A number of studies across different model systems revealed that chromatin undergoes significant changes in dynamics in response to DNA damage. These include local motion changes at damage sites, increased nuclear exploration of both damaged and undamaged loci, and directed motions to new nuclear locations associated with certain repair pathways. These studies also revealed the need for new analytical methods to identify directed motions in a context of mixed trajectories, and the importance of investigating nuclear dynamics over different time scales to identify diffusion regimes. Here we provide an overview of the current understanding of this field, including imaging and analytical methods developed to investigate nuclear dynamics in different contexts. These dynamics are essential for genome integrity. Identifying the molecular mechanisms responsible for these movements is key to understanding how their misregulation contributes to cancer and other genome instability disorders. Drosophila cells  repair of double-strand breaks (DSBs) . First, la cells  , and on monitoring repair sites with mponents .SceI, or HO), which recognizes a target sequence proximal to tetO or lacO arrays (foci)  . Given ts (foci) . In yeass (foci) , unrepais (foci) , sub-tels (foci) , and rDNs (foci) . Similars (foci) . A variaged loci . These sged loci  revealedged loci . Here, rged loci . These aged loci , nuclearged loci , and theged loci  , and detecting repair sites using fluorescent-tagged HR components . This hala cells . A majorla cells . Furtherla cells , b. A pola cells .Alternative approaches employed lasers, alpha-particles, or heavy ions to induce damage along linear tracks in the nucleus of mammalian cells , and repThus, several approaches have been developed to characterize the dynamics of repair foci. The preferred method depends on the type of question and the features of the motion under investigation.A traditional approach to characterize the dynamics of damaged DNA is the mean-square displacement (MSD) analysis of the positional data of repair sites  the coordinates of the locus in 3-dimensions, and Δt the time interval of the acquisition. MSDs are typically calculated for several trajectories across distinct nuclei, and averaged to extract a time-ensemble-averaged MSD. The data are then fitted to a curve to characterize the type of diffusion. In the following sections, we present different models used in the literature to fit averaged MSD curves.where When a molecule freely diffuses, its MSD curve is linear at increased time intervals and its motion is called Brownian . In thisd is the dimension of the movement, D is the diffusion coefficient of the locus, and Δt is the time interval. The term 2dDΔt is the theoretical MSD for Brownian motion in the absence of any experimental noise. However, when measuring the position of a molecule in living cells, the experimental location accuracy can strongly affect the experimental MSD. The error in location for molecules detected by live imaging can be divided into two components (where mponents :•Error in the determination of the position due to convolution of the sample with the point spread function (PSF). This depends on imaging conditions and microscope features . This error is higher at short acquisition times since the number of photons collected is small.•Error due to the movement of the spot during the acquisition. This error, referred as “motion blur” is higher at longer exposure times.Experimental MSD curves for Brownian motion, taking into account the location errors, can be fitted by where tExp is the exposure time of the camera.In living cells, DNA motion is often slower than Brownian and is called “sub-diffusive” . This isWhen a chromosomal locus stays confined inside a sub-volume of the nucleus, its motion is called confined sub-diffusion . The MSDR∞ is the measured plateau of the MSD, D is the diffusion coefficient of the locus and ε is the noise due to the experimental measurements. The confinement radius (Rc) of the motion is given by the relation: d is the dimension of the motion. It refers to the radius of a sphere inside which the motion is contained. The MSD curve starts to bend at time where When the force or structure that restricts the motion is not a simple confinement but is modulated in time and space with scaling properties, the motion is called anomalous sub-diffusion  Figure . In thiswhere α, the anomalous exponent, is smaller than 1.A represents the amplitude of the motion; it is proportional to the diffusion coefficient only in the case of normal diffusion (when α = 1), which is rarely observed in biological systems  chromosomal loci  before and after damage suggest that global chromatin mobilization is also conserved .Multi-scale motion section, below) and polymer simulations also suggest the importance of chromatin stiffening in local and global chromatin dynamics (What promotes the dynamics of undamaged loci in response to DSBs? Different contributing mechanisms have been identified: (i) the release of structures that anchor chromosomal loci to the nuclear periphery, (ii) repair and checkpoint proteins; (iii) the transfer of cytoplasmic forces to the chromatin through the LINC complex; and (iv) global chromatin modifications. Specifically, anchoring of centromeres, telomeres and the nucleolus to the nuclear envelope provides constraints to the motion of interphase chromosomes in budding yeast, limiting chromosome dynamics . Releasidynamics . This isOf note, studies in yeast revealed that increased nuclear exploration does not correlate with higher speed of locus movement. In fact, the diffusion coefficient does not significantly change in response to damage, both at damaged and undamaged loci . In otheOverall, MSD analyses have been an invaluable tool for identifying damage-induced nuclear dynamics, revealing a significant increase of nuclear exploration in response to DSBs for both damaged and undamaged chromatin, and identifying several molecular mechanisms responsible for these dynamics.micro, in a region that itself moves with a diffusion coefficient Amacro . This is in part to compensate for the location measurement errors mentioned above, and in part to enable the use of relatively short trajectories limited by photo-bleaching and photo-toxicity effects. However, averaging the behavior of several trajectories affects the ability to detect the differences between them, i.e., it does not account for heterogeneity across different cells and break sites. Second, MSD calculations assume that each site undergoes homogenous motion during the time of acquisition, which is rarely the case. For example: (i) repair sites can be transiently bound to the nuclear periphery or other nuclear structures; (ii) their motion can be different inside or outside phase separated domains; and (iii) directed motions can occur for limited time periods , left. At Amacro , right. t Amacro  and Chint Amacro  showed tAccordingly, simulations of a particle moving with different types of motions: confined, directed, and a combination of confined and directed (mixed trajectory), show how MSD curves can mask the presence of directed motions –S3 Bach. The simIn the following sections, we will illustrate two major types of complex motions occurring in response to DNA damage , and we A major question in the field of nuclear dynamics is whether repair focus motion is driven by active forces, or alternatively subdiffusive motions followed by anchoring to subnuclear structures are sufficient to generate these dynamics. Recent studies revealed the existence of directed motions in a context of mixed trajectories for at least some damage-induced responses  and myosins  in revisiae  , nuclear flows, and phase separation of nuclear domains, add complexity to the trajectories. Additionally, distinct diffusion regimes typically occur at different time scales, likely reflecting different level of chromatin organization. A simple MSD analysis is not adapted for such composite motions, as it assumes a homogenous mode of diffusion during the acquisition. Additionally, time-ensemble MSD analyses mix different type of motions that start asynchronously and occur for different durations. New analytical methods enabled the dissection of some of these dynamics. To reveal the existence of several diffusion regimes, multi-scale tracking, simulations, and mathematical models of complex motions need to be performed. The identification of several contexts where nuclear dynamics is dependent on nuclear actin filaments or microtubules, and characterized by short or long tracts of directed motions, suggests the existence of forces that drive the motion in more situations than initially thought. The development of new dedicated analytical methods started unlocking the door toward a deeper understanding of these dynamics, and the discovery of the molecular mechanisms responsible for their regulation. Nuclear dynamics facilitate DNA repair in different contexts, but nuclear exploration of damaged sequences is also responsible for chromosome rearrangements (A large number of studies in the past decade have shown that DSBs trigger a larger exploration of the nucleus for damaged and undamaged chromatin sites, and this response is conserved from yeast to mammalian cells. Increasing chromatin confinement radius, or changing the nature of its motion, dramatically enhances the ability of a locus to sample neighboring DNA sequences during homology search. In addition to this response, recent studies have shown that chromatin motion is more complex than initially anticipated. Relocalization of repair sites ngements . Defectsngements  (reviewengements , and estBoth authors equally contributed to this review and approved the submitted version.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.


