the cell-cycle, the combination resulted in most cells entering the sub-G1 phase, where they subsequently underwent apoptosis . While iPreclinical studies have investigated selinexor in combination with the proteasome inhibitor bortezomib, which antagonizes the catalytic activity of the 26S proteasome and prevents activation of pro-inflammatory transcription factor NFκB . This drIκB-α is also a known cargo molecule of XPO1 in hematological malignancies . Even thDue to their anti-cancer effects via IκB-α activation and NFκB downregulation, Kashyap et al. investigated the efficacy of combining bortezomib and selinexor in vitro and in vivo . In vitrInterestingly, contrasting results were observed by Abeykoon et al. , who fouSalicylates are a class of anti-inflammatory drugs that have been found to relocalize p65, one of the subunits of NFκB, to the nucleolus in cancer cells . AlthougThe interactions between these two drugs and the mechanisms behind their synergy may be explained by their mutual actions in restoring nuclear localization of various cargo molecules. This is further supported by the investigators reporting that the combination therapy resulted in complete nuclear localization of an engineered reporter construct encoding green fluorescent protein (GFP) with a NLS, whereas selinexor alone resulted in incomplete nuclear localization. CH also acts as a modulator of nuclear export in, but the mechanisms underlying this process are poorly understood. However, its potential to restore nuclear localization of various proteins makes it an interesting candidate to potentiate selinexor’s mechanism of action. The combination also enhanced the degradation of XPO1 in a proteasome-dependent mechanism, as reduced protein levels were detected via Western blotting, and concurrent treatment with bortezomib prevented this reduction . AdditioVenetoclax is a B cell lymphoma-2 (BCL-2) inhibitor used to treat various types of leukemia and is but actively being investigated as a potential treatment for DLBCL due to the role of BCL-2 in this malignancy. The BCL-2 protein is involved in inhibiting apoptosis and is normally expressed during B-cell maturation . Due to Recognizing that the NFκB pathway is involved in both the nuclear export and activation of BCL-2, Elloul et al. conducted in vitro and in vivo studies examining the effect of combining selinexor with venetoclax . The comChemotherapy treatments such as bendamustine work by damaging the cells’ DNA. Bendamustine specifically alkylates bases and creates cross-links between DNA strands to cause breaks that the cancer cells may not be able to repair and may drive them to undergo apoptosis ,52. BendPreclinical studies have determined that nuclear localization of p53 can be restored with selinexor , promptiGemcitabine is a treatment commonly used as a salvage therapy for relapsed NHL . This chFinally, positive preclinical studies investigating selinexor combined with an immuno-chemotherapy combination used as part of the current standard of care for NHL  have inspired early phase clinical trials for this combination . FrontliIn vitro, the combination of cyclophosphamide, doxorubicin, and vincristine (CHO) enhanced apoptotic effects and inhibited cell proliferation and resulted in increased in PARP cleavage to initiate cell death . When exThe combination of selinexor and CHO was also effective in vivo as demonstrated by xenograft mouse models . SCID miThe positive results from preclinical studies have prompted the investigation of selinexor in small phase I/II clinical trials for various types of NHL. A full list of completed clinical trials is available in The first in-human phase I study investigating selinexor as a monotherapy enrolled 79 patients with relapsed/refractory (R/R) NHL ,61. DLBC2 resulting in significantly increased incidences of adverse events and reduced time on therapy, the recommended phase II dose (RP2D) identified was 35 mg/kg2, simplified to a flat dose of 60 mg [Several toxicities were noted in this trial that necessitated dose reductions. These toxicities were mainly hematologic in nature, including thrombocytopenia, neutropenia, and anemia. However, there was only 1 dose-limiting toxicity (DLT) of grade 4 thrombocytopenia, and the maximum tolerated dose (MTD) was not reached. Due to doses above 40 mg/kgof 60 mg Similar to the preclinical studies, PD analyses on the tumour biopsies found that selinexor decreased protein expression as measured by immunofluorescent staining of BCL-2, BCL-6, and c-Myc, and restored nuclear localization of XPO1 cargo such as p53 . The resThe subsequent phase II trial SADAL evaluated weekly 60 mg and 100 mg doses of selinexor in patients with R/R DLBCL . SimilarAEs were seen in 98% of patients but were reversible with supportive care or dose modification . HoweverThe promising results from the preliminary clinical trials using selinexor monotherapy in NHL supported subsequent investigations for use of selinexor in combination with other anti-cancer agents. Based on the positive results of preclinical studies examining the combination of selinexor and BTK inhibitors, ref.  a recentThe recent Phase I clinical trial testing selinexor and R-CHOP in NHL involved patients receiving six 21-day cycles of R-CHOP and either 60 mg or 80 mg of weekly selinexor, followed by weekly selinexor maintenance for 1 year . 