Low-income and middle-income countries are aligning their National Action Plans on antimicrobial resistance with WHO’s 2015 Global Action Plan.Regulation is a key tool for operationalising national standards aimed at optimising the use of antimicrobial medicines.On its own, the traditional command-and-control approach to regulation is poorly suited to this challenge.‘Smart regulation’ can be used to supplement, fine-tune and improve on more traditional regulatory approaches.Antimicrobial resistance (AMR) contributes to over 700 000 annual deaths globally. According to the O’Neill report, an estimated 10 million lives a year and a cumulative US$100 trillion of economic output will be at risk by 2050 if we do not put in place proactive solutions now. Tackling AMR is a multifaceted task that requires a One Health approach encompassing human, animal and environmental health as suggested in the WHO’s 2015 Global Action Plan (GAP) on AMR. Countries around the world have aligned their National Action Plans (NAPs) on AMR with this international guidance.One of the important links for various activities for AMR containment is the appropriate use of antibiotics to reduce selection pressure on microbes. In low-income and middle-income countries (LMICs), however, antibiotic consumption rates have been converging towards (and in some countries surpassing) levels typically observed in high-income countries.WHO’s GAP identified regulation as a key tool for ensuring the operationalisation of national standards to optimise the use of antimicrobial medicines in human and animal health. More recently, the United Nations Inter-Agency Coordination Group on AMR also recommended the development, maintenance, strengthening and implementation of AMR regulation for human, animal and environmental health.Traditionally, the term ‘regulation’ refers to the top-down command-and-control model of regulation, which imposes standards and sanctions in case of non-compliance with the standards.When viewed from a traditional regulatory perspective, legislators can solve the problem of gaps in the law by formulating appropriate regulatory standards. Once appropriate laws are on the statute books, non-compliance can be addressed through measures including greater investment in regulatory infrastructure, increasing the numbers of trained regulators and resolving instances of confusion, overlap or fragmentation of responsibilities among the array of regulatory bodies charged with enforcing the laws. Although these ways of bolstering the traditional regulatory approach are indeed valuable and necessary, they may not be sufficient to address the complex challenges at hand.One problem with the traditional regulatory perspective is that it envisages a top-down relationship between the regulator and the regulated entity. It often does not allow space for meaningful engagement between the two, or with the range of other actors who directly or indirectly influence and/or are affected by regulation. This is problematic, particularly in LMICs, as regulations may be drafted without fully appreciating situated interests and how socioeconomic and structural factors underpin behaviour. The regulatory standards that emerge may therefore fail to secure widespread support. Private stakeholders may resist or actively contest regulatory policies if these are perceived as threatening their interests.et al coined the term ‘smart regulation’ ‘to overcome the inefficiencies of traditional regulation on the one hand, and the pitfalls of deregulation on the other’.Gunningham Designing smart regulation involves two stages. First, the policymakers should identify the desired policy goal(s) and the trade-offs necessary to achieve it, the unique characteristics of the problem, the range of potential actors and instruments and opportunities for consultation and public participation. Then, they should apply five enabling core regulatory design principles sequentially to arrive at solutions .Box 1Incorporate a broad range of complementary instruments and actors.Prefer least interventionist (prescriptive or coercive) but viable instruments.Create a ‘regulatory enforcement pyramid’ that comprises three faces representing first parties (government), second parties (business) and third parties  and set out progressively serious penalties for non-compliance across several instruments and different faces.Empower second and third parties to act as surrogate regulators.Optimise the opportunity for win-win outcomes.et al originally developed smart regulation in the context of environmental regulation, but they recognised that the implications of their research could extend to other domains. Subsequently, scholars have considered the application of smart regulation to other fields, including e-waste,Gunningham To illustrate, we discuss how the above five core regulatory design principles of the smart regulation approach might be applied in practice, using OTC sales of antibiotics without a valid prescription (which is rampant in LMICs) as an example. For the first principle, a broad range of instruments and actors could be involved in the design and implementation of regulation. Actors could include government/regulators, pharmacists, associations of pharmacists, consumers, representatives of consumers, non-governmental organisations (NGOs), area resident welfare associations, community/religious leaders, local government bodies, pharmaceutical companies and their sales representatives, importers, wholesalers and distributors of antibiotics. In terms of instruments, traditional regulation, voluntary codes of conduct, awareness/information campaigns and the use of technology could all be considered. For the second design principle, the least interventionist but viable instrument could take the form of education and awareness instead of immediate imposition of a penalty. For the third principle, the regulatory enforcement pyramid could comprise the government/regulator (first face), pharmacists and associations of pharmacists (second face) and consumers, representatives of consumers, NGOs, area resident welfare associations, community/religious leaders, local government bodies, pharmaceutical companies and their sales representatives, importers and wholesalers and distributors of antibiotics (third face). Progressively serious penalties for non-compliance could be set out across several instruments and different faces. This could take the form of: education and awareness, then a warning, followed by a second warning and a nominal fine, then temporary closure of pharmacy and a higher fine, then ultimately imprisonment. For the fourth principle, surrogate regulators could include the actors represented in the second and third faces, harnessing their ability to implement forms of regulation including self-regulation. Fifth, all the above-mentioned actors could work together to explore and optimise opportunities for win-win opportunities. In this case, this could mean attempting to satisfy public health interests  alongside ensuring that the economic interests of pharmacists as business owners and members of the community are also supported.Some existing regulatory arrangements and policy proposalsSmart regulation utilises the existing institutional, legal and governance framework, including the traditional command-and-control approach, but also expands options for regulators. It could encourage behaviour change, incentivise regulatory compliance and ensure the most efficient and effective application of the resources of different actors as well as greater acceptance and smoother implementation of regulatory instruments. By taking into account the consequences of regulation on different actors from the outset, it might also be possible to avoid or mitigate problems arising from the unintended consequences of AMR regulation. This would also represent an advance from the perspective of equity, which is critical in LMICs.Of course, the successful operationalisation of smart regulation of AMR would be contingent, among other factors, on sequential application of all of the core regulatory design principles, including ensuring the complementarity of actors and instruments and viability of instruments, as well as effective monitoring mechanisms to avoid unintended consequences, in addition to avoiding regulatory capture. With awareness of these issues, LMICs can harness the advantages offered by smart regulation to develop more efficient, workable and effective regulatory frameworks for tackling AMR.


The extent of tobacco cultivation remains substantially high in Bangladesh, which is the 12th largest tobacco producer in the world. Using data from a household survey of current, former, and never tobacco farmers, based on a multi-stage stratified sampling design with a mix of purposive and random sampling of households, this study estimated the financial and economic profitability per acre of land used for tobacco cultivation. The environmental effects of tobacco cultivation on land and water resources were estimated using laboratory tests of sample water and soil collected from tobacco-cultivating and non-tobacco cultivating areas. The study finds that tobacco cultivation turns into a losing concern when the opportunity costs of unpaid family labour and other owned resources, and the health effects of tobacco cultivation are included. Tobacco cultivation poses a significantly high environmental cost that causes a net loss to society. Nevertheless, the availability of unpaid family labour and the options of advanced credit as well as a buy back guarantee from the tobacco companies attract farmers to engage in and continue tobacco cultivation. Therefore, supply side interventions to curb the tobacco epidemic in Bangladesh need to address major drivers of tobacco cultivation to correct the wrong incentives and motivate tobacco farmers to switch to alternative livelihood options. Tobacco use is one of the major risk factors for noncommunicable diseases. About 46.0% of adult males and 25.2% of adult females in Bangladesh use tobacco . In 2018To meet the unyielding demand for tobacco leaf from both domestic and foreign manufacturers of tobacco products, the extent of tobacco cultivation remains considerably high. Bangladesh is the 12th largest tobacco producing country in the world ,4,5. TobThe tobacco industry publicly makes the claim that tobacco cultivation generates gainful employment and income for tobacco farmers, while global evidence suggests otherwise. Tobacco cultivation is highly labour intensive, as evident in similar studies conducted for Kenya , IndonesAlong with generating health hazards for tobacco growers, tobacco cultivation leaves significant detrimental effect on the surrounding environment, including deforestation resulting from the demand for wood to cure tobacco leaves , degradaEven with so many negative effects, tobacco cultivation is keeping its pace in Bangladesh, partly because, like in many other countries, tobacco farmers in Bangladesh are provided with soft loans, inputs, and forward prices by the tobacco industry for their produce. Poor farmers, constrained by cash and both fixed and working capital, are easily lured by these incentives for their survival. Furthermore, tobacco is considered a normal agricultural good and a cash crop and therefore it enjoys almost all sorts of government subsidies such as lower electricity tariff for irrigation and support meant for poor and marginal farmers. As a result, the private cost of tobacco farming in many instances remains understated. If we account for all the social costs , the total gains over the competing crop production will wither away. In a country with limited land resources and food deficit, the alternative use of land for anything other than essential and staple crops tends to threaten the long-term food security of the nation.Tobacco farmers are encouraged by the tobacco industry to continue and expand tobacco cultivation by offering various incentives including loan and buy back guarantee. On one hand, farmers become entrapped in a vicious cycle of debt, and on the other hand, they consider tobacco farming profitable without taking into consideration the private costs, including unpaid family labour, health hazards caused by tobacco handling as well as the social costs, including environmental damage to soil, water, and forest resources imposed by tobacco cultivation.Existing scientific studies have, however, not considered the harmful effects of tobacco cultivation on the environment. The exclusion of environmental cost from the calculation of the costs of tobacco cultivation has resulted in incomplete assessment of the true cost to society. The present study fills this gap by measuring the environmental costs of tobacco cultivation, as well as the implicit costs , alongside the accounting costs of cultivation in a quasi- experimental set up.Tobacco farming is more popular among farmers in the lowest socio-economic status . MeasuriDrope et al.  conducteSimilar types of studies were conducted in Zambia  and KenyAll these three studies mentioned are very extensive in terms of exploration of tobacco farmers’ livelihood and their characteristics. However, one major limitation of these studies is that they compared the economic costs of current and former tobacco farmers in the tobacco-growing areas without consideration of never tobacco farmers from either tobacco-growing or non-tobacco growing areas as a control group. It is possible that the regions from where data were collected might be responsible for low income from tobacco cultivation. Unless we know what the counterfactual would have been with cultivation of other non-tobacco crops by never tobacco farmers, the estimates of the economic costs of tobacco farming might be biased. The present study seeks to correct this bias. The study on Indonesia overcomes this limitation to some extent by comparing the economic costs between current and former tobacco farmers. Moreover, even though the studies mention the detrimental effects on health and environment, limited effort has been made in estimating those effects. Therefore, these studies are confined to the direct and indirect private costs and benefits only. One previous study conducted by Akter  was veryTo support policy makers to adopt appropriate tobacco control measures, reliable information on the economic costs of tobacco including environmental costs of tobacco farming is crucial. The WHO Framework Convention on Tobacco Control (FCTC), through article 18, calls for the protection of environment and health of persons involved in tobacco cultivation, and through article 17, provides emphasis on alternative livelihoods to tobacco farmers and workers . BangladThis is a cross-sectional study that included tobacco farmer households (treatment group) classified by tobacco farming status, such as current and former tobacco farmers and never tobacco farmers (control group), from both tobacco and non-tobacco cultivating areas. The study was undertaken by the Bureau of Economic Research of the University of Dhaka; which received funding and technical support from the World Health Organization (WHO) and technical support from the American Cancer Society (ACS). The primary data were collected from a multi-stage stratified sampling design with a mix of purposive and random sampling of households of current, former and never tobacco farmers using a structured questionnaire through face-to-face interviews by trained interviewers. The samples of current and former tobacco farmers were selected from two purposively selected major tobacco growing Upazilas (lowest level of administrative unit) in each of four purposively selected major tobacco-growing districts . The sample of never tobacco farmers was selected from both the above tobacco farming Upazilas and four non-tobacco farming Upazilas randomly selected from four randomly selected non-tobacco farming districts . The tobacco-growing areas, level of production, and study sample areas are shown in the maps of Bangladesh in Inclusion of both tobacco and non-tobacco growing areas gave us the opportunity of examining the differences in costs between the treatment and the control groups situated within tobacco growing areas and beyond it. It allowed us to isolate the external effects of being in the neighbourhood of tobacco growing areas for never tobacco farmers. For example, the contamination of soil and water in the tobacco growing areas can raise the cost of production and reduce the return of growing other crops in the tobacco growing areas for farmers regardless of their tobacco farming status.Under the overall supervision of the principal investigator and the co-investigators, and the direct supervision of the field coordinator, interviewers collected data from households, with support from the local health authorities. The head of the family or any other adult family member having enough knowledge on tobacco/paddy farming and marketing by his/her family was interviewed from each household. Only paddy growing was taken into consideration as the principal economic activity of the control group of never tobacco farmers. The interviewers visited each selected household three times to complete the interview before declaring it ‘not completed’.n = required sample size; z = 1.96 ; σ =population standard deviation = BDT 15,000; d = margin of error (2.5% of mean profit) = BDT 1500. Therefore, n = (1.96 × 1.96) × /(1500 × 1500) = 384. Adding 20% nonresponse, n = (384/0.8) = 480. Therefore, the study targeted interviewing 480 current tobacco farmers from 480 tobacco farming households. Similarly, 480 former tobacco farmers and 480 never tobacco farmers were planned for interview from the tobacco-growing areas.As the objective of the study was to estimate the cost and return to tobacco cultivation, we used available data on profit made by tobacco farmers to calculate the sample size of farmer households that would be sufficient to estimate the population mean of annual profit with precision. On average, a tobacco farmer makes a profit of BDT 60,000 annually from harvesting one hectare of land . The samWhen we consider tobacco farmers, former tobacco farmers and never tobacco farmers as mutually exclusive groups, then 480 households from each group might appear to be a reasonable sample size. However, upon piloting, we realized that former and never tobacco farmers possessed similar characteristics, and so collecting a substantial number of samples from former tobacco and never tobacco farmers will not add much value in terms of precision. Rather, having more samples from the current tobacco farmers appeared to be more useful. Therefore, we decided to increase the sample size for the current tobacco farmer group keeping total samples of all groups very much like the initial plan. In addition, 240 samples of never tobacco farmers from the non-tobacco-growing areas were selected for interview. The final total sample size, after allowing for data validation and cleaning, was 1549 comprising 625 current, 327 former and 597 never tobacco farmers. The detailed distribution of final sample size by districts is provided below .The survey collected two sets of data. First, household level data were collected on demographics, socio-economic profile of respondents, data related to farming including resource endowments, land allocation, labour allocation, other technological and management decisions, costs related to farming including labour costs, other factor prices, environmental factors , asset distribution of the farmers, area of tobacco and paddy farming land, condition of land, loan facility, input requirement, trend in input use, tobacco processing related data, such as wood, labour, price of raw tobacco, finished tobacco, and profitability of tobacco and paddy cultivation.Second, to understand the impact of tobacco cultivation on soil and water quality, soil and water samples were collected from both tobacco-cultivating (treatment) and non-tobacco-cultivating (control) areas by trained collectors of specimen suitable for laboratory analysis. The soil and water samples were tested in scientific laboratories of the Department of Soil Science and Pharmaceutical Technology of the University of Dhaka to evaluate the impact of tobacco cultivation on the chemical composition of soil and water in tobacco cultivating areas.The primary data were supplemented with secondary data collected from documents published by the Government agencies, including Bangladesh Bureau of Statistics (BBS) and Ministry of Agriculture, and various studies related to the environmental effects of cultivation on air, water, and soil quality.The study estimated the profitability of tobacco cultivation per acre of land in three stages. First, the financial profit per acre was estimated by using the difference of the sales revenue of tobacco leaf and the costs of explicit inputs used in tobacco cultivation. Second, the implicit costs of tobacco cultivation, such as the opportunity costs of unpaid family labour, own land and fuelwood from own sources, were estimated and subtracted from the estimated financial profit to obtain the estimate of private economic profit per acre. Finally, other indirect costs, including healthcare costs, costs of environmental impact through carbon emission from wood burning, deforestation, and sequestration, and detrimental effects of pesticide and chemical fertilizer on soil and water bodies in tobacco cultivating areas were estimated and subtracted from the private economic profit to obtain the estimate of social profit per acre.We estimated the total wood required to process different types of tobacco in Bangladesh. The total wood required was then converted to equivalent measure of deforestation, and the magnitude of deforestation was converted into dollar value. The calculation of solid wood required for tobacco processing is based on the following formula: C = F × H × M, where C is the quantity of firewood used in tons per year, F is the quantity of firewood in tons required to cure one metric ton of tobacco leaves, H is the average production of tobacco leaves in tons per hectare of tobacco cultivated land, M is the total number of hectares of tobacco cultivated land . Then, C3). Due to irregular gaps and air spaces, only approximately 60–70% of the volume is made up of solid wood, so that the weight of wood in one stack will range from approximately 250–600 kg. This translates into a mean stacking factor of 425 (kg) or 0.43 (tonnes). An equivalent ratio of 2.33 t could thus be used to convert solid wood into a stack of wood [A stack of wood is one metre long by