12 patiPatients also had blood samples taken throughout the course of their treatment for PD and biomarker analysis to determine the combination’s mechanism of efficacy. In keeping with previous preclinical studies , this coFinally, a phase I study investigated the combination of selinexor with dexamethasone, ifosfamide, carboplatin and etoposide (DICE) in 11 patients with R/R peripheral T cell lymphoma (PTCL) or natural killer/T cell lymphoma (NKTL) . The comXPO1 functions as an exporter of a wide range of cargo molecules that aid in the growth and progression of cancer. SINE compounds, such as selinexor, that are potent XPO1 inhibitors are able to simultaneously target numerous pathways in NHL cells, leading to cytotoxic effects and sensitization to other anti-cancer agents. The pleotropic properties of XPO1 inhibitors results in the modulation of multiple downstream pathways by restoring imbalances in cargo protein and RNA localization. These properties make them interesting candidates for combination therapies. Defining appropriate and rational combination therapies using selinexor or other SINE compounds in NHLs is dependent on a multitude of factors and will need to be determined empirically.NHL is a highly heterogeneous disease, with many patients presenting with a spectrum of genetic abnormalities and protein expression profiles that affects response to treatment and prognosis. For example, each case of the DLBCL subtype of NHL presents with an estimated 75 different mutations, indicating a prominent role for genetic and biomarker testing to inform individual treatments for this disease . It is e


Autism spectrum disorder (ASD) is a neurodevelopmental disorder that impairs normal brain development and socio-cognitive abilities. The pathogenesis of this condition points out the involvement of genetic and environmental factors during in-utero life. Placenta, as an interface tissue between mother and fetus, provides developing fetus requirements and exposes it to maternal environment as well. Therefore, the alteration of DNA methylation as epigenetic consequence of gene-environmental interaction in the placenta could shed light on ASD pathogenesis. In this study, we reviewed the current findings on placental methylation status and its association with ASD. Differentially methylated regions (DMRs) in ASD-developing placenta were found to be mainly enriched in ASD gene loci affecting synaptogenesis, microtubule dynamics, neurogenesis and neuritogenesis. In addition, non-genic DMRs in ASD-placenta proposes an alternative contributing mechanism for ASD development. Our study highlights the importance of placental DNA methylation signature as a biomarker for ASD prediction. Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by persistent disturbances in social interaction and communication, restricted interests and repetitive behaviours   was found to be hypermethylated in ASD , specifically for the induction of the mesencephalon and cerebellum  angiogenesis and blood–brain barrier regulation  shores: regions up to 2 kb from CpG island, (b) shelves: regions from 2 to 4 kb from CpG island and (c) open sea: the rest of the genome (Sandoval et al. As the pathology of ASD is mostly limited to the development of the brain, the most appropriate approach to investigate DNA methylation in this condition should be through samples of brain tissue. However, analysing brain tissue is challenging due to small sample size, limited replication capacity, timing of collection after disease onset and aging (Bakulski et al. DDL1, AUTS2, CYP2E1, and IRS2 have been emphasized in more than one study. In perspective, aggregating additional datasets such as mRNA and miRNA expression with placental DNA methylation data (Santos Jr et al. NHIP that had been missed by standard array-based methods (Zhu et al. The placenta, as an accessible tissue with distinctive global and site-specific DNA methylation profiles, could provide important information about ASD development as it is a more precise catalogue of obstetric, perinatal and labor influences than other tissues and could have precise diagnostic value, however, following confirmation of the available predictive evidence by replication studies with larger sample size as most of the current studies suffer from limited sample size or being underpowered. In addition, there are heterogeneity in study design of the available reports which limits providing top differentially methylated loci as a few of them such as


The human body generates 10–100 billion cells every day, and the same number of cells die to maintain homeostasis. The genetically controlled, autonomously ordered cell death mainly proceeds by apoptosis. Apoptosis is an important way of programmed cell death in multicellular organisms, timely and effective elimination of apoptotic cells plays a key role in the growth and development of organisms and the maintenance of homeostasis. During the clearance of apoptotic cells, transcription factors bind to specific target promoters and act as activators or repressors to regulate multiple genes expression, how transcription factors regulate apoptosis is an important and poorly understood aspect of normal development. This paper summarizes the regulatory mechanisms of transcription factors in the clearance of apoptotic cells to date. In multicellular organisms, billions of cells are fated to be dead and cleared away for the normal development, and the excessive or harmful cells undergo a programmed progress which is activated and regulated by a series of genes . ProgramApoptosis is an important cell biological process in animal development, which is considered be essensial for organogenesis and maintaining daily tissue homeostasis. Once the apoptosis program is initiated, the signals released from ACs rapidly recruit and be captured by the professional phagocytes or neighboring cells, ultimately promoting the engulfment and elimination of cell corpses . GeneralCaenorhabditis elegans, in which there are 1090 somatic cells produced and 131 cells dead through apoptosis procedure. Observation on mutants which affected the 131 somatic cell deaths during worm development discovered a series of specific genes controlled the apoptosis process, which were proved to exist and play important roles as a similar manner in mammals  extrinsic apoptotic pathway and the mitochondria-triggered endogenous pathway . The extACs that undergo programmed cell death are usually eugulfed by “professional” phagocytes or other cells timely and effectively, to avoid inflammatory responses or maintain homeostasis. Failure in this process would cause redundant ACs accumulation in the organ, ultimately lead to the development of inflammatory autoimmune or neurodegenerative diseases. Therefore, timely and effective clearance of apoptotic cells in multicellular organisms is of great importance for the homeostasis of the organism .C.elegans, Drosophila melanogaster, researchers have uncovered the molecular mechanism of ACs clearance by phagocytes. Differently, mammalian macrophages are divided into two sub groups according to their functions and levels of inflammatory cytokines   and sphiund area . During ruitment .The cellular environment is very complex, including healthy cells, ACs, lymphocytes, and phagocytic cells. Therefore, the recognition of ACs by phagocytes is very crucial, and this process mainly depends on the molecules exposed to the apoptotic cell surface such as phosphatidylserine, Intercellular adhesionmolecule 3 (ICAM3) , Calretiv β3 integrin binds to ACs through PS-dependent bridging molecule MFG-E8; TAM receptor  recognizes PS through interacting with growth arrest-specific gene 6 (Gas6) or protein S , phagocytes directly or indirectly recognize and bind these signals through their own PS recognizing membrane receptor (PSR), thereby promoting the recognition and engulfment during apoptosis and preventing inflammation. The trigger receptor and advanced glycation end product receptor of Brain Angiogenesis Inhibitor 1/3(BAI1/3), T cell immunoglobulin mucin receptor 4 (TIM4), Stabilin-1/2 (Stab2), (TREM)-like protein 2 (TLT2) are identified as directly binding to PS . The recrotein S . Other brotein S . These rDrosophila Draper , integrin and Croquemort  and other phagocytosis receptors, which were found to function in different ways during phagocytosis: BAI1 is essential for phagosome formation, while TIM-4 stabilizes phagosomes  P2 and PtdIns3P phosphatidylinositol is a very important event in the sealing of phagocytic vesicles . PtdIns turation .S. hidradiata remains to be determined  and DNase II(NUC-1) to control the digestion and degradation of ACs  by repressing the transcription of both the egl-1 and ced-3 genes , and erythropoietin EPO is also upregulated, thereby stimulating the anti-inflammatory macrophage phenotype, promoting the polarization of macrophage M2, and enhancing efferocytosis  acting on sphingosine as an important “find me” signal to recruit phagocytes . S1PR, tocytosis . S1P alsocytosis .Panx1 was initiated and activated by transcription factors CREB and ETV4 in the rat epididymis  and LXR  are ligand-activated transcriptional activators with different tissue expression. After binding to ligands such as fatty acids and oxysterols, PPAR and LXR then form heterodimers with the retinoid X receptor (RXR) and recruit co-activators to induce transcription of various genes involved in lipid and cholesterol metabolism (The peroxisome proliferator-activated receptor (PPAR) and the liver x receptor (LXR), which belong to the nuclear receptor superfamily, are involved in cellular lipid homeostasis. PPAR  and complement binding to ACs provide eat me signals for macrophages. Increased PPARγ and RXR ligands promote IgG or IgM recognition by ACs, while the absence of PPARγ or RXRα reduces the expression of complement factors , thus inhibits the binding and uptake of ACs by macrophages .In the last few decades, we have made great progress in the study of apoptotic cell clearance. In living organisms, molecules, proteins and signalling pathways interact with each other, and the signalling pathways and functions involved are complex and diverse. However, there are still many regulators involved in apoptotic cell clearance waiting to be discovered and clarified. At the same time, the presence of transcription factors, a hot topic of research in recent years, regulates gene expression and affects the function of the organism.The regulatory role of transcription factors is not entirely point-to-point; it may be a one-to-many or many-to-one process, hence the complexity of its study. In previous studies, numerous transcription factors such as the nuclear receptor superfamily, IRF, AP-1, NF-κB and STAT family have been identified as regulating the clearance of ACs. Through the regulation of genes participating in the clearance of ACs, transcription factors directly or indirectly influence the recognition of ACs, the maturation of macrophages and the degradation of ACs, leading to the development of organs and maintenance of the immune system. As impaired clearance of apoptotic cells leads to human diseases, the NR superfamily and transcription factors such as AhR have been documented as important targets for the prevention and treatment of hyperlipidaemia, diabetes and chronic inflammatory diseases including atherosclerosis, as well as autoimmune diseases.In conclusion, transcription factors play important roles in the regulation of ACs clearance. There are still some unexplored signaling pathways between ACs and macrophages, and the current articles on the involvement of transcription factors in the process of apoptotic cell clearance are relatively superficial, and there are still many questions waiting to be explored, such as how transcription factors regulate how transcription factors regulate the shutdown of efferocytosis when apoptotic cell clearance is completed and how the high load/continuous b efferocytosis is transcriptionally regulated, etc. Therefore, finding new signaling pathways through transcription factors would be a valuable approach to broaden the field of apoptotic cell clearance. At the same time, with the advances in big data analysis and experimental techniques, it is hopeful that researchers will be able to broaden the field of research while working together to provide new insights and avenues to treat human diseases. There is an intriguing, but still not fully understood transcriptional mechanism between ACs and macrophages, which requires for further studies in the revelation of transcriptional regulation in ACs clearance and its therapeutic use.