one metre wide by one metre high, giving a total volume of one (stacked) cubic metre (stm3 /ha per year (mean = 15 m3 /ha) [The next step is to translate the annual amount of solid wood required (in tonnes and in cubic meter) into the equivalent area of woody biomass needed (in hectares) and either managed on a sustainable basis using the mean annual increment (MAI) approach or deforested using the growing stock (GS) approach. MAI means the annual increase in the aggregate volume of trees, commonly expressed in solid volume per hectare. It is often used to indicate the yield, since it represents the long-term sustainable quantity of wood which can be harvested. MAI specifications have fixed range and are dependent on the temperate plantation and tropical plantation. The tropical plantations normally give an MAI in the range of 6–24 m m3 /ha) . Using t3 of pine wood per hectare. Deforestation means complete removal of the natural woody biomass (depletion of growing stock). GS specifications are fixed and dependent on the ecosystem of a continent. Using GS values as specified for forest and woodlands, the wooded area needed to be completely removed (deforested) was calculated.The growing stock (GS) of woody biomass is the (solid) volume of wood standing on a given area such as one hectare (0.01 square km). The estimated coefficient of woody mass per hectare of land has a range. For example, pine forest has 85–120 MOnce the total equivalent deforestation area in hectares was obtained, the carbon sequestration approach was used to estimate the environment related costs. The carbon sequestration method estimates the emission of carbon to the environment from burning the wood, and dissipation of carbon from soil due to deforestation. Firstly, it is standard that about 50% of biomass is the amount of carbon emitted from burning wood. From the data already available on total amount of wood burnt annually in metric tonnes, the amount of carbon emitted was calculated.2 from soil are 975 t/hectare from bare-land and 868 t/hectare from arable land in 20 years. Using the total equivalent area of deforestation obtained in the previous step, we calculated the total carbon as CO2 emitted from soil due to deforestation caused by tobacco cultivation.Secondly, sequestration is the stock of organic carbon stored in the soil. Carbon is emitted naturally from the soil and it is accelerated due to deforestation. It is found that the standard rates of emission of carbon as COAdding the amount of carbon emitted due to burning of firewood and the amount of carbon emitted due to deforestation, the total amount of carbon emitted was calculated. There is substantial variation in the value of environmental costs of emission ranging from USD 100 to 1000 . HoweverIn Bangladesh, tobacco cultivation mainly replaces other essential crops like paddy. The deforestation, therefore, mainly takes place to cure and process tobacco leaves, and therefore land cleared of forest to grow tobacco was not considered.There is no established study or technique for estimating the cost of topsoil contamination and water degradation attributable to tobacco cultivation. This study is the first of its kind to quantify the cost of damage of water quality and soil quality by tobacco farming. We measured the degree of contamination due to use of pesticides and fertilizers as well as the depletion of nutrients due to tobacco cultivation. Based on information from secondary sources, we assessed the total cost to restore the soil and water condition in tobacco cultivating areas to the level in the control areas (paddy cultivating area). For that purpose, 80 soil samples, 80 surface water samples, and 32 ground water samples (from tube well) were collected from tobacco growing fields and nearby water bodies of 16 tobacco growing Upazilas. Further, 72 soil samples, 72 surface water samples and 24 ground water samples (from tube well) were collected from paddy growing fields and nearby waterbodies of 24 non-tobacco growing Upazilas.+), organic carbon (OC%), magnesium (Mg), nickel (Ni), zinc (Zn), copper (Cu), aldicard, chlorpyrifos, and 1,3-D .Soil samples were collected from 0–15 centimetres (topsoil) in clean polythene bags, properly labelled, and taken to the labouratory for analysis. Topsoil has the highest concentration of organic matter and microorganisms and is where most of the biological soil activity occurs. The quality of soil from tobacco cultivation and control areas, in terms of mineral particles, organic matters, and pesticides, was compared. The variables used to assess the soil quality include pH Level, phosphate (PO4), total nitrogen (N%)/nitrate (NO3), exchangeable potassium  and tube wells (ground water) of tobacco farming and control areas to check its physical, chemical and biological quality.The variables used to assess the water quality included water clarity , dissolved oxygen (to be measured by DO meter), water temperature, pH, specific conductance , nitrates (NO3), phosphorus (P), potassium (K), aldicard, chlorpyrifos, and 1,3-D .To come up with an estimate of the damage to soil and water from tobacco cultivation, we used secondary sources to get the parameters to restore the soil and water quality to the level observed in the control areas. We also estimated the loss of fisheries and faunas due to water contamination from tobacco cultivation compared to the control areas, and the loss of grazing land by comparing livestock density in tobacco growing and control areas. Primary data collected from household survey and secondary data (on fishery and livestock) were used to obtain the estimates.Adding the soil and water quality reversal cost, the fish stock loss due to water quality degradation and the economic loss from anticipated lower production of livestock in the tobacco cultivating area, we estimated the environmental cost due to water and soil quality degradation.The financial profit made by the farmers was estimated by using the difference of the sales revenue of tobacco leaf and the costs of explicit inputs used in tobacco cultivation, including fertilizer, seed, and hired factor (labour) costs. The private economic profit from tobacco farming was estimated by the difference in financial profit and implicit costs, such as the opportunity costs of unpaid family labour, own land, and fuelwood from own sources. In case of family labour, market wage rate in tobacco farming was used to estimate the opportunity costs, and market rate of leasing cost for land was used to measure forgone opportunity costs of own land use. Similar approach was applied for estimating the implicit costs of fuelwood used from own source. The productivity lost due to sick days was used to estimate the opportunity cost of time lost due to sickness. The accounting of health care spending for treatment of illnesses was, however, beyond the scope of this study.The tobacco farming-attributable average environmental cost (TFAEC) per acre was estimated using the following model:CECFWB = average cost of carbon dioxide emission from wood burningCSCCFDF = average cost of carbon sequestration from deforestationCSQR = average cost of soil quality reversalCWQR = average cost of water quality reversalLFF = average cost of loss of fish and faunaThen, combining all costs, both direct  and indirect , we finally estimated the social cost of tobacco farming per acre of land. Subtracting this cost from the revenue earned by tobacco farming per acre resulted in social profit or the net economic gain of tobacco farming per acre.The private economic profit from tobacco farming was compared with that of paddy cultivation, considering that paddy would have been farmed had tobacco been not grown in the available land. This provided an opportunity to estimate the opportunity cost of tobacco farming over paddy cultivation or the gain from shifting from tobacco cultivation to other major crops.The study used the Cashpor housing index (CHI) to determine the socio-economic status (SES) of the respondents. CHI is believed to be one of the cost-effective ways for identifying the poor. It uses housing condition to understand the socio-economic status of the respondents. Since low income households spend substantial amounts of household resources on daily necessities and other social obligations (such as weddings of children), they do not have enough saving to invest in improving housing conditions. Though a good housing condition helps to increase social status, not being able to invest in housing indicates that the residents are poor. This index is widely used in the context of developing countries .Current tobacco farmers (contract and independent), former tobacco farmers, and never tobacco farmers (both from tobacco farming and non-tobacco farming districts) possess very similar characteristics in terms of maximum level of education of household members, household size and primary occupation . The aveBased on the Cashpor index, 32% of the current tobacco farmers fall under the high SES category, while 35% of never tobacco farmers belong to this group . HoweverThe average size of total landholding of the current tobacco farmers  is lower than the never tobacco farmers , but higher than the former tobacco farmers  . Out of While the proportion of cultivation in own land is similar (50% vs. 52%) between the contract and independent tobacco farmers, some noticeable variations exist in the proportion of land under sharecropping (44% vs. 20%) and cultivation with lease (5.57% vs. 28%) between these two groups . The shaCurrent tobacco farmers have lower household monthly income and higher household monthly expenses and assets compared to the never tobacco farmers . The aveEven though the number of work hours per day, number of days per month and the number of months per year in non-farm employment do not vary significantly across current, former and never tobacco farmers, the total annual work hours is significantly higher for former and never tobacco farmers in non-tobacco growing areas compared to current tobacco farmers. The high labour intensity of tobacco farming may limit the hours that tobacco farming households can allocate to other non-farm income generating activities. As shown in the The cost of inputs, namely fertilizers, pesticides, and other raw materials, is significantly higher for tobacco farmers than the costs of inputs for non-tobacco farmers . MoreoveIn tobacco cultivation, especially during curing leaves, farmers must work for weeks at a stretch, and so the use of family labour is very common. This study finds substantial use of family labour for tobacco cultivation. While yearly family labour hours used by never tobacco farmers is 934, it is 722 h more for tobacco cultivation, and for contract tobacco farmers the difference is even higher . The useIn the last few years, the price of paddy especially during harvest times was low. Many marginal farmers usually sell the paddy during that season and sales revenue could be highly sensitive to prices. The negative profit for never tobacco farmers in non-tobacco growing areas is likely subject to this seasonal fluctuation at the time of the survey. In addition, we do not have counter-factual data for that area so there is no way to know what the profit would have been if tobacco were cultivated in that area. Therefore, a comparison of non-tobacco farmers of tobacco areas with tobacco farmers of those same areas appears to be more reasonable.Tobacco cultivation not only requires extensive labour of household members, it also exposes them to health hazards from handling tobacco. https://www.reference.com/science/much-cubic-foot-soil-weigh-7b509c02c5101291). One bigha is equivalent to 14,400 square feet or one-third acre). Since 6″ depth of soil is considered for treatment, the total soil amount per bigha to be treated is 0.5 × 14,400 cft. The cost of treatment is 0.0002 dollar per kg of soil. The cost per bigha is equal to /2.2 = BDT 5498 .Various forms of contamination were observed in water and the level of contamination was higher for water collected from the water bodies adjacent to tobacco cultivating land. Various forms of soil contamination were also evident in tobacco cultivating land. The most important parameter significantly higher in tobacco cultivating land was the presence of toxic aldicarb. A neutralization process of aldicarb is proposed with a cost of USD 0.0002 per kg of soil. We estimated the cost of treatment of soil to revert the soil quality to the aldicarb level of other crops. The cost per bigha (approximately one-third acre) was estimated at BDT 5498  per acre of land used for tobacco cultivation. The steps involved in the estimation are illustrated in As shown in the All the cost differences among various groups presented in the previous tables are descriptive. Therefore, these could have been tagged as sampling variations. However, we have also applied statistical tests (t-tests) to verify whether that is the case. As shown in The top three reasons why former tobacco farmers discontinued tobacco cultivation were extensive labour requirements, the availability of alternative livelihood options, and negative effects on land fertility . While aBoth tobacco and non-tobacco farmers believed that tobacco cultivation requires relatively more chemical fertilizers compared to other crops. In addition, tobacco plants attract more pests and insects and therefore larger amounts of chemical pesticides are being used for tobacco cultivation. Despite such knowledge and perceptions, some farmers continue to grow tobacco. Based on the responses of tobacco farmers on what motivate them to continue to grow tobacco, the key drivers appeared to be easy access to market and high profitability that topped the list of the number one influencing factor . Thus, iWith a well-designed survey and analytical approach, the current study fills a significant knowledge gap in measuring the economic costs of tobacco cultivation. The study provides concrete evidence against the most-circulated myth of prosperity offered in tobacco cultivation. Most importantly, the study finds that tobacco cultivation is not a profitable undertaking for farmers if the opportunity costs of unpaid family labour and own land and other resources are taken into consideration. Even though the financial profit from tobacco cultivation is found to be positive, it is much lower than that gained from alternative crops. Furthermore, when the environmental costs posed by tobacco cultivation to water, soil, and forest resources are considered, the broader society incurs a huge net loss from tobacco cultivation.Tobacco cultivation contributes to the short- and long-term loss of soil quality, water quality, and deforestation, thus slowing down the current income of the farmer as well as increasing the life cycle cost through loss of life cycle income. This in turn affects inter-generational equity and slows down economic progress of the community dependent on tobacco cultivation for their livelihood, in both the short and the long run.Contract farming is a common feature in tobacco cultivation in Bangladesh, although the prevalence is low compared to Indonesia, Kenya, and Zambia. The low number could be due to the strict eligibility requirement of tobacco companies. Anecdotal evidence suggests that independent farmers can sell tobacco leaf through other contract farmers. Moreover, since contract famers have to employ both extensive and intensive labour hours, some tobacco farmers might be reluctant to get involved in contracts with tobacco companies.Contract famers are different from the independent farmers in many respects. They tend to spend significantly more time in farming and a significantly higher proportion of family labour is used in contract farming. Though they earn larger financial profit than the independent farmers, if opportunity costs of unpaid family labour and other own resources are taken into account, their profitability gets lower. Nevertheless, they continue contract farming due to the availability of various incentives  from tobacco companies. A similar conclusion is drawn in the studies of tobacco farming in Kenya , IndonesOne major limitation of this study is that the estimates of private and social costs and profitability were obtained per acre of land used for cultivation. In the absence of reliable data on the aggregate acreage of land used for tobacco cultivation, a national level estimate of farmer’s and society’s costs and benefits could not be obtained. However, by making the per acre estimates available, this study creates the scope for making national level estimates in the future when data on total area of tobacco cultivation, such as a proper agricultural census, becomes available. In addition, we did not cover all the diseases attributable to tobacco farming due to limited resources-time and fund-wise. As the disease-specific health cost has not been estimated, the retrospective reporting of treatment cost can potentially be an understatement. Hence, reporting that cost would be an understatement of the true cost. That is one of the limitations of the paper which calls for further research to depict the incidence of tobacco farming related diseases and disease-specific cost to get a comprehensive estimate of the health cost.Tobacco cultivation poses a significantly high environmental cost that causes a net loss to society. From a household survey of current, former, and never tobacco farmers, with laboratory tests of soil and water of the respective areas, this study estimated the financial and economic profitability, accounting for implicit costs, as well as environmental costs per acre of land used for tobacco cultivation. The study finds that it is less profitable than other crops for the farmers and turns into a losing concern when the opportunity cost of unpaid family labour and other owned resources and the health effects of tobacco cultivations are included. Nevertheless, the availability of unpaid family labour and the options of advanced credit as well as a buy back guarantee from the tobacco companies attract farmers to engage in and continue tobacco cultivation. Supply side interventions to curb the tobacco epidemic in Bangladesh need to address these factors to correct the wrong incentives and motivate tobacco farmers to switch to alternative livelihood options.


In recent years, precision medical detection techniques experienced a rapid transformation from low-throughput to high-throughput genomic sequencing, from multicell promiscuous detection to single-cell precision sequencing. The emergence of liquid biopsy technology has compensated for the many limitations of tissue biopsy, leading to a tremendous transformation in precision detection. Precision detection techniques contribute to monitoring disease development more closely, evaluating therapeutic effects more scientifically, and developing new targets and new drugs. In the future, the role of precision detection and the joint detection in epigenetics, rare gene detection, individualized targeted therapy, and multigene targeted drug combination therapy should be extensively explored. This article reviews the changes in precision medical detection technology in the era of precision medicine, as well as the development, clinical application, and future challenges of liquid biopsy. Precision medicine development should be based on accurate detection and should take into account various factors, including genetic and environmental susceptibly factors including living habits in order to identify disease etiology, pathogenesis, and therapeutic targets. The aim of precision medicine is to eventually improve the benefits of disease in diagnosis, treatment, and prevention, as well as achieve optimal personalized precision treatment . The eraGiven that different pathological tumor types have different gene mutations, precision medical detection technology has been revolutionized from the detection of mutations at a single site to multiple sites simultaneously, from low-throughput to high-throughput sequencing and from multicell hybrid detection to single-cell precision detection. It has achieved the simultaneous detection of multitarget detection of genes, which promotes the development of precision medicine.The first-generation sequencing technology (Sanger sequencing) was difficult to universalize as it was expensive and time consuming. The constant demand of the clinic led to the emergence of next-generation sequencing (NGS) that can obtain all the genetic information and provide a “real-time” snapshot of the tumor genetic landscape by combining polymerase chain reaction (PCR) with fluorescent labeling imaging technology . CurrentThe third-generation single-molecule sequencing technology has gradually developed . It compTraditional genetic testing is based on mixed-cell DNA samples. The results of this analysis represent the characteristics of most cells in the tissue or the average of the whole sample, making it impossible to characterize the intercellular differences and interpret the growth and development mechanism of the organism and the features of a small number of special cells. In recent years, single-cell sequencing technology, based on high-throughput sequencing analysis of the genome, transcriptome, and epigenetics of single cells, has allowed to analyze tumor heterogeneity and clonal evolution and to reveal the molecular mechanisms of tumor chemotherapy resistance . Single-Single-cell sequencing allows the construction of immune cell maps and the study of the tumor immune microenvironment –16. Guo Due to the continuity of spatiotemporal and dynamic evolution, tumor cells and genomic variation appear highly heterogeneous. Tumor heterogeneity is broadly classified into inter- and intratumor heterogeneity , 19. TheIn recent years, the emergence of liquid biopsy technology seems to enable the precision detection from static to dynamic. It allows to repeatedly and dynamically monitor the evolution of tumors over time and become the most powerful tool for studying intratumor heterogeneity by simple noninvasive blood sampling and analysis , 23. ThiQuantitative information can be obtained from the measurement of the mutant allele fraction (MAF), which is a reflection of the tumor burden. It is used to detect minimal residual disease (MRD) and occult metastases; it is also a high-sensitivity, high-specificity predictor in monitoring therapeutic effectiveness and relapse. Qualitative information can be sourced through the profiling of mutations, amplifications, deletions, and translocations in ctDNA. This allows the identification of genetic alterations associated with response, hence supporting decision making for personalized management . In receIn addition to single-gene detection, ctDNA-based multigene analysis techniques, such as blood TMB (bTMB) and microsatellite instability (MSI) tests, show more convenient, fast, and dynamic characteristics compared with histological detection . Recent (1) Early Diagnosis of Tumor. Early screening of tumors through screening tests is a key step in early diagnosis and treatment of tumor. Early screening tests should be highly sensitive, specific, and cost-effective. However, a concern in early detection is the predictive value of single or small sets of mutations, since cancer-associated mutations can also be found in the plasma of healthy individuals as a result of clonal hematopoiesis , and matched serum [ctDNA persistence (CP): CP1 and CP3] after alloSCT was associated with a higher 3-year cumulative recurrence rate, confirming the effectiveness of ctDNA in predicting the risk of disease recurrence [s et al.  performes et al. . In thiscurrence .DNA methylation is one of the earliest epigenetic changes observed in tumor development, and its abnormal pattern is highly specific in different tumor tissues , 42. PreCTCs are defined as cells that shed from the primary tumor site or from metastases and enter the blood circulation through the vascular or lymphatic system . Their pp = 0.25), while AR-V7-negative patients treated with ARS inhibitors had a higher OS than those treated with taxanes. It is suggested that future the research studies of CTCs should look into the analysis of macromolecules expressed by CTCs and the design of therapeutic measures to target them.CTCs can reflect the tumor tissue and can help carry out pathological diagnoses, molecular sequencing, and disease monitoring, as well as providing treatment decision making and prognosis information for the clinic through noninvasive dynamic monitoring . Scher eEVs are lipid vesicles secreted by cells that carry various proteins and nucleic acids, which can provide tumor-related information , 55. StuGenomics combined with transcriptomics or proteomic analysis extends the application of precision medicine to provide the best treatment options for cancer. The WINTHER trial  recommenAt present, there are several technical limitations in liquid biopsy. Firstly, plasma can be used as a substitute biomaterial instead of tissue to detect tumor information, but its detection sensitivity still needs to be optimized. In clinical practice, it is necessary to fully evaluate its false negative rate, and repeated liquid biopsies or tissue repeat tests should be obtained from patients with negative plasma tests. Secondly, clinical practice of ctDNA analysis lacks a standardized protocol for preanalytical sample preparation and purification. The current analytical testing procedures are complex and may lead to ctDNA degradation and cytolysis. The analytical platform for rapid purification from the blood is a difficult problem to be solved in the future. In addition, challenges in detecting CTCs include extreme rarity, fragility, and physical and phenotypic heterogeneity. Finally, the predictive value of single or small sets of mutations in early detection is limited, and joint detection methods, such as the combination of genomics and transcriptomics or proteomics, may be a new technique to improve tumor-specific detection, like the CancerSEEK platform. Although some studies have confirmed the potential of liquid biopsy to change the mode of cancer treatment, a lot of research is still required in the future to fully define its role in tumor diagnosis, monitoring, and prognosis.Accurate detection techniques promote the development of individualized cancer diagnosis and treatment in the era of precision medicine. At the same time, the demand for clinical precision diagnosis and treatment can further drive the improvement and application of precision detection technology. Precision medicine relies on a thorough understanding of tumor biology. New technologies allow us to map tumors, identify key carcinogenic drivers, and formulate treatments that specifically target tumor cells to achieve clinical benefits. In recent years, precision medical detection techniques promoted the emergence and development of new technologies, new targets, and new drugs in the times of precision medicine. This has accelerated research progress and improved the understanding of disease risk mechanisms and predictions of optimal treatment options for multiple diseases.At present, there are still many challenges in precision detection techniques and platform construction. Firstly, the results obtained from different NGS-based technologies are inconsistent, which may relate to sample preprocessing, NGS platform, and algorithm differences. Secondly, the sensitivity and accuracy of liquid biopsy need to be verified by further evidence, and it cannot yet be used for early screening and diagnosis of tumors . ThirdlyIn the future, the role of precision detection and combined detection in epigenetics, rare gene detection, individualized targeted therapy, and multigene targeted drug combination therapy should be extensively explored . The acc


The mechanisms associated with the transport of these LMW (poly)phenol metabolites across the BBB and possible transporters will be discussed. Overall, the transport of these LMW (poly)phenol metabolites is crucial to elucidate which compounds may exert direct neuroprotective effects, so it is imperative to continue dissecting their potential to cross the BBB and the mechanisms behind their permeation.The world of (poly)phenols arising from dietary sources has been significantly amplified with the discovery of low molecular weight (LMW) (poly)phenol metabolites resulting from phase I and phase II metabolism and microbiota transformations. These metabolites, which are known to reach human circulation have been studied to further explore their interesting properties, especially regarding neuroprotection. Nevertheless, once in circulation, their distribution to target tissues, such as the brain, relies on their ability to cross the blood-brain barrier (BBB), one of the most controlled barriers present in humans. This represents a key step of an underexplored journey towards the brain. Present review highlights the main findings related to the ability of LMW (poly)phenol metabolites to reach the brain, considering different studies:  Phenolic compounds and polyphenols, commonly referred to as (poly)phenols, constitute a group of small molecules widely spread mostly in plants, being found in many fruits and vegetables. Their structures having at least one hydroxyl group and one aromatic ring, together with functional groups have been linked to a huge variety of beneficial properties to human health, some of them related with brain health .Since they easily reach our organism through our daily diet, their impact on human’s health may be relevant. Therefore, to reveal their potential effects, several studies focused on understanding their routes after being ingested, the metabolism they are submitted, the distribution to reach the target tissues, and ultimately, the mechanisms behind their action. It has been established that the (poly)phenols influence the brain by modulating receptors function, interacting with neuronal signaling pathways, and by promoting the expression of proteins mainly involved in synaptic plasticity and neuronal repair . The outin vivo, where the influence of the described metabolic events over the compounds occurs.Importantly, dietary (poly)phenols are subjected to several biotransformations after oral intake. Phase I and phase II enzymes are responsible for intensive catabolism over the parent compounds both in the intestine and in the liver, leading to a broad range of (poly)phenol metabolites . MoreoveμM) than their parent compounds [Overall, colonic microbiota and liver metabolism originates a huge variety of metabolites. Some of them are unique metabolites derived from a specific parent compound   but the ompounds .in silico, in vitro, and in vivo.Concerning the effect of (poly)phenol metabolites on brain function, it is still unclear whether their effect derives from direct action on brain cells or/and an indirect mechanism by affecting peripheral and cerebrovascular blood flow . ConsideBefore emphasizing the data related to brain bioavailability of LMW (poly)phenol metabolites derived from the diet, it is important to state all possible origins of these compounds within the human body and the implication for brain function. Some of the gut microbes and host enzymes involved in the described biotransformations are responsible for the general metabolism of any chemical substance not naturally produced by or expected to be present within the human organism (xenobiotic metabolism). Therefore, it is not surprising that these LMW (poly)phenol metabolites could be obtained in humans not only through the regular diet, but also by metabolizing other ingested compounds, like residues of industrial chemicals and pharmaceuticals .In fact, many of these compounds appear described as intermediates of xenobiotics biodegradation and metabolism pathways like the bacterial aminobenzoate degradation pathway . On the Regardless of the mode of occurrence within the human body (by endogenous production or by dietary exposure to parent compounds) for some LMW (poly)phenol metabolites their presence in different human organs is already described in the human metabolome database (Table1) . MoreoveNotably, the ability of metabolites in circulation to reach the target tissues is very important when considering their final effect. Their distribution relies on their capability to cross several barriers they may encounter during their journey. Regarding the nervous system, they will face one of the most regulated and controlled barriers in the human body, the blood-brain barrier (BBB), that must be crossed to directly influence the brain .BBB is a complex and highly selective semi permeable barrier found in most of the blood vessels that irrigate the brain, acting as a border between the circulating blood and the extracellular space of the central nervous system (CNS) . It is fHaving this in mind, phenolic metabolites that reach the systemic circulation towards neuronal tissue are faced with this selective barrier that separates them from the brain. Meaning that, crossing the BBB represents a crucial step for metabolites in order to directly affect our brain.in silico, cell-based, and in vivo assays have been used to address that issue.In fact, this issue is not fully understood, and the mechanisms by which phenolic metabolites permeate the BBB are still unknown. The structure of metabolites should affect the effective uptake of these metabolites by the brain. Also, the routes used to access the brain might differ due to the structural differences of metabolites. Youdim et al showed that permeation across the BBB is associated with lipophilicity, meaning polar metabolites like sulfate and glucuronide conjugates will have reduced passage through the BBB by comparison with less polar methylated conjugated . Thus thAlthough the presence of the LMW (poly)phenols metabolites has been confirmed in circulation as mentioned, the mechanisms by which these molecules cross the cell membranes into circulation and reach target organs are still quite underexplored, especially in the case of the brain. Several types of membrane transport have been considered for (poly)phenols. For flavonoids, several mechanistic studies on their transport have been proposed, due to their molecular weight and other chemical properties like polar surface and number and position of the oxygen atoms among others discussed below. The main idea of a transport, based on passive transport, has been substituted by the concept of flavonoids transport be mediated by ATP-binding cassette transporters (ABC) and organic anion transporters (OAT)  and alsoin silico methods. Yet, a limited number of studies have been conducted on LMW (poly)phenol metabolites molecules. These methods rely on physical-chemical parameters denominated by molecular descriptors, that are intrinsic to a molecule, for example molecular weight, number of hydrogen acceptor and donor bounds, volume, dipole moment, and many others [in silico models are reported in in silico predictions projected as not probable to reach the brain. However, this molecule has been detected in the rat brain and its presence was associated with transport-mediated mechanisms [in silico predictions since are less labor-intensive, inexpensive and high throughput, are quite useful for initial information about the potential of a metabolite to cross the BBB. Initial in silico screening in a set of compounds allows refining further analysis in the more technically challenging process of obtaining detailed in vivo BBB permeability data for the most promising compounds. A good example of this rationale was the comprehensive study that was done for the flavan-3-ols metabolites, phenyl-γ-valerolactones, phenylvaleric acids, and their conjugates. This study explored through an in silico analysis the most promising molecules which were tested in a cell-based assay. Finally, the presence of the above mentioned compounds was validated in the brain in three different in vivo studies [Considering that the transport of (poly)phenol metabolites across cell membranes is known to be either through passive permeation, or active transport , this coy others . All they others . Due to chanisms . For som studies . It willIn vitro cell-based studies, mostly taking advantage of brain endothelial cellular models, have been a very important tool to address the ability of metabolites permeation across the BBB endothelium in a more realistic way. For some flavonoids and their known circulating metabolites, the permeability was tested on in vitro models of the BBB [in vivo glucuronides, as well as the anthocyanins cyanidin-3-rutinoside and pelargonidin-3-glucoside has been reported in two brain endothelial cell lines from mouse (b.END5) and rat (RBE-4). In this study flavonoids permeation across in vitro BBB mono layers was compared to the potential for permeation for their more polar glucuronidated conjugates and to specific phenolic acids derived from colonic biotransformation of flavonoids. The authors concluded that hesperetin and naringenin present a high apparent permeability [In vitro transmembrane transport of flavonols, flavan-3-ols and anthocyanins and some of their methylated and glucuronidated metabolites were also observed in the hCMEC/D3 cells [ the BBB . The upteability . Moreoveeability . In vitrD3 cells . InteresD3 cells . Overallin vivo validation [O-sulfate, pyrogallol-2-O-sulfate, 1-O-methylpyrogallol-O-sulfate, 2-O-methylpyrogallol-1-O-sulfate, vanillic acid-4-O-sulfate, 4-O-methylgallic acid, and 4-O-methylgallic acid-3-O-sulfatehave been tested for BBB permeability and all of them were detected in the abluminal side [In a recent study, the endothelium transport of some known LMW (poly)phenol metabolites, products of sulfate conjugations was addressed by using a human brain micro vascular endothelial cell (HBMEC) line . The samlidation . The BBBnal side . Howevernal side . One of nal side . Interesnal side .in vitro model of BBB (RTU), consisting of bovine endothelial cells, was used to test the potential of ferulic acid, dihydroferulic acid, and dihydrocaffeic acid to cross the BBB [–3 cm.min–1 phenol metabolites BBB permeability. In that sense, resorting to the BBB kit (RBT-24), made of primary cultures of rat brain endothelial cells, pericytes and astrocytes, the transport for gallic acid, pyrogallol and pyrogallol-1-O-glucuronoide was demonstrated . Despite the BBB . In factcm.min–1 . This mein silico predictions about the permeability of the specific molecules with available information show corroborating data between in vitro and in silico predictions. However, the number of in vitro studies is still limited to generalize this validation of the in silico data.Interestingly, Still, although the data available regarding LMW (poly)phenols BBB permeability is scarce, it has gained a lot of attention since the BBB represents the entrance door to reach the brain. Noteworthy, sometimes the goal of the research is to find methods to avoid brain penetration of toxins and potentially dangerous molecules but sometimes it is convenient to promote the entrance of drugs. An interesting branch of research has been focusing on increasing the efficiency of drug delivery with the purpose of overcoming the tight regulation characteristic of the BBB. In that sense, sinapic acid, conjugated with a zwitterionic polymer in an encapsulated bovine serum albumin-based nanoparticles, proved to be a novel bioinspired BBB-permeable ligand for delivery of cargo into the brain , which iin vivo experiments are crucial since they are the only way to fully recapitulate the complexity of the BBB. Being this a very emergent issue, several studies have been interested in confirming the brain bioavailability of (poly)phenols, having already been identified some of them in different brain regions of rats [For a full comprehension regarding the brain uptake of phenolic metabolites,  of rats  and pigs of rats  which ca of rats . Micro d of rats . The exa of rats . Also, t of rats , epicate of rats , anthocy of rats , quercet of rats  and nari of rats , were de of rats  as well  of rats . However of rats  but were of rats . For ana of rats . This wain vitro studies that can give us a wide-ranging clue about their permeation, both approaches are still limited. On the other hand, in vivo models, despite being based on rodent and pig models, are those that get closest to recapitulate the human physiology, undergoing the influence of natural processes of a living organism. This means that in animal studies the brain bioavailability of phenolic metabolites will be influenced by different cell types, enzymes, transporters, endogenous molecules, etc. mimicking in a better way the organism environment.By contrast to the dietary (poly)phenols, only a very limited number of studies have focused on LMW (poly)phenol metabolites, although some evidence can be found of their potential to cross the BBB and reach the brain. Among predictive studies, whose outcomes provide an idea about the ability of metabolites to cross the BBB endothelium by passive diffusion and in vivo models have been increasing in the literature. However the main findings attained in these studies, have different experimental designs, particularly regarding the animal model, the gender, the type of administration, the dose, and the compounds sourced, hampers an exact comparison. Within this section, we review some of these studies, highlighting the compounds that were tested to cross the BBB, the dosage, and the brain uptake, using in vivo models. An important difference in these studies, that lead to different conclusions, is the type of administration of the (poly)phenols. Compounds were sourced either by extract form or pills being administered intragastrically or orally mixed with the diet  associated with their presence. On the other hand, if the administration is directly in the blood stream it will bypass most hepatic metabolic pathways, reaching the tissues faster and making it easier to know with more accuracy the percentage that reached the brain.Although limited, evidence of the BBB capacity to uptake LMW (poly)phenol metabolites using the diet , or theythe diet . The difin vivo peaks around 6 hours post-consumption and have longer elimination half-life [vs. acute ingestion could impact the levels detected since there is no a direct comparison for the same time point of tissue collection after the last intake. Another aspect that may account for different results is the fact that these studies have used different rat strains (Sprague-Dawley vs. Wistar rats) whose endocrine system was described as different and this causes differences in food conversion and food intake [Margalef et al. have administered orally different acute doses  of grape seed (poly)phenol extract (GSPE) to the male rats . The totalf-life . Accordid intake . AltogetConcerning the presence of gallic acid in the brain, another study also using Sprague-Dawley rats, likewise has not detected this metabolite in the brain when rats were orally gavaged with acute doses of GSPE  . Only upProtocatechuic acid was detected in the brain of rats that received Danshen extract intragastrically  . AdditioAlthough not derived from colonic metabolism, metabolites derived from intake of hydroxytyrosol food sources, like virgin olive oil, are also LMW (poly)phenol metabolites. Homovanillic acid and some related metabolites detected in rodents like homovanillic acid sulfate and homovanillic alcohol sulfate, were detected in the brains of the rats 5 hours after an acute high dose of hydroxytyrosol (100 mg/Kg BW) . All thein vitro models of BBB that estrogens may play a role in modulating free flavanol uptake, and it has been further suggested that progesterone can act as an endogenous factor that modulates P-glycoprotein ability to serve as transporters of flavan-3-ol’s across the BBB [Interestingly a significant gender effect in the plasma, liver, and kidney concentrations of hydroxytyrosol metabolites was observed . However the BBB .in vivo is its direct administration to the circulation bypassing the gastrointestinal tract phenol metabolites al tract . In thatal tract . Among t14C hydroxytyrosol in Sprague-Dawley rats [In an attempt to identify circulating hydroxytyrosol metabolites and their deposition in tissues, D'Angelo, and colleagues injected intravenously radiolabelled ley rats . Althougley rats . In factley rats . All comley rats . The samley rats .Another study has addressed the penetration of catechol and pyrogallol into male mouse brain after intraperitoneal injection of 60 mg/Kg BW and 120 mg/Kg BW respectively, being both compounds detected in the brain . SimilarConcerning these data, different conclusions can be drawn if the proper interpretation is not made. There are many variables between studies regarding their experimental designs that influence the amount and type of LMW (poly)phenol metabolites detected in the brains that should be taken into consideration. From the studies herein presented, the source of (poly)phenols is quite wide and variable and even the regular diet given to the rats can be different. The time of fasting and the time after the last (poly)phenol administration and the quantification are critical parameters that generate different results. As we already highlighted, the rat strain and gender, the concentration of the compound, as well as the route and the frequency of the administration have also a strong influence on the amount and type of LMW metabolites able to reach the brain.The mechanisms by which LMW (poly)phenol metabolites cross the BBB are still quite underexplored. As stated previously, passive permeation and carrier mediated transport could constitute a potential mechanism by which these molecules could cross the BBB and potentially reach the brain. However, other mechanisms of transport are also considered, such as paracellular transport and vesicular transport . StudiesOther types of transport may also be considered, especially in the disease context. These include vesicular transcytosis and paracellular transport that are strictly regulated in the BBB and are a less probable method for these types of compounds to reach the brain. Nevertheless, it has been proven that permeation through these two methods can be compromised in diseases within which inflammation occurs leading to an increase permeability of BBB to small molecules .in vitro when incubated in the presence of the human brain microvascular cells [in vivo, yet much is awaiting to be discover regarding (poly)phenol metabolism in the brain.There is still huge gap in knowledge about the fate of LMW (poly)phenol metabolites in the brain, especially due to the diversity of brain resident cells . The fatar cells . Also, sar cells . These tin silico, in vitro and in vivo methods. Understanding which are the metabolites from the dietary (poly)phenols that can in fact cross the BBB and reach the brain is imperative to further explore their potential benefits to the brain.(Poly)phenol’s brain effects research reached a moment where it is crucial to embrace and clarify the discovery of which metabolites are reaching the brain. Several approaches have been used to understand this potential ranging from in silico predictions of brain permeability were validated in in vivo studies. Also, the predicted inability to cross the BBB by sinapic acid could reflect its absence in the rat brain [In silico models only reflect passive permeation and other possibilities should also be consider in vivo. Also the conclusions made from in vivo studies should be take with precaution, different conclusions about the percentage of brain permeability of (poly)phenols can be drawn if the proper interpretation is not made. There are many variables between studies regarding their experimental designs that influence the amount and type of LMW (poly)phenol metabolites detected in the brains that should be taken into consideration. The source of (poly)phenols is quite wide and variable and even the regular diet given to the rats can differ. Food complexity and interactions among the different components can affect the release of the compounds from the matrix and the uptake leading to different pharmacokinetic profiles. Moreover, the experimental design concerning the time of collecting the tissues for analysis after the last (poly)phenol administration will define the metabolites that will be detected and quantified in the brain. Additionally, as already highlighted, the rat strain, the concentration, the frequency and the route of the administration have also a strong influence on the amount and type of LMW (poly)phenol metabolites able to reach the brain. Sex differences may have also an important impact and it is a factor that most studies do not account. (Poly)phenols that target the brain are probably the physiologically active forms.For some metabolites  the at brain . But thiat brain . In siliUnderstanding the mechanisms by which LMW (poly)phenol metabolites reach the brain will also impact the development of (poly)phenol derivatives as potential drugs with better pharmacokinetic properties. Another aspect, still in its infancy, is the knowledge on the potential enzymes and bio transformations that these compounds may be submitted to when entering the brain and once inside the brain. These further metabolic reactions may be decisive for their potential activity. Altogether, the presence and concentration in circulation, the percentage of crossing the BBB and the metabolism of LMW (poly)phenol metabolites is crucial if the objective is to design nutritional and pharmacological approaches to create a link between dietary factors and the prevention or treatment of neurodegenerative diseases.The authors declare no conflict of interest to report.


Human astrocytic tumors are primary central nervous system (CNS) tumors that arise either from astrocytes or from precursor cells. A growing number of epidemiological and incidence studies in different countries underlined that, in addition to increasing economic costs for health systems, these cancers are still representing one of the main hurdles in developing a successful therapeutic goal for patients. On the other hand, new-omics technologies are offering customized instruments and more and more advantageous results toward personalized medicine approaches, underlining the concept that each tumor mass undergoes a peculiar transformation process under the control of specific genes’ and proteins’ functional signatures. The main aim of this Special Issue was to collect novel contributions in the wide field of human tumor astrocytic basic and translational research, to suggest further potential therapeutic targets/strategies that might interfere, possibly at the earliest stage of transformation, with the tumor progression, and to increase the molecular-based arsenal to counteract the prognostic poverty of high-grade astrocytic tumors. Malignant glial tumors  are, after cerebral stroke, the second cause of death in CNS diseases. The current standard clinical interventions are based on specifically designed combinations of tumor resection, radio-, and chemo-therapy protocols. However, at present, these therapeutic approaches do not result in significant improvement of the average survival of patients with, in the case of the highest malignant GBM, a median survival of 12–15 months. Consequently, this Special Issue has collected different multidisciplinary research articles describing novel potential therapeutic strategies against gliomas ,2, the iIn the first contribution, Bilmin and collaborators  focused In a different contribution, always on the therapeutic side, Guidotti and co-workers  dissect Gliomas are specifically characterized by the presence of hypoxic regions, especially within the tumor core, leading to an increase in vascularity. This increased vascularization is mainly driven by the expression of the major angiogenic inducer vascular endothelial growth factor (VEGF) and by the epidermal growth factor (EGF), which in turn stimulates VEGF expression. With their contribution, Nicolas and collaborators  analyze EGFR gene amplification and poor prognosis in the parental astrocytic tumors. Altogether, according to the authors, these results could have prognostic and therapeutic relevance, identifying NG2/CSPG4 as a promising tumor-associated antigen for future antibody-based immunotherapy in patients with malignant gliomas. Particularly for gliomas, novel biomarkers associated with the progression of malignancy degrees and with poor prognosis are especially required. For this reason, the group lead by Prof. Schiffer  exploit It is becoming more and more evident that the capacity of infiltration of GBM cells into the unaffected brain tissue surrounding the tumor mass is largely responsible for tumor reoccurrence, thus reflecting on the limited efficacy of current standard-of-care treatments. In relation to this important topic, Thompson and Sontheimer  perform One of the main adverse clinical features of gliomas is the high probability of tumor recurrence, mainly mediated by a cellular subpopulation with stem cells characteristics known as glioblastoma stem-like cells (GSCs) . These cDSG3, FN1, IGFBP2, CLDN1, HDAC7, and L1CAM, useful for predicting the prognosis of GBM, based on the status assessment of EMT and GSCs. As a future prospective, the authors underline the potentiality of the clustered gene expression data in evaluating the survival of glioma patients, which would help to develop novel therapeutics strategies and to provide de novo marker candidates to increase the prediction of the prognosis of high-grade gliomas.As already stated, the invasiveness of high-grade gliomas depends on their high infiltration capacity to invade the basement membranes of the surrounding brain tissues. Glioma cells are reprogrammed to have increased motility via weakened cell adhesions and through molecular mechanisms that results in a dysregulated cytoskeleton, a process overall known as epithelial–mesenchymal transition (EMT), further characterized by multiple biochemical changes that culminate in mesenchymal phenotypes . This remut/IDHwt) sub-types has provided additional criteria in a better definition of the patients’ outcome [mut GSCs lines, perform a large-scale screening of 147 Food and Drug Administration (FDA)-approved anticancer compounds that are able to promote inhibition of cell growth and induce programmed cell death processes. In particular, within their in vitro analysis, the authors identify seven promising FDA-approved drugs that should be further taken into clinical investigations for the treatment of IDHmut glioma sub-types. These drugs include the proteasome inhibitors bortezomib and carfilzomib, three anthracyclines , as well the antineoplastic antibiotic plicamycin and the protein translation inhibitor omacetaxine. As a future therapeutic perspective, the authors stress the fact that the tested drugs are already FDA approved, and, therefore, they might enable faster translation into in vivo preclinical studies and subsequent clinical trials.In relation to the prognosis of gliomas, the genetic characterization of glioma patients into isocitrate dehydrogenase (IDH) mutant and wildtype  and gliomas, specifically in the maintenance and expansion of GSC population [C, the physiological counterpart of the pathogenic prion protein (PrPSc), has become a relevant player in the oncology field. In fact, several pieces of evidence provided a pivotal role of PrPC in tumorigenesis, cancer progression, acquisition of a multidrug resistance phenotype, and metastatic propagation, even in glial tumors [C in particular is overexpressed in cancer stem cells (CSCs) from different tumors, including gliomas, and that this molecular signature is predictive for poor prognosis and correlates with tumor relapses. At a cellular level, the authors illustrate the possible mechanistic contribution of PrPC to multipotency, invasiveness, and tumorigenicity features of GSCs. However, due to the high number and complexity of PrPC interactors and molecular pathways, future progress is required in understanding the function of PrPC and of its paralogue prion Doppel protein  within In conclusion, I believe that the papers collected in this Special Issue, each addressing a specific aspect of glial tumor biology, will help the scientific community to better understand the complex underlying mechanisms of glioma tumorigenesis and will help to design more effective therapeutic strategies that are able to interfere with a tumor showing a very poor prognosis